There are no population-based data on the relative importance of specific causes of hypercapnic respiratory failure (HRF). We sought to quantify the associations between hospitalisation with HRF and potential antecedent causes including chronic obstructive pulmonary disease (COPD), obstructive sleep apnea, and congestive cardiac failure. We used data on the prevalence of these conditions to estimate the population attributable fraction for each cause.2 > 45 mmHg. Controls were randomly selected from the study population using a cluster sampling design. We collected self-reported data on medication use and performed spirometry, limited-channel sleep studies, venous sampling for N-terminal pro-brain natriuretic peptide (NT-proBNP) levels, and sniff nasal inspiratory pressure (SNIP) measurements. Logistic regression analyses were performed using directed acyclic graphs to identify covariates.A case–control study was conducted among residents aged ≥ 40 years from the Liverpool local government area in Sydney, Australia. Cases were identified from hospital records based on PaCOWe recruited 42 cases and 105 controls. HRF was strongly associated with post-bronchodilator airflow obstruction, elevated NT-proBNP levels, reduced SNIP measurements and self-reported opioid medication use. There were no differences in the apnoea-hypopnea index or oxygen desaturation index between groups. COPD had the highest population attributable fraction .COPD, congestive cardiac failure, and self-reported use of opioid medications, but not obstructive sleep apnea, are important causes of HRF among adults over 40 years old. No single cause accounts for the majority of cases based on the population attributable fraction.The online version contains supplementary material available at 10.1186/s12890-023-02639-6. Hypercapnic respiratory failure (HRF) is a commonly encountered clinical scenario for hospital clinicians in a wide range of disciplines. Many patients are known to have a predisposing condition such as severe chronic obstructive pulmonary disease (COPD). However, in some patients presenting with HRF, the underlying cause is not apparent at initial presentation, and there may be multiple underlying potential causes existing concurrently. Although each cause requires disease-specific therapies, most patients require hospitalisation and many benefit from ventilatory support in dedicated respiratory and critical care units. Hence, HRF can be considered a single, albeit heterogeneous entity, that constitutes a significant problem for health facilities worldwide.Previous studies examining the underlying causative conditions among patients with HRF have typically included participants identified following admission to an intensive care or respiratory admission and requiring ventilatory support therapy –5. ThreeUsing a community-based case control study design, we sought to determine the strength of association between hospitalisation with HRF and the following conditions: COPD, congestive cardiac failure (CCF), obstructive sleep apnea (OSA), respiratory muscle weakness, and the use of opioid and benzodiazepine medications. We selected these causes in consensus based on previous studies and our clinical experience. In addition to estimating the strength of association with HRF, we used data on the prevalence of these conditions in the general community to estimate, for each cause, the population attributable fraction (PAF), an epidemiologic measure to describe the relative importance and public health impact of a risk factor in a population.This prospective study was based in the City of Liverpool, a metropolitan area within Sydney, Australia . The stu2 > 45 mmHg and pH ≤ 7.45. We anticipated the number of cases of HRF missed by this screening method to be low due to the increased risk of hospitalisation associated with hypercapnia, the public healthcare scheme in Australia that provides free hospital services to all citizens and most permanent residents, and local data that indicate most people with respiratory conditions from this population who require hospitalisation attend Liverpool Hospital. Medical records were reviewed to exclude suspected nosocomial cases of HRF, or instances where the person had suffered an out-of-hospital cardiac arrest or traumatic injury. Cases were invited to participate first by mail and then by follow-up telephone calls.Cases were patients who attended Liverpool Hospital between 2016 and 2018. Potential cases were identified based on an ABG, collected within 24 h of presentation, demonstrating PaCOPopulation-based controls were randomly selected using a two-stage geography-based cluster sampling design, implemented from 2018 to 2019. First, we randomly selected 40 census tracts from the 449 comprising this region, the City of Liverpool. The probability of tract selection was proportional to the number of eligible residents in each tract. Next, we undertook ‘random walks’ to select households units within each tract, from which control participants were recruited. Investigators starting from the geographical centre of each tract walked along streets in directions guided by a computer-based random number generator. This method of population-based sampling is a practical method for random selection of participants from a large population when a population list is incomplete or unavailable, and is modified from methods used by the World Health Organization . LettersStandardised questionnaires were administered by members of the research team. Data were collected on sociodemographic factors, comorbidities, and medications, including the use of opioids and benzodiazepines, in the preceding two years.Spirometry was performed using the EasyOne spirometer , before and after administration of salbutamol 200 μg via metered dose inhaler and spacer. All spirograms were reviewed by the author (Y.C.) for acceptability and repeatability using published criteria . N-termi1)/forced vital capacity (FVC) ratio, NT-proBNP levels, maximum SNIP value (SNIPmax) and apnea-hypopnea index (AHI). Receiver operator characteristic (ROC) curves were generated from these models to determine the area under the ROC curve (AUC) in order to assess the predictive value of each of these variables for HRF. For subsequent analyses, participants were classified as having COPD if post-bronchodilator FEV1/FVC was below the lower limit of normal, using Global Lung Initiative reference values [max was less than 70 cmH2O and 60 cmH2O, among males and females, respectively. A diagnosis of moderate-to-severe OSA was recorded if the AHI was ≥ 15 events per hour.Continuous variables are summarised as means with standard deviations (SD) and medians with interquartile ranges (IQR). Groups were compared using independent t-tests or Mann–Whitney U tests, as appropriate. Frequencies and percentages are used to describe categorical variables, and Fisher’s chi-square test used to compare groups. Baseline logistic regression models were used to assess the relationship between the presence or absence of HRF and continuous variables: the post-bronchodilator forced expiratory volume  with 95% confidence intervals (CI). Covariates for each model were informed by a directed acyclic graph (DAG) developed by the authors using the web-based program ‘daggity’  showing We estimated 205 participants would be required in each group to detect an odds ratio of 2.8 and 2.0 for risk factors with prevalence values of 5% and 15%, respectively, with 80% power and two-sided alpha of 0.05. Study recruitment was stopped early due to low response rates and the COVID-19 pandemic.One hundred and forty-seven subjects (42 cases and 105 controls) completed the study, as shown in Fig. p < 0.001). The Charlson comorbidity index, a composite predictor of mortality [p < 0.001).Demographic and clinical characteristics are shown in Table ortality , was sigp < 0.001) (Table 1 in cases was 51 (21) percent predicted. The most frequent potential cause for HRF among controls was moderate-to-severe OSA (34%). The median (IQR) AHI among all controls was 6.6  events per hour.At least one pre-specified cause for HRF was identified in 42 (100%) cases, and 66 (63%) controls . Post-hoc analysis showed that when a lower NT-proBNP cut-off value of 35 pmol/L (296 pg/mL) was used to determine the presence of CCF, the magnitude of association was attenuated but remained statistically significant with an OR of 4.25 (95% CI 1.16 – 15.6).The risk of HRF associated with each cause is shown in Table Moderate-to-severe OSA did not appear to be associated with increased risk of HRF, with more controls having this condition compared with cases. There was no difference between groups based on mean AHI, using a 4% desaturation threshold, or using the oxygen desaturation index. There were no significant differences in age, BMI, degree of comorbidity, reported sleepiness, and estimated risk of OSA based on the STOP-Bang score  between No single cause was identified as being responsible for more than 50% of HRF cases, based on the PAF, as shown in Table In this population-based case–control study, we show that HRF is a multifactorial condition with no single disease responsible for the majority of cases. In addition to chronic health conditions such as COPD and CCF, opioid use and respiratory muscle weakness are significantly associated with HRF hospitalisations. Interventions to reduce the prevalence of these causes have the potential to substantially reduce HRF-associated hospitalisations in this and other comparable populations.Of the hypothesised causes, COPD had the highest population attributable fraction. Most previous surveys have shown COPD to be the dominant cause of hospitalisation with HRF –3, 7, 8,In this population, CCF was an important contributor to hospitalisations with HRF. Patients with heart failure have reduced lung compliance and increased airways resistance, experiencing greater mechanical costs of breathing resulting in muscle fatigue and inability to maintain adequate ventilation . HypercaSelf-reported use of opioid medications contributed significantly, with a PAF comparable to that of COPD. In recent decades, Australia and other high-income countries have documented a marked increase in the use of prescription opioids . The cliThere was a relatively high prevalence of respiratory muscle weakness among cases, and there was a significant association between reduced muscle strength and HRF. Neuromuscular disease including motor neuron disease, polio and muscular dystrophy accounts for a substantial proportion of home mechanical ventilation users . We did Our study did not show a significant association between moderate-to-severe OSA and hospitalisation with HRF. Our analysis was based on the assumption that OSA could lead to HRF via sleep-related hypoventilation, or via an alternate pathway involving the development of CCF, with or without central sleep apnea and sleep hypoventilation  Fig. 1)1). The aThis study has some weaknesses. We were unable to achieve the recruitment target, but in our analysis we found that our sample size was sufficient to detect with statistical confidence the high magnitude of associations observed between HRF and the causes COPD, CCF and opioid use. Rather than the small groups per se, the primary limitation of this study is the potential selection bias induced by low response and non-participation rates. A relatively small proportion of potentially eligible people proceeded to study participation, despite attempts made by the investigators to minimise inconveniences and provide financial reimbursement for the time provided. Controls may have been more likely to participate if they had symptoms of, or were concerned about, OSA. If this effect had occurred in controls but not cases, it would have led to an underestimation of the association with OSA. Potential cases may have been excluded due to frailty or death. The effect of this selection bias would have been under-estimation of the association with causes known to increase the risk of death, such as CCF. Due to the case–control design, risk factors for the outcome are measured after the occurrence of HRF, although we expect most to be chronic conditions that would have been present prior to hospitalisation. Finally, our results may not be generalisable to other populations depending on socioeconomic and other factors affecting the prevalence of each cause.Nevertheless, our work has several strengths distinguishing it from previous studies. This is one of few studies that have selected cases based on ABG results. We have provided objective measurements of causes rather than self-reported diagnoses or medical records which may be incomplete. We employed causal diagrams to inform our statistical analysis in keeping with modern epidemiological theory. Importantly, this is the first study of HRF to provide data on a control group, allowing estimation of the association between specific causes and HRF at a population level as well as the relative importance of each cause as reflected in the population attributable fraction.In summary, our study provides evidence for the multifactorial nature of HRF and the range of potential contributing factors. In addition to COPD, other causes including CCF, opioid use and respiratory muscle weaknesses are significantly associated with HRF hospitalisations. These findings have important implications for the assessment and management of patients with HRF and highlight the need for comprehensive evaluation to ensure that all treatable factors are addressed.Additional file 1: Table E1. Details of regression models used to determine the associations between each cause and the outcome of hypercapnic respiratory failure.


Background: Eating disorders are a problem that is becoming more and more common among younger and younger age groups. Many studies examine the risk factors for EDs, however, the treatment of these diseases is very complicated and requires dietary, psychological and medical intervention. Methods: 233 primary and secondary school students aged 12 to 19 were surveyed using the EAT-26 (Eating Attitudes Test-26) questionnaire, the self-esteem Scale SES and the Cantril scale for life satisfaction. Results: Women, when compared to men, showed lower self-esteem, satisfaction with their appearance, body weight and their lives and at the same time a higher risk of eating disorders in all three areas. Low life satisfaction is often correlated with weight loss greater than 10 kg. Low self-esteem correlated positively with significant weight loss (>10 kg) and more frequent uncontrollable binge eating and exercising (more than 60 min a day) to influence appearance. People with low self-esteem were more likely to be treated for EDs. Subjects dissatisfied with their lives binged, feeling that they could not stop. Conclusion: The younger the person, the more likely they are to develop eating disorders. This is closely correlated with low self-esteem and negative life satisfaction. Men were more likely to be satisfied with their weight, appearance, and life, and were less likely to show ED symptoms. Eating disorders (ED) are a complex group of mental disorders that result in pathological eating behaviors that can lead to serious complications . IncorreAtypical anorexia nervosa ;Nervosa bulimia (of low frequency and/or limited duration);Binge eating disorder (of low frequency and/or limited duration);Purging disorders;Midnight Eating Syndrome.In the Diagnostic and Statistical Manual of Mental Disorders DSM-4), eating disorders include anorexia nervosa (AN), bulimia nervosa (BN), and eating disorder not otherwise specified (EDNOS). EDNOS is a composite diagnosis that includes all eating disorders that do not meet the AN or BN diagnostic criteria [, eating The DSM-5 also includes a category called unspecified feeding or eating disorder (UFED), which includes people who do not fit into any of these five categories or for whom there is not enough information to make a specific diagnosis of OSFED . In addiAlthough the different forms of eating disorders differ in course and treatment, they share common psychological and behavioral symptoms as well as risk factors. It has been shown that experiencing negative emotions increases the risk of losing self-esteem and thus losing control over your eating behavior . Excess The criteria for diagnosing eating disorders have changed over the years. One of the screening tools commonly used to detect eating disorders is the EAT-26 . EAT-26 The aim of this study was to determine the relationship between the impact of self-esteem and life satisfaction on the occurrence of eating disorders among adolescents in three areas, such as control, weight loss, and bulimia and food preoccupation.2, and the average BMI desired by the subjects was 19.72 ± 3 kg/m2. Each of the participants provided informed consent to participate in the study, and the consent of the legal guardian was also obtained. Lack of consent and an age under 12 or over 19 excluded participation in the research. The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Bioethics Committee at the University of Rzeszow . The study was conducted in 2022, when restrictions related to the COVID-19 pandemic were lifted. Isolation had no effect on the test results.In total, 233 primary and secondary school students aged 12 to 19 were surveyed. They all came from the Subcarpathian region. The average age of the respondents was 15.46 ± 2.33 years. Among the respondents, 37% were boys and 63% were girls. The mean body mass index (BMI) of the subjects was on average 20.84 ± 4 kg/mTo examine the risk of eating disorders, the Eating Attitudes Test-26 questionnaire was used. It explores the risk of eating disorders in three areas: dieting, bulimia and food preoccupation, and oral control. It is a standardized survey consisting of 26 questions relating to feelings and 4 questions related to behaviors occurring during the last 6 months in the examined person, such as losing more than 10 kg of body weight or a sense of loss of control over food consumption. This scale was developed in 1982 by David M. Garner for the early detection of eating disorders. The study used the Polish adaptation from 2016, developed by Rogoza et al. .The Self-esteem Scale (SES) of M. Rosenberg consists of 10 statements that relate to your beliefs about yourself. It is a tool that allows you to assess levels of self-esteem. The respondent is asked to indicate to what extent he or she agrees with each of the statements . This stThe Cantril scale has the form of a ladder, with degrees from 0 to 10 defining the level of satisfaction, with 0 being the worst and 10 the best satisfaction. It is assumed that a score <6 is defined as dissatisfaction, and >6 means satisfaction with one’s life .In addition, the respondents were asked to provide their height and current and desired body weight. On this basis, the current and desired Body Mass Index (BMI) was calculated, and their values were found on the WHO charts .p < 0.05.Statistical analysis of the collected data was performed in the Statistica 13.3 package. Only non-parametric tests were used for the analysis of variables. The choice of this type of test was conditioned by the failure to meet the basic assumptions of parametric tests, i.e., compliance of the distributions of the examined variables with the normal distribution, which were verified by the Shapiro–Wilk W test. For most numerical variables, descriptive statistics were calculated: mean, median, minimum, maximum, first and third quartiles, and standard deviation. The Mann–Whitney U test was used to assess the differences in the average level of a numerical trait in the two populations. The correlation of two variables that did not meet the criterion of normal distribution was determined using Spearman’s rank correlation coefficient. The level of statistical significance was p < 0.001), body weight (p = 0.007) and life (p < 0.001) compared to women. Compared to men, women scored more on the SES scale (p = 0.001); therefore, they had lower self-esteem and a higher risk of eating disorders—EAT scale (p < 0.001). Regarding eating disorders, women also scored higher on dieting (p = 0.034), bulimia and food preoccupation (p = 0.005) and oral control (p = 0.001) (It was shown that men obtained higher results of satisfaction with their appearance (= 0.001) .p = 0.035; R = −0.14), and a positive relationship between the age of the subjects and bulimia and food preoccupation . The older the respondents were, the lower their score in the area of eating disorders—oral control. Conversely, older people scored higher in the category of bulimia and food preoccupation among ED areas . The influence of age on losing more than 10 kg is shown in the It was shown that people who lost 10 kg or more were on average younger (14.87 years old) than people who did not lose such an amount of body weight (15.64 years old). The described difference in the age of the subjects from the two groups was statistically significant (p = 0.052), although this result was close to the threshold of significance. People treated for this reason were on average slightly older (16.5 years old) than those not treated (15.37 years old).There were no differences in the ages of people treated and not treated for eating disorders  were less satisfied with their appearance and their body weight. It also showed that people with lower self-esteem were more likely to overeat in the last 6 months when they felt they could stop, vomit or exercise more than 60 min a day to lose weight or control weight. People with lower self-esteem had a higher risk of eating disorders—EAT-26 scale in all three areas .p = 0.010). The descriptive data related to weight loss of 10 kg or more and self-esteem are in People who had lost 10 kg or more in the last 6 months had lower self-esteem  than people who had not lost weight (23.53 points). This difference was statistically significant (p = 0.023). The correlation between treatment for eating disorders and self-esteem is shown in In addition, it was shown that people who were treated for eating disorders had lower self-esteem  than people who were not treated for this reason (23.68 points). This difference was statistically significant (p < 0.001), satisfaction with their body weight (p < 0.001), risk of eating disorders—based on the EAT-26 scale in the area of slimming (p < 0.001), and controls (p = 0.017). The higher the respondent’s self-esteem, the higher their satisfaction with their appearance and body weight was. Risk of eating disorders—the EAT-26 scale was statistically significantly more frequent in people with low self-esteem than in people with average and high self-esteem. This was similar in the ED area—slimming. The degree of control, however, differed statistically significantly among people with low and high self-esteem  from 1980 to 2019. It was found that over the years the incidence among persons aged under 15 years has increased. The reason may be the more effective diagnosis of the disease and taking into account younger and younger subjects . A studyIt should be noted that people with abnormal eating habits are at high risk of eating disorders, which are characterized by a psychopathological attitude and accompanying groups of syndromes involving weight changes or physiological disorders, including anorexia nervosa, bulimia and atypical eating disorders . For exaTo a large extent, self-esteem is related to satisfaction with the appearance. In particular, low self-esteem determines dissatisfaction with the appearance of the body, and vice versa, low self-esteem leads to dissatisfaction and a negative perception of the figure . In our Satisfaction with life plays a significant role as a risk factor for eating disorders. Satisfaction with appearance and self-confidence affects this indirectly. Doornik et al. found that life satisfaction is a plastic factor that changes with the severity of anorexia symptoms. To obtain effective treatment, you also need to improve your commitment to specific domains of life . Eating The present study has some limitations. Respondents could subjectively complete the questionnaires, which may change the results. Another limitation is the small number of men in the study. This study, however, provides some direction for future research. All people from primary and secondary schools in the Subcarpathian region who gave their consent were tested. Each respondent had different conditions of private life, knowledge about the state of physical health, and emotional approach. We believe that it would be interesting to study a group in a lower age range, with the exclusion criteria presented above. It is also worth diversifying the income levels and living environments of children’s carers in terms of the impact on the quality and quantity of nutrition. Considering further research on this topic, researchers should increase the study group and include adolescents from different regions of the country.Gender plays a significant role in the risk of eating disorders. Women, compared to men, showed lower self-esteem and at the same time a higher risk of eating disorders in all three areas. Men showed greater satisfaction with their appearance, body weight and their lives, and they also had higher self-esteem.Episodes of significant weight loss (>10 kg) concerned a group of younger students. The younger the person, the greater the probability of eating disorders, especially in the area of bulimia and food preoccupation and control.Low self-esteem correlated positively with significant weight loss (>10 kg), more frequent uncontrollable binge eating and exercising more than 60 min a day to influence appearance. People with lower self-esteem were more likely to be less satisfied with their appearance and weight and were more likely to be treated for eating disorders.Among adolescents, satisfaction with appearance and weight affects life satisfaction, as measured with the Cantril ladder, which showed a positive relationship with the risk of eating disorders in all three areas. Subjects dissatisfied with their lives binged, feeling that they could not stop.


Scutellariae Barbatae Herba and Scutellariae Radix and is demonstrated to have anti-tumor properties in colon cancer. Notwithstanding, the function and mechanism of Salvigenin in hepatocellular carcinoma (HCC) are less well studied. Different doses of Salvigenin were taken to treat HCC cells. Cell viability, colony formation ability, cell migration, invasion, apoptosis, glucose uptake, and lactate production levels were detected. As shown by the data, Salvigenin concentration dependently dampened HCC cell proliferation, migration, and invasion, weakened glycolysis by abating glucose uptake and lactate generation, and suppressed the profiles of glycolytic enzymes. Moreover, Salvigenin strengthened HCC cells’ sensitivity to 5-fluorouracil (5-FU) and attenuated HCC 5-FU-resistant cells’ resistance to 5-FU. Through network pharmacological analysis, we found Salvigenin potentially regulates PI3K/AKT pathway. As shown by the data, Salvigenin repressed the phosphorylated levels of PI3K, AKT, and GSK-3β. The PI3K activator 740Y-P induced PI3K/AKT/GSK-3β pathway activation and promotive effects in HCC cells. However, Salvigenin substantially weakened 740Y-P-mediated effects. In-vivo assay revealed that Salvigenin hampered the growth and promoted apoptosis of HCC cells in nude mice. Collectively, Salvigenin impedes the aerobic glycolysis and 5-FU chemoresistance of HCC cells by dampening the PI3K/AKT/GSK-3β pathway.Salvigenin is a Trimethoxylated Flavone enriched in The online version contains supplementary material available at 10.1007/s12010-023-04511-z. Hepatocellular carcinoma (HCC), a primary malignancy of hepatocytes, has high-level invasiveness . In the Metabolic reprogramming, particularly aerobic glycolysis , was first detected in HCC . As HCC Scutellariae Barbatae Herba (SBH), a dried whole plant of Scutellaria Barbata D. Don, is referred to as Ban-Zhi-Lian in traditional Chinese medicine (TCM). SBH, equipped with anti-cancer, anti-oxidant, and anti-angiogenic activities, enjoys a history of thousands of years in China [Scutellariae Radix, the root of Scutellaria Baicalensis, boasts anti-inflammatory and anti-cancer functions [Scutellariae Radix exerts a remarkable anti-cancer function in liver cancer [Scutellariae Barbatae Herba and Scutellariae Radix, is reported to show the dual activities of reducing lipid and boosting mitochondrial functions [in China –15. The in China , 17. Scuunctions . Scutellr cancer . Salvigeunctions . Furtherunctions . Salvigeunctions .Network pharmacology is an emerging field in terms of the pharmacological research of modern Chinese medicine. It reveals the underlying molecular mechanisms of traditional Chinese medicine potency via the establishment and visualization of the “drug-target-disease” interactive network , 24. Herhttps://www.genecards.org/) database and obtained Salvigenin-associated targets from SwissTarget (http://www.swisstargetprediction.ch/) [https://pubchem.ncbi.nlm.nih.gov/). Venn’s diagram online tool (https://bioinfogp.cnb.csic.es/tools/venny/index.html) was adopted to analyze the crossing targets of the disease and drug. The Cytoscape software (version 3.9.1) was harnessed to visualize the drug-target network diagram. We uploaded the common targets to the String database (https://cn.string-db.org/cgi/input.pl) to produce the protein-protein interaction (PPI) network diagram. The DAVID database (https://david.ncifcrf.gov/home.jsp) was introduced for the GO and KEGG enrichment analyses of the common targets.We acquired the targets related to hepatocellular carcinoma (HCC) from the GeneCards (ion.ch/) . The Iso2, humid air).The Human HCC cell lines  were ordered from American Type Culture Collection. The cells were cultured with an RPMI1640 medium  filled with 10% fetal bovine serum  and 1% penicillin/streptomycin in an incubator  for overnight culture. Predicated on prior studies, we treated the cells with Salvigenin  or 5-FU  for 24 h. Next, 10 μL of CCK-8 solution was administered to each well for 2 h of further incubation . At the end of the culture, a microplate reader  was utilized to gauge the absorbance value at 450 nm. Santa Cruz Biotechnology, Inc.  was the supplier of Salvigenin (CAS number: 19103-54-9). Cell death was assessed using the Calcein/PI Cell Viability/Cytotoxicity Assay Kit . Huh7 and HepG2 cells were seeded into 96-well plates (density: 1×103 cells/well) for overnight culture. Followed by Salvigenin  treatment, Calcein AM/PI working solution was added into each well (100 per well), and the plates were put in a 37 °C incubator for 30 min. The Calcein AM/PI fluorescence signals were observed and taken using a microscope .The CCK-8 kit  was adopted to examine cell viability. We inoculated HCC cells into 96-well plates , 740Y-P (50 μg/mL) or 5-FU (100 μM) for two days. After the medium was removed, the cells were cultured in a fresh culture medium without the above drugs. The medium was exchanged every 3 days; 10 days later, the colonies were fixed using 70% methanol for 30 min. Then, they were stained in 0.5% crystal violet at room temperature (RT) for 30 min. A light microscope  was utilized for counting the number of cell colonies.Huh7 and HepG2 cells were seeded onto 60-mm culture dishes with a density of 1×10−ΔΔCT approach was employed to calculate the relative expression of the target gene . The primers were designed via Primer3 (https://primer3.ut.ee/). The primer sequences are exhibited in Table TRIzol reagent (Thermo Fisher Scientific) was taken to extract total RNA from the cells. Nano Drop 2000  was adopted to measure the purity and concentration of the total RNA. As per the manufacturer’s recommendations, the RevertAid First Strand cDNA Synthesis Kit  was exploited to reverse-transcribe 1 μg of the total RNA into cDNA, which was utilized as the template to amplify the target gene and reference gene GAPDH. SYBR GreenPCR (MedChemExpress) was introduced for PCR. Thermal cycling was implemented with these conditions: 3 min of initial degeneration at 95 °C, 10 s of denaturation at 95 °C, 30 s of annealing at 60 °C, and 30 s of extension at 60 °C . The 2We harvested the tumor tissues from the nude mice and HCC cells treated with varying doses of Salvigenin (25–100 μM) and/or 740Y-P (50 μg/mL). RIPA lysis buffer  supplemented with a mixture of protease and phosphatase inhibitors  was adopted to extract total protein from the cells and tumor tissues, respectively. The Bicinchoninic Acid (BCA) protein quantification kit  was harnessed for protein quantification. Then, 50 μg of the total protein samples from each group were isolated through 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and moved onto polyvinylidene difluoride (PVDF) membranes ; 5% skimmed milk was given to seal the membranes at RT for 2 h. Next, the membranes were incubated with primary antibodies , Anti-p-PI3K , Anti-AKT , Anti-p-AKT , Anti-GSK-3β , Anti-p-GSK-3β , Anti-HK2 , Anti-PFK1 , Anti-PKM2 , and Anti-β-actin ) at 4 °C overnight. Later, the horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody  was added to the membranes for 2 h of incubation at RT. Color and image development was done with the use of ECL  in darkness. β-actin served as the internal parameter.5 cells/mL), 200 μL of the cell suspension was given to the upper chamber, whereas 600 μl of a medium with 15% FBS was administered to the lower compartment. A sterile, thermostatic incubator was adopted to culture the cells for 24 h at 37 °C with 5% CO2. The compartments were taken out. The cells that failed in migration and invasion were wiped off using cotton swabs. Transwell membranes were immobilized in 4% paraformaldehyde for 10 min and dyed in 0.1% crystal violet. They were observed and photographed employing a microscope.Transwell compartments  were placed in 24-well plates; 50 μL of diluted Matrigel  was added to the upper compartment in the invasion experiment instead of the migration assay. With the cells suspended in a medium without serum . Then, the supernatant was harvested. As instructed by the manufacturer, the glucose uptake detection kit  and the lactate production detection kit  were taken to determine the levels of glucose uptake and lactate generation.6 cells/mL, Huh7 cells were suspended in a serum-free medium and subcutaneously transfused into the right abdomen of the mice (0.1 mL each). In this way, a liver cancer xenograft model was built. A vernier caliper was taken to gauge the length (L) and width (W) of transplanted tumors every three days. As the tumor volume attained around 50 mm3, the animals were randomized to different groups (n = 5 per group). Salvigenin  was intraperitoneally injected into the mice for treatment. Normal saline of the identical amount was given to the control group. The mouse tumor bodies were examined every week for 4 weeks running. V (mm3) = L × W2/2. When the experiment came to an end, the mice were sacrificed through the inhalation of excessive CO2. The tumors were separated and photographed. Their volume and weight were gauged. The tumors were harvested for the following experiments. All experiments, authorized by the Ethics Committee of Zhejiang Taizhou Hospital, affiliated with Wenzhou Medical University, were implemented in conformity with the Guidelines for the Care and Use of Laboratory Animals .Male BALB/c nude mice, 6–8 weeks of age and 20 ± 2 g in weight, were ordered from the Animal Experimental Center of Wenzhou Medical University. They were reared under specific-pathogen-free (SPF) conditions  with a 12-h light/dark cycle. They could access food and water at will. With the cell density adjusted to 4×102O2 was applied to block the activity of endogenous peroxidase. After antigen repair, the slices were sealed using 5% goat serum for 20 min, flushed in PBS three times, and then incubated along with Anti-Ki67  antibody overnight at 4 °C. Afterward, the sections were incubated with the HRP-conjugated goat anti-rabbit IgG secondary antibody  for 2 h. Diaminobenzidine (DAB) was taken for color development. Hematoxylin was used for redyeing. The staining outcomes were observed employing a microscope  after the slices were dehydrated, made transparently, and sealed by neutral gum.The mouse tumor tissues were immobilized using 4% paraformaldehyde, dehydrated with alcohol of gradient concentrations, made transparent with xylene, embedded in paraffin, and severed into slices (4-μm thick). The sections were dewaxed and hydrated; 3% HThe paraffin slices of tumor tissues were dehydrated using gradient alcohol, washed in distilled water, dyed with the hematoxylin solution for 8 min, flushed in running water, and turned blue with 0.6% ammonia. Subsequently, the sections were stained in the eosin solution for 2 min, dehydrated, and made transparent with the use of xylene. Finally, they were dried and sealed with neutral gum. A microscope  was utilized for observation and photography.The paraffin slices were routinely dewaxed to water and subjected to antigen repair. After PBS washing, the sections were sealed using 5% bovine serum for 30 min. We added the primary antibodies Anti-PI3K  and Anti-AKT  to incubate them overnight at 4 °C. Next, the fluorescence-labeled goat anti-rabbit IgG secondary antibody  was applied for 1-h incubation at RT. DAPI was utilized for redyeing. An anti-fluorescence quenching agent was adopted to seal the sections. A fluorescence microscope  was taken for observation and imaging.After the HCC cells were treated with Salvigenin (100 μM), 740Y-P (50 μg/mL), or 5-FU (100 μM), as mentioned before, the medium was removed. The cells were flushed in pre-cooled PBS, fixed with 4% polyformaldehyde for 60 min, and rinsed in PBS once. Later, an immunostaining detergent was applied for 2 min of incubation in an ice bath. The samples were incubated for an hour in darkness at 37 °C with 50 μL TUNEL solution. After being flushed in PBS three times, the slices were sealed using an antifade mounting medium  and observed by a fluorescence microscope .As per the instructions of the TUNEL apoptosis kit , the tumor slices were routinely dewaxed, hydrated, and flushed using PBS three times. The Protease K working solution (20 μg/mL) without any DNase  was added for 15 min of reaction at 37 °C. The slices were rinsed in PBS three times. Next, 50 μL TUNEL solution was added and evenly spread over the samples. After being sealed with an anti-fluorescence quenching agent, the slices were observed by a fluorescence microscope.The mouse blood samples were harvested. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and serum creatinine (Scr) were through the Fuji Dri-Chem 3500i Biochemistry Analyzer .t-test was implemented to compare data differences between two groups. P < 0.05 held statistical significance.GraphPad Prism 8.0  was introduced for analyzing statistics. Measurement statistics were exhibited as mean ± standard deviation (SD). One-way ANOVA was taken for comparison among multiple groups, followed by post hoc Tukey’s test, while a To investigate the pharmacological function of Salvigenin Fig. B. We alsP < 0.05, Fig. P < 0.05, Fig. Aerobic glycolysis, a symbol of cancer metabolism, features glucose uptake and lactate generation. This prompted us to probe the influence of Salvigenin on the aerobic glycolysis of HCC cells. Corresponding kits were taken to examine the levels of glucose uptake and lactate generation in Huh7 and HepG2 cells. As a result, Salvigenin vigorously attenuated glucose uptake and lactate production  and 5-FU  were applied to treat Huh7 and HepG2 cells for the purpose of studying the influence of Salvigenin on HCC cells’ chemoresistance. CCK-8 tested cell viability, with the IC50 value calculated. The outcomes displayed that Salvigenin dramatically fortified HCC cells’ sensitivity to 5-FU (versus the con group) (https://www.genecards.org/). The SMILES number of Salvigenin obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) was inputted into the SwissTarget database (http://www.swisstargetprediction.ch/). We selected Homo Sapiens and downloaded 100 Salvigenin-concerned targets. The targets of the disease and drug were uploaded to Venn’s diagram online tool (https://bioinfogp.cnb.csic.es/tools/venny/index.html), and we obtained 32 common targets . We selected Homo Sapiens to generate the PPI network diagram  and visualized the top 20 signaling pathways and the top 10 terms of biological process (BP), cellular component (CC), and molecular function (MF)  and The Human Protein Atlas (HTTPS://www.proteinatlas.org/) databases revealed that the expressions of PIK3CA, AKT1, and GSK3B in HCC tissues were enhanced   Fig. A, B throP < 0.05, Fig. P < 0.05, Fig. To better understand the anti-cancer function of Salvigenin in HCC, we treated Huh7 cells with Salvigenin and/or 740Y-P (the PI3K activator). CCK-8, colony formation assay, Transwell migration and invasion experiments, and TUNEL were implemented. Figure in vivo. Salvigenin dose-dependently hampered tumor growth  was taken to treat the mice. In this way, we probed the influence of Salvigenin on HCC cell growth wth Fig. A, B. HE wth Fig. A, B. Thein vivo.The profiles of HK2, PFK1, PKM2, and the PI3K/AKT/GSK-3β pathway were determined by western blot. The experimental statistics unveiled that Salvigenin restrained their expressions Fig. A, B. Tisin-vivo and in-vitro experiments. Salvigenin exerted its cancer-suppressing function in the context of HCC by hampering the PI3K/AKT/GSK-3β signaling pathway.HCC development, a sophisticated biological process, concerns multiple molecular and cellular signaling pathways . Conventin-vitro outcomes, Salvigenin concentration dependently hampered HCC cell proliferation, migration, and invasion, elicited apoptosis, and weakened cell aerobic glycolysis and chemoresistance. In-vivo experiments unraveled that Salvigenin repressed the growth of tumors  . The PI3Glycogen synthase kinase (GSK)-3β is a classical downstream target of the AKT pathway. Reportedly, GSK-3β has two phosphorylation sites (Ser9 and Tyr216). The phosphorylation of the Ser9 site can culminate in GSK-3β inactivation, whereas the phosphorylation of the Tyr216 site leads to GSK-3β activation . The funAll in all, through network pharmacological analysis and experiments, we have probed the underlying mechanism of Salvigenin in HCC. As a result, Salvigenin may dampen HCC cell proliferation, migration, and invasion and weaken cell glycolysis and chemoresistance mainly by modulating the PI3K/AKT/GSK-3β pathway. Nonetheless, our study still has some limitations. In more animal studies, we will confirm the function and molecular mechanism of Salvigenin in the context of HCC.Supplementary Figure 1http://gepia.cancer-pku.cn/) database. D-G: The Human Protein Atlas (https: //www. proteinatlas.org/) was adopted to check the profiles of PIK3CA, AKT1, and GSK3B proteins in HCC tissues and normal liver tissues. (PNG 2290 kb)The profiles of PIK3CA, AKT1, and GSK3B in HCC tissues and adjacent normal tissues. A-C: The profiles of PIK3CA, AKT1, and GSK3B in HCC tissues and adjacent normal tissues were determined through the GEPIA (High resolution image (TIF 8192 kb)Supplementary Figure 2http://gepia.cancer-pku.cn/) was introduced to verify the relationship between the levels of PIK3CA, AKT1, and GSK3B and the OS and RFS rates of HCC patients. (PNG 495 kb)The correlation between the expression levels of PIK3CA, AKT1, and GSK3B and prognosis in HCC. A-C: The GEPIA database (High resolution image (TIF 1670 kb)Supplementary Figure 3The toxicity of Salvigenin in tumor-bearing mice. A-B: ELISA ascertained ALT, AST, Scr, and BUN levels in the mouse serum. C: HE staining monitored pathological alterations in the heart, liver, spleen, lung, and kidney tissues of mice. (PNG 4175 kb)High resolution image (TIF 14644 kb)


Specifically, the research topics “new bio-ink investigation,” “modification of extrusion-based bioprinting for cell viability and vascularization,” “application of 3D bioprinting in organoids and in vitro model” and “research in personalized and regenerative medicine” were predicted to be hotspots for future research.Three-dimensional (3D) bioprinting is an advanced tissue engineering technique that has received a lot of interest in the past years. We aimed to highlight the characteristics of articles on 3D bioprinting, especially in terms of research hotspots and focus. Publications related to 3D bioprinting from 2007 to 2022 were acquired from the Web of Science Core Collection database. We have used VOSviewer, CiteSpace, and R-bibliometrix to perform various analyses on 3,327 published articles. The number of annual publications is increasing globally, a trend expected to continue. The United States and China were the most productive countries with the closest cooperation and the most research and development investment funds in this field. Harvard Medical School and Tsinghua University are the top-ranked institutions in the United States and China, respectively. Dr. Anthony Atala and Dr. Ali Khademhosseini, the most productive researchers in 3D bioprinting, may provide cooperation opportunities for interested researchers. Tissue Engineering Part A contributed the largest publication number, while Frontiers in Bioengineering and Biotechnology was the most attractive journal with the most potential. As for the keywords in 3D bioprinting, Bio-ink, Hydrogels , Scaffold , extrusion-based bioprinting, tissue engineering, and  Three-dimensional (3D) bioprinting is a technology that enables the 3D printing of various cells, biocompatible materials, and supporting components into complex 3D functional living tissues . This tein vitro models and organoids  database has been widely used in bibliometric studies . We perfSeveral software are frequently used in bibliometric analysis, including VOSviewer, CiteSpace, and HistCite . Two sofVOSviewer 1.6.18 is a widely used software for constructing knowledge-maps based on a co-occurrence matrix . The genCiteSpace 6.1. R2 is another bibliometric software for creating visualization maps based on the data retrieved from the database . Severalhttps://bibliometric.com/app_v0) was used for the comparison analysis of the annual number of papers among the top 10 countries. GraphPad Prism 8 and Microsoft Office Excel 365 were respectively used to draw a column chart and provide a descriptive analysis for annual publications. Impact factor (IF) scores were extracted from 2021 Journal Citation Reports (JCR).Moreover, an online platform (R2 = 0.9873) of the trend was created, where X represents the year and Y indicates the amount of annual publications.Based on the search strategy, a total of 3,327 papers were identified. The number of annual publications regarding 3D bioprinting is illustrated in n = 1,069), followed by China (n = 733), South Korea (n = 288), Germany (n = 210), and India (n = 166). As shown in All publications regarding 3D bioprinting were published by 2,733 institutions in 83 countries/regions in total. The top 10 prolific countries and institutions are listed in n = 86) is the institution with the largest number of publications, followed by Tsinghua University (n = 75), Chinese Academy of Sciences (n = 74), Zhejiang University (n = 69), and Wake Forest School of Medicine (n = 56). University of California, Los Angeles, has the highest centrality of 0.18. Of the top 10 most prolific institutions, four were from China and four were from United States. Moreover, an institution co-authorship analysis is shown in The co-occurrences of institutions are presented in n = 63) contributed the largest number of papers, followed by Lee SJ (n = 53), Gatenholm P (n = 41), Zhang YS (n = 39), and Yoo JJ (n = 39). These and the rest of the authors are listed in The top 25 most prolific authors are shown in n = 1,139), followed by Ozbolat IT (n = 806), Skardal A (n = 691), Kolesky DB (n = 650), and Pati F (n = 589). These and the rest of the top 10 co-cited authors are listed in The density maps of co-cited authors based on co-citations are shown in n = 225), followed by Biofabrication (n = 191), Advanced Healthcare Materials (n = 82), International Journal of Bioprinting (n = 81), and Frontiers in Bioengineering and Biotechnology (n = 65). All the top 10 most productive journals had an IF (2021) of >4. Seven of these journals were categorized in the Q1 JCR division.The articles included in the analysis were published in 752 journals. Of these, 33 published a minimum of 20 papers and were included and visualized in The dual-map overlay of journals created using CiteSpace is shown in in vitro and in vivo applications (ications . In 2006ications . In 2019ications  proposedn = 470) and the highest strength of citation bursts (Strength = 120.73) was published in Nature Biotechnology by Murphy SV et al. in 2014 , Bio-ink was defined as materials which are capable to include cells and other bioactive components for the use in biofabrication . Bio-inkBased on the material requirements, hydrogels are gradually being used in bio-inks. Hydrogel biomaterials include alginate, gelatin, collagen, fibrin/fibrinogen, gellan gum, hyaluronic acid, agarose, chitosan, silk, decellularized extracellular matrix (dECM), poly (ethylene glycol), and Pluronic. Hydrogels have many attractive features for use as bio-inks. As they are biocompatible and typically biodegradable, most of them are easy for cells to adhere, grow, spread and proliferate on . FurtherTissue engineering is one of the hottest research fields in recent years, and it has been widely applied to musculoskeletal tissue, oral tissue, cardiovascular tissue, urogenital tissue, ocular tissue, and so on . Based oThere are four major 3D bioprinting methods, namely, extrusion, inkjet, stereolithography, and laser-assisted printing . EBB extDECM scaffold refers to biomaterials formed by human or animal organs/tissues with the removal of immunogenic cellular components using decellularized technologies . A dECM In vitro models are important tools to study the occurrence and development of diseases , is the research Frontier in this field, and it is currently in an explosive period. Last but not least, the application of 3D bioprinting in personalized and regenerative medicine will continue to develop in the future, but the important issues of cell viability and vascularization need to be addressed first.Using information visualization technology, this study fully summarizes the research progress, hotspots, and frontiers in the 3D bioprinting field. Exciting findings can provide a foundation for further research in this field and inspire the collaboration among potential partners and institutions. Research on 3D bioprinting has greatly developed and still has huge prospect in the future. The United States and China have absolute advantages in this field, and interested researchers may find cooperation opportunities in Harvard Medical School, Tsinghua University, Chinese Academy of Sciences, University of California-Los Angeles, and Wake Forest School of Medicine. Tissue Engineering Part A published the most articles in this field, and Dr. Anthony Atala was the most productive author. The academic exchange and cooperation between countries, institutions, and authors has promoted the development of this research field. Bio-inks and scaffolds have become a hotspot recently. The development of different hydrogels  and dECM is expected to become an attractive direction in the next years. In addition, further studies on the modification of the EBB technique are key to promoting the progress of 3D bioprinting. Notably, the application of 3D bioprinting, especially in tissue engineering and