Crocus sativus L. (Iridaceae), or saffron, has been used as food additives and spices. In the traditional medicine of Iran, C. sativus has been used for the treatment of liver disorders, coughs, and as an anti-inflammatory agent for eyes.C. sativus stigma and its petal flavonoid (kaempferol) on respiratory disorders with several mechanisms such as anti-inflammatory, and smooth muscle relaxant effects. The current study reviewed the possible therapeutic effects of Crocus sativus’, ‘saffron’, ‘kaempferol’, ‘airway inflammation’, and ‘smooth muscle relaxant’ were searched. This review article searched databases including PubMed, Google Scholar, and ScienceDirect, up to November 2019. The keywords including; ‘C. sativus reduced nitric oxide (NO), inducible nitric oxide synthase (iNOS) levels and inflammatory cytokines in the lung tissue. Saffron and kaempferol reduced white blood cells (WBCs) and the percentage of neutrophils and eosinophils in bronchoalveolar lavage fluid. Moreover, saffron reduced tracheal responsiveness to methacholine and ovalbumin on tracheal smooth muscles. In addition, kaempferol reduced the total leukocyte and eosinophil counts similar to the effect of dexamethasone and also showed relaxant effects on smooth muscle.Crocus sativus and its petal flavonoid, kaempferol, showed relatively potent therapeutic effects on respiratory disorders by relaxation of tracheal smooth muscles via stimulatory or blocking effects on β-adrenoceptor and muscarinic receptors, respectively. Saffron and kaempferol also decreased production of NO, inflammatory cytokines and chemokines in respiratory systems. Asthma is a complex inflammatory disorder characterised by airway inflammation and hyperresponsiveness, hypersecretion of mucus by goblet cells and eosinophilic inflammation -sensitised animal model , or saffron, is commonly cultivated in Iran, Afghanistan, Turkey and Spain  with several mechanisms, such as anti-inflammatory and smooth muscle relaxant effects on inflammatory respiratory diseases.The current study reviewed the potential therapeutic effects of Crocus sativus stigma and its petal flavonoid, kaempferol on respiratory disorders were selected in PubMed, Google Scholar, and ScienceDirect, up to November 2019. The keywords including; ‘Crocus sativus’, ‘saffron’, ‘Kaempferol’, ‘airway inflammation’, and ‘smooth muscle relaxant’ were searched individually or combined.All literature that reported the effects of C. sativus  on cell viability and cytokine release of stimulated peripheral blood mononuclear cells (PBMC) by phytohemagglutinin (PHA) and non-stimulated cells significantly inhibited cell viability of lymphocytes and secretion of interferon-γ (IFN-γ) in stimulated cells and also inhibited interleukin (IL)-10 secretion in stimulated and non-stimulated cells  with crocin  for 24 and 48 h reduced cell viability at dose dependently. Crocin also significantly decreased IL-8 secretion and protein levels of TNF receptor 1 (TNFR1) by HepG2 cells at 6 and 12 h after treatment  and its constituents safranal resulted in a decrease of nitric oxide (NO), inducible nitric oxide synthase (iNOS) levels, peroxynitrite ion generation, and prevented cytochrome c release  and safranal in the OVA-induced murine model of asthma (in vitro) study, reduced iNOS levels and inflammatory cytokines such as; L-5 and IL-13 levels in the lung tissue. Airway hyper‐responsiveness and airway cellular infiltration to the lungs as well as, bronchial epithelial cell apoptosis was decreased in treated mice with C. sativus and safranal  was higher effective than dexamethasone (50 µg/mL)  on OVA-sensitised induced asthma in guinea pigs significantly reduced serum levels of endotheline-1 (ET-1) and total protein (TP) compared to untreated OVA-sensitised guinea pigs  prevented the increase in total white blood cells (WBC), eosinophil and lymphocyte numbers in OVA-sensitised animals. The extract reduced WBC count similar to dexamethasone. In addition, the low concentration of extract (0.1 mg/mL) reduced eosinophil count more than dexamethasone treatment  also significantly decreased WBC count, eosinophil percentage, neutrophil percentage, red blood cell (RBC) and platelet count in the blood of sensitised rats. Lymphocyte percentage was increased in animals receiving 100 mg/kg of saffron hydroethanol extract  remarkably reduced tracheal responsiveness, as main characteristic of obstructive respiratory diseases especially asthma, to both methacholine and OVA and also decreased serum levels of inflammatory mediators compared to untreated sensitised animals. In addition, C. sativus extract (80 mg/kg) was more effective than those of dexamethasone (10 mg/kg). These findings indicated that the extract of the plant could attenuate serum levels of inflammatory mediators as well as increase tracheal responsiveness to methacholine and OVA  reduced neuropathic pain in the chronic constriction injury (CCI) model through attenuation levels of pro-inflammatory factors including: tumour necrosis factor α (TNF-α), IL-1 β and IL-6 , on the lumbar spinal cord  intraperitoneal administrated in mice showed antinociceptive activity against acetic acid-induced writhing. Also, the plant extracts showed mild to moderate effects against acute inflammation caused by xylene in mice ear edoema. In addition, both aqueous and ethanol C. sativus extracts, showed anti-inflammatory effects by formalin-induced edoema in rat paw in induced chronic inflammation  in the treatment of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice significantly reduced the clinical symptoms in C57BL/6 mice with EAE. Also, treated mice displayed a delayed disease onset compared with control mice. Antioxidant capacity production was significantly elevated in saffron treated mice. Furthermore, mice treated with saffron reduced typical spinal cord leukocyte infiltration compared with control mice. These results suggested that saffron is effective in the prevention of symptomatic EAE by inhibition of oxidative stress and leukocyte infiltration to the central naevus system and may be potentially useful for the treatment of multiple sclerosis (MS)  twice daily compared with patients receiving a placebo in schizophrenia in which WBC count was in the normal range. Moreover, the other haematologic components and markers did not change significantly during 3 months of the study  were significantly increased in patients receiving the C. sativus,trans-crocetin  enhanced amyloid-β 42 (Aβ42) degradation in AD monocytes through the up-regulation of the lysosomal protease cathepsin B  of both healthy donors and Alzheimer’s disease (AD) subject with a constituent of These results indicated that saffron and its components may be useful in treating CNS disorders by reducing oxidative stress and reducing inflammation. Anti-inflammatory effects of saffron is summarised in Kaempferol is a polyphenolic compound isolated from the fresh flower petals of saffron  also suppressed mRNA expression of MMP-2 to restrain the migration of oral cancer cells via inhibiting the c-Jun pathway and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) phosphorylation in a dose-dependent manner  inhibited secretion of β-hexosaminidase and histamine, and reduced the production and mRNA expression of inflammatory cytokines (IL-4 and TNF-α) in immunoglobulin E (IgE)-sensitised RBL-2H3 cells. Kaempferol also inhibited (IgE)-mediated phosphorylation of phospholipase Cγ, protein kinase C (PKC), and the mitogen-activated protein kinases, extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase  decreased the levels of TNF-ɑ and IL-1β in serum of high cholesterol-fed rabbits. In addition, kaempferol respectively down-regulated mRNA and protein expression of inflammatory molecules such as E-selectin (E-sel), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and MCP-1 in the aorta of rabbits  on inflammation in human airway epithelial cells (BEAS-2B) showed kaempferol inhibited the expression of Toll-like receptor 4 (TLR4) a promotor of inflammatory mechanisms which significantly increased by LPS. Kaempferol also decreased the cellular expression of IL-8 through stimulating  inhibited mast cell degranulation and prostaglandin release, leading to the development of aberrant airways in basophilic leukaemia (RBL-2H3) mast cells obtained from dinitrophenylated bovine serum albumin (DNP-BSA)-sensitised rat. Kaempferol suppressed β-hexosaminidase release and COX-2-mediated production of prostaglandin D2 (PGD2) and prostaglandin F2α (PGF2α) in sensitised mast cells. Kaempferol prevented the antigen-induced mast cell activation of cytosolic phospholipase A2 (cPLA2) responsive to protein kinase Cμ (PKCμ) and extracellular signal-regulated kinase (ERK). These results demonstrated that kaempferol inhibited ERK-cPLA2-COX-2 signalling in mast cells  significantly decreased LPS-induced cellular levels of transforming growth factor beta 1 (TGF-β1) in a dose-dependent manner in BEAS-2B cells. Furthermore, LPS stimulation significantly induced TGF-β RI and TGF-β RII, which was reversed by kaempferol. Epithelial E-cadherin expression was substantially increased by kaempferol, which was dampened by TGF-β. Kaempferol (1–20 μM) suppressed the epithelial production of collagen IV, which was enhanced by LPS in BEAS-2B cells. Thus, kaempferol may improve epithelial barrier function by cell-cell adhesion  pathway in human monocytic cell line THP-1 were reviewed  on lipopolysaccharide (LPS)-induced lung injury in BALB/c mice strongly reduced overproduction of pro-inflammatory cytokines in BALF, including TNF-α, IL-1β and IL-6. Kaempferol also significantly inhibited LPS-induced alveolar wall thickness, leukocytes infiltration and alveolar haemorrhage in lung tissue with evidence of reduced myeloperoxidase (MPO) activity and increased superoxide dismutase (SOD) activity. Furthermore, kaempferol significantly blocked the activation of mitogen-activated protein kinases (MAPKs) and NF-κB signalling pathways induced by LPS. These results suggested that kaempferol exhibits a protective effect on LPS-induced acute lung injury by suppressing MAPKs and NF-κB signalling pathways, involved the inhibition of oxidative injury and inflammatory process  and eotaxin-1 protein in the lung tissue were enhanced in OVA-exposed mice, but the supplementation of kaempferol  dose-dependently eliminated the production levels of CCR3 and eotaxin-1. OVA challenge also increased macrophage inflammatory protein 2 (MIP-2) and C-X-C chemokine receptor type 2 (CXCR2) production in mouse lung tissue which kaempferol supplemented markedly reduced MIP-2 and CXCR2 production  either as a preventive (administered 1 h before OVA-sensitisation) or curative (OVA-sensitisation at day 18–21) treatment in sensitised mouse models  and MAPKs signal pathways in aged rat kidney  markedly inhibited the passive cutaneous antigen-induced anaphylaxis (PCA) response in IgE-sensitised mice  suppressed bovine serum albumin inhalation-induced epithelial cell excrescence and hypertrophy in smooth muscle by attenuating the induced COX-2 and the formation of PGD2 and PGF2α, as well as reduced the expression of anti-α-smooth muscle actin in mouse airways. These results demonstrated that kaempferol inhibited airway wall thickening through disturbing Syk-PLCγ signalling and suggested it may be a potent anti-allergic compound that targeting of allergic asthma  to OVA-challenged mice induced pulmonary TGF-β1 induction reduced TGF-β RI and levels of the epithelial marker E-cadherin in lung tissues of OVA-challenged mice. In addition, kaempferol (10 or 20 mg/kg) significantly reduced the lung tissue level of fibrogenic collagen IV which increased in mice sensitised with 5% OVA  reduces omega-6 and ovalbumin-induced allergic reactions at lung and trachea in BALB/c mice. It also inhibited the increased histamine level and expression level of COX-2 which increased in the lung and trachea after OVA sensitisation. The results of this study suggested kaempferol might have a positive effect in mitigating allergic inflammatory response of the respiratory tract  1 h before caecal ligation and puncture surgery in mice significantly decreased water content in the lungs. Pre-treatment with kaempferol also reduced cytokines, such as IL-6, IL-1β, and TNF-α in the plasma and in the lung tissue compared to the untreated mice. Kaempferol increased SOD and catalase and non-enzymatic antioxidant glutathione (GSH) activities in septic mice. Additionally, kaempferol reduced the lung tissue nitrite level and iNOS level and down regulated mRNA expression of intercellular adhesion molecule 1 (ICAM-1) and iNOS in septic mice  activity and increased superoxide dismutase (SOD) activity. Kaempferol also reduced the total leukocyte and eosinophil counts similar to dexamethasone used for the treatment of allergic inflammatory diseases. The anti-inflammatory effects of kaempferol are summarised in C. sativus  showed a potent relaxant effect on guinea‐pig precontracted tracheal chains (in vitro) by methacholine (10 μM) when compared with saline treatment. Furthermore, there was a positive correlation between increasing concentrations of the extract and the relaxant effects  reduced tracheal responsiveness to methacholine in ovalbumin (OVA) sensitised guinea pigs  by reducing the number of cough was observed, which could be due to its relaxant effect on airway smooth muscle (Hosseinzadeh and Ghenaati C. sativus (0.1 and 0.2%), and 10 nM propranalol (β-blockers) precontracted isolated guinea pig tracheal smooth muscle showed clear leftward shifts in isoprenaline (β adrenoreceptor agonist) curves obtained in the presence of saffron extract compared to that of saline, while propranolol caused rightward shift in isoprenaline response curve. This result indicated a potent stimulatory effect for C. sativus extract on β2-adrenoreceptors  on muscarinic receptors of guinea pig tracheal chains showed the functional antagonistic effect of the extracts on muscarinic receptors on smooth muscle of tracheal chain  on 10 nM chlorpheniramine, precontracted isolated guinea pig tracheal smooth muscle showed inhibitory effect of the plant extract on histamine H1 receptors , in normotensive and hypertensive rat model, reduced the mean arterial blood pressure and heart rate in a dose‐dependent manner. Additionally, administration of 10 mg/kg of saffron reduced the mean systolic blood pressure (MABP) by 60 ± 8.7 mmHg. These results suggested that C. sativus extract and its two active components have hypotensive properties in an animal model of hypertensive  showed a concentration-dependent inhibitory effect on heart rate and contractility, which was comparable to the effect of diltiazem. The effect of plant extract on heart contractility could be due to its muscle relaxant effect  in desoxycorticosterone acetate (DOCA) salt induced hypertensive in rats resulting in reduced MSBP in a dose-dependent manner, but this hypotensive effect was not observed in normotensive rats. Data showed that antihypertensive effects of saffron did not last long, so it could be postulated that long-term blood pressure regulation systems are not affected by saffron , on isolated rat thoracic aorta rings (tissue) were contracted by 10−6 M phenylephrine (PE) or KCl 80 mM showed vasodilatory effect on intact and denuded endothelium aortic rings. The extract induced relaxation in a dose-dependent manner in endothelium-intact aortic rings precontracted with PE. The relaxation effects of plant extract in precontracted tissue with KCl was less than that of precontracted tissue with PE. The relaxant activity of C. sativus was abolished by incubation of tissue with L-NAME but not in the presence of indomethacin. These results suggested C. sativus induced relaxation in isolated rat aortic tissue due to its effect on endothelium via nitric oxide synthase and the effect on vascular smooth muscle cells via L-type voltage-dependent calcium channels  , kaempferol (3–60 µM) and quercetin (1–100 µM) in rat uterus incubated in medium in the presence of different inhibitors proposed that cyclic adenosine monophosphate (cAMP) contributed to the relaxant effect of quercetin and kaempferol on induced tonic contraction by KCL. Furthermore, polyamins contributed to the relaxant effects of kaempferol on KCL-induced tonic contraction but not on CaCl2-induced contraction in the depolarised uterus. The other results of this study also indicated that flavonoids have a calcium antagonist action  through cAMP produces transcriptional events and polyamines have a relaxant effect on KCL induced tonic contraction in isolated rat uterus  on KCl (60 mM)-induced contraction in isolated rat uterus showed relaxant effect in a dose-dependent manner. This relaxing effect was antagonised by cAMP-dependent protein kinase inhibitors (Rp-cAMPS and TPCK) and adenylyl cyclase inhibitor  . Thus, results suggested that kaempferol glycosides are not responsible for the extract induced relaxation  on isolated pulmonary artery of rats was investigated. Experiments were done using the isolated organ bath system by recording tension with the help of data acquisition system, Power lab. Results of this study demonstrated that kaempferol caused concentration-dependent relaxation of endothelium intact pulmonary artery. In endothelium-denuded arterial rings, relaxation produced by kaempferol was not different from the intact artery. L-NAME (NOS inhibitor), indomethacin had not any effect on kaempferol induced relaxation. Furthermore, the administration of other drugs together with kaempferol was done. The results suggested that kaempferol relaxes rat pulmonary artery in endothelium-independent manner through involvement of BKca channel, sGC, PKA pathways and inhibition of Ca2+-influx through l-type calcium channels  was constructed. Kaempferol at high concentration has significant relaxation but this effect was not shown at low concentration. At low concentration (10 µM), kaempferol enhanced relaxation produced by isoproternole, bradykinin, sodium nitroprusside and calcium ionophore A23187 in endothelium intact arteries. In endothelium disrupt rings, kaempferol (10 µM) enhanced relaxation caused by isoproternole, sodium nitroprusside and nifidipine. According to these results, kaempferol at low concentration (10 µM) has no significant vascular effect, but it can enhance endothelium-dependent and endothelium-independent relaxations  on inflammatory respiratory diseases is shown in in vivo and in vitro studies. Saffron and kaempferol showed smooth muscle relaxant effects on tracheal smooth muscle (in vitro studies) by stimulatory and inhibitory effects on β-adrenoceptor and muscarinic receptors, respectively. The results of this review article indicate C. sativus (saffron) and its constituent (kaempferol) have potent therapeutic effects that may be useful for attenuating inflammation in some respiratory diseases such as asthma and COPD.There are two types of common respiratory disorders, including asthma and COPD, which is characterized by airway inflammation, generates excess mucus and smooth muscle dysfunction, making it hard to breathe. Saffron and kaempferol reduced the production of NO, and inflammatory mediators including; IL-4, IL-1β, TNF-α, and MMP-9 in the serum and broncho-alveolar lavage fluid. This plant and its active constituent also increased anti-inflammatory mediators including IFN-γ and IL-10 in


Type 2 diabetes mellitus (T2DM) is an age-related metabolic disease that is of increasing concern. Gut microbiota might have a critical role in the pathogenesis of T2DM. Additionally, Hippo signaling has been associated strongly with the progression of T2DM and the aging process. We adopted db/db male mice as a T2DM model, and the gut microbiota of db/db and m/m mice were transplanted successfully into pseudo germ-free mice. Furthermore, Hippo signaling, including mammalian sterile 20-like protein kinases 1 (MST1), large tumor suppressors 1 (LATS1), Yes-associated protein (YAP), and phosphorylation of YAP (p-YAP) in peripheral tissues were significantly altered and highly correlated with blood glucose in db/db mice. Interestingly, the host after gut microbiota transplantation from db/db mice showed decreased MST1 and LATS1 levels, and p-YAP/YAP ratio in the heart, liver, and kidney compared to those from m/m mice. Negative correlations between fasting blood glucose and Hippo signaling levels in selected peripheral tissues also were identified. These findings suggest that alterations in Hippo signaling in selected peripheral tissues may contribute to the development of T2DM, and that therapeutic interventions improving Hippo signaling by gut microbiota transplantation might be beneficial for the treatment of T2DM and other age-related metabolic diseases. Worldwide, type 2 diabetes mellitus (T2DM) in individuals older than 65 years is gradually becoming a prevalent public concern , 2 becauWe previously reported that abnormal composition of gut microbiota greatly contributed to cognitive decline in streptozotocin-induced diabetic mice and Alzheimer’s disease’s (AD) mouse model-senescence-accelerated mouse prone 8 mice , 10. AltThere also is a causal linkage between T2DM characterized by insulin resistance and obesity in pathology and gut microbiota dysbiosis –19. AccoHippo signaling is recognized as a key regulator of organ size and tissue homeostasis , 24. It Although recent work has uncovered novel mechanisms of mitochondrial dysfunction in peripheral tissues implicated in T2DM progression , the rolWe adopted db/db mice as a T2DM mouse model and m/m mice as control subjects. Metabolic parameters, including blood glucose, body weight, and food and water intake, between the db/db and m/m mice were evaluated after 1 week of acclimation. Fasting blood glucose, body weight as well as food and fluid intake were significantly increased in db/db mice than those of m/m mice –1D.Expressions of Hippo signaling, consisting of MST1, LATS1, p-YAP, and YAP were determined in selected peripheral tissues between db/db and m/m mice –2E. CompConsidering the pivotal role of fasting blood glucose in the progression of T2DM, correlations between fasting blood glucose and Hippo signaling levels were analyzed –3E. IntrAfter the establishment of pseudo germ-free mice, fecal transplantation were conducted from db/db and m/m mice . Then, mAfter fecal transplantation, MST1 and LATS1 levels, and p-YAP/YAP ratio in selected peripheral tissues were determined by Western blot in pseudo germ-free mice –5E. IntrA significant correlation was noted between fasting blood glucose and MST1, LATS1 levels, and p-YAP/YAP ratio in the heart, liver, and muscle tissues, as analyzed in selected peripheral tissues after fecal transplantation from db/db and m/m mice into pseudo germ-free mice , 6B, 6D.It is generally acknowledged that aging plays a predisposing role in the dysregulation of glucose metabolism . Age-relIn fact of growing incidence of T2DM in elderly populations, more attentions have focused on the intricate pathogenesis of T2DM that mainly attributed to genetic susceptibility and environmental risk factors , 43. RecPeripheral tissues, such as skeletal muscle and liver, are considered as vital tissues in regulation of glucose metabolism . In thisin vitro trials of multiple cell lines [It is acknowledged that Hippo signaling is mediated by G protein coupled receptor pathway, and epinephrine or glucagon can stimulate Gs-coupled receptors, thus activating Lats1/2 kinase and inhibiting YAP function ll lines . TherefoOur study has several limitations. First, we should adopt more mice to minimize the difference among the groups. Second, all age groups especially the aged ones are needed. Third, tissues, including adipose tissues and various brain tissues, also should be evaluated by various techniques, including immunohistochemistry and quantitative polymerase chain reaction. Moreover, antagonists of Hippo signaling, or interference plasmid with lentivirus vectors that knockdown Hippo signaling were not conducted in this study. It is therefore further investigations are urgently needed.To conclude, our results revealed that the aberrant expressions of Hippo signaling in selected peripheral tissues may trigger the onset of T2DM. Thus, therapeutic interventions improving Hippo signaling by gut microbiota transplantation might be beneficial to the treatment of T2DM, which provides a new insight into the study of age-related metabolic disorders in the near future.n = 6), m/m mice  of the C57 BL/KS strain, and C57BL/6J male mice (n = 40)  were housed 3-5 per cage under controlled lighting conditions (12 h light: 12 h darkness cycle), with free access to rodent feed and water. Housing conditions were in a specific pathogen–free (SPF) facility at a consistent temperature (22° C ± 2° C) combined with a relative humidity of 60% ± 5%. All experimental protocols and animal handling procedures were conducted strictly according to the recommendations in the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health . This study was approved by the Experimental Animal Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology .8-week-old male db/db mice  was measured from a tail vein blood sample via a OneTouch Ultra blood glucose meter .ad libitum for 2 weeks before fecal transplantation [C57BL/6J male mice (n = 40) weighing 20-25 g were treated with large doses of broad-spectrum antibiotics  by antation , 10, 20.antation , 52.During the establishment of pseudo germ-free mouse model, fecal samples can be prepared and stored at −80° C , 53. EacTo prepare total protein extracts, selected peripheral tissues  were quickly collected after sacrifice and lysed in RIPA buffer  mixed with protease and phosphatase inhibitors. The concentration of proteins in the supernatants was quantified through a BCA protein assay kit . Protein samples were denatured in loading buffer and resolved by 10% SDS-polyacrylamide gel electrophoresis, and transferred onto polyvinylidene difluoride membranes . Membranes were blocked in 5% bovine serum albumin for 1 hour before incubation with primary antibody overnight at 4° C. Membranes were washed with TBST and incubated with secondary antibodies at room temperature for 1.5 hour. The signals of proteins were then visualized by an ChemiDocXRS chemiluminescence imaging system . The following antibodies were used: rabbit anti-MST1 , rabbit anti-LATS1 , rabbit anti-p-YAP  and rabbit anti-YAP , and horseradish peroxidase-conjugated goat anti-rabbit IgG antibody .t-test. Correlation analyses were performed using Pearson’s product–moment coefficient. The P < 0.05 was considered statistically significant.Data are shown as mean ± standard error of the mean. Statistical analyses were conducted using GraphPad Prism 7 . Data were analyzed by 1- or 2-way analysis of variance or unpaired


Discrepancies exist between healthcare provider and patient perceptions surrounding breast cancer treatment. Significant treatment changes in the last 10 years have made re-evaluation of these perceptions necessary.Physicians and nurses involved in breast cancer treatment, and patients who had received breast cancer chemotherapy (past 5 years), were questioned using an Internet survey. Participants ranked physical concerns (treatment side effects), psychological concerns, priorities for treatment selection, and side effects to be avoided during treatment. Patients were asked about desired treatment information/information sources. Rankings were calculated using the mean value of scores. Spearman’s rank correlation was used to determine the concordance of rankings among groups.Survey respondents included 207 patients, 185 physicians, and 150 nurses. Patients and nurses similarly ranked distressing physical concerns; physician rankings differed. Quality of life (QoL) and treatment response ranked high with physicians and patients when considering future treatment; nurses prioritized QoL. All three groups generally agreed on ranking of psychological concerns experienced during chemotherapy, explanation of treatment options, and how treatment decisions were made, although more patients thought treatment decisions should be made independently. Healthcare providers reported providing explanations of treatment side effects and information on physical/psychological support options while patients felt both were lacking. Concordance was calculated as 0.47 (patient–physician), 0.83 (patient–nurse), and 0.76 (physician–nurse). Patients desired additional information, preferring healthcare providers as the source.Specific areas for improvement in breast cancer patient care were identified; programs should be implemented to address unmet needs and improve treatment in these areas.The online version of this article (10.1007/s00520-019-05029-z) contains supplementary material, which is available to authorized users. The progress made in breast cancer treatment over the last decade, including new diagnostic methods and approval of targeted therapeutic agents, has improved patient survival rates . These tRegarding disease awareness in patients, a survey conducted in 2008 reported differences between major concerns of patients and those of physicians and nurses , 4. OtheIn Japan, the major target of the medical care system had been shifted to “reduction of burden among all cancer patients and their families and improvement of quality of life (QoL),” and “building a society in which cancer patients can live peacefully” . HoweverIn the current therapeutic environment, it is clinically important to clarify the priority of the unmet medical information needs of breast cancer patients and to detect discrepancies in perceptions between physicians or nurses. This will maximize the effectiveness of communication between patients and healthcare professionals, making the best use of limited resources. We conducted a survey of physical and psychological perceptions toward treatment between breast cancer patients and healthcare professionals using an Internet questionnaire that was given to patients, physicians, and nurses in Japan.For the survey, patients aged 20–69 years with a diagnosis of breast cancer who underwent chemotherapy within the past 5 years were recruited from a Medilead patient panel . This general consumer panel consisted of 2 million people, 300,000 of whom have verified medical conditions and diseases, including cancer. From this panel, we contacted Japanese patients with breast cancer who met our study criteria. Physicians and nurses were recruited from the M3 panel  which includes 250,000 physicians and 80,000 nurses in Japan. Doctors and nurses with a background suitable for this survey  received study questionnaires. Specifically, physicians (oncologists or surgeons) who belonged to a department of breast oncology/surgery, department of general surgery, or department of oncology, who had experience treating more than 10 breast cancer patients per month with chemotherapy, and nurses who had managed at least one breast cancer patient in the past month were recruited. Participant recruitment occurred between 14 May 2018 and 1 June 2018. Within this period, participants were recruited for each group until the target sample size for that group  was reached. The number of participants was defined by the feasibility of each research panel.An Internet survey aimed at thorough identification of circumstances surrounding common breast cancer treatments in Japan was implemented by Medilead, Inc. The survey questionnaire was developed with reference to previously published questionnaires , 10, 11 Background information and the following survey items were asked of all survey participants: (1) physical symptoms during chemotherapy, (2) psychological anxiety, (3) priorities at treatment selection, and (4) side effects considered most desirable to avoid when selecting treatment. Patients were surveyed regarding: (1) the most necessary information on breast cancer treatment (treatment-related), (2) the most necessary information on breast cancer treatment (other than treatment-related), and (3) desirable information sources and their usefulness.Hormone receptor and HER2 status were assessed based on the American Society of Clinical Oncology–College of American Pathologists guideline definitions  in cliniAnswers to questions, other than single-choice questions, were rated on a 5-point scale, and rank was calculated using the mean value of scores. The concordance of rankings among the three groups was examined using Spearman’s rank correlation, and side effects and concerns regarding chemotherapy were cross tabulated with screening items. Statistical analyses were performed by Medilead, Inc.Study participants comprised individuals who responded to screening questions. Informed consent was obtained from all individual participants included in the study as part of the survey. Although no personal information was collected, disease information was carefully handled. Medilead, Inc. stored the results of the survey on a server housed inside Medilead, Inc.n = 45); fluorouracil, epirubicin, and cyclophosphamide (FEC) (n = 33); paclitaxel (n = 29); nab-paclitaxel (n = 17); epirubicin and cyclophosphamide (EC) (n = 17); docetaxel and cyclophosphamide (TC) (n = 16); doxorubicin and cyclophosphamide (AC) (n = 10), all injections; and capecitabine (n = 12), an oral formulation.A total of 207 patients were included in the analyses. Their regional distribution reflected the general Japanese population. Mean age was 50.8 years (range 26–69 years), and approximately 80% of patients did not experience recurrence or metastasis Table . Fifty-sA total of 185 physicians including those practicing in breast oncology/surgery (81 [43.8%]), general surgery (81 [43.8%]), and oncology (23 [12.4%]) responded to the survey. Affiliated institutions included university hospitals (24.3%), designated cancer care hospitals (29.2%), and general hospitals (42.2%) , general outpatient units (48 [32.0%]), outpatient chemotherapy units (45 [30.0%]), and breast cancer outpatient units (13 [8.7%]). Some nurses had multiple affiliations  experienced by patients during chemotherapy, the most distressing was hair loss, followed by skin and nail problems, weariness/fatigue, taste disorder, numbness in hands and legs , and edema Fig. . The sevWhen asked about side effects most desirable to avoid when selecting treatment, nausea/vomiting and hair loss were ranked high by both patients and nurses. However, physicians most highly ranked pain, followed by nausea/vomiting, then fever; hair loss ranked low (data not shown). Skin and nail problems were a major concern for nurses but not for patients and physicians (data not shown).Regarding psychological concerns experienced during chemotherapy, patients were highly concerned about “changes in appearance and mood,” “fear for metastases and recurrence,” “uncertainty about the future,” and “vague anxiety and depressed mood.” This was similar to the results of physicians and nurses Fig. . ConcernThe results of rank correlation for concern analyzed by Spearman’s rank correlation coefficient Fig.  demonstrFor the questions regarding the priority of treatment attributes important in determining future treatments for the patient, approximately 47% of patients chose items related to QoL (QoL or mild side effects) and approximately 40% of patients chose items related to treatment response  (Online Resource provided by most physicians and nurses.Information related to chemotherapy that patients perceived as having been received included “treatment-associated side effects” (97%), “drugs to reduce treatment-associated side effects” (86%), “all available treatments” (78%), and “risk-benefit balance of treatment” (71%). These informational items were all expected to be received by patients and perceived as provided by physicians Fig. . HoweverMost physicians and nurses thought that treatment decisions should be made either through discussion between patients and physicians or by patients with consideration of the opinion of their treating physician. However, a higher percentage of patients (25%) thought that treatment should be determined by physicians (data not shown). This trend was higher in elderly patients (data not shown).Regarding the most desirable person for consultation regarding chemotherapy, most patients thought that the physician was the only person who should be consulted  between patients and physicians.Regarding physical concerns, “hair loss” and “skin and nail problems” were highly ranked by patients as both the most distressing chemotherapy side effects and most desirable to avoid when selecting treatment, but these were ranked as less important by physicians. This result was reflected in the higher ranking of “changes in appearance (hair loss and complexion) and mood” in patients and nurses regarding psychological anxiety. With an improved survival rate for breast cancer , more paDistress caused by changes in appearance is reported to be particularly intense in patients with breast cancer compared with patients with other types of cancer . For phyContrary to the concerns of patients and nurses, physicians perceived the most important side effects for concern as fever, numbness in hands and legs , and pain. Physicians consider these of high concern because fever may be a sign of serious complication and peripheral neuropathy may cause a dose reduction or discontinuation of chemotherapy. Nausea/vomiting was also recognized as important to manage because it can cause a reduction in both QoL and the continuation of chemotherapy. As a result of the high priority placed on the management of nausea/vomiting by physicians and nurses, these side effects are relatively controlled in clinical practice. The resulting decreased occurrence may have led to reduced concerns in patients.Regarding psychological concerns experienced during chemotherapy, patients were highly concerned about “changes in appearance and mood.” We speculate this may be related to hair loss; this point differed from the previous survey .Regarding treatment efficacy, concerns of “fear for metastases and recurrence,” “anxiety about dying,” and “anxiety about failure to achieve expected treatment responses” were ranked similarly high by patients, physicians, and nurses. In other words, these concerns might reflect longer OS as an attribute for future treatment and were consistent with the results of the previous survey . In the Regarding the difference between the information which patients perceived they had received and the information that was necessary for them to receive (information that should be provided), the core therapeutic information (risk and benefit of chemotherapy) was considered as necessary information, as information having been received by patients, and as information considered by physicians and nurses to have been provided to patients. However, patients felt there was a shortage of information regarding cost (treatment or other), pain relief, professional and social rehabilitation, and information provided during their recovery period despite the fact that patients felt this information should be provided to them. The types of information perceived to be lacking by the patients suggest that patients realized the need for this information after receiving chemotherapy.Over half of physicians and nurses believed they had fully explained physical and psychological support available to relieve symptoms, but patients reported that the information was inadequate. “Provision of palliative care from the time of diagnosis” is a mainstay of cancer control so it may be necessary to provide this information through multiple channels, encouraging patients to use palliative care and to liaise with palliative care specialists wherever possible. This study also showed that there is a need for better provision of information regarding the use of the social security system for help with treatment costs, particularly because the agents used to treat breast cancer are known to be expensive .The discrepancy between physician and nurse beliefs around their explanations of the support available and patient perceptions about these explanations could arise because the explanations really are insufficient. Alternatively, the mental state of the patient at the time of explanation could impact how well an explanation is understood. Regarding information other than that on treatment, patients strongly wished to obtain “information on how to communicate with healthcare professionals,” further suggesting that patients may have felt there was a communication gap. In terms of health information, relationships between health literacy and physical/mental activity have been reported , 20. ThiRegarding consultation for the choice of chemotherapy, patients thought that physicians were both the most appropriate and most important person to consult. Both physicians and nurses expressed a desire for patients to consult nurses and pharmacists more actively. This suggests that while healthcare professionals understand that nurses and pharmacists can provide information regarding the management of side effects, patients do not know that consulting with nurses and pharmacists is a viable option.Our study had several limitations resulting from the use of a questionnaire survey. These limitations included that the responses were chosen from a list, therefore limiting responses; the questionnaire required the time and effort of respondents; there was a possible bias in that respondents may not have disclosed inconvenient information; and there was uncertainty about the credibility of answers received. The survey respondents may have been limited to those who are highly conscious of breast cancer treatment.The results of this survey revealed some differences between patients and physicians in the perception of chemotherapy. While healthcare providers believed they were appropriately sharing information with patients, patients felt that they had not received adequate information regarding pain relief, treatment side effects, and options for physical and psychological support. Both patients and physicians were highly aware of the importance of shared decision-making about treatment selection and of the provision of necessary basic information for decision-making, suggesting that the overall awareness of these points in Japan is good.Specific areas to focus on for improving overall communication between healthcare providers and patients include the establishment of educational programs to inform healthcare professionals that more patients may need appropriate informational and psychological support. Healthcare providers should strive to provide a wide range of information and take time to fully understand and address the sentiments of patients regarding their treatment and care.Online Resource 1Questionnaire completed by participants. (PDF 94 kb)Online Resource 2Priority of treatment attributes for the future treatment of breast cancer. Answers chosen by < 3% of respondents are not listed. QoL: quality of life. (PDF 314 kb)Online Resource 3Preferred source of consultation regarding breast cancer chemotherapy. Patients with breast cancer who had received chemotherapy within the past 5 years, physicians (oncologists or surgeons) who had treated patients with breast cancer and nurses who had at least one breast cancer patient in their care were surveyed and asked who they considered to be the preferred source of consultation regarding breast cancer chemotherapy. Results are shown for each survey group and are represented as a percentage of the total responses. (PDF 320 kb)


In partnership with communities of the Williams Treaties First Nations in southern Ontario (Canada), we describe an approach to work with communities, and highlight perspectives of food security and sustainability, including priorities and opportunities to revitalize local food systems as a pathway to food security and food sovereignty. The objectives of our project were: (1) to build a shared understanding of food security and sustainability; and (2) to document community priorities, challenges and opportunities to enhance local food access. Utilizing an Indigenous methodology, the conversational method, within the framework of community-based participatory research, formative work undertaken helped to conceptualize food security and sustainability from a community perspective and solidify interests within the four participating communities to inform community-led action planning. Knowledge generated from our project will inform development of initiatives, programs or projects that promote sustainable food systems. The community-based actions identified support a path towards holistic wellbeing and, ultimately, Indigenous peoples’ right to food security and food sovereignty. So if they’re hunting muskrat there’s no muskrat left here really. There might be a few beavers.”Community advisor 8: Concerns were shared related to the high cost of food within the community, including high costs associated with travelling to grocery stores outside of the community. In addition, the costs to engage in traditional food activities related to travel to hunting areas, and access to tools or equipment to hunt was a concern. Amplifying these challenges is the dependency on social assistance due to low employment opportunities and rates in the community.“Food insecurity is huge for us. What is the root problem? It’s income basically.”Community advisor 2: “I think also because we live in the cottage country that really determines the price especially meat and produce. I think sometimes we end up as during summer months like we are paying more for stuff that you know if you lived in the city you wouldn’t be paying that price. I think costs is the biggest factor.”Community advisor 6: The loss of traditional knowledge, language and skills to engage in traditional food acquisition practices was emphasized as a barrier to food security. In particular, reflections were made on how the loss of traditional knowledge is tied to colonization and the Indian Act which have contributed to a lack of sovereignty and control over access to land to hunt, fish and gather (The Constitution Act (1876), Canada has the responsibility for ‘Indians and Lands Reserved for Indians’ under the Indian Act . The Ind“I think we need to start with the younger generations because I think with the older generation we’re slowly losing that link to the past especially the ones that remember what it’s like way back when times were simpler, when people helped themselves, help each other, they didn’t have to rely with a lot of programs.”Community advisor 6: Emphasized, was the implication of limited traditional knowledge, in particular the lack of opportunities for traditional knowledge of food and culture to be passed down to younger generations on how to engage in community food gathering and harvesting practices, including knowing the location of hunting grounds and how to use tools or equipment to hunt.“I think there’s a gap that needs to be bridged somehow because we are losing our traditions and cultures within our own community. Because of that we are having to bring other people in that are teaching the same skills, but they are not our community’s skills.”Community advisor 9: Priorities and actions were identified within each community to enhance food security . These rAccess to grocery stores outside of the community was emphasized. Within each community, the importance of having access to means of transportation  was commonly expressed, as this was recognized as essential to accessing food, and especially healthy food of good quality.Having a community-based travel system in place with appropriate storage was identified as an opportunity to support access to grocery stores outside of the community. For example, having a community-based bus that could accommodate large grocery portions and preserve freshness of vegetables in the summer was discussed.“…my one Aunt she won’t even buy bananas in the winter because they are brown by the time they get on the boat across the bay, but I think too, a lot of people like having to transport and so many people are on the boat. So if the ice starts building up then we use our passenger ferry only, so if you have 70 people riding a ferry then all their food and stuff like you kinda have to watch yourself.”Community advisor 5: “But again it’s just our access has always been an issue because you know you have to bring stuff over. We do have farmers market in the summer, sometimes they are well received and sometimes they are not. It depends who’s bringing stuff over; sometimes it’s fresh, sometimes it’s not.”Community advisor 6: Also highlighted as key to supporting access to food, was ensuring sufficient income and employment opportunities within the community. Creating jobs within the community to support economic growth and security was emphasized.“And we know about [how] income and accessing affordable fresh produce in general, changes their body. Chronic disease goes down, you feel good. You’re not losing limbs because you’re eating sugary bread all the time.”Community advisor 2: The importance of making fresh produce available and affordable for communities to access at wholesale costs was emphasized. Having food markets in the community with a variety of vendors of locally produced food and other essential products was identified as an action that could support greater access to fresh food. Having stable and reliable access to enough healthy and affordable food year-round without worry was broadly understood by communities as having secure access to food.Support for health promoting activities to increase food literacy and knowledge of healthy foods and recipes was also identified as a priority that can support community in healthy eating practices. Developing incentives to have community members engaged at local markets and community-based food initiatives was also discussed.“I think with our community, I’ve heard from [a] few people that the issue too is education and equipment right. So, I mean, just finding out from people like, Ellen, you know getting that knowledge in order [to know] ‘where do I start?’. Some areas in our community are so sandy that you probably couldn’t get a garden, ‘so what do you have to do, okay fine you need to build up, so you have to build a crib right.’ ‘Who do I talk to?’ ‘Who has the skills to build this crib right?’Community advisor 7: “I think it’s trying to find ways to provide fresh foods and vegetables. Cause like I said, they have this program called, the good food box, but that’s only catered towards certain people right; it’s not available to everybody.”Community advisor 3: An existing initiative within one of the partnering communities, Nourish, provides affordable access to local produce and market foods, was identified as an opportunity to support local food production and enhance community food security.“Nourish is a collaboration from Peterborough and public health from the YMCA which we are housed out of. We are a small group. We try to bring people into the system to help with system change…It’s all about food. We have people entering into our programs so we do cooking, we do growing, we have community meals, and we do advocacy work. Basically, the bottom line is to empower people to make decisions for themselves in order to make system changes.”Community advisor 2: Accelerating the uptake of existing projects within the community to support scale-up efforts, including support for local business to enhance traditional food supply  ,46 is anRestoring and maintaining access to traditional food sources and distribution systems, such as wild rice production within the community, was emphasized. Communities shared the importance of promoting traditional food acquisition activities such as fishing, hunting and cultivating . This was discussed as an opportunity to re-establish connections to traditions, culture and the environment . This included promoting greater engagement with youth in traditional food teachings and encouraging younger generations to be advocates for local food projects and traditional food activities. In addition, utilizing social media platforms to engage youth and generate interest in learning more about traditional food and ways to contribute to food security in their community through gardening, gathering of traditional medicines, fishing, hunting, and harvesting and production , was emphasized. An approach identified to support this was having hunters and trappers from the community share their knowledge and skills to other members of the community , including teachings of food as medicine. In addition, having a communal resource for members of the community to access tools and equipment for hunting was also identified as an opportunity to enable greater engagement in traditional food acquisition practices.“I think if we are able somehow to find a way to get the kids excited about farming, gardening, and fishing that we might see a difference, you know? Them bringing that home to their own parents and then getting them interested as well, but I think we need to offer some, I don’t know some ways to get them involved.”Community advisor 8: Creating a sustainable food system, that can support healthy eating in the community and opportunities to achieve food security through approaches that enable local food production , was discussed. Communities highlighted the importance of having policies that enable better management, protection and preservation of the land from pesticides/herbicides, and industry related activities. This was identified as an important process to ensure food safety and minimize food system exposure to environmental contaminants.Regulations in place, with community input to support ongoing environmental assessment of water and soil quality, was identified as a priority to support local food production and a way to build legislative trust within the community. As invasive species can pose a potential threat to traditional food systems, programs and services in place that can remove invasive species were identified as an opportunity to support long-term environmental health in the community. Holding workshops that can enhance community members’ skills and knowledge to engage in local food production and activities that promote energy sustainability was also emphasized.“…well I think there is movement already to tell you the truth. From what I see from the people who have access to programs and their wants and their needs. I think even the younger are more [willing] now to try things. We’ve done canning workshops here. The only thing that I think of is if people were to grow their own food and they have all this extra produce. They get overwhelmed by okay, what do I do with all this kale? So, there’s education in that and how to do that or maybe have a barter system set up or whatever trading and things.”Community advisor 7: The formative work undertaken in this participatory project aimed to facilitate the building of shared understandings of food security and food sustainability, including opportunities to strengthen access to nutritious and sustainable food systems. The Indigenous approaches utilized for knowledge generation and sharing helped to ensure appropriate engagement that centred the priorities of First Nations communities at the core. In addition, the findings will support community-led action planning and will be used to inform next steps for implementation. Knowledge generated from our project is intended to help develop initiatives, programs or projects that promote sustainable food systems. Such community-based action supports a path towards holistic wellbeing and, ultimately, Indigenous peoples’ right to food security and food sovereignty.Food security was broadly understood from a holistic wellness perspective by all partnering communities. It was viewed as both an outcome and determinant of wellbeing. Participants highlighted the importance of access to food, knowledge of how to grow your own food, and the skills and resources to prepare food. Also emphasized was the importance of food procurement from the land, whether through hunting, fishing or gardening, should honour traditional practices and be carried out in ways that protect the environment.Conceptualizations of food security underscored the importance of considering unique dimensions as related to access, availability and use of market and traditional food systems in efforts aimed at enhancing food security in Indigenous communities . For IndFood sovereignty is closely aligned with how partnering communities described food security, food sustainability and opportunities to improve access to food ,49,50,51present day strategies that enable and support the ability of Indigenous communities to sustain traditional hunting, fishing, gathering, farming and distribution practices, the way we have done for thousands of years prior to contact with the first European settlers” .While partnering communities recognize the need to mobilize interests towards creating more sustainable and accessible food systems, moving from priorities to actions requires a level of readiness and capacity to plan, develop and implement initiatives. In addition, it requires working within the specific context of each community’s traditional and market food systems. Building on the momentum to better understand their local context as it relates to challenges and opportunities to strengthen access and availability of food, communities can begin thinking about how to create an environment for transformation and meaningful change. Existing implementation and change frameworks have highlighted the importance of understanding local context to build shared knowledge, assumptions and practices to define the change pursuits of interest as an initial step before effective implementation can take place ,52,53.The community-identified priorities from this formative work therefore present opportunities for change, whereby communities mobilize their interest for sustainable food systems, food sovereignty and food security by identifying and building on existing strengths within the community to enhance readiness and capacity for action. This may include identification of key system actors and champions, along with existing projects, initiatives and programs that can be scaled-up within the community. In addition, successes and lessons identified from existing or previous projects can also be leveraged. For example, an existing initiative, Nourish, was identified by community members as an opportunity for scale-up; extending its reach to nearby First Nations communities and incorporating traditional food could support their access to nutritious, culturally-relevant food. Strengthening relational capacities with key system actors has been identified as a critical process to support successful change pursuits ,53. WithOngoing engagement with key community members from this preliminary work will be essential to identifying key system actors and champions that can lead and sustain implementation efforts and ensure success is achieved in the community. Furthermore, such engagement, driven by the community, will be fundamental to ensuring that actions, including partner-support of implementation strategies, are taken in a culturally safe way that best serves the interests of communities.Community-based participatory work, though well respected and recognized as an appropriate approach to working with Indigenous communities, is not without challenges or shortcomings. Short-term funding cycles may not support additional efforts to plan and implement community-identified projects of interest. While time and resource constraints limited the immediate uptake of the current project, partners continue to identify opportunities to utilize and leverage the findings from this project to inform other areas of existing or future funded projects.We use Indigenous methodologies and western approaches to partner with communities and engage with community advisors to solicit input on priorities and opportunities to enhance food security, strengthen sustainability of food systems, and in doing so support the promotion of food sovereignty. The shared understanding of community perspectives, priorities, challenges and opportunities to strengthen food security will benefit participating Williams Treaties First Nations communities directly. Moreover, the approach taken in this formative initiative will help to inform other work to integrate robust Indigenous practices and approaches to community engagement critical to building shared knowledge and understanding as an initial step and requirement of implementation of programs intended to improve Indigenous health. Hence, the contributions made to understanding ways to support the wellbeing of partnering Williams Treaties First Nations by taking a community driven and participatory approach to identifying priorities and opportunities to revive local food systems, will promote greater food security, sustainability and food sovereignty that may resonate with other Indigenous communities.The current research can serve as an important foundation for planning Indigenous community-based projects and initiatives to strengthen food security, create more sustainable food systems and work towards food sovereignty.


Children with persistent obesity, newly developed obesity, or converting from obese to non-obese had significantly higher HBP risk. For children with GOB or AOB, reduction of <3.6% in BMI or <4.5% in WHtR could decrease the HBP risk.This study aimed to examine the impact of short-term adiposity change on risk of high blood pressure (HBP), and to assess the low limit range of body mass index (BMI) and waist-to-height ratio (WHtR) reduction proposed to decrease the HBP risk in children. Children were longitudinally surveyed at baseline and after a short-term follow-up. General obesity (GOB) is categorized by age and gender-specific BMI cut-off points, abdominal obesity (AOB) by WHtR. Logistic regression model was used to estimate relations between adiposity change and HBP risk with adjustment of covariates. A total of 28,288 children (median of baseline age:10 years) were involved with follow-up of 6.88±1.20 months. After the follow-up, 9.4% of the children had persistent general obesity (GOB), 2.8% converted from GOB to non-GOB, 0.9% had newly developed GOB. When compared with children remained non-GOB, children with continuous GOB status, newly developed GOB, converting from GOB to non-GOB had 5.03-fold (95%CI: 4.32~5.86), 3.35-fold (95%CI: 1.99~5.65), 2.72-fold (2.03~3.63) HBP risk, respectively. Similar findings were observed for abdominal obesity (AOB). Reduction of 0.21–0.88 kg/m The prevalence and magnitude of pediatric high blood pressure (HBP) increased dramatically in recent years, which poses a heavy burden for cardiovascular diseases (CVDs) prevention . As the The sharp increase of overweight and obesity prevalence contributes greatly to the epidemic of HBP among children . The assIn addition, a national secular trend study of BP and obesity among Chinese children showed that the magnitude of impact of obesity on HBP increased sharply in recent years . TherefoA previous weight management study among overweight children show that the reduction of just 5% of body mass index (BMI) is associated with improvements in lipid profiles and insulin sensitivity in six months , and a mhttps://www.clinicaltrials.gov/, Registration number: NCT02343588) which involved more than 70,000 children aged 6–17 years based on a random sampling method from 7 provinces  in China at autumn of 2013. A detailed description of the program was published before Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The statistics used in this paper, both univariate and multivariate, are fairly routine and the sample size is certainly large enough. The paper, given the size of the sample, is more descriptive than inferential and the conclusions follow from the analyses performed.Reviewer #2: An important issue, the one the Authors decided to confront with, and given the big sample they could put together, I think their results are worth being widespread.Here just a few remarks:- It's a pity you used "standard “BMI Reference for Screening for overweight and obesity among school-age children and adolescents” published by National Health Commission of the People‘s Republic of China" instead of more internationally accepted ones: thinking of future research, it makes difficult to compare your results with other obtained in different countries. I believe that this point deserves to be added to the limitations of the study, and possibly counter-explained.- At lines 224-225 the sentence "A reduction of at least 0.21~0.88 kg/m2 of BMI (only 0.86%~3.59% of the baseline BMI) in children with GOB..." is not clear to me; specifically, what do you mean with "(only 0.86%~3.59% of the baseline BMI)"?- At lines 274-276, the meaning of the sentence "those converted from obese to non-obese doubles the HBP risk compared with the persistent non-obese children" is clear enough, but I believe it should be re-phrased.- There is some other minor revision of English to be made; in the attached file I highlighted the line numbers where some correction is needed, IMHO. There are possibly more than those ones, but they are the ones I easily spotted.Reviewer #3: 1. Did the Authors use some exclusion criteria to enrol the subjects?2. All the investigated variables were normally distributed? Which kind of test the Authors used to verify data normality?3. It would be better use BMI-SDS instead of BMI considering the age of subjects.4. Why the Authors decided to categorize the adiposity parameter (BMI or WHtR) change during the follow-up as quintiles?5. In logistic regression, in addition to OR they have to show and mention also the goodness of fit test of the model. To adjust a regression for multiple variables without to verify if these variable are possible confounders is not correct. Mantel–Haenszel method could be used to verify if a variable is a possible confounder.6. Could the Authors clarify the sentence a” reduction of at least 0.21-~0.88 kg of BMI (only 0.86%~3.59% of the baseline BMI) in children with GOB (baseline BMI=24.49 kg) at lines 224. It is not very clear: only 0.86%~3.59% of the baseline BMI.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, AttachmentPONE-D-21-04822 - Impact of short-term change of adiposity on risk of high blood pressure in children, follow-up study in China.pdfSubmitted filename: Click here for additional data file. 17 Aug 2021Response letter to the Editors’ suggestions1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdfResponse: Thanks for the suggestion. We have revised our paper according to PLOS ONE’s style requirements.2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction noResponse: Thanks for the suggestion. We have checked all the references in the manuscript, no cited papers have been retracted.3. Please ensure you have included the registration number for the clinical trial referenced in the manuscript. Moreover, please clarify 1) the inclusion criteria used for recruitment, as we note that the trial registry indicates that children aged 7-18, not 6-17, were included; 2) where the study took place; 3) the length of the follow-up .https://www.clinicaltrials.gov/, Registration number: NCT02343588) in the Method Section (Line 89).Response: Thanks for the suggestion. We have added the registration number for this national multi-centered cluster randomized controlled trial (1) the inclusion criteria used for recruitmentFor the inclusion criteria used for our recruitment, the inclusion and exclusion criteria were listed as follows:Inclusion Criteria:Grade of 1 to 5 in primary schools and grade of 6 to 8 and 10 to 11 in secondary schools.Exclusion Criteria:Suffering or any history of disabilities, cardiovascular diseases, asthma, metabolic diseases, etc.Actually, before the study began, we planned to recruit grade 1 to 5 in primary schools, grade of 6 to 8 and 10 to 11 in secondary schools. For students in grade 1 to 5 in primary schools, the age of them were supposed to be 7-11 years old. But actually, in the real-life study, some children aged 6 years old also started primary school and the intervention is school based. Therefore, there is slight difference in the age range in the registration trial (7-18 years old) and the present study (6-18 years old), also the age range of our paper is same with another paper about the effectiveness of this lifestyle intervention study (1).https://doi.org/10.3389/fped.2021.615283.[1] Dong Y, Zou Z, Wang H, Dong B, Hu P, Ma Y et al. National School-Based Health Lifestyles Intervention in Chinese Children and Adolescents on Obesity and Hypertension. Front Pediatr. 2021; 9(615283. 2) where the study took placeThe study was conducted in 7 provinces  in China. We have added the description of study location in the Method Section (Line 98).3) the length of the follow-up The length of follow up was 6.88 months , which is shorter than the length of follow up described in the study registry (9 months). This is because our study was conducted in 7 provinces , the length of baseline physical examination and survey was almost a month, and the follow-up physical examination and survey also take a month to finish, so the range of the follow-up is set at 9 months, but for every participant at different schools the length of follow-up might be slightly different, and the mean value of follow-up is about 7 months.We have added more description of the length of follow-up and age of the study population in the Method section as follows (Line 104-119).For the enrollment of subjects, the inclusion criteria were as follows: grade of 1 to 5 in primary schools and grade of 6 to 8 and 10 to 11 in secondary schools. And the exclusion criteria were as follows: Suffering or any history of disabilities, cardiovascular diseases, asthma, etc. For students in grade 1 to 5 in primary schools, the age of them were supposed to be 7-11 years old. But actually, in the real life, some children aged 6 years old also started primary school. Therefore, the age range in the registration trial (7-18 years old) is slightly different that of the present study (6-18 years old), also the age range of our paper is same with another paper about the effectiveness of this lifestyle intervention study. The length of follow up was 6.88 months , which is shorter than the length of follow up described in the study registry (9 months). This is because our study was conducted in 7 provinces , the length of baseline physical examination and survey was almost a month, and the follow-up physical examination and survey also take a month to complete, so the range of the follow-up is set at 9 months, but for every participant at different schools the length of follow-up might be slightly different, and the mean value of follow-up is 6.88 months.4. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service.Response: Thanks for the suggestion. We have thoroughly copyedited our manuscript for language usage, spelling, and grammar.5. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.Response: Thanks for the suggestion. We have revised the “Funding information” and “Financial Disclosure” accordingly.6. Thank you for stating the following in the Acknowledgments Section of your manuscript: We would like to thank all the children for their participation in our study, also we would like to thank all the head teachers of the schools for their help and cooperation in the research. This research was funded by the National Natural Science Foundation of China , the Hunan Provincial Natural Science Foundation of China , Scientific Research Project of Hunan Health Committee , and a Project Supported by Scientific Research Fund of Hunan Provincial Education Department . The funders had no role in the design, analysis or writing of this article.We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: This research was funded by the National Natural Science Foundation of China , the Hunan Provincial Natural Science Foundation of China , Scientific Research Project of Hunan Health Committee , and a Project Supported by Scientific Research Fund of Hunan Provincial Education Department . The funders had no role in the design, analysis or writing of this article.Please include your amended statements within your cover letter; we will change the online submission form on your behalf.Response: Thanks for the suggestion. We have deleted the funding information in the Acknowledgments Section. Please help revise the funding information in the online submission as follows:FundingThis research was funded by the National Natural Science Foundation of China (81903336), the Hunan Provincial Natural Science Foundation of China (2019JJ50376 & 2020JJ5386), Scientific Research Project of Hunan Provincial Health Commission (202112031516), Key Project of Hunan Provincial Science and Technology Innovation (2020SK1015-3) and a Project Supported by Scientific Research Fund of Hunan Provincial Education Department (18A0028). The funders had no role in the study design, data collection, data analysis, writing of this article or interpreting the results.http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. 7. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see In your revised cover letter, please address the following prompts:a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail  and who has imposed them . Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of acceptable repositories, please see We will update your Data Availability statement on your behalf to reflect the information you provide.Response: Thanks for the suggestion. The datasets generated and/or analyzed during the present studyare not publicly available, since ethics approval and participants’ consent does not allow public sharingbindong@bjmu.edu.cn) upon reasonable request.of data, only available from the institute , which involving six large nationally representative cross sectional growth studies  [1], which was also used in previous studies [2-4]. And also similar results based on the World Health Organization (WHO) standard were observed. Although lower prevalence of obesity was observed when two international definitions for child obesity was used (S2 Table), the associations between different obesity patterns with BP level or HBP risk were observed were similar with the results when Chinese BMI Reference was used . We have added these results as part of the sensitivity analysis, and the description of the method and results were added the text  as follows. In the Method section (Line 195-198)(4) We used an international definition of child obesity developed by the International Obesity Task Force (IOTF) and the World Health Organization (WHO) standard of obesity for 5-19 years old children to test the associations[18-24].In the result section (Line 212-217 and line 291-293)The prevalence of obesity based on the international definition of child obesity developed by IOTF was 6.1% at the baseline  and 5.1% at the follow-up survey . The prevalence of obesity based the WHO standard was 10.1%  at baseline and 8.8% at the follow-up survey .We also used two international definitions of child obesity (ITOF standard and WHO standard) to test the associations between BP level or HBP risk and obesity change pattern, similar results were observed . 18. Cole TJ, Bellizzi MC, Flegal KM, Dietz WH. Establishing a standard definition for child overweight and obesity worldwide: international survey. BMJ. 2000; 320: 1240-1243. 19. Deren K, Wyszynska J, Nyankovskyy S, Nyankovska O, Yatsula M, Luszczki E, Sobolewski M, Mazur A: Assessment of body mass index in a pediatric population aged 7-17 from Ukraine according to various international criteria-A cross-sectional study. PLoS One 2020, 15(12):e0244300.20. Oosterhoff M, Over EAB, van Giessen A, Hoogenveen RT, Bosma H, van Schayck OCP, Joore MA: Lifetime cost-effectiveness and equity impacts of the Healthy Primary School of the Future initiative. BMC Public Health 2020, 20(1):1887.21. Martos-Moreno GA, Martinez-Villanueva J, Gonzalez-Leal R, Barrios V, Sirvent S, Hawkins F, Chowen JA, Argente J: Ethnicity Strongly Influences Body Fat Distribution Determining Serum Adipokine Profile and Metabolic Derangement in Childhood Obesity. Front Pediatr 2020, 8:551103.https://www.who.int/tools/growth-reference-data-for-5to19-years/indicators/bmi-for-age]22. World Health Organization (WHO). Growth reference data for 5-19 years: WHO . Then for the obese children, we categorized the then into 5 quintiles with P20, P40, P60, and P80 of BMI SDS change from baseline to the follow-up, similar results were observed. When compared with the reference group (quintile 5), quintile 1 group had significantly low risk of HBP . For quintile 2 group, OR was also <1 , but it didn’t reach statistical significance. We have added this as a part of our sensitivity analysis as follows. In the method section (Line 187-190)(5) We also used BMI standard deviation score (BMI SDS) instead of BMI to compare the risk of HBP in different groups with multivariate logistic models. Age- and sex-specific BMI SDS was calculated using the World Health Organization (WHO) child growth charts[25-27].In the result section (Line 282-284)In addition, BMI SDS was used instead of BMI to test the association of different quintiles of BMI SDS change and risk of HBP, similar results were observed (S8 Table).25. McLaughlin EJ, Hiscock RJ, Robinson AJ, Hui L, Tong S, Dane KM, Middleton AL, Walker SP, MacDonald TM: Appropriate-for-gestational-age infants who exhibit reduced antenatal growth velocity display postnatal catch-up growth. PLoS One 2020, 15(9):e0238700.26. Zhang L, Huang L, Zhao Z, Ding R, Liu H, Qu W, Jia X: Associations Between Delivery Mode and Early Childhood Body Mass Index Z-Score Trajectories: A Retrospective Analysis of 2,685 Children From Mothers Aged 18 to 35 Years at Delivery. Front Pediatr 2020, 8:598016.27. Poh BK, Lee ST, Yeo GS, Tang KC, Noor Afifah AR, Siti Hanisa A, Parikh P, Wong JE, Ng ALO, Group SS: Low socioeconomic status and severe obesity are linked to poor cognitive performance in Malaysian children. BMC Public Health 2019, 19(Suppl 4):541.4. Why the Authors decided to categorize the adiposity parameter (BMI or WHtR) change during the follow-up as quintiles?Response: We thank the reviewer for this comment. We categorized (BMI or WHtR change) as quintiles [1] because we want to find the potential BMI or WhtR reduction range of obese children to get an improved BP profile. We further categorized the reduction range of obese children into ten equal groups with similar results observed.[1] Carthy P, Lyons S, Nolan A. Characterising urban green space density and footpath-accessibility in models of BMI[J]. BMC Public Health, 2020, 20(1).5. In logistic regression, in addition to OR they have to show and mention also the goodness of fit test of the model. To adjust a regression for multiple variables without to verify if these variables are possible confounders is not correct. Mantel–Haenszel method could be used to verify if a variable is a possible confounder.Response: Thanks for this comment. For logistic regression models exploring the association between risk of high blood pressure and different general or abdominal obesity status change, from model 1 to model 4, the goodness of fit test improved gradually, the Nagelkerke R2 of model 1 and model 4 for general obesity change pattern were 5.9% and 13.4%, and 4.8% and 13.2% for model 1 & 4 for abdominal obesity change patterns. For the categorical covariates in the logistic regression models, we used Mantel–Haenszel method to verify whether sex, age group(<=13 years old and >13 years old), and area  are potential cofounders, the results showed that they were possible confounders which were also found in previous studies[1-3]. These variables were adjusted when the risk factors for high blood pressure were analyzed.[1] Yang YD, Song J Y, Wang S, et al. Combined effects of the rs9810888 polymorphism in calcium voltage-gated channel subunit alpha1 D (CACNA1D) and lifestyle behaviors on blood pressure level among Chinese children[J]. PLoS ONE, 2019, 14(5):e0216950-.[2] Yang Y, Dong B , Wang S , et al. Prevalence of high blood pressure subtypes and its associations with BMI in Chinese children: a national cross-sectional survey[J]. BMC Public Health, 2017, 17(1):598.[3] Yang Y, Dong B , Zou Z , et al. Association between Vegetable Consumption and Blood Pressure, Stratified by BMI, among Chinese Adolescents Aged 13–17 Years: A National Cross-Sectional Study[J]. Nutrients, 2018, 10(4):451.6. Could the Authors clarify the sentence a” reduction of at least 0.21-~0.88 kg of BMI (only 0.86%~3.59% of the baseline BMI) in children with GOB (baseline BMI=24.49 kg/m2) at lines 224. It is not very clear: only 0.86%~3.59% of the baseline BMI.Response: We apologize for this and have clarified this sentence. This sentence means that a reduction of 0.21-0.88 kg/m2, and the average baseline BMI of children with general obesity was 24.49kg/m2, so 0.86%-3.59%  of the baseline BMI will beneficial to the BP profile. We have clarified it in the text as follows (Line 277-279).A reduction of at least 0.21~0.88 kg/m2 of BMI  in children with GOB (baseline BMI=24.49 kg/m2) could be significantly beneficial to the BP profile compared with the reference group (Table 4).AttachmentCover letter+Response letter to reviewers 20210816.docxSubmitted filename: Click here for additional data file. 25 Aug 2021Impact of short-term change of adiposity on risk of high blood pressure in children: Results from a follow-up study in China.PONE-D-21-04822R1Dear Dr. Yang,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Raffaella Buzzetti, M.D.Academic EditorPLOS ONE 1 Sep 2021PONE-D-21-04822R1 Impact of short-term change of adiposity on risk of high blood pressure in children: Results from a follow-up study in China. Dear Dr. Yang:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. 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Background: As a plant-derived polycyclic phenolic carboxylic acid isolated from Salvia miltiorrhiza, lithospermic acid (LA) has been identified as the pharmacological management for neuroprotection and hepatoprotection. However, the role and mechanism of lithospermic acid in the pathological process of myocardial ischemia-reperfusion injury are not fully revealed.Methods: C57BL/6 mice were subjected to myocardial ischemia and reperfusion (MI/R) surgery and pretreated by LA  for six consecutive days before operation. The in vitro model of hypoxia reoxygenation (HR) was induced by hypoxia for 24 h and reoxygenation for 6 h in H9C2 cells, which were subsequently administrated with lithospermic acid (100 μM). Nrf2 siRNA and dorsomorphin (DM), an inhibitor of AMPKα, were used to explore the function of AMPKα/Nrf2 in LA-mediated effects.Results: LA pretreatment attenuates infarct area and decreases levels of TnT and CK-MB in plasm following MI/R surgery in mice. Echocardiography and hemodynamics indicate that LA suppresses MI/R-induced cardiac dysfunction. Moreover, LA ameliorates oxidative stress and cardiomyocytes apoptosis following MI/R operation or HR in vivo and in vitro. In terms of mechanism, LA selectively activates eNOS, simultaneously increases nuclear translocation and phosphorylation of Nrf2 and promotes Nrf2/HO-1 pathway in vivo and in vitro, while cardioprotection of LA is abolished by pharmacological inhibitor of AMPK or Nrf2 siRNA in H9C2 cells.Conclusion: LA protects against MI/R-induced cardiac injury by promoting eNOS and Nrf2/HO-1 signaling via phosphorylation of AMPKα. Myocardial ischemia-reperfusion (MI/R) injury has a significant impact on the prognosis of patients with revascularization . MoreoveSalvia miltiorrhiza has been widely utilized to prevent cardiovascular diseases (CVDs) in China and Asia -induced hepatic oxidative damage  is implicated in antioxidant defense mechanisms in the myocardium, which is usually activated by increased reactive oxygen species (ROS) production . Nrf2 upIn the present study, we demonstrate that LA improves cardiac function and attenuates myocardial injury during MI/R. Moreover, LA suppresses oxidative stress and apoptosis by promoting activation of endothelial nitric oxide synthase (eNOS) and Nrf2/HO-1 pathway via phosphorylation of AMPKα in MI/R injury.TableAll animal experimental procedures followed the National Institutes of Health (NIH) guidelines and were approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University. Lithospermic acid  was obtained from Shanghai Winberb Medical Technology Co., Ltd .n = 10), Sham-LA (n = 10), MI/R-NS (n = 15) and MI/R-LA (n = 15). Mice were pretreated with LA  for six consecutive days before MI/R surgery or sham, and poured into the normal saline control group processing.C57BL/6 male mice , obtained from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences , were randomly separated into 4 groups: Sham-NS   was utilized to determine the area at risk and infarcted area following MI/R operation. More details are provided in the supplement material. The isolated heart tissue was immobilized with 4% paraformaldehyde and dehydrated with gradient alcohol. Then, 5 μm-thick sections were paraffin-embedded. Immunohistochemistry (IHC) of 4-hydroxynonenal and Nrf2 was performed to detect oxidative stress and the location of Nrf2 in the heart. Dihydroethidium (DHE) fluorescence was also performed to assess oxidative. Moreover, a terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay was used to determine apoptotic cells in the heart after MI/R surgery. The density of IHC and DHE staining was assessed using Image-Pro Plus version 6.0. More details are provided in the supplement material.in vivo and in vitro using the Nuclear and Cytoplasmic Protein Extraction kit  and protein concentration was determined by bicinchoninic acid assay . Subsequently, 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to separate protein samples (40 μg), and then proteins were transferred to polyvinylidene difluoride (PVDF) membranes. After incubation with blocking buffer and targeted antibodies overnight, the PVDF membranes were subsequently incubated with a corresponding secondary antibody. Afterward, targeted protein bands were examined using a chemiluminescence method, and the density of target bands was evaluated by AlphaEaseFC software processing system  and Image Lab software. Primary antibodies used in this study are provided in The heart sample was collected after reperfusion for 24 h. Protein was extracted from heart homogenates by radioimmunoprecipitation assay (RIPA) lysis buffer. Nuclear protein was obtained Total RNA was extracted from mouse hearts and H9C2 cells using Trizol reagent  and cDNA was produced using the Transcriptor First Strand cDNA synthesis kit . Meanwhile, SYBR Green (04707516001) was utilized to amplify transcripts, and GAPDH was the endogenous reference. All primers used are listed in 2 at 37°C, whereas for HR, cells were subjected to hypoxia for 24 h and reoxygenation for 6 h with 5% CO2, 94% N2 and 1% O2 at 37°C in tri-gas incubators. H9C2 cells in the LA group were subjected to LA (100 μM) treatment for 24 h after Nor or HR.H9C2 cells were obtained from the Cell Bank of the Chinese Academy of Sciences  and cultured in Dulbecco’s modified Eagle’s medium . Afterward, H9C2 cells in good growth condition were divided into 4 groups: Normoxia (Nor)-PBS group, Nor-LA group, hypoxia-reoxygenation (HR)-PBS group and HR-LA group. For Nor, cells were incubated under 5% COFor small interfering RNA (siRNA)-mediated knockdown experiments, Nrf2-siRNA (sc-156128) or Scr-siRNA was transfected with Lipofectamine 6,000  at 40 nM concentration in culture medium following the manufacturer’s protocol. Western blot analysis was performed to assess the efficiency of knockdown. For the AMPK inhibitor, dorsomorphin  was incubated with H9C2 cells for 18 h after Nor or HR. Immunoblotting was performed to assess the efficiency of blockage.After washing with phosphate-buffered saline (PBS), H9C2 cells were fixed by 4% paraformaldehyde for over 15 min. Afterward, permeabilization was performed with 0.1% Triton X-100 (Amresco) in PBS. Then cells were incubated with 10% goat serum and subsequently stained with anti-Nrf2 overnight at 4°C. Next, cells scrubbed with PBS were then subjected to the secondary antibody goat anti-rabbit Alexa Fluor™ 488  for 1 h. SlowFade® Gold anti-fade reagent with DAPI  was utilized for mounting as previously described .p-value <0.05 was considered statistically significant.Data are expressed as mean ± standard error of the mean (SEM) and evaluated using SPSS version 22.0 . Comparison of multiple groups was performed using one-way analysis of variance (ANOVA) and two group comparisons were analyzed by unpaired Student’s t-test. A MI/R injury is characterized by a significant increase in oxidative stress, inflammation and apoptosis in the heart tissues . LA has in vivo.To determine the effects of LA on cardiac function, echocardiography and hemodynamic assessment were performed in mice. It was found that LA attenuated MI/R-induced left ventricle systolic dysfunction ; Table 1To evaluate whether LA regulates oxidative stress and apoptosis in the MI/R heart, immunohistochemistry staining of 4-hydroxynonenal (4-HNE) in the heart section was performed. The results showed a higher expression of 4-HNE after MI/R operation, which was significantly suppressed by LA pretreatment . SimilarTUNEL staining was employed to determine the role of LA MI/R-induced apoptosis in heart tissues. TUNEL-positive cells were remarkably increased after MI/R surgery; however, LA treatment attenuated MI/R-induced apoptosis in the heart . Subsequin vitro experiments were performed with the HR model  production in heart tissues , which wowing HR , which wowing HR . Overallin vitro were evaluated. Congruous with in vivo experiments, Western blotting showed that LA promoted the activation of the Nrf2/HO-1 pathway in H9C2 cells under HR  kinase group, AMPK is widely distributed in various organs . AMPK isve genes . FurtherIn summary, this study demonstrates that LA improves cardiac function and attenuates myocardial injury in mice. It also suppresses oxidative stress and apoptosis following hypoxia-reoxygenation. Mechanistically, LA promotes the activation of the eNOS and Nrf2/HO-1 pathway by enhancing phosphorylation of AMPKα, providing a novel therapeutic strategy for reperfusion in AMI patients.