| Significance of research:The incidence of diabetic nephropathy and its treatment are currently topics of interest and difficult clinical issues.Various studies on the pathogenesis of diabetic nephropathy have been reported,and they have focused on inflammatory mechanisms,immune mechanisms,oxidative stress,high metabolism,high filtration,hemodynamic changes,etc.Among these traits,inflammation and the immune response are widely recognized as being associated with the pathogenesis.This is related to the discovery of a large number of inflammatory cells,inflammatory mediators,immune cells,etc.in the kidney tissue of diabetic nephropathy patients.In addition to classic immune cells and inflammatory mediators,the immune response of the kidney’s congenital cells is also activated and is actively involved in immune and inflammatory responses,leading to autophagy,apoptosis and other cytological changes in the kidneys,which leads to the occurrence of this disease clinically.In this study,we studied the autophagy,apoptosis and regulation pathway of one of the intrinsic cell types of diabetic nephropathy from the perspective of immunology,and we intervened with rapamycin to explore its pathogenesis and intervention effect to provide an experimental basis for the clinical treatment of diabetic nephropathy.In recent years,the mammalian target of rapamycin(mTOR)has been a research hotspot.Many studies have reported that mTOR proteins play a key role in autophagy and apoptotic signaling pathways.The mTOR protein complex is divided into mTORC1 and mTORC2,which have different compositions,structures and functions.Upstream signaling proteins can induce phosphorylation.The two mTOR proteins,after being activated,induce phosphorylation and panto-acidification of downstream molecules,thereby regulating cell growth cycle and mRNA transcription,leading to changes in autophagy and apoptosis.At present,there are many studies on the upstream pathway of mTOR activation,such as the classic PI3K-Akt-TSC1/2-mTOR signaling pathway and LKB1-AMPK-TSC1/2-mTOR signaling pathway.However,there are relatively few studies on the expression and regulation of downstream proteins after the activation of mTOR occurs,and there are few studies on the pathogenesis of renal foot cell injury.As a receptor binding agent for specific mTOR,rapamycin can inhibit the activation of the mTOR protein,thereby regulating the growth cycle and function of renal podocytes in diabetic nephropathy.However,the receptors for mTORC1 and mTORC2 have different sensitivities to rapamycin and produce different regulatory effects.The study focused on the downstream regulatory channel of the mTORC1 protein.Autophagy is a process of degrading part of the cytoplasm and organelles in cells by lysosomes and fine-tuning by autophagy-related genes(ATG).Autophagy is an important degradation mechanism in cells and an intrinsic physiological function of eukaryotic cells.It is essential for maintaining the intracellular environment and cell survival,and it is involved in antiaging of the body,cell differentiation,immune response,and tumor pathology.Factors that induce autophagy are starvation,organelle damage,DNA damage,radiation therapy,drugs,and so on.The cells are destroyed by these factors,forming a two-layer structural separation membrane around the cell components to be degraded,then gradually separating the separation membrane,and finally completely blocking the cytoplasmic portion to be degraded to form autophagosomes.After the autophagosome is formed,it will be transported to the lysosome through the cytoskeleton microtubule system,and the two will fuse to form autophagosomes;finally,the contents will be degraded by the lysosomal enzyme.The current study found that autophagy regulation involves multiple signaling pathways,with the adenosine monophosphate-activated protein kinase(AMPK)and mTOR signaling pathways serving as regulatory cores.In this study,we observed the changes of autophagy and the expression of the mTOR protein in the kidney tissue of diabetic nephropathy in animals and confirmed the mTOR protein pathway regulation on autophagy.Apoptosis plays an important role in the evolution of organisms,the stability of the internal environment,and the development of multiple systems.Apoptosis is a special type of cell death,but it also has important biological significance and complex molecular biological mechanisms.Apoptosis is a process of strict control of multiple genes,such as those from the Bcl-2 family,caspase family,oncogenes such as C-myc,and the tumor suppressor gene P53.With the development of molecular biology technology,there is considerable understanding of the process of apoptosis,but the exact mechanism of apoptosis has not yet been fully understood.Disorder of the apoptotic process may be directly or indirectly related to the occurrence of many diseases,such as tumors,autoimmune diseases,etc.,and many factors that can induce apoptosis,such as radiation,drugs,etc.Studies have reported that podocytes encourage abnormal apoptotic processes in diabetic nephropathy,and their regulatory mechanisms,including classical membrane receptor pathways,cytochrome c and caspases pathways,etc.,are complex.There are also apoptosis-inhibiting molecules,such as P53,CrmA,IAPs,FLIPs and those in the Bcl-2 family,which regulate the process of apoptosis.Studies have reported that there is apoptosis in renal podocytes of diabetic nephropathy,and whether the mTOR protein plays a role in this process and its mechanism of action deserves further study.We investigated the relationship between the apoptosis of renal podocytes and the mTOR protein in diabetic nephropathy animals and found evidence that the mTOR protein regulates podocyte apoptosis.Purpose:1.To observe the changes of autophagy and apoptosis of podocytes in diabetic nephropathy,detect the autophagy proteins mTOR,S6K1,LC3II and the apoptosis-related proteins Bax,Bcl-2,c-caspase3,c-caspase9,and confirm that there has the pathological process of abnormal cell death in the diabetic nephropathy.2.To clarify the regulation mechanism of the mTOR protein on the autophagy and apoptosis of podocytes.3.RAPA binds to the mTOR protein receptor and inhibits mTOR protein activation,thereby improving autophagy and apoptosis while protecting podocytes.Research methods:1.Zoological research1.1 Observation of clinical indexes of rats with diabetic nephropathy:Male Sprague-Dawley rats weighing 150-200 g received an intraperitoneal injection of STZ to produce the rat model of diabetic nephropathy.The rats were randomly divided into three groups:the normal control group(NC group),diabetic nephropathy group(DN group),and rapamycin group(RAPA group).Blood creatinine level,24-h urine volume and urine protein quantitation were measured at 0,4,8 and 12 weeks,and the disease changes in rats were observed as clinical evaluation indicators.1.2 Expression of podocyte marker proteins podocin and nephrin in the rat renal cortex:Immunohistochemistry and immunofluorescence quantitative PCR were used to detect the expression of the podocin and nephrin proteins.1.3 Detection of mTOR protein expression in the renal cortex of rats with diabetic nephropathy:At the end of the experiment,the rats were sacrificed and kidney samples were obtained.The expression of the mTOR protein in the kidney tissues of each group was detected by immunofluorescence quantitative PCR and western blot.1.4 Effect of the mTOR protein on downstream protein expression after activation:At the end of the experiment,the rats were sacrificed.The expression of the S6K1 protein and LC3II protein in the renal cortex were detected by immunofluorescence quantitative PCR and western blot.The changes of podocyte injury were detected by electron microscopy and immunohistochemistry.1.5 Expression of podocyte apoptosis in renal tissues of rats with diabetic nephropathy:Western blot analysis of apoptosis-related proteins Bax,Bcl-2,c-caspase3,c-caspase9 was performed.2.In vitro experimental study2.1 Pretest:The podocytes were cultured in different concentrations of glucose medium to observe the growth of podocytes.The concentration of cells suitable for cell experiments was selected by the MTT method as the glucose concentration standard for subsequent experiments.Different concentrations of RAPA were added to the medium to observe the growth of podocytes,and the MTT method was used to screen out the concentration of RAPA intervention suitable for the experimental requirements.2.2 Effect of high glucose on the apoptosis of renal podocytes in vitro:The kidney podocytes were cultured in vitro,and the podocytes were divided into two groups:the normal control group(NC group)and the high glucose group(HG group).The NC group podocytes were placed in 5 mmol/L glucose RPMI1640 medium,and the HG group podocytes were placed in the 350 mmol/L glucose RPMI1640 medium.The apoptosis of the podocytes was detected by the TUNEL method 24 hours later.2.3 Flow cytometry observed changes in the podocyte apoptosis induced by different concentrations of RAPA:The podocytes were divided into 4 groups and placed in a medium of 35 mmol/L glucose,and the cells were treated with RAPA(concentrations,ng/ml:0,5,10,15)for 24 h.The grouping was set to blank(normal complete medium),Ong/mlgroup(35 mmol/L complete glucose medium),5 ng/mlgroup(35 mmol/L complete glucose medium + 5 ng/ml rapamycin),10 ng/mlgroup(35 mmol/L glucose complete medium +10 ng/ml rapamycin),and 15 ng/mlgroup(35 mmol/L complete glucose medium+ 15 ng/ml rapamycin).Flow cytometry was performed to detect changes in podocyte apoptosis.2.4 TUNEL detects changes in podocyte apoptosis in a high glucose state:According to the results of the flow cytometry,the groups were set to 0 ng/mlgroup(35 mmol/L sugar complete medium)and 15 ng/mlgroup(35 mmol/L sugar complete medium +15 ng/ml RAPA).TUNEL detects changes of podocyte apoptosis and the protective effect of RAPA in a high glucose state.2.5 Western blot analysis of autophagy protein expression:According to the results of flow apoptosis,15 ng/ml rapamycin was selected for subsequent western blot experiments.The podocytes were plated at a density of 60-80%.After.24 h,35 mmal/L glucose medium was directly added,and the cells were treated with rapamycin(final concentration of 15 ng/ml)for 24 h.Grouping:normal control group(NC group),HG group(complete medium of 35 mmol/L glucose),HG+RAPA group(complete medium of 35 mmol/L glucose + 15 ng/ml of rapamycin).Total protein was extracted,and the following indicators were detected by western blot:mTORC1,S6K1,LC3II,and Beclin-1.Results1.Renal lesions and podocyte injury in diabetic nephropathy rats1.1 Detection of biochemical indicators in rats:The urine volume,urine protein and serum creatinine levels of the three groups were measured before,during and at the end of the experiment(0,4,8 and 12 weeks).The results showed that the urine volume of the rats in the DN group was increased compared with that of the other two groups.A quantitative increase in urinary protein and serum creatinine level and impaired renal function;the above indicators in the RAPA group were higher than those in the NC group but improved compared with the DN group.1.2 Pathological changes in rats:The pathological changes of renal podocytes were observed under electron microscopy.The podocytes of the DN group were changed compared with those of the NC group.The foot was ablated,the number was decreased,and stromal hyperplasia of the mesangial area,mesangial cell proliferation,glomerular capillary filtration and focal stage hardening occurred in the network;in the RAPA group,the podocytes and glomerular filtration membrane were significantly more obvious than those in the NC group.The podocytes showed ablation and structural changes,and the mesangial area and glomerular filtration membrane lesions were obvious.However,there was significant improvement compared to the DN group.1.3 Changes in podocyte marker proteins in rats:The results showed that the expression of the podocin and nephrin proteins in the renal tissue of the DN group was significantly decreased,and the fold expansion decreased.The expression of podocin and nephrin protein in the RAPA group was lower than that in the NC group and better than that in the DN group.1.4 Expression of autophagy in the renal cortex of rats in each group and the protective effect of RAPA:The expression of mTORC1,S6K1 and LC3II was detected by western blot and quantitative immunofluorescence PCR.The results showed that the mTORC1 protein in the kidney tissue of the DN group was significantly higher than that of the NC group.The RAPA group had a better result than the DN group,and the autophagocytic LC3II expression was compared.In contrast,the LC3II in the kidney tissue of the DN group was lower than that in the NC group,and that in the RAPA group was improved.The S6kl protein in the kidney tissue of the DN group was significantly increased compared with the NC group,and the RAPA group showed better results than the DN group.1.5 Apoptosis expression in the renal cortex of rats in each group and the protective effect of RAPA:The apoptosis-related proteins Bax,Bcl-2,c-caspase3 and c-caspase9 were detected by western blot.The results showed that Bax,Bcl-2,c-caspase3 and c-caspase9 were significantly increased in the kidney tissue of the DN group compared with the NC group.The RAPA group showed better results than the DN group,and the apoptotic factor was expressed in the rat model of diabetic nephropathy.In the RAPA group,after the intervention,the expression of apoptotic factors decreased,which had a certain protective effect on renal podocytes.2.In vitro cell assay to study the changes of renal podocyte apoptosis and the expression of autophagy in cells under high glucose2.1 Pretest result:The medium was screened for high sugar concentration by the MTT method.Glucose concentration settings:0,5,10,15,20,25,30,35,and 40 mmol/L.The results show that when the sugar concentration is higher than 35 mmol/L,the inhibition rate of cells reached 100%,which is not suitable for cell experiments.The screening was continued in the range of 0-35 mmol/L.When the glucose concentration was 35 mmol/L,the cell inhibition rate was approximately 20%,while when it was 5 mmol/L or 10 mmol/L,the inhibition rate was very low.MTT was used to screen the effects of different concentrations of RAPA on podocytes.The results showed that when the concentration of glucose was 35 mmol/L,the cell viability increased obviously when the rapamycin was increased from 5 to 15 ng/ml and the timepoint was 24 h.2.2 Changes in phenotypic function for podocytes at high glucose:Flow cytometry was used to detect changes in the podocyte phenotype function.When the glucose concentration was 35 mmol/L,the density of renal foot cells was 60-80%.After 24 h,35 mmol/L high glucose medium was added directly,and RAPA was added to the medium(concentration,ng/ml:0,5,10,15)The cells were treated for 24 h,and flow apoptosis was then delivered and grouping was set up as follows:blank(normal complete medium),0(35 mmol/L sugar complete medium),5(35 mmol/L sugar complete medium +5 ng/ml of RAPA),10(35 mmol/L sugar complete medium + 10 ng/ml RAPA),and 15(35 mmol/L sugar complete medium+ 15 ng/ml RAPA).Experiments have shown that when the concentration of RAPA is 5-15 ng/ml,apoptosis is indeed relieved and the proportion of apoptotic cells is significantly reduced.In the subsequent experiments,15 ng/ml RAPA was used to treat the cells,and the related proteins were detected.2.3 TUNEL detects changes in podocyte apoptosis in a high glucose state:According to the results of flow cytometry,a grouping was set:HG group(35 mmol/L sugar complete medium)and HG+RAPA group(35 mmol/L sugar complete medium +15 ng/ml RAPA).The results showed that apoptotic bodies were observed in the podocytes of the HG group under 200-fold and 400-fold fluorescence microscopy,and the number of apoptotic bodies was decreased in the HG+RAPA group under the same microscopy methods.This shows that RAPA can improve the apoptosis of podocytes in a high glucose state.2.4 Expression of autophagy protein in podocytes in a high glucose state:Western blot was used to detect autophagy proteins,such as mTORC1,S6K1,LC3II/LC3I and Beclin-1,and grouping was:HG group(35 mmol/L sugar complete medium)and HG+RAPA group(35 mmol/L sugar complete medium +15 ng/ml of RAPA).The results show that the expression of mTORCl and Beclin-1 in the podocytes of the HG group increased,the expression of the S6K1 and LC3II/LC3I proteins decreased,the expression of the autophagy protein in podocytes changed significantly,and autophagy decreased.The expression of mTORCl and Beclin-1 in podocytes of the HG+RAPA group was lower than that in the HG group.The expression of S6K1,LC3II/LC3I protein was higher than that in the HG group.RAPA improved the autophagy of podocytes.Conclusion and innovation1.This study clarified that high glucose can induce an increase in the mTORCl protein expression in the pathogenesis of diabetic nephropathy,and the mTORCl protein can also be used as an independent risk factor for inducing the expression of autophagosome marker protein LC3II,suggesting a link between the two.At the same time,we also detected increased expression of apoptotic factors such as Bax,Bcl-2,c-caspase3 and c-caspase9 in animal models,suggesting that there is a specific relationship between autophagy and apoptosis.2.As a specific inhibitor of the mTORC1 protein,RAPA can bind tightly to the mTORC1 protein and regulate the expression of the mTORC1 protein to protect diseased podocytes.3.Signal transduction between the mTORC1 protein and LC3II is required.S6K1,as one of the downstream signaling proteins of the mTORC1 protein,can regulate expression of the LC3II protein,thus demonstrating the existence of the mTORC1-S6K1-LC3II signaling pathway.4.The expression of the mTORC1 protein was positively correlated with the expression of apoptotic factors,such as Bax,Bcl-2,c-caspase3 and c-caspase9,and it was suggested that the mTORCl protein can regulate the expression of apoptotic proteins,suggesting that the mTORCl protein plays an important role in podocyte changes. |
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