| Background:(1)To observe the changes of autophagy,inflammation and epithelial-to-mesenchymal transition(EMT)of renal tubular epithelial cells(RTECs)through experiments in vitro,so that clearly identified the mechanism of regulating autophagy and improving inflammation and EMT of RTECs in high glucose(HG)concentration.(2)NRK-52 E were cultured in HG environment in vitro to regulate the expression of SIRT1.The changes of autophagy,inflammation and EMT indexes of NRK-52 E were observed to clarify SIRT1/NF-κB signaling pathway is involved in the mechanism of autophagy and inflammatory injury of NRK-52 E induced by high glucose.(3)Explored the potential targets of Astragalus membranaceus in treating DN through network pharmacology analysis and molecular docking,and elucidated the mechanism of Astragalus in treating DN from various aspects.(4)Explored the intervention of Astragaloside IV to improve autophagy disorder,inhibit inflammatory reaction,ameliorate EMT of RTECs in high glucose environment,improve the mechanism of renal interstitial fibrosis in DN via SIRT1/NF-κB signaling pathway through in vitro and in vivo experiments.Methods:Part Ⅰ: NRK-52 E were cultured in vitro.Combined with the cell proliferation of NRK-52 E under different HG concentration culture conditions in domestic and foreign studies and previous studies of the research group,30mmol/L glucose concentration was selected as the concentration of high glucose group for the next experiment.The cells were divided into normal group,HG group,HG+autophagy activator(Rapamycin)group and HG+autophagy inhibitor(3-methyladenine)group.After 24 hours,the total protein and RNA were extracted.The expression of autophagy indexes(P62,Beclin-1,LC3),inflammation indexes(ace NF-κB p65,MCP-1,IL-1β)and EMT indexes(FN,α-SMA)were detected by Western blotting and quantitative RT-PCR.Part Ⅱ: NRK-52 E were cultured in glucose medium and divided into normal group,HG group,HG+SIRT1 activator(Resveratrol)group and HG+SIRT1 inhibitor(Ex-527)group.Cultured cells for 24 hours,extracted total protein and total RNA.The expression of SIRT1 was detected by cellular immunofluorescence.The expression of SIRT1,autophagy indexes(P62,Beclin-1,LC3),inflammatory indexes(ace NF-κB p65,MCP-1,IL-1β)and EMT indexes(FN,α-SMA)were detected by Western blotting and quantitative RT-PCR.Part Ⅲ: The main active components and targets of Astragalus membranaceus were obtained through TCMSP,TCMID and other databases.Retrieved the main targets of Diabetic Nephropathy through Gencards,OMIM,TTD,Dis Ge NET and other databases.Took the intersection of drug and disease targets for protein interaction analysis,and String platform was used to build PPI network.The PPI network was visualized and analyzed by Cytoscape 3.7.2 software,and PPI core network protein was screened out.The Metascape platform was used for GO and KEGG enrichment analysis,obtained the biological processes and signal pathways involved in “Drug-Ingredients-Target” network.The Cytoscape3.7.2 network was used to construct the network of “Astragalus membranaceus-Target of DN-Signal Pathway” and to select the main active components and core targets of Astragalus membranaceus.Finally,the molecular docking was verified by Auto Dock Tools 1.5.6 software.Part Ⅳ: NRK-52 E were cultured in vitro and divided into normal group,HG group,HG+AS-IV group and HG+SIRT1 activator(Resveratrol)group.After 24 hours,total protein and total RNA were extracted.The expression of SIRT1 was detected by cellular immunofluorescence.The expression of SIRT1,autophagy indexes(Beclin-1,LC3,P62),inflammatory indexes(ace NF-κB p65,MCP-1,IL-1β)and EMT indexes(FN,α-SMA)were detected by Western blotting and quantitative RT-PCR.Part Ⅴ: Forty male SD rats aged 8 weeks were randomly divided into normal group(N,n=10)and model group(DN rats).DN rats were induced by right kidney resection combined with intraperitoneal injection of STZ.The rats were randomly divided into group DN(n=10),group AS-IV(n=10),and group R(n=10).Rats in group N and group DN were given 5ml/kg/d of water for injection,Astragaloside IV gavaged 40mg/kg/d in group AS-IV,and Resveratrol 20mg/kg/d in group R for 8 weeks.(1)The general conditions of rats in each group before treatment,4 weeks and 8 weeks after treatment,including hair status,urine,defecation,activity,reactivity were dynamically observed.(2)Biochemical indexes including blood glucose(Glu),24-hour urinary protein(24UTP),SCr and BUN were measured before treatment,4 weeks and 8 weeks after treatment.(3)At the end of 8 weeks of treatment,the renal tissues of rats in each group were taken for HE,PAS,PASM and Masson staining.The changes of renal tubules in each group were observed under the light microscope.(4)At the end of 8 weeks of treatment,the renal tissues of rats in each group were taken,and the total protein and RNA of renal tissues were extracted.The expression of SIRT1,autophagy indexes(Beclin-1,LC3,P62),inflammation indexes(ace NF-κB p65,MCP-1,IL-1β)and fibrosis indexes(FN,α-SMA)were detected by immunohistochemistry,Western blotting and quantitative RT-PCR.Results:Part Ⅰ:(1)After the intervention of NRK-52 E cells with 30mmol/L glucose medium,the results of Western blotting and quantitative RT-PCR showed that compared with group N,the expression of Beclin-1 and LC3 decreased(P<0.05),the expression of P62 increased(P<0.05),ace NF-κB p65,MCP-1 and IL-1β increased(P<0.05),and FN,α-SMA increased(P<0.05)in group HG.(2)Under the condition of HG culture,after the intervention on NRK-52 E cells by autophagy activator Rapamycin,the results of Western blotting and quantitative RT-PCR showed that compared with group HG,the expression of Beclin-1 and LC3 increased(P<0.05),the expression of P62 decreased(P<0.05),MCP-1,IL-1β and ace NF-κB p65 decreased(P<0.05),while the expression of FN and α-SMA decreased(P<0.05).(3)Under the condition of HG culture,after the intervention of autophagy inhibitor 3-MA on NRK-52 E cells,the results of Western blotting and quantitative RT-PCR showed that compared with group HG,3-MA could inhibit the expression of Beclin-1 and LC3(P<0.05),increased the expression of P62(P<0.05).The expression of ace NF-κB p65,MCP-1 and IL-1β increased(P<0.05),while the expression of FN and α-SMA increased(P<0.05)compared with group HG.Part Ⅱ:(1)The results of immunofluorescence showed that SIRT1 was scattered in the cytoplasm and nucleus of NRK-52 E.The fluorescence intensity of SIRT1 in group HG was weaker than that in group N(P<0.05),enhanced in group HG+R and decreased in group HG+Ex-527(P<0.05).(2)After Resveratrol intervention,the results of Western blotting and quantitative RT-PCR showed that compared with group HG,the expression of SIRT1 in NRK-52 E cells was increased(P<0.05),the expression of Beclin-1 and LC3 were increased(P<0.05),and the expression of P62 was decreased(P<0.05),the inflammatory indexes MCP-1,IL-1β and ace NF-κB p65 were decreased(P<0.05),while the expression of FN,α-SMA were decreased(P<0.05).(3)After intervention with Ex-527,Western blotting and quantitative RT-PCR showed that compared with group HG,Ex-527 could inhibit the expression of SIRT1(P<0.05),reduce the expression of Beclin-1 and LC3(P<0.05),increase the expression of P62(P<0.05).The expression of MCP-1,IL-1β and ace NF-κB p65 were increased(P<0.05),while the expression of FN,α-SMA were increased(P<0.05)compared with group HG.Part Ⅲ: The core active ingredients of Astragalus membranaceus in treating DN are Astragaloside IV,quercetin,isorhamnetin,calycosin,formononetin,kaempferol,etc.The core target of Astragalus membranaceus in treating DN are PTGS2,IL6,AKT1,RELA,CDKN1 A,TP53,etc.The key signal pathways involved are AGE-RAGE,PI3K-Akt,HIF-1,FOXO,JAK-STAT,p53 signaling pathway,etc.Its functions mainly involved in biological processes such as apoptosis signaling pathway,regulation of DNA binding transcription factor activity,inflammatory response,positive regulation of cell death,epithelial cell proliferation,reactive oxygen species metabolism,regulation of cell adhesion and so on.Molecular docking verification showed that the target genes RELA(NF-κB p65),IL1 B,NFKBIA and IKBKB(target gene related to NF-κB signaling pathway)have good binding activity with Astragaloside IV,an effective active component of Astragalus membranaceus.Part Ⅳ:(1)Under the condition of HG culture,NRK-52 E cells were intervened with15ug/ml AS-Ⅳ and SIRT1 activator for 24 hours.Cellular immunofluorescence detection showed that the fluorescence intensity of SIRT1 in group HG was weaker than that in group N(P<0.05),and that in group HG+R and group HG+AS-Ⅳ was stronger than group HG(P<0.05).There was no significant difference between group HG+R and group HG+AS-Ⅳ(P>0.05).(2)The results of Western blotting and quantitative RT-PCR showed that compared with group HG,the expression of SIRT1 in NRK-52 E cells in group HG+R and group HG+AS-Ⅳ were increased(P<0.05),the expression of Beclin-1,LC3 were increased(P<0.05),the expression of P62 was decreased(P<0.05),the expression of MCP-1,IL-1β and ace NF-κB p65 were decreased(P<0.05),and the expression of FN,α-SMA were decreased(P<0.05).Part Ⅴ:(1)General conditions of rats in each group: the hair of rats in group N is neat and shiny,the reaction is quick,and there is no abnormality in diet,drinking water and defecation.In group DN,the hair gradually fell off,sparse and dull,the activity decreased,the reactivity decreased,the amount of diet and drinking water increased,and the amount of urine increased.Compared with group N,the feces were dry,gradually emaciated and poor mental state.The activity,reaction and mental state of rats in group R and group AS-Ⅳ were better than those in group DN,which was still worse than that in group N,and the urine volume was reduced.(2)Biochemical indexes: The random Glu of tail vein in model group were more than 16.7mmol/L,which was significantly higher than that in group N(P<0.05).With the extension of intervention period,the Glu of group DN increased further,which was significantly higher than that of group N(P<0.05);The Glu level in group R and group AS-Ⅳ was higher than that in group N(P<0.05).At the end of the 8th week,the Glu level decreased compared with that in group DN(P<0.05).The24 UTP in model group were significantly higher than that in group N(P<0.05).At the end of the 8th week,the 24 UTP in group DN was higher than that in group N(P<0.05).Compared with group DN,group R and group AS-Ⅳ were decreased(P<0.05).At the end of 4 and 8 weeks,the levels of BUN and Scr in group DN were higher than those in group N(P<0.05).After AS-Ⅳ and Resveratrol intervention,the levels of BUN and Scr were lower than those in group DN(P<0.05).(3)The results of HE,PAS,PASM and Masson staining showed that the RTECs in group N were the same size and arranged neatly,and the structures of mesangium and basement membrane were normal.Masson staining rarely stained collagen tissue with blue.Compared with group N,the basement membrane of renal tissue in group DN was thickened,mesangial cells and mesangial matrix proliferated,RTECs were hypertrophic,lumen narrowed,PAS positive protein deposition was seen in glomerular mesangial area and RTECs,and blue staining collagen tissue was seen in Masson staining of RTECs.But there was no renal interstitial fibrosis.After Astragaloside IV and Resveratrol intervention,mesangial hyperplasia,basement membrane thickening and RTECs hypertrophy were reduced in varying degrees,PAS positive protein deposition and blue stained collagen tissue were reduced.(4)The immunohistochemical results of renal tissue of rats in each group showed that SIRT1 was expressed in brown granules in the nucleus and cytoplasm of renal tubules.There was a certain amount of SIRT1 expression in renal tissue of rats in group N.The expression of SIRT1 in DN group was lower than that in group N(P<0.05),and the expression of SIRT1 in group R and group AS-Ⅳ was higher than that in group DN(P<0.05).Ace NF-κB p65 protein was diffusely expressed in the nucleus and cytoplasm of renal tubules in brown.There was a small amount of expression in the renal tissue of rats in group N.The expression of ace NF-κB p65 in group DN was higher than that in group N(P<0.05),decreased in group R and group AS-Ⅳ compared with group DN(P<0.05).The autophagy indexes Beclin-1 and LC3 were brown granules,which were mainly expressed in the cytoplasm.The expression of Beclin-1 and LC3 in group DN were lower than that in group N(P<0.05),and the expression of Beclin-1 and LC3 in group R and group AS-Ⅳ were higher than that in group DN(P<0.05).Inflammatory indexes MCP-1 and IL-1β were brown granules expressed in nucleus and cytoplasm.The expression of MCP-1 and IL-1β in group DN were higher than group N(P<0.05),the expression of MCP-1 and IL-1β in group R and group AS-Ⅳ were lower than in group DN(P<0.05).Fibrosis indexes FN,α-SMA were brown granules expressed in nucleus and cytoplasm.The expression of FN and α-SMA were increased compared with group N(P<0.05),and decreased in group R and group AS-Ⅳ compared with group DN(P<0.05).(5)The results of Western blotting and quantitative RT-PCR showed that compared with group N,the protein and m RNA expressions of SIRT1 and autophagy indexes(Beclin-1,LC3)in group DN were decreased(P<0.05),the expression of P62 was increased(P<0.05).The expression of the inflammatory indexes(ace NF-κB p65,MCP-1,IL-1β)and fibrosis indexes(FN,α-SMA)were increased(P<0.05).Compared with group DN,the protein and m RNA expressions of SIRT1,autophagy indexes(Beclin-1,LC3)were increased(P<0.05),the expression of P62 was decreased(P<0.05),the inflammatory indexes(ace NF-κB p65,MCP-1,IL-1β)and fibrosis indexes(FN,α-SMA)were decreased(P<0.05)in group AS-Ⅳ and group R.Conclusion:High glucose intervention can inhibit autophagy activity,induce the expression of inflammatory factors in RTECs in vitro,and promote mesenchymal transformation of renal tubular epithelial cells.The mechanism of above functionis is to regulate autophagy and inflammation by inhibiting the expression of SIRT1 and regulating SIRT1/NF-κB signaling pathway.Based on the results of network pharmacology analysis,Astragaloside IV is effective components of Astragalus membranaceus in treating DN.RELA(NF-κB p65)and IL1 B are the key targets of Astragaloside IV in treating DN.Studies showed that Astragaloside IV could enhance autophagy and inhibit inflammation via regulated SIRT1/NF-κB signaling pathway in vivo and in vitro,thereby improving inflammation and EMT of RTECs in high glucose concentration and improving renal interstitial fibrosis in DN rats. |
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