| Objective:After entering the 21st century,population aging is the main problem facing the world.As one of the fastest aging organs,the kidney is characterized by renal cortex,glomerulosclerosis,interstitial fibrosis,renal tubular atrophy and arteriosclerosis.In addition to physiological aging,many chronic diseases can accelerate renal aging,and these diseases may induce renal oxidative stress,DNA damage and mitochondrial damage,leading to stress-induced aging.Thus exploring the mechanism of its occurrence and progression will help to target and delay the process of stress-induced aging.Studies have found that cellular senescence is widely involved in the occurrence and progression of various diseases,and autophagy is also closely related to aging.The aging of renal cells mainly affects human glomerular mesangial cells(HGMC)and renal tubular epithelial cells.Signal transducer and activator of transcription 3(STAT3)is a member of the STAT protein family of transcription factors.Some studies have shown that STAT3 plays an important role in regulating cell senescence,and STAT3 can alleviate aging by regulating autophagy.During autophagy,p62 forms a complex with ubiquitin protein and LC3II in the cytoplasm and is degraded in autophagy lysosomes.Therefore,p62 is a marker protein that reflects autophagy activity.p53 is a tumor suppressor,which is involved in regulating the cell cycle and inducing cell senescence and apoptosis.Recent studies have shown that p53 plays an important role in the aging process of hepatic stellate cells(HSC).As a downstream target gene of p53,when cell DNA is damaged,cell cycle inhibitor p21 is activated.It binds tightly with the cyclin-CDK complex,induces cell cycle arrest and inhibits cell proliferation.Accordingly,we speculate that STAT3/p62/p53/p21 may be involved in renal aging.The chemical name of PRO,probucol,has the effect of lowering blood lipids and antioxidants.It can enhance the antioxidant capacity of the kidneys of diabetic rats and delay the occurrence and development of diabetic nephropathy,thereby playing a role in kidney protection.Losartan(LOS)is a non-peptide Angiotensin Ⅱ receptor antagonist,which can protect the kidneys by antagonizing AT1 receptors.Studies have found that both PRO and LOS can delay the senescence of HGMC,and are closely related to reducing the phosphorylation of STAT protein.Metformin(MET)is currently the first-line drug for the treatment of type 2 diabetes.It can effectively reduce liver gluconeogenesis,reduce intestinal glucose absorption and increase insulin sensitivity in peripheral tissues.Studies have found that metformin can counteract stress aging in diabetic kidney,such as accumulation of advanced glycation end products(AGEs),oxidative stress and inflammation.We use streptozotocin(STZ)injection to create a diabetic rat model to study stress-induced aging.Glomerular mesangial cells(HGMC)are the main intrinsic cells of the kidney and the most active cells in the glomerulus.HGMC senescence induced by high glucose is an important pathophysiological basis of renal senescence.In this study,we combined experiments in vivo and in vitro to construct high glucose models in rats and HGMC cells to study the effects and mechanisms of PRO,LOS and MET on renal aging through the STAT3/p62/p53/p21 signaling pathway.The mechanism of occurrence provides a theoretical basis and an experimental basis for the development of specific target drugs that delay kidney aging.Methods:1.Human glomerular Mesangial cells(HGMC)were used in the experiment,and the senescent cell model was established by the intervention of high glucose in vitro.According to the needs of the experiment,the cells were divided into control group(Con)and high glucose group(HG).After all the cells were synchronized with serum-free medium for 24 hours,the cells were treated with different treatments.The control group was cultured in normal HGC medium,while the high glucose group(HG)was cultured in the medium with the final concentration of glucose 30mmol/L for 24,48 and 72 hours,respectively,and the cells were collected for follow-up experiment.The effect of high glucose exposure on the viability of HGMC cells was detected by CCK-8.The LDH release of HGMC cells treated with high glucose was detected by LDH method.The level of apoptosis was detected by flow cytometry.WesternBlotting and Real-TimeQuantitativePCR were used to analyze the protein level and mRNA expression of STAT3 in HGMC cells exposed to high glucose.Construction of STAT3 silencing cell model:STAT3 silencing lentivirus was transfected into HGMC cells and STAT3 silenced HGMC cells were obtained after 7 days of puromycin selection.The exposed groups were as follows:control group(Con-NC),control group(Con-Si),30mM high glucose group(HG-NC)and 30mM high glucose STAT3 silence group(HG-Si).Collect cells of each group to select RNA-seq test to screen the potential autophagy target genes of STAT3,and use Real-TimeQuantitativePCR to analyze the expression of STAT3 potential target gene mRNA to verify the results of RNA sequencing,so as to screen the key factors downstream of STAT3.2.In this part of the study,the cells were divided into the control group(cultured in HGMC medium without high sugar),PRO group(40 μmol/L PRO stimulated cells in HGMC medium without high sugar for 2 h),LOS group(10-5 mol/L LOS stimulated the cells for 2 h,then cultured in HGMC medium without high glucose),MET group(50 mmol/L MET stimulated cells for 2 h,cultured in HGMC medium without high glucose),high Glucose group(30 mmol/L glucose stimulated cells for 72 h),high glucose+PRO group(add 40 μmol/L PRO 2 h before adding 30 mmol/L glucose and culture for 72 h),high glucose+LOS group(30 mmol/L 10-5mol/L(10μmol/L)LOS was added 2 h before glucose addition and cultured for 72 h),high glucose+MET group(50 mmol/L MET was added 2 h before 30 mmol/L glucose was added and cultured 72 h).By detecting cell morphology,cell viability,LDH release,changes in STAT3,p62,p53 and p21 protein and mRNA expression,and apoptosis.The aim was to explore the effects of three drugs on STAT3/p62/p53/p21 signaling pathway activity changes and protein expression in the aging process of HGMC.3.Fifty rats(weight 210±10 g at the beginning of the experiment;N=50;the same number of males and females)were randomly divided into 5 groups by weight,with 10 rats in each group.The five groups included the control group,50 mg/kg STZ group,PRO+STZ,LOS+STZ and MET+STZ group.STZ(50 mg/kg body weight citrate buffer,pH=4.0)was injected intraperitoneally(st.ip.)for 3 consecutive days,and the control group was given the same dose of citrate buffer.The study only included animals with non-fasting blood glucose levels>16.7 mmol/l for two consecutive mornings.After the STZ model was successfully established,drug treatment was performed.Rats in the control group and STZ group received intraperitoneal injections with 0.9%sodium chloride.Rats in the PRO+STZ group received intraperitoneal injection of 100 mg/kg PRO.Rats in the LOS+STZ group received intraperitoneal injection of 25 mg/kg LOS.Rats in the MET+STZ group received an intraperitoneal injection of 250 mg/kg MET.Urine was collected in the 10th week.After measuring body weight and muscle content,all rats were sacrificed 10 weeks after treatment.At the same time,collect kidney tissue and blood for further analysis.The histological changes of the kidneys of four rats in each group were observed.Extract total RNA and protein from the remaining kidneys to test target mRNA and protein levels.The collected blood was used to test blood lipids,glucose,urine protein,urea nitrogen,urine albumin and creatinine.Results:1.The results of CCK8 showed that compared with the normal control group,the proliferation inhibition rate of 10,20,30,40 and 50mmol/LHG groups decreased significantly after 24 h,48 h and 72 h culture.Compared with the control group,the LDH release of the HG group increased at three different time periods,and the LDH release of the 72hHG group was significantly higher than that of the control group(P<0.01).The results of flow cytometry showed that the early apoptosis rate and total apoptosis rate in the high glucose model group were higher than those in the control group.Westernblotting and RT-qPCR results showed that high glucose exposure significantly increased the expression of STAT3 in HGMC cells.Next,after STAT3 silencing treatment on HGMC cells,the potential key factors were screened by RNA-seq experiment.The results showed that p62,p53 and p21 may be the potential downstream key factors of STAT3 in HGMS cell model exposed to high glucose.In order to verify this conjecture,we used RT-qPCR experiment to observe the expression of mRNA of the above three factors.The results showed that the mRNA levels of p62,p53 and p21 in the HG model group were higher than those in the control group.The mRNA expression levels of p62,p53 and p21 in STAT3 silencing HG model group were lower than those in HG model group.2.Compared with the control group,with the prolonged stimulation time,the polymorphonuclear and binuclear cells of the high-glucose(HG)group gradually increased,the cell volume became larger,and the cytoplasmic area was flat.Among them,there were common granules or vacuoles with irregular arrangement.Compared with the HG group,after the treatment of PRO,LOS and MET,the cell arrangement was more regular,the volume increased,and the cells with multiple nuclei and binuclear decreased significantly.Compared with the control group,the cell viability and the LDH release amount of the HG group,HG+PRO group,HG+LOS group and HG+MET group all increased and the difference was statistically significant.Compared with the HG group,the cell viability and the LDH release amount of the HG+PRO group,the HG+LOS group and the HG+MET group decreased and the difference was statistically significant.Compared with the control group,the expression levels of STAT3,p62,p53 and p21 protein and mRNA in the HG group,HG+PRO group,HG+LOS group and HG+MET group increased significantly.Compared with the HG group,the expression levels of STAT3,p53 and p21 protein and mRNA in the HG+PRO group,HG+LOS group,and HG+MET group were reduced.Compared with the HG group,the p62 protein and mRNA expression levels of the HG+PRO group and the HG+LOS group were reduced.Compared with the control group,the early apoptosis and total apoptosis rate of cells in the HG group,HG+PRO group,HG+LOS group and HG+MET group were all increased.Compared with the HG group,the early apoptosis and total apoptosis rate of cells in the HG+PRO group,HG+LOS group and HG+MET group all decreased.3.Compared with the control group,the body weight and muscle content of rats in other groups decreased.In addition,the body weight and muscle content of the intervention group increased.The MET group had the largest weight,followed by the LOS group and the PRO group,but it was significantly higher than the STZ group.The kidney organ index of rats in the STZ group increased significantly.In contrast,the kidney organ index of each group in the intervention group decreased significantly.For Upro,ALB,BUN and Cr of DN rats,compared with the model group,each administration group has different degrees of recovery,but there is still a certain gap compared with the control group.HE staining showed that the structure of the kidney in the control group was complete,the cortex and medulla had clear boundaries,the glomeruli had no obvious atrophy or swelling,and there was no thickening and inflammatory exudation of the capillary base.The renal pathological changes of the model group and each treatment group were different.The renal tubule interstitial damage in the model group was the most serious,the expression of inflammatory cells in the renal interstitium increased,and fibrous connective tissue hyperplasia was seen.On the contrary,the pathological damage of the intervention group was significantly reduced.At the same time,blood pressure,blood lipids and blood sugar in the STZ group increased significantly.On the contrary,PRO,LOS and MET can significantly reduce blood pressure,lipid and blood sugar levels.STZ induced increased expression of STAT3 and p62 protein and mRNA.This study found that PRO,LOS and MET alleviated the premature kidney aging induced by STZ,PRO,LOS and MET also inhibit STZ-induced activation of STAT3 and p62.Conclusion:1.High glucose upregulates renal signal transducer and transcriptional activator STAT3 to induce senescence of HGMC cells.2.High glucose regulates the expression of autophagy signal pathway in p62/p53/p21 cells through STAT3,which leads to cell senescence 3.The treatment of PRO,LOS and MET can antagonize the destruction of STAT3/p62/p53/p21 signal pathway and delay renal senescence in rats. |
PDF Full Text Request