| Diabetic kidney disease(DKD)is an ordinary complication of diabetes mellitus(DM).It is characterized by the presence of renal syndrome and diffuse glomerular sclerosis.DKD belongs to the category of“quenching thirst”and“kidney elimination”of traditional Chinese medicine which is an important factor causing end-stage renal disease(ESRD).Podocyte injury is considered to be the central link in the pathogenesis and development of DKD.It is always the focus of DKD treatment to explore the pathological damage and functional changes of podocytes and inhibit this progression.Traditional Chinese medicine plays an important role in the treatment of DKD.Cordyceps sinensis can benefit kidney,nourish yang,lung and kidney,which has a good effect on DKD.Cordyceps militaris polysaccharide(CMP)is an important active substance of Cordyceps miitaris(CM).The previous study of the reporter found that CMP can alleviate renal function damage in DKD mice.The exact regulation mechanism needs further study.Autophagy is a lysosomal-dependent intracellular degradation system ubiquitous in eukaryotic cells.It can remove damaged or aging organelles and biological macromolecules in cells,and maintain the homeostasis of cells.Studies have shown that autophagy is closely related to glomerular podocyte injury,and is widely involved in the occurrence and development of various kidney diseases,which may be a new way to treat kidney disease.In addition,studies have shown that Janus kinase/signal transducer and transcriptional activator(JAK/STAT)signaling pathway mediates renal cell processes during hyperglycemia injury,and negative regulation of JAK/STAT can effectively inhibit the pathogenesis of DKD.However,the study of JAK/STAT signaling pathway regulating autophagy and affecting DKD has rarely been reported and needs further elaboration.Therefore,we hypothesized that JAK/STAT signaling pathway can promote autophagy in podocytes by regulating autophagy-related genes,thereby reducing podocyte injury and protecting kidney function of DKD.The protective effect of CMP may be related to this mechanism.In order to verify the above hypothesis,this project intends to use STZ-induced DKD mice and immortalized podocyte injury model to clarify the role of JAK/STAT signaling pathway in regulating autophagy-related gene expression in podocyte injury and DKD disease development,in order to explore JAK/STAT-autophagy plays a key role in the protection of DKD renal function in CMP,providing new ideas for DKD prevention and treatment.Objective:1.By studying the relationship between JAK/STAT signaling pathway regulation of autophagy in DKD,revealing the specific molecular mechanism of JAK/STAT signaling pathway regulating autophagy activity,expanding the pathogenesis of DKD,and providing important basic research for its therapeutic drug development.2.By studying the trend of JAK/STAT-autophagy in the intervention of CMP,from the perspective of JAK/STAT signaling pathway regulation of autophagy,the mechanism of DKD improved by CMP was explained,which provided a new idea for DKD prevention and treatment.Methods:1.Study on the protective effect of CMP on renal function in STZ-induced diabetic nephropathy model(1)The polysaccharide of Cordyceps militaris was extracted by hot water extraction method.The polysaccharide obtained was detected by phenol-sulfuric acid method,the polysaccharide structure was detected by infrared,and the composition of polysaccharide was detected by high performance liquid phase.(2)Male C57BL/6J mice were intraperitoneally injected with STZ 50 mg/kg·d-1 for 5 consecutive days to induce DKD model.At the beginning of the 14th week,each group was given low and high doses of CMP(200mg/kg,400mg/kg)for 6 weeks.24h urine protein,blood glucose,body weight,kidney index,blood biochemical indicators such as serum creatinine(Scr),urea nitrogen(BUN),total protein(ALB),triglyceride(TG),total cholesterol(TC)content.The pathological changes of renal tissues were observed by HE,PAS and Masson staining.The ultrastructural changes of renal tissues were observed by transmission electron microscopy(TEM).The changes of desmin were detected by immunofluorescence,immunohistochemistry and western blot assay.The IL-1β,IL-6 and MCP-1 in renal tissues were detected by qRT-PCR assay.(3)The changes of autophagy in renal tissues were observed by transmission electron microscopy.The changes of autophagy-related proteins such as LC3,atg5,atg12,beclinl and p62 in renal tissues were detected by immunofluorescence and Western blot assay.(4)JAK/STAT pathway protein such as JAK1,JAK2,STAT1,STAT3,STAT5,STAT6 expression were detected by Western blot assay.2.Mechanism of polysaccharides from CMP on podocyte injury induced by high glucose(1)In podocyte experiments,the groups were as follows:NG group(low sugar complete medium);HG group(glucose was 50 mM concentration of complete medium);CMP(25)group(glucose was 50 mM concentration of complete medium+25 p g/ml CMP);CMP(50)group(glucose is 50 mM complete medium+50 μg/ml of CMP);CMP(100)group(glucose is 50 mM concentration of complete medium+100 p g/ml of CMP).MTT assay was used to detect the optimal concentration of high glucose-induced podocyte injury.Western blot assay was used to determine the optimal high glucose concentration and stimulation time for podocyte apoptosis.Anexin V/FITC kit was used to detect apoptosis.qRT-PCR assay was used to detect the levels of cytokines IL-1β,IL-6 and MCP-1.Podocyte cells were transfected with autophagy double-labeled adenovirus(mRFP-GFP-LC3)to observe autophagic flow.(2)In the podocyte experiment,the autophagy inhibitor 3-MA was added to the CMP,and the changes of autophagy-related proteins LC3,atg5,atg12,beclinl and p62 were detected by Western blot and immunofluorescence.Podocytes were transfected from The phagocytic adenovirus(mRFP-GFP-LC3)was observed for autophagic flow.(3)In the podocyte experiment,the JAK/STAT pathway inhibitor Ruxolitinib was added to the CMP,and the expression of JAK/STAT pathway protein STAT2 in podocytes was detected by immunofluorescence;the autophagy-associated protein LC3 was detected by Western blot and Changes of atg5,atg12,beclinl and p62 were detected by immunofluorescence.Podocytes were transfected with autophagy double-labeled adenovirus(mRFP-GFP-LC3)to observe autophagic flow.Results:1.Study on the protective effect of CMP on renal function in STZ-induced diabetic nephropathy model(1)The concentration of polysaccharides of CM was 73.55%by phenol-sulfuric acid method.Infrared spectroscopy measured the stretching vibration of polysaccharide OH at 3289.87 cm-1,with a CH absorption peak at 2923.56 cm-1,and absorption at 1100.00 cm-1 indicating that the polysaccharide contained C-OH side groups and COC glycosides.High performance liquid chromatography(HPLC)results show that CMP contains five kinds of monosaccharides such as mannose,rhamnose,glucose,galactose and xylose.(2)The results of mouse body weight and kidney index showed that CMP had no effect on improving the body weight of DKD mice,but it could improve the kidney enlargement of DKD mice.it also could reduce the 24-hour urinary protein excretion,and reduce the serum Scr,BUN,MDA,TC and TG levels,increased the expression level of ALB in DKD mice.HE,PAS and Masson staining showed that CMP can alleviate renal tissue damage and improve mild hyperplasia of basement membrane and mesangial proliferation.TEM results show that CMP can improve DKD Mouse glomerular podocyte foot process fusion.Immunofluorescence,immunohistochemistry and Western blot results showed that CMP can reduce the expression of desmin in renal tissue.QRT-PCR results show that CMP can reduce expression of IL-1β,IL-6 and MCP-1 mRNA.(3)The results of TEM showed that CMP could promote the expression of autophagy in DKD mice.Immunofluorescence and Western blot showed that the expression of autophagy-related proteins LC3,atg5,atg12 and beclinl were significantly inhibited in the DKD group,and the expression of p62 was increased.After giving CMP,there has been improvement.(4)Western blot analysis showed that JAK/STAT pathway-related proteins JAK1,JAK2,STAT1,STAT3,STAT5 and STAT6 were significantly increased in the DKD group,and JAK/STAT pathway protein expression was decreased after CMP administration.2.Protective mechanism of CMP on high glucose-induced podocyte injury(1)In the podocyte experiment,MTT and Western blot results showed that the optimal condition for podocyte injury was 48 h stimulation with 50 mM glucose.Flow cytometry results showed that high glucose can induce apoptosis of mouse podocytes,and CMP can reduce the apoptosis of podocytes induced by high glucose.QRT-PCR results showed that high glucose can induce the expression of inflammatory factors IL-1β,IL-6 and MCP-1 in podocytes,and treatment with CMP can improve high glucose-induced podocyte inflammatory injury.Autophagy double-labeled adenovirus(mRFP-GFP-LC3)transfected podocytes showed that CMP can promote the autophagic flow of podocytes induced by high glucose.(2)Immunofluorescence and Western blot were used to detect the expression of autophagy in podocytes.The results showed that the polysaccharides from CM could promote the expression of autophagy-associated proteins LC3,atg5,atg12,beclinl and reduce the expression of p62 to improve podocyte injury.Transfection of autophagy double-labeled adenovirus(mRFP-GFP-LC3)into podocytes showed that CMP could promote the autophagic flow of podocytes induced by high glucose to improve podocyte injury.(3)Immunofluorescence and Western blot were used to detect the expression of autophagy in podocytes,and the JAK inhibitor Ruxolitinib was used as a control group.The results showed that the CMP could inhibit JAK/STAT pathway-related proteins expression of JAK1,JAK2,STATI,STAT3,STAT5,and STAT6 to promote podocyte autophagy to improve podocyte injury.Transfection of autophagy bispecific adenovirus(mRFP-GFP-LC3)into podocytes showed that promoting the expression of podocyte autophagic flow improved podocyte injury.Conclusion:1.CMP can improve the urinary protein elevation,kidney enlargement,renal function damage and pathological changes in DKD mice,delaying the onset of DKD mice.2.In vitro experiments confirmed the activation of JAK/STAT signaling,which has a regulatory effect on the level of autophagy.After inhibition of JAK,autophagy activity showed an increasing trend,and apoptosis of podocytes decreased,which provided an important research perspective for the pathological progression of diabetic nephropathy.3.In vitro and in vivo experiments show that CMP can effectively prevent and treat DKD.CMP can improve the autophagy level of podocytes,reduce the release of inflammatory factors and reduce kidney damage by inhibiting JAK/STAT signal,and provide new ideas and methods for prevention and treatment of DKD by Chinese medicine. |
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