Protective Effects of Naringenin in a Rat Model of Sepsis-Triggered Acute Kidney Injury via Activation of Antioxidant Enzymes and Reduction in Urinary Angiotensinogen
Authors: Guoxin Hu
Lin Mu
Yan Chen
Wenjuan Cui
Lujun Qiao
Jian Liu
Journal: Medical Science Monitor (2019)
Discipline: CLINICAL MEDICINE
Keywords: Sepsis; Naringenin; Kidney injury; Rats; Apoptosis
Affiliations: Department of Critical Care Medicine, Shengli Oilfield Central Hospital, Dongying, Shandong, China (mainland).

Abstract:  BACKGROUND Sepsis is a devastating medical condition. In the USA, about 745 000 people are diagnosed with sepsis annually. Although many anti-inflammatory drugs have been used to manage sepsis, the treatment success rate is very low. This study was undertaken to examine the protective effects of naringenin on sepsis-induced kidney injury in rats. MATERIAL AND METHODS Sepsis was induced in Wistar albino rats by cecal ligation and puncture methods. Histological analysis was performed with hematoxylin and eosin (HE) staining. Reactive oxygen species (ROS) levels were determined by flow cytometery. TUNEL assay was used to demonstrate apoptosis. Sandwich ELISA method was used for the determination of urinary angiotensinogen, and protein expression was determined by Western blot analysis. RESULTS We found that naringenin decreased atrophy in the glomerulus and enabled maintenance of the capsule area and normal tubular cavity of the septic rats. Admistration of naringenin at the dosage of 10 and 20 mg/kg to sepsis rats caused significant reduction in the sepsis-induced apoptosis of kidney cells, accompanied by decrease in Bax and increase in Bcl-2 expression. Moreover, naringenin also decreased the ROS levels in septic rats and downregulated the expression of SOD, CAT, and APX. The effects of naringenin were also examined on the levels of urinary angiotensinogen in sepsis rats. We found that naringenin caused a significant decrease in urinary angiotensinogen levels of septic rats. CONCLUSIONS Naringenin appears to have potential in the treatment of sepsis. 

Retracted: No

PubPeer comments:
  The cells isolated from the kidney tissues from all the treatment groups were washed twice in PBS and re-suspended in 500 u03bcl of DCFH-DA (10 u03bcM) at 37u00b0C in a dark room for 30 min. The samples were then examined instantly using flow cytometry as described previously [15]. [...] As ROS has been implicated in sepsis-induced kidney injury [15], flow cytometery was used to examine the ROS levels in all 4 rat groups. The reference is to  15. Hua F, Li CH, Chen XG, Liu XP: Daidzein exerts anticancer activity towards SKOV3 human ovarian cancer cells by inducing apoptosis and cell cycle arrest, and inhibiting the Raf MEK ERK cascade. Int J Mol Med, 2018; 41(6): 3485u201392 . The authors appear to be under the impression that [Hua et al (2018)] mentioned or quantified ROS levels. They did not. ------------------------------------------------ The influence of [Hua et al (2018)] is surprisingly far-reaching. Google Scholar informs us that their in-vitro study of Daidzein has been cited **71** times. Many of these are review papers, but we also find a surprising number of papers using it in  Apoptosis Assay paragraphs to illustrate the use of flow-cytometry and Annexin V PI staining to quantify cell apoptosis. Indeed, Hua et al did apply the method, but they were not pioneers, nor is their explanation superior to many others. Alternatively, the paper is cited as a precedent for a phytochemical (daidzein) inhibiting cancer proliferation by interrupting the cell cycle. Given all the earlier precedents, the choice again invites some explanation. It is almost as if all these papers were prepared by a third-party service (a papermill ), following a template from one paper to the next, without checking its contents. Consistent with this notion, the papers came to my attention by recycling the same figures (despite a lack of authors in common). - [Jia et al (2019)] .  The slides were covered with a covers lip and examined with a fluorescent microscope. Annexin V PI staining of the OVACAR3 cells was performed as described previously [17]. - [Luo et al (2019)] .  Many plant-derived molecules have been reported to interfere with the **ROS** level in the cancer cells [13]. ... The slides were then covered with a covers lip and examined with a fluorescent microscope. Then 4u2019,6-diamidino-2-phenylindole (DAPI) and annexin V propidium iodide (PI) staining was performed as described previously [13]. - [Zeng et al (2019)] .  The slides were then covered with a covers lip and examined with a fluorescent microscope (BDBiosciences, San Jose, CA, USA). The annexin V PI staining was performed as described previously [8]. - [Wu et al (2019)] *[retracted]*,  The slides were covered with a coverslip and examined with a fluorescent microscope (BD Biosciences, San Jose, CA, USA). Annexin V PI staining was performed as described previously [13]. - [Yang, Chen Wang (2019)] .  The slides were covered with cover slips and examined with a fluorescent microscope. Annexin V PI staining of the SW-948 cells was performed as described previously 16 . - [Yang et al (2019)] .  The slides were covered with cover slips and examined with a fuorescent microscope. DAPI and annexin V propidium iodide (PI) staining were performed as described previously [14] *[14 is a misprint for 13]*. - [Huang et al (2019)] .  The slides were covered with cover slip and examined under fluorescence microscope. Annexin V propidium iodide (PI) staining of the Icariin-treated HeLa cells was performed as described previously [18]. - [Liu et al (2019)] .  The distribution of the breast cancer SK-BR-3 cells in different cycle phases was performed by flow cytometry after PI staining, following the method reported by Hua et al [11]. ...After overnight incubation at 4u00b0C, the cells were subjected to PI staining and flow cytometry. - [Liu et al (2019)] .  After covering the glass slides with cover slips, the cells were analyzed under a fluorescent microscope (Nikon Instruments, Inc., NY, USA). For determining percentage of apoptotic cells, Annexin V PI staining assay using a FACSCalibur flow cytometry (BD Biosciences, NY, USA) was carried out using a previously reported method [12] - [Yin et al (2019)] .  The slides were covered with covers slips and examined with a fluorescent microscope. Annexin V PI staining of the SW-948 cells was performed as previously described [13] . - [Zhang et al (2019)] .  The slides were cover-slipped and examined with a fluorescent microscope. Annexin V PI staining of the SW48 cells was performed as described previously [11] . - [Ma et al (2019)] .  The slides were cover-slipped and examined under a fluorescent microscope. Annexin V PI staining was performed as described previously [15]. - [Li et al (2019)] .  The slides were cover-slipped and assessed by fluorescent microscopy. ROS estimation was performed as described previously [13]. - [Zhang et al (2019)] .  The slides were covered with a cover slip and examined with a fluorescent microscope. Annexin V PI staining was performed as previously described [12]. - [Wu et al (2020)] .  The slides were covered with a covers lip and examined with a fluorescent microscope. Annexin V PI staining of the AGS cells was performed as described previously [15]. - [Yu et al (2020)] .  The slides were cover-slipped and examined with a fluorescent microscope. Annexin V PI staining was performed as described previously [9]. - [Yin et al (2020)] .  The slides were cover-slipped and examined with a fluorescent microscope. Annexin V PI staining was performed as described previously [10]. - [Wei et al (2019)] .  The slides were covered with cover slips and examined with a fluorescent microscope. Annexin V PI staining of the OVACAR-3 cells was performed as described previously [11]. - [Wen et al (2020)] .  The glass slides laden with cell cultures were then covered with cover slips and then using a fluorescence microscope (BioTek Instruments, Inc., Winooski, VT, USA,) the cells were analysed. Quantification of apoptosis was done with annexin V PI staining assay which was performed as reported already (Hua et al., 2018). -------------------------------------------------------------- Cell-cycle arrest: - [Li Han (2019)] .  Cell cycle arrest is another important mechanism by which anticancer molecules exert their effects [16] . - [Mao et al (2019)] .  The arrest of the cancer cells at G2 M phase prevents the cancer cells from entering mitosis and as such the molecules that trigger the cell cycle arrest prevent the proliferation and tumourigenesis of cancer cells (Hua et al., 2018). - [Si et al (2019)] .  Previous studies have shown that depletion of cyclin A and B1 and upregulation of p27 and p21 results in the G2 M arrest of the cells during cell division [21]. - [Zhou et al (2019)] .  The arrest of cancer cells at G2 M phase prevents the cancer cells from entering mitosis, so the molecules that trigger cell cycle arrest prevent the proliferation and tumorigenesis of cancer cells [20]. - [Tan et al (2019)] .  Cell cycle arrest caused by several anticancer agents is an essential mechanism in the growth inhibition of cancer cells [19] . - [Wei et al (2019)] .  Furthermore, arrest of the cells at different phases of the cell cycle is an important route by which anticancer agents halt the growth of cancer cells [18]. - [Zeng et al (2019)] .  Besides autophagy, cell cycle arrest is another mechanism by which anticancer agents exert their anticancer effects [19] . -------------------------------------------------- Another list of papers cite Hua et al (2018) as precedents for methods which those authors did not in fact apply. - [Zhang et al (2019)] .  For determination of the ROS and mitochondrial membrane potential levels, the A549 cells were treated with 0, 9, 18, and 36 u00b5M concentrations of murrayanine for 24 hours and then the ROS and mitochondrial membrane potential levels in the A549 cells were determined, as described previously [11] *(in fact Hua et al did not measure or even mention ROS or mitochondrial membrane potentials)*. - [Guo et al (2019)] .  Plant-derived molecules have been reported to affect ROS levels in the cancer cells [15] . - [Xu et al (2019)] . Hua et al appear in the References but not in the text at all. - [Du et al (2019)] .  **Osteosarcoma** causes significant mortality world over and the treatments available are not efficient and are associated with a number of side-effects [11]. - [Yu et al (2019)] .  Several of the natural products have been reported to induce apoptosis and autophagy through production of ROS [18]. - [Liu et al (2019)] .  The colony formation assay was carried out as described previously [15] *[Hua et al did not perform colony-formation assays]* - [Zhu et al (2020)] .  Estimation of ROS was measured according to a method previously described [8]. - [Du et al (2020)] . Hua et al appear in the References but not in the text at all. ,