Corilagin alleviates podocyte injury in diabetic nephropathy by regulating autophagy via the SIRT1-AMPK pathway

BACKGROUND Diabetic nephropathy(DN)is the most frequent chronic microvascular consequence of diabetes,and podocyte injury and malfunction are closely related to the development of DN.Studies have shown that corilagin(Cor)has hepatoprotective,anti-inflammatory,antibacterial,antioxidant,anti-hypertensive,antidiabetic,and anti-tumor activities.AIM To explore the protective effect of Cor against podocyte injury in DN mice and the underlying mechanisms.METHODS Streptozotocin and a high-fat diet were combined to generate DN mice models,which were then divided into either a Cor group or a DN group(n=8 in each group).Mice in the Cor group were intraperitoneally injected with Cor(30 mg/kg/d)for 12 wk,and mice in the DN group were treated with saline.Biochemical analysis was used to measure the blood lipid profiles.Hematoxylin and eosin staining was used to detect pathological changes in kidney tissue.Immunohistochemistry and Western blotting were used to assess the protein expression of nephrin and podocin.Mouse podocyte cells(MPC5)were cultured and treated with glucose(5 mmol/L),Cor(50μM),high glucose(HG)(30 mmol/L),and HG(30 mmol/L)plus Cor(50μM).Real-time quantitative PCR and Western blotting RESULTS Compared with the control group,the DN mice models had increased fasting blood glucose,glycosylated hemoglobin,triglycerides,and total cholesterol,decreased nephrin and podocin expression,increased apoptosis rate,elevated inflammatory cytokines,and enhanced oxidative stress.All of the conditions mentioned above were alleviated after intervention with Cor.In addition,Cor therapy improved SIRT1 and AMPK expression(P<0.001),inhibited reactive oxygen species and oxidative stress,and elevated autophagy in HG-induced podocytes(P<0.01).CONCLUSION Cor alleviates podocyte injury by regulating autophagy via the SIRT1-AMPK pathway,thereby exerting its protective impact on renal function in DN mice.

Huangqin decoction alleviates lipid metabolism disorders and insulin resistance in nonalcoholic fatty liver disease by triggering Sirt1/NF-κB pathway

BACKGROUND Nonalcoholic fatty liver disease(NAFLD)is a clinicopathological entity characterized by intrahepatic ectopic steatosis.As a consequence of increased consumption of high-calorie diet and adoption of a sedentary lifestyle,the incidence of NAFLD has surpassed that of viral hepatitis,making it the most common cause of chronic liver disease globally.Huangqin decoction(HQD),a Chinese medicinal formulation that has been used clinically for thousands of years,has beneficial outcomes in patients with liver diseases,including NAFLD.However,the role and mechanism of action of HQD in lipid metabolism disorders and insulin resistance in NAFLD remain poorly understood.AIM To evaluate the ameliorative effects of HQD in NAFLD,with a focus on lipid metabolism and insulin resistance,and to elucidate the underlying mechanism of action.METHODS High-fat diet-induced NAFLD rats and palmitic acid(PA)-stimulated HepG2 cells were used to investigate the effects of HQD and identify its potential mechanism of action.Phytochemicals in HQD were analyzed by highperformance liquid chromatography(HPLC)to identify the key components.RESULTS Ten primary chemical components of HQD were identified by HPLC analysis.In vivo,HQD effectively prevented rats from gaining body and liver weight,improved the liver index,ameliorated hepatic histological aberrations,decreased transaminase and lipid profile disorders,and reduced the levels of pro-inflammatory factors and insulin resistance.In vitro studies revealed that HQD effectively alleviated PA-induced lipid accumulation,inflammation,and insulin resistance in HepG2 cells.In-depth investigation revealed that HQD triggers Sirt1/NF-κB pathwaymodulated lipogenesis and inflammation,contributing to its beneficial actions,which was further corroborated by the addition of the Sirt1 antagonist EX-527 that compromised the favorable effects of HQD.CONCLUSION In summary,our study confirmed that HQD mitigates lipid metabolism disorders and insulin resistance in NAFLD by triggering the Sirt1/NF-κB pathway.

Effect and Mechanism of Dicliptera chinensis Polysaccharide on miR-141/AMPK/SIRT1 Signaling Pathway in Rats with NAFLD

[Objectives]Non-alcoholic fatty liver disease(NAFLD)rat model was established by feeding high-fat and high-sugar fodder to rats,and the protective effect of Dicliptera chinensis polysaccharide(DCP)on NAFLD rats was studied to explore its potential mechanism.[Methods]45 SD rats were randomly divided into 4 groups:normal control group,model control group and DCP treatment groups(100 and 300 mg/kg).The rats in the normal control group were fed with ordinary fodder,and the rats in other groups were fed with high-fat and high-sugar diet for 14 weeks to establish NAFLD model.From the 9^(th)week,the rats in the DCP treatment groups were given different doses of DCP by intragastric administration(5 mL/kg)for 6 weeks.After the last intragastric administration,the rats fasted for 16 h,and the serum and liver of rats were collected for detection.Hematoxylin-eosin(HE)staining was conducted to observe the histopathological changes of rat liver,and alanine aminotransferase(ALT),aspartate aminotransferase(AST),superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),malondialdehyde(MDA),triglyceride(TG),total cholesterol(TC),low density lipoprotein cholesterol(LDL-C),and high density lipoprotein cholesterol(HDL-C)were detected by biochemical method.Interleukin-6(IL-6),interleukin-1β(IL-1β),tumor necrosis factor(TNF-α)and micrornA-141(micro RNA-141)were detected by reverse transcription-polymerase chain reaction(RT-PCR).The expression of SIRT1 and adenosine 5'-monophosphate(AMP)-activated protein kinase(AMPK)in rat liver was detected by western blot.[Results]Compared with the model control group,the inflammatory damage and steatodegeneration of rats in the DCP groups were relieved to varying degrees,and the number of lipid vacuoles significantly reduced.The ALT,AST,TC,TG and LDL-C content in the serum and MDA content in the liver tissue decreased to varying degrees,while the HDL-C,SOD and GSH-Px content increased.The expression of SIRT1 and AMPK increased,while the expression of miR-141,TNF-α,IL-6 and IL-1βdeclined,and the DCP 300 mg/kg treatment group had better improvement effect.[Conclusions]DCP had a certain protective effect on NAFLD rats,which may be related to the regulation of miR-141/AMPK/SIRT1 signaling pathway.

Quercetin ameliorates oxidative stress-induced senescence in rat nucleus pulposus-derived mesenchymal stem cells via the miR-34a-5p/SIRT1 axis

BACKGROUND Intervertebral disc degeneration(IDD)is a main contributor to low back pain.Oxidative stress,which is highly associated with the progression of IDD,increases senescence of nucleus pulposus-derived mesenchymal stem cells(NPMSCs)and weakens the differentiation ability of NPMSCs in degenerated intervertebral discs(IVDs).Quercetin(Que)has been demonstrated to reduce oxidative stress in diverse degenerative diseases.AIM To investigate the role of Que in oxidative stress-induced NPMSC damage and to elucidate the underlying mechanism.METHODS In vitro,NPMSCs were isolated from rat tails.Senescence-associatedβ-galactosidase(SA-β-Gal)staining,cell cycle,reactive oxygen species(ROS),realtime quantitative polymerase chain reaction(RT-qPCR),immunofluorescence,and western blot analyses were used to evaluated the protective effects of Que.Meanwhile the relationship between miR-34a-5p and Sirtuins 1(SIRT1)was evaluated by dual-luciferase reporter assay.To explore whether Que modulates tert-butyl hydroperoxide(TBHP)-induced senescence of NPMSCs via the miR-34a-5p/SIRT1 pathway,we used adenovirus vectors to overexpress and downregulate the expression of miR-34a-5p and used SIRT1 siRNA to knockdown SIRT1 expression.In vivo,a puncture-induced rat IDD model was constructed,and X rays and histological analysis were used to assess whether Que could alleviate IDD in vivo.RESULTS We found that TBHP can cause NPMSCs senescence changes,such as reduced cell proliferation ability,increased SA-β-Gal activity,cell cycle arrest,the accumulation of ROS,and increased expression of senescence-related proteins.While abovementioned senescence indicators were significantly alleviated by Que treatment.Que decreased the expression levels of senescence-related proteins(p16,p21,and p53)and senescence-associated secreted phenotype(SASP),including IL-1β,IL-6,and MMP-13,and it increased the expression of SIRT1.In addition,the protective effects of Que on cell senescence were partially reversed by miR-34a-5p overexpression and SIRT1 knockdown.In vivo,X-ray,and histological analyses indicated that Que alleviated IDD in a punctureinduced rat model.CONCLUSION In summary,the present study provides evidence that Que reduces oxidative stress-induced senescence of NPMSCs via the miR-34a/SIRT1 signaling pathway,suggesting that Que may be a potential agent for the treatment of IDD.

Mechanism of hesperidin improving myocardial ischemia/reperfusion injury in type 2 diabetic rats through SIRT1/Nrf2/HO-1 signaling pathway

Objective:To observe the protective effect of hesperidin on myocardial ischemia/reperfusion injury in type 2 diabetes mellitus and its effect on SIRT1/Nrf2/HO-1 signaling pathway.Methods:50 Sprague-Dawley(SD)rats were randomly assigned to the normal control group(NC),model group,ischemia-reperfusion group(IR),hesperidin group,SIRT1 inhibitor group and hesperidin plus SIRT1 inhibitor group.In addition to NC,the rats in the remaining groups were replicated by intraperitoneal of high-fat diet combined with injection of streptozotocin for type 2 diabetic rats.After then,the myocardial ischemia/reperfusion injury(MIRI)rat model was established by LAd for 30 minutes with 2 hours reperfusion.He staining was used to observe the pathological changes of myocardial tissue,and the levels of serum LDH,CK-MB and SOD,GSH and MDA in myocardial tissue were detected by kit methods,and the expression abundance of related proteins in 4-HNE and SIRT1/Nrf2/HO-1 signal pathway were detected by immunohistochemistry and Western blot;Results:Hesperidin could significantly inhibit cardiomyocyte necrosis and inflammatory cell infiltration,reduce LDH activity,CK-MB and MDA level,and increase SOD activity,GSH and 4-HNE level,the differences were statistically significant when compared with IR group(P<0.01).In addition,compared with the ischemia-reperfusion group,the expressions of SIRT1,Nrf2 and HO-1 proteins in hesperidin group were significantly up-regulated,the differences were statistically significant(P<0.01);Conclusion:Hesperidin inhibits oxidative stress by activating SIRT1/Nrf2/HO-1 signaling pathway,and play a protective effect of myocardial ischemia reperfusion injury in diabetic rats.

Mechanism of Resveratrol on autophagy mediated by Mst1/Sirt3 signaling pathway in diabetic cardiomyopathy

Objective:To observe the effects of resveratrol on myocardial cell injury and Mst1/Sirt3 signaling pathway mediated autophagy in type 2 diabetic mice. Methods:C57 BL/KSJ db/db mice were allocated to the normal control group,the model group,and the resveratrol group;C57 BL/KSJ db/m mice served as the melbine group,with 10 mice each. The resveratrol group and the melbine group were treated with resveratrol and metformin by gavage,respectively. The normal control group and the model group were treated with equal volume of normal saline by gavage,for 8 consecutive weeks. H & E staining,transmission electron microscopy and immunofluorescence were used to observe the pathological morphology,ultrastructure and apoptosis levels of myocardial tissues,respectively. RT-qPCR method was used to detect the expression levels of apoptosis genes Bax and Bcl-2 in myocardial tissues,and Western-blot method was used to detect the expression levels of autophagy proteins(LC3 and p62),Mst1 and Sirt3 proteins in myocardial tissue. Results:Compared with the model group,resveratrol can significantly reduce the body weight,blood glucose level and serum CK and LDH levels of db/db mice,and the differences were statistically significant(P<0.05;P<0.01). Meanwhile,after resveratrol treatment,myocardial inflammation score,apoptosis rate,Bax mRNA expression level and Bax/Bcl-2 ratio in myocardial tissue were significantly reduced,and Bcl-2 mRNA expression level was significantly increased,and the differences were statistically significant(P<0.01). In addition,compared with the model group,the expression level of p62 and p-Mst1 protein in the myocardial tissue of the resveratrol group was significantly reduced,and the expression level of Sirt3 protein and the ratio of LC3Ⅱ/LC3Ⅰ were significantly increased,and the differences were statistically significant(P<0.01). Conclusion:Resveratrol promotes the autophagy level of cardiomyocytes by activating the Mst1/Sirt3 signaling pathway and inhibits cardiomyocyte apoptosis to play a protective role in diabetic cardiomyopathy.

Protective Effect of SGLT2 Inhibitor on D-Galactose-Induced Senescence in Mice and Its Mechanism

Objective: To observe the cerebral protective effect of dagliflozin, a sodium-glucose co-transport protein 2 (SGLT2) inhibitor, in aging mice and to explore its molecular mechanism. Methods: 1. 66 male C57BL/6 mice were divided into control group (13) and model group (53), and the model group was moulded by subcutaneous injection of D-galactose into the back of the neck, while the control group was treated with equal amount of saline for 8 weeks. The weight of each group of mice was observed and recorded every 7 days, and two groups of mice were randomly selected for frozen sections of brain tissue at the end of the modelling period to verify the aging model. 2. After the aging model was successfully established, the aging groups were divided into 5 groups: model group, dagliflozin-treated group (high and low dose), and dagliflozin + ex527-inhibited group (high and low dose). Fasting blood glucose was measured in each group every 2 weeks for 8 weeks. At the end of treatment, Morris water maze was performed at the end of the treatment. After execution of the mice, the organ indices of heart, brain, liver, kidney and spleen were measured;the levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in serum were determined. Results: After the successful establishment of the aging model, it was found that during the treatment phase of dagliflozin. 1) The organ indices of mice in the aging group were significantly lower than those of other groups, and no significant hypoglycemia was observed throughout the treatment process. 2) In the water maze test, mice in the aging group had a significantly longer latency in the plateau phase compared to the control and treatment groups, while the number of times the mice crossed the original plateau and the percentage of time spent exploring the original plateau quadrant were reduced after the plateau was removed. 3) The nerve cells in the aging mice were disorganized and the nuclei of the mice were deeply stained;the dagliflozin group improved the morphological changes in the brain of aging mice. 4) In addition, compared with the control mice, the serum MDA level was significantly increased and the antioxidant enzyme SOD activity was significantly decreased in the aging group, while compared with the aging group, dagliflozin significantly decreased the MDA level and increased the SOD activity. 5) The expression of SIRT1 and PGC-1α was significantly upregulated in the low and high doses of dagliflozin compared to the aging group. Conclusion: The present study suggests that dagliflozin can delay organ aging, improve the learning and memory ability of aging mice, and exert antioxidant effects, probably through upregulating the SIRT1/PGC-1α signaling pathway.

A soy glycinin derived octapeptide protects against MCD diet induced non-alcoholic fatty liver disease in mice

Soy glycinin derived octapeptide(SGP8)is a peptide obtained from degradation of the soy glycinin,whose amino acid sequence is IAVPGEVA.To determine the effect of SGP8 on non-alcoholic fatty liver disease(NAFLD),steatosis Hep G2 cells were induced by 1 mmol/L free fatty acid(FFA)and C57 BL/6 J mice were fed with methionine-choline defi cient(MCD)diet for 3 weeks to establish NAFLD model.The results of oil red O staining and total cholesterol(TC)/triglyceride(TG)contents showed that SGP8 could signifi cantly reduce the lipid content of steatosis Hep G2 cells.In vivo,SGP8 lowered plasma alanine aminotransferase(ALT)and low density lipoprotein(LDL)content,normalized hepatic superoxide dismutase(SOD)and malondialdehyde(MDA)production,and reduced the severity of liver infl ammation.The results of Western blotting showed that SGP8 increased expression of Sirtuin-1(SIRT1)and phosphorylation level of AMP activated protein kinase(AMPK)in hepatocytes.Through activation of SIRT1/AMPK pathway,SGP8 downregulated the expression of sterol regulatory element binding protein 1 c(SREBP-1 c)and its target genes ACC and FAS expression levels,and increased the phosphorylation level of acetyl Co A carboxylase(ACC).Furthermore,SGP8 also upregulated the expression of transcription factor peroxisome proliferator activated receptorα(PPARα),which was regulated by SIRT1/AMPK pathway,and its target gene CPT1 level.In conclusion,SGP8 might improve NAFLD by activating the SIRT1/AMPK pathway.Our data suggest that SGP8 may act as a novel and potent therapeutic agent against NAFLD.

L-leucine stimulates glutamate dehydrogenase activity and glutamate synthesis by regulating mTORC1/SIRT4 pathway in pig liver

The liver is the most essential organ for the metabolism of ammonia, in where most of ammonia is removed by urea and glutamine synthesis. Regulated by leucine, glutamate dehydrogenase(GDH) catalyzes the reversible inter-conversion of glutamate to ammonia. To determine the mechanism of leucine regulating GDH, pigs weighing 20 ± 1 kg were infused for 80 min with ammonium chloride or alanine in the presence or absence of leucine. Primary pig hepatocytes were incubated with or without leucine. In the in vivo experiments with either ammonium or alanine as the nitrogen source, addition of leucine significantly inhibited ureagenesis and promoted the production of glutamate and glutamine in the perfused pig liver(P < 0.05). Similarly, leucine stimulated GDH activity and inhibited sirtuin4(SIRT4)gene expression(P < 0.01). Leucine could also activate mammalian target of rapamycin complex 1(m TORC1) signaling(P < 0.05), as evidenced by the increased phosphorylation levels of ribosomal protein S6 kinase 1(S6 K1) and ribosomal protein S6(S6). Interestingly, the leucine-induced m TORC1 pathway activation suitably correlated with increased GDH activity and decreased expression of SIRT4.Similar results were observed in primary cultured hepatocytes. Notably, leucine exerted no significant change in GDH activity in SIRT4-deficient hepatocytes(P > 0.05), while m TORC1 signaling was activated.Leucine exerted no significant changes in both GDH activity and SIRT4 gene expression in rapamycin treated hepatocytes(P > 0.05). In conclusion, L-leucine increases GDH activity and stimulates glutamate synthesis from different nitrogen sources by regulating m TORC1/SIRT4 pathway in the liver of pigs.

MicroRNA-23a reduces lipopolysaccharide-induced cellular apoptosis and inflammatory cytokine production through Rho-associated kinase 1/sirtuin-1/nuclear factor-kappa B crosstalk

Background:MicroRNAs are closely associated with the progression and outcomes of multiple human diseases,including sepsis.In this study,we examined the role of miR-23a in septic injury.Methods Lipopolysaccharide(LPS)was used to induce sepsis in a rat model and H9C2 and HK-2 cells.miR-23a expression was evaluated in rat myocardial and kidney tissues,as well as H9C2 and HK-2 cells.A miR-23a mimic was introduced into cells to identify the role of miR-23a in cell viability,apoptosis,and the secretion of inflammatory cytokines.Furthermore,the effect of Rho-associated kinase 1(ROCK1),a miR-23a target,on cell damage was evaluated,and molecules involved in the underlying mechanism were identified.Results:In the rat model,miR-23a was poorly expressed in myocardial(sham vs.sepsis 1.00±0.06 vs.0.27±0.03,P<0.01)and kidney tissues(sham vs.sepsis 0.27±0.03 vs.1.00±0.06,P<0.01).Artificial overexpression of miR-23a resulted in increased proliferative activity(DNA replication rate:Control vs.LPS vs.LPS+Mock vs.LPS+miR-23a:H9C2 cells:34.13±3.12 vs.12.94±1.21 vs.13.31±1.43 vs.22.94±2.26,P<0.05;HK-2 cells:15.17±1.43 vs.34.52±3.46 vs.35.19±3.12 vs.19.87±1.52,P<0.05),decreased cell apoptosis(Control vs.LPS vs.LPS+Mock vs.LPS+miR-23a:H9C2 cells:11.39±1.04 vs.32.57±2.29 vs.33.08±3.12 vs.21.63±2.35,P<0.05;HK-2 cells:15.17±1.43 vs.34.52±3.46 vs.35.19±3.12 vs.19.87±1.52,P<0.05),and decreased production of inflammatory cytokines,including interleukin-6(Control vs.LPS vs.LPS+Mock vs.LPS+miR-23a:H9C2 cells:59.61±5.14 vs.113.54±12.30 vs.116.51±10.69 vs.87.69±2.97 ng/mL;P<0.05,F=12.67,HK-2 cells:68.12±6.44 vs.139.65±16.62 vs.143.51±13.64 vs.100.82±9.74 ng/mL,P<0.05,F=9.83)and tumor necrosis factor-α(Control vs.LPS vs.LPS+Mock vs.LPS+miR-23a:H9C2 cells:103.20±10.31 vs.169.67±18.84 vs.173.61±15.91 vs.133.36±12.32 ng/mL,P<0.05,F=12.67,HK-2 cells:132.51±13.37 vs.187.47±16.74 vs.143.51±13.64 vs.155.79±15.31 ng/mL,P<0.05,F=9.83)in cells.However,ROCK1 was identified as a miR-23a target,and further up-regulation of ROCK1 mitigated the protective function of miR-23a in LPS-treated H9C2 and HK-2 cells.Moreover,ROCK1 suppressed sirtuin-1(SIRT1)expression to promote the phosphorylation of nuclear factor-kappa B(NF-κB)p65,indicating the possible involvement of this signaling pathway in miR-23a-mediated events.Conclusion:Our results indicate that miR-23a could suppress LPS-induced cell damage and inflammatory cytokine secretion by binding to ROCK1,mediated through the potential participation of the SIRT1/NF-κB signaling pathway.