| Diabetic nephropathy(DN),as a kind of diabetic microvascular complication,is spreading all over the world.As the main cause of end-stage renal diseases,it seriously endangers the quality of human life and has become a worldwide public health problem.Since no specific therapy exists in clinical practice,it is an urgent problem to explore the pathogenesis of diabetic nephropathy and effective drugs for DN.In recent years,premature renal aging has been considered to be an important mechanism of the onset and development of DN.The occurrence of premature senescence in the intrinsic cells of kidney,especially in the renal tubular epithelial cells is the core factor to promote premature renal aging.Downregulation of a-Klotho and inhibition of autophagy activity are two mechanisms related to aging and DN.Traditional Chinese medicine(TCM)has been used to treat diabetes and its complications for thousands of years,which has been proved to be curative effect and unique advantages.Professor Yaoxian Wang innovatively puts forward the theory of "Gathering and dispersing of minature mass in renal collateral" by inheriting the theory of "miniature mass of renal collateral" by professor Lv Renhe and then the method of "eliminating masses and accumulation" and Shenyan fangshuai Decoction are formulated.Consequently,it has formed a complete theoretical system of principles,methods,prescriptions and medicines to guide the clinical diagnosis and treatment of DN in TCM.The previous basic research suggested that Shenyan fangshuai decoction might resist diabetic renal fibrosis by improving oxidative stress and inflammatory response,two key factors to induce senescence.So if Shenyan fangshuai decoction plays the role of renal protection by regulating the premature senescence of renal inherent cells has been brought out However,the active components and mechanism of Shenyan fangshuai decoction in DN are not clear,so it is necessary to further study.Objective:1 To analyze the chemical components of Shenyan fangshuai decoction by ultra-high performance liquid chromatography-time of flight high resolution mass spectrometry,and predict the related targets in the treatment of DN through network pharmacology,and screen the related targets of renal aging by combining the front of the discipline and the basis of team research.2 To elucidate whether the down regulation of α-Klotho and the obstruction of autophagy pathway are the material basis of "imbalance of aggregation and dispersion" of DN pathogenesis,whether the dredging of autophagy pathway via up regulation of α-Klotho expression is the material basis of "Fuzheng Xiaozheng",and whether Shenyan fangshuai decoction can delay the premature senescence of renal tubular epithelial cells in DN by regulating α-Klotho/autophagy pathwayMethods:1 Gradient elution was performed on UHPLC-Q-TOF MS column with acetonitrile-0.1%formic acid water as mobile phase.Under the mode of positive and negative ions,collect the data of UHPLC-Q-TOF MS,analyze the effective components in Shenyan fangshuai decoction according to retention time information,high-resolution mass spectrometry precise molecular weight and MSN multi-level fragment information.Screen the target protein of the effective components through Binding database,stitch(5.0)database,ETCM database and TCMIP 2.0,and use the network visualization software Cytoscape 3.7.0 to construct the effective component-target interaction network of Shenyan fangshuai decoction for topology analysis.Obtain the treatment target of DN in DisGeNET database.In String online database,construction and analysis of PPI network for the potential target of Shenyan fangshuai decoction in the treatment of DN are carried out.Go enrichment analysis and KEGG pathway enrichment analysis are also carried out for the target protein to explore the mechanism and principle of Shenyan fangshuai decoction in the treatment of DN.2 A single intraperitoneal injection of streptozotocin combined with unilateral nephrectomy was used to establish a DN model in rats.The experiment was divided into control group,model group,Chinese medicine group and benazepril positive control group.After the intervention with 12 weeks,24h urine and blood were collected and kidneys were retained.ELISA method was used to detect urinary albumin and serum creatinine.The blue staining area of SA-β-Gal was detected by the specific kit Cyclin dependent kinase inhibitor p21,DNA double strand break marker yH2AX and α-Klotho were detected by immunohistochemistry and Western blot,respectively.Immunofluorescence and WB were used to detect the expression of autophagy marker protein LC3 and autophagy substrate protein p62.3 Human proximal renal tubular epithelial cell line(HK-2)was stimulated by glycosylation end products(AGEs)to simulate the pathological factor of DN.The experiment was divided into blank group,blank plus Chinese medicine group,model group,model plus Chinese medicine group.After 48 hours of intervention,the number of SA-β-gal blue staining cells was detected.Immunofluorescence and WB were used to detect the expression levels of p21,γH2AX,LC3,p62 and α-Klotho in different groups of cells to investigate whether Shenyan Fangshuai decoction can regulate the TECs premature senescence,autophagy activity inhibition and α-Klotho down regulation induced by AGEs.Furthermore,mRFP-GFP-lc3 double fluorescence system combined with autophagy inhibitors was used to screen the blocking sites of autophagy flow in AGEs induced-HK-2 cells.Autophagy downstream was blocked by autophagy blocker CQ to observe the regulation of Shenyan fangshuai decoction on premature cellular senescence via regulating autophagy.Finally,the stable HK-2 strain was established by overexpression of α-Klotho lentivirus.The expression of LC3,p62 and premature cellular senescence related proteins was observed to verify whether Shenyan fangshuai decoction could up regulate α-Klotho to restore autophagy activity and delay premature cellular senescence.Results:1 138 potential active components were identified,647 corresponding targets,560 related genes of DN and 58 potential targets of Shenyan fangshuai decoction in the treatment of DN were screened out.PPI analysis showed eighteen core targets of Shenyan fangshuai decoction in the treatment of DN.The enrichment analysis of Go indicated that the main biological processes included inflammation,oxidative stress,hypoxia response,apoptosis,aging and so on.The enrichment analysis of KEGG pathway suggested that nephritis anti-aging liquid could play a role in renal protection by regulating PI3K-Akt,AMPK,mTOR and other nutrition related pathways.2 Compared with the control group,the weight of rats in model group decreased significantly(P<0.01),the ratio of kidney weight to body weight increased significantly(P<0.01),24-hour urine protein and serum creatinine were significantly increased(P<0.01).After treatment with Shenyan fangshuai decoction,the body weight of DN rats was recovered(P<0.01),the ratio of kidney weight to body weight was decreased(P<0.01),24-hour urine protein and serum creatinine were significantly decreased(P<0.01).After benazepril treatment,24-hour urine protein and serum creatinine of DN rats decreased significantly(P<0.01),while body weight and kidney weight to body ratio had no significant changes(P>0.05).It is suggested that Shenyan Fangshuai decoction can effectively protect the renal function of DN rats,which has no statistical difference with western medicine.At the same time,it has potential advantages in improving survival qualities.3 Compared with the control group,the area of SA-β-gal blue staining in DN rats was increased(P<0.05),and the expression of cyclin dependent kinase inhibitor p21 and DNA double strand break marker yH2AX were increased(P<0.01).After drug treatment,compared with the model group,the area of SA-β-gal blue staining was decreased(P<0.05),and the expression of p21 and yH2AX protein was decreased(P<0.01).The results of in vitro experiments showed that the blue staining rate of SA-β-gal and the expression of p21 and γH2AX protein in HK-2 cells stimulated by AGEs were increased(P<0.001),and Shenyan Fangshuai decoction could reverse the above changes.These results suggest that Shenyan Fangshuai decoction may delay the premature senescence of renal cells in DN.4 In the aspect of autophagy,compared with the control group,the expressions of LC3 and p62 in kidney tissue of DN rats were significantly increased(P<0.05),while the expressions of Beclinl,ATG5 and Atg7 did not change significantly(P>0.05).Compared with the normal group,the turnover rates of LC3 and p62 protein were significantly decreased(P<0.001),and after the intervention of Shenyan fangshuai decoction the turnover rates could be restored(P<0.001).Furthermore,the changes of autophagy flow were detected via mRFP-GFP-LC3 double fluorescence labeling system combined with autophagy inhibitor.Compared with the control group,the expression of red fluorescence in HK-2 cells stimulated by AGEs was decreased and the expression of yellow fluorescence was increased(P<0.001).After the treatment of Shenyan fangshuai decoction,the yellow fluorescence expression decreased and the red fluorescence expression increased(P<0.001).After the autophagy pathway was blocked by autophagy inhibitor CQ,the regulation of Shenyan fangshuai decoction on HK-2 premature senescnece induced by AGEs was reversed,which suggested that Shenyan fangshuai decoction could restore autophagy activity by dredging autophagy pathway,thus delaying the premature senescence of HK-2 induced by AGEs.5 In kidney of DN rats,the expression of anti-aging protein α-Klotho was significantly down-regulated(P<0.001),and Shenyan fangshuai decoction could up regulated the expression of α-Klotho(P<0.01).In vitro,the results of experiments also confirmed that AGEs could down regulate the expression of α-Klotho in HK-2 cells(P<0.05),while the expression of α-Klotho was up-regulated after the intervention of Shenyan fangshuai decoction(P<0.001).The results showed that up-regulation of α-Klotho could reverse the blocking of autophagy pathway.Compared with blank AGEs Group,the expression of LC3 inα-Klotho overexpression AGEs group was significantly decreased(P<0.001).Compared withα-Klotho overexpression control group,AGEs did not change the expression of LC3 in group significantly.Compared with α-Klotho overexpression control group,AGEs significantly increased the expression of p62(P<0.05),but compared with AGEs,the expression of p62 was significantly decreased via α-Klotho overexpression(P<0.001).Conclusion:1 Shenyan Fangshuai decoction can effectively protect kidney function of DN rats,and its effect is similar to Benazepril.Furthermore,it has potential advantages in improving the quality of life.2 The HK-2 cells stimulated by AGEs and the kidney of diabetic rats showed the phenomenon of premature cellular senescence.Shenyan fangshuai decoction might play a protective role in the kidney by delaying the premature cell failure of TECs.3 Shenyan fangshuai decoction may delay the premature senescence of DN renal cells by regulating α-Klotho/autophagy pathway.4 This study suggests that the two mechanisms closely related to cell senescence,"α-Klotho downregulation expression " and "autophagy activity inhibition" are the material basis of "imbalance of aggregation and dispersion "in DN.The up regulation of α-Klotho expression to restore activity of autophagy may be the material basis for the treatment of"Fuzheng Xiaozheng". |
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