Study On The Mechanism Of Bu-Yang-Huan-Wu Decoction On Diabetic Kidney Injury Based On MiR-218/GPRC5A Pathway

Background:Diabetic nephropathy is an important microvascular complication secondary to diabetes mellitus,which is a major cause of end-stage renal disease.The hyperglycemia has been shown to have a profound effect on all types of renal intrinsic cells,leading to glomerulosclerosis and tubulointerstitial Fibrosis,ultimately leading to irreversible renal failure.Recent studies have shown that tubular lesions sometimes occur independently of glomerular lesions.Tubular injury is more closely correlated with the deterioration of renal function and play a key role in the progression of DN.Searching biomarkers that targeting renal tubule cells may contribute to the research and clinical treatment of DN.Most studies have confirmed that miRNAs are involved in regulating multiple aspects of DN pathogenesis,while in vivo and in vitro experiments in high glucose environments have also confirmed that renal intrinsic cells can induce miRNA expression.Identifying potential specific miRNAs biomarkers is helpful to forecast or test the development and progress of diabetes,and its complications in an early phase.Bu-yang huan-wu Decoction was written by Wang Qingren in the Qing Dynasty which is a representative prescription for invigorating qi and promoting blood circulation.The Decoction is mostly used in the treatment of symptoms caused by Qi deficiency and Blood stasis.Numerous studies have shown that Buyang huanwu Decoction can reduce the clinical symptoms of diabetic nephropathy and improve the pathological damage to the intrinsic cells of the kidney,thereby delaying the decline of renal function.Objective:1.To explore the significance and possible mechanisms of miR-218/GPRC5A pathway in high glucose-induced renal tubular epithelial cell injury through in vitro experiments.2.A db/db mouse model was used to validate the interventional effect of the Bu-Yang-Huan-Wu Decoction on kidney injury.Explore its regulatory effect on miR-218/GPRC5A pathway and further explain its molecular biological mechanism.Provide new ideas for revealing the pathogenesis of diabetic nephropathy and future therapeutic strategies.Methods:1.The mechanism of miR-218/GPRC5A pathway in HG-induced HK-2 cell injury1.1 Bioinformatics analysis Data were downloaded from the GEO database,and differential gene analysis was performed using the GEO online analysis tool GEO2R to confirm miR-218 for study.Target prediction of MIR218 was performed using target gene website Target Scan and miRDB.1.2 Mechanism of miR-218/GPRC5A pathway in high glucose-induced HK-2 cell injury(1)The HK-2 were cultured in DEME medium supplemented with 10%FBS at 37°C.When growing to 60%and after 12 h of serum starvation,they were divided into HG group?NG group and Con group.(2)Cell transfection:The miR-218 mimic/inhibitor and negative control?pc DNA3.1-GPRC5A and corresponding control vector were transfected into the cells by Lipofectamine 3000.48h later transfection efficiency was measured by q RT-PCR.(3)The whole RNA was extracted by Trizol and reverse transcribed to c DNA using Prime Script RT Reagent Kit.miRNA expression was detected using SYBR Premix Ex Taq II in a 7500 real-time PCR system,with GAPDH as an internal reference and 2^-??Ct was used to analyse relative miRNA expression.(4)Extraction of proteins for Western blot with RIPA lysate and heated 5 min at 95°C.The protein concentration was measured by BCA method.Equal amount of protein was added in a vertical electrophoresis tank and protein samples were separated by SDS polyacrylamide gel electrophoresis.Transfer to PVDF membrane,add 5%skimmed milk powder to close for 1h,incubate primary antibody at 4°C overnight,wash 3×5min with TBST,incubate secondary antibody at room temperature for 1 h,wash the membrane and add ECL to develop.QUANTITY ONE software scans the grey scale values,use GAPDH as internal reference control,target protein/internal reference to calculate the relative expression of each protein.(5)CCK-8 assay:The prepared cell suspension was seeded into 96-well plates at a standard of 1000 cells per well and routinely incubated in a CO2 incubator.Cell viability was tested every 24 h.The proliferation curve was plotted using an enzyme marker with450 nm excitation light to detect the OD value.(6)Apoptosis assay:Collect cells into a centrifuge tube,centrifuge at 1000 rpm for 5min,add pre-cooled PBS at 4°C to resuspend the cells,centrifuge again to precipitate the cells and carefully aspirate the supernatant.Add 1X binding buffer to resuspend the cells and adjust the cell density to 1-5 x 10⁶/ml.100 ul of cell suspension was added to a 5 ml flowtube,5 ul of Annexin V/FITC was added and incubated for 5 min at room temperature and protected from light.10 ul of PI dye was added and 400 ul of PBS was added,followed by the assay.Flowjo software was used to analyse the results.(7)Luciferase assay Cells were inoculated into 24-well plates.The 3'-UTRs of GPRC5A-WT and GPRC5A-Mut were cloned into the pmiR-RB-REPORTTM dual luciferase reporter vector.The cells were gathered and submitted for luciferase analysis using the Dual-Luciferase Reporter Assay Kit post 48 h transfection.2.Bu-Yang-Huan-Wu Decoction regulates miR-218/GPRC5A pathway to improve kidney injury in db/db mice.(1)Experimental mice:4W-old male spontaneous type II diabetic db/db mice were randomly divided into model group(DKD group),low-dose BYHWT group(DKD+BYHWT-l group),and high-dose BYHWT group(DKD+BYHWT-h group).the male same genetic background without disease db/m were randomly divided into control group(Control),control with low-dose BYHWT group(Control+BYHWT-l group),control with high-dose BYHWT group(Control+BYHWT-h group).(2)Drug intervention:DKD+BYHWT-l group and Control+BYHWT-l group were given 2.7g/kg/d of Bu-Yang-Huan-Wu Decoction.DKD+BYHWT-h group and Control+BYHWT-h group were given 5.4g/kg/d of Bu-Yang-Huan-Wu Decoction.The control and DKD group were given equal volume of saline respectively.(3)The body weight,blood glucose,urinary protein excretion rate(UAE),serum creatinine(Scr)and urea nitrogen(BUN)were measured every 4W during drug intervention.The mice were executed after feeding until 16W.(4)Pathological observation of renal tissue One side of renal tissue was fixed in 4%paraformaldehyde,and PAS and Masson staining were performed to observe the pathological morphology and structural changes of renal tissue of mice in each group.(5)The other side of renal tissue was grounded,q RT-PCR was used to detect the expression of miRNA,and Western blot was used to detect the expression of GRPC5a,a-SMA,Vimentin and Col-1 proteins.The expressions of apoptosis-related proteins and inflammatory cytokines were also detected..the method was same as in Part 1.3.Statistical analysis SPSS22.0 and GRAPHPAD Prism 6.0 were used for all statistical analyses.T-test is used for comparison between the two groups and one-way ANOVA variance analysis and Tukey's test were used to compare the mean values among three or more groups.Results were expressed as mean±standard deviation(SD).P<0.05was considered to be significantly different.Results:1.The mechanism of miR-218/GPRC5A pathway in HG-induced HK-2 cell injury1.1 Bioinformatics analysis:Data were downloaded from the GEO database(access number:GSE114477)and differential gene analysis was performed using the GEO online analysis tool GEO2R to analyse the differential expression of miR-218 in DN patients and controls.miR-218 were confirmed combined with literature studies for the study.Target gene website,were used for miR218 target prediction,while differential genes in the database(access number:GSE30528)were analysed.The miRNA-predicted target genes were intersected with the down-regulated genes analysed in the GSE30528 dataset to obtain a total of 17 common genes.The bioinformatics analysis was combined with literature studies to finally identify GPRC5A as the target gene for miR-218 for the next research.1.2 Mechanism of miR-218/GPRC5A pathway in high glucose-induced HK-2 cell injury.(1)The q RT-PCR method was used to detect the expression levels of miR-218 and inflammatory factors in the NC control,NG and HG groups.The results showed that the expression levels of miR-218,inflammatory factor TNF-?and IL-1?were significantly increased under high glucose conditions(P<0.01).(2)The protein expression levels of miR-218,inflammatory factor TNF-?and IL-1?in HK-2 cells under high glucose conditions were significantly higher than those in NC control and NG groups(P<0.01).(3)The effect of transfection of miR-218 mimics,miR-218 inhibitor or NC miRNA on the expression level of miR-218 in cells under high glucose conditions was shown in the experiment:miR-218 mimics could up-regulate the expression level of miR-218,while miR-218 inhibitor could decrease the expression level of miR-218(P<0.01).The cell viability of HK-2 cells was detected using CCK-8.OD results showed that the cell viability of HG group was significantly lower than that of the control group at 48h and 72h(P<0.01).While transfected by miR-218 inhibitor,the cell viability at 48h and 72h was significantly higher in miR-218 expression down-regulation than in HG group(P<0.01).These results suggest that down-regulation of miR-218 promoted the viability of renal proximal tubular cells under high glucose conditions.(4)The results of Annexin v/FITC flow cytometry showed that apoptosis was significantly increased in the HG group compared with the control group,while the apoptosis rate was significantly lower in the HG group after miR-218 deletion(P<0.01).(5)Western blot showed that the expression of caspase-3 and caspase-9 was significantly enhanced in HK-2 cells under the effect of high glucose,and their expression levels were significantly down-regulated after treatment with miR-218 inhibitor.Compared with the control group,the expression of pro-apoptotic proteins(cleavage caspase-3 and cleavage caspase-9)in HK-2 cells was significantly reduced after treatment with miR-218inhibitor.(6)GPRC5A WT/Mut and miR-218 mimic/Control were cotransfected with Lipofectamine 3000 in HK-2 cells and validated using a dual luciferase reporter assay,showing that enhanced miR-218 expression was followed by a significant reduction in luciferase activity of the WT GPRC5 3?-UTR reporter gene(P<0.0 1).It was also confirmed that miR-218 mimic significantly reduced the expression levels of GPRC5A protein and miRNA(P<0.01).(7)Co-transfection experiments with miR-218 inhibitor and pc DNA3.1-GPRC5A showed that overexpression of GPRC5A enhanced the attenuating effect of miR-218inhibitor on high glucose-induced HK-2 cell injury.miR-218 inhibitor and pc DNA3.1-GPRC5A co-transfection of HK-2 cells resulted in a significant increase in cell survival,along with a significant decrease in apoptosis and inflammatory factor levels were significantly reduced(P<0.01).2.Bu-Yang-Huan-Wu Decoction regulates miR-218/GPRC5A pathway to improve kidney injury in db/db mice.(1)After Bu-Yang-Huan-Wu Decoction intervention,the body weight,fasting blood glucose,UAE and renal function index levels of db/db mice were significantly improved compared to the model group(p<0.01,p<0.05).(2)The degree of renal pathomorphological damage in db/db mice after Bu-Yang-Huan-Wu Decoction intervention was lower than that in the model group.(3)The expression of miR-218 was significantly decreased and the GPRC5A protein levels were significantly increased in kidney tissues of db/db mice after different doses of Bu-Yang-Huan-Wu Decoction intervention(P<0.01).(4)The intervention of Bu-Yang-Huan-Wu Decoction significantly reduced the expression levels of renal fibrosis marker protein a-SMA,Vimentin and Col-1 in db/db mice(p<0.01,p<0.05),and inhibit the expression of apoptotic protein of renal tissue to cleavage Caspase-3,cleavage Caspase-9 and inflammatory cytokines protein of TNF-?and1L-1?(p<0.01,p<0.05).Conclusion:1.The expression of miR-218 and inflammatory factor TNF-?and IL-1?were significantly elevated in high glucose-induced HK-2 cells.2.Inhibition of miR-218 expression promotes the viability of renal proximal tubular cells under high glucose conditions and reduces high glucose-induced apoptosis in renal tubular epithelial cells.3.miR-218 directly targets GPRC5A and negatively regulates the expression of GPRC5A.4.The miR-218/GPRC5A pathway regulates high glucose-induced renal tubular cell proliferation and apoptosis and serves as a potentially effective target for the treatment of diabetic nephropathy.5.Bu-Yang-Huan-Wu Decoction can improve blood glucose and excessive weight gain in db/db mice,while reduce urinary protein excretion rate,lower Scr and BUN to improve renal function.6.Bu-Yang-Huan-Wu Decoction can reduce the pathological damage of DN renal tissue,inhibit the expression of renal fibrosis related proteins,reduce the expression of apoptotic protein and inflammatory factor protein in renal tissue,and delay glomerulosclerosis and renal tubulointerstitial fibrosis.7.Bu-Yang-Huan-Wu Decoction can exert a protective effect against DN kidney injury by regulating the miR-218/GPRC5A pathway,providing a new possible pathway for the prevention and treatment of DN.