| Objective: This study focused on chronic kidney disease(CKD)and renal fibrosis(RF)to carry out clinical and experimental research.First,through prospective randomized controlled trials,the efficacy and safety of Qihuang Siwu Decoction in improving CKD and RF were preliminarily discussed.On this basis,cyclosporin A(CsA)was used to induce RF models in mice and human renal tubular epithelial cells(HKC)in vivo and in vitro.We first observed the protective effect of Qihuang Siwu Decoction on renal structure and function of RF mice by regulating regulating the co-expression network of miRNA-mRNA in vivo,and then observed the effect of Qihuang Siwu Decoction on inhibiting epithelial mesenchymal transition(EMT)of HKC by regulating the representative miRNA-mRNA in the co-expression network to improve the effect of RF in vitro.We will provide theoretical basis and experimental support for the prevention and treatment of CKD and RF by Qihuang Siwu Decoction through clinical and experimental research.Methods:1.Study on the efficacy and safety of Qihuang Siwu Decoction in the treatment of CKD3-5 patients: According to the calculated sample size and selection criteria,a total of 80CKD3-5 patients were enrolled,40 cases in each of the experimental group and the control group.The control group was given conventional symptomatic treatment with western medicine,and the test group was given Qihuang Siwu Decoction on the basis of the control group.The treatment period was 8 weeks.Spss19.0 was used for statistical analysis.The changes of serum creatinine(Scr),urea nitrogen(BUN),uric acid(UA),estimated glomerular filtration rate(e GFR),urinary ?1-microglobulin(?1-mg),urinary NAG enzyme and urinary protein / creatinine ratio(UACR)were observed at 4 and 8 weeks.At 8 weeks,the changes of hemoglobin(HGB),blood calcium,blood phosphorus,parathyroid hormone(PTH),transforming growth factor-?1(TGF-?1),interleukin-6(IL-6),superoxide dismutase(SOD)and TCM syndrome score were observed,and the TCM syndrome effective rate and clinical total effective rate of the two groups were compared.2.Study on the protective effects of Qihuang Siwu Decoction on the kidneys of CsA-induced RF mice: Establishment of mouse RF models induced by CsA(divided into normal group,model group,Qihuang Siwu Decoction low,medium and high dose groups and valsha Tan control group).Observe the general conditions of RF mice's body weight and24-hour urine output after the intervention of Qihuang Siwu Decoction.Observe the pathological changes of kidney by HE,PAS,Masson staining,observe the apoptosis of kidney tissue by TUNEL method,and detect renal function and RF related indexes by biochemical analysis or ELISA method: Scr,BUN,UA,UACR,NAG,MDA,IL-6.3.The regulatory effect of Qihuang Siwu Decoction on the miRNA-mRNA co-expression network of RF mice: This study further performed miRNA and mRNA combination sequencing on the kidneys of the above-mentioned mice(normal group,model group,and Qihuang Siwu Decoction high-dose group).Firstly,the target difference(DE)miRNAs of Qihuang Siwu Decoction for improving RF were screened and verified.At the same time,bio-analysis(Target Scan and other software)was used to predict the target DE mRNAs of DE miRNAs,and the two co-expression network were constructed by Cytoscape.(normal group vs model group;Qihuang Siwu Decoction high-dose group vs model group).Furthermore,KEGG was used to enrich the top 20 pathway of DE mRNAs in the two co-expression networks,and screened out the common pathway,that is,the target pathway of Qihuang Siwu Decoction improving RF,and then further screened the DE mRNAs in the target pathway,and qRT-PCR,Western Blot,immunofluorescence and ELISA methods were used to verify the RF-related DE mRNAs at gene and protein levels.At the same time,representative miRNA-mRNA was selected for further study.4.The effect of Qihuang Siwu Decoction on HKC EMT induced by CsA by regulating the miR-21-5p/SMAD7 axis: CsA was used to intervene in HKC to establish an RF model,and the MTT method,Transwel experiment and flow cytometry were used to observe the effect of Qihuang Siwu Decoction on the influence of HKC proliferation,apoptosis and migration.Using qRT-PCR and Western Blot methods to observe the effects of Qihuang Siwu Decoction on E-cadherin and a-SMA related to EMT,and observe its effects on some of the target miRNAs screened in animal experiments(miR-21-5p,miR-375-3p and miR-802),some target mRNAs(SMAD7,TGF?1)and SMAD3.It was predicted by Target Scan that miR-21-5p and SMAD7 are targeted regulatory pairs,and miR-21-5p was further simulated or inhibited to observe the expression of SMAD7,E-cadherin,a-SMA and the change of HKC migration ability.Results:1.Qihuang Siwu Decoction was safe and effective in treating CKD 3-5 patients::(1)Total clinical efficacy: After treatment,the total clinical effective rate of the control group was 62.16%,and the total clinical effective rate of the experimental group was 78.38%.The therapeutic effect of the experimental group was better than that of the control group(P<0.05).(2)TCM syndrome scores and efficacy: After treatment,the TCM syndrome scores of the two groups were significantly reduced(P<0.01).The total effective rate of TCM syndromes in the control group was 43.24%,and in the experimental group was 67.57%.The test group was better than the control group in improving the scores of TCM syndromes(P<0.01)and the total effective rate of TCM syndromes(P<0.05).(3)Renal function related indicators: After treatment,the control group effectively improved BUN,UA(P<0.05),NAG(P<0.01),and the Scr,e GFR,a1-MG,and UACR were not significantly different from those before treatment;the experimental group effectively improved BUN,UACR(P<0.01),NAG,UA,Scr and e GFR(P<0.05),and there was no significant difference in a1-MG compared with before treatment;the experimental group was better than the control group in reducing BUN and UACR(P <0.05).(4)CKD complication indicators: after treatment,the control group effectively improved HGB,PTH(P<0.01),blood phosphorus(P<0.05),and the test group effectively improved HGB,PTH and blood phosphorus(P<0.01).There was no significant difference between the two groups of blood calcium.The test group was better than the control group in increasing HGB(P<0.05).(5)Fibrosis,oxidative stress and inflammation indicators: after treatment,the control group effectively improved TGF-?1(P < 0.05)and IL-6(P < 0.01),and there was no significant difference in SOD between before and after treatment;the experimental group was effectively improved With TGF-?1,SOD(P<0.01)and IL-6(P<0.05),the test group was better than the control group in reducing TGF-?1(P<0.05).(6)The liver function of the two groups before and after treatment was in the normal range and there was no significant difference(P>0.05).There were no adverse events in the two groups,and the test process was safe and effective.2.Qihuang Siwu Decoction could regulate miRNA-mRNA co-expression network and improve RF in mice:(1)Compared with the model group,the body weight and 24-hour urine output in each dose group of Qihuang Siwu Decoction increased significantly(P<0.01),and also improved Scr(P<0.05),BUN(P<0.01),UACR(P<0.01)<0.01),NAG(P<0.01 or P<0.05),MDA(P<0.01).Compared with the model group,the high-dose group improved UA and IL-6(P<0.05).(2)Compared with the model group,each dose group of Qihuang Siwu Decoction reduced the degree of basement membrane thickening,renal tubule dilatation,cytoplasmic vacuolation,arteriolar hyalineosis,and interstitial fibrosis,and significantly improved collagen volume fraction(P<0.01)and renal cell apoptosis(P<0.01).(3)The miRNA sequencing results showed that the model group had 55 and 42 DE miRNAs(FC>1.5or<0.67,P<0.05)respectively compared with the normal group and the Qihuang Siwu Decoction high-dose group.Finally,14 target miRNAs were screened out for Qihuang Siwu Decoction to improve RF,including protective miRNAs(down-regulation in the model group,callback with Qihuang Siwu Decoction): miR-677-3p,miR-138-2-3p,miR-3087-3p,miR-874-5p,miR-2137,miR-551b-3p,miR-187-3p,and damaging miRNAs(up-regulated in the model group,Qihuang Siwu Tang callback): miR-692,miR-137-3p,miR-21a-5p,miR-375-3p,miR-802-3p,miR-802-5p,miR-7b-5p,the verification of qRT-PCR was also highly consistent with the sequencing results.(4)The mRNA sequencing results showed that there were 2463 DE mRNAs(FC>1.5 or<0.67,P<0.05)in the model group compared with the normal group,of which 1324(54%)were predicted by 55 DE miRNAs.Compared with the Qihuang Siwu Decoction high-dose group,the model group had 1898 DE mRNAs(FC>1.5 or<0.67,P<0.05),of which 679(36%)were predicted by 42 DE miRNAs.We constructed two co-expression networks and selected 11 target pathways of Qihuang Siwu Decoction to improve RF after enrichment of target DE mRNAs by KEGG: ECM-receptor interaction ? Sphingolipid metabolism ? Leukocyte transendothelial migration ? TGF-beta signaling pathway?PPAR signaling pathway?Biosynthesis of unsaturated fatty acids?Protein processing in endoplasmic reticulum ? Peroxisome ? Tryptophan metabolism ? Butanoate metabolism?Propanoate metabolism.(5)Among the 11 target pathways that Qihuang Siwu Decoction improves RF,there were a total of 37 target DE mRNAs.Their expression in the model group was disordered,and they were called back by Qihuang Siwu Decoction.14 of them were closely related to RF(EMT and ECM deposition,TGF-? signal regulation,endoplasmic reticulum stress,fatty acid metabolism,mitochondrial oxidative stress).We have verified the 14 target DE mRNAs at the gene and/or protein level.Compared with the model group,Qihuang Siwu Decoction decreased the gene and protein expression of COL1A1,COL3A1,FN1,CD44,SPP1,MMP2,TGF-?1,PDIA6 and DDIT3 in a dose-dependent or optimal concentration(P<0.01 or P<0.05),increased the gene and protein expression of SMAD7,PCK1,ACOX1,MPV17 L and AGPS(P<0.01 or P<0.05).In addition,Qihuang Siwu Decoction increased the protein expression of SOD(P<0.01),and decreased the protein expression of 8-OHd G(P<0.01).3.Qihuang Siwu Decoction could regulate HKC EMT through miR-21-5p/SMAD7 axis to improve RF:(1)CsA dose-dependently inhibited HKC proliferation.On the basis of half inhibition(10mg/L,the concentration of the follow-up experiment),Qihuang Siwu Decoction15mg/m L(the concentration of the follow-up experiment)promoted the proliferation of HKC most significantly(P<0.01).(2)The apoptosis and migration of HKC increased under the induction of CsA.Qihuang Siwu Decoction reduced the apoptosis of HKC(P<0.01)and inhibited the migration ability of HKC(P<0.01).(3)Compared with the model group induced by CsA,Qihuang Siwu Decoction increased the expression of E-cadherin gene(P<0.01)and protein(P<0.05),and decreased the gene of a-SMA(P<0.05)and protein(P<0.01)expression.(4)Consistent with the results of animal experiments,Qihuang Siwu Decoction significantly reduced the expression of HKC miR-21-5p,miR-375-3p and miR-802 induced by CsA(P<0.01),and promoted the gene and protein expression of SMAD7(P<0.01),inhibited the expression of TGF-?1 gene(P<0.05)and protein(P<0.01),and at the same time inhibited the SMAD3 gene(P<0.01)and protein(P<0.05)expression.(5)SMAD7 was predicted to be a potential target gene of miR-21-5p by Target Scan and other bio-info software.After transfection of miR-21-5p mimics,inhibitors and corresponding negative controls to HKC,the expression of miR-21-5p inhibitor increased by 20 times(P<0.01)and the expression of miR-21-5p inhibitor decreased by 5 times(P<0.01),indicating that the transfection was successful.(6)After successful transfection,CsA was used to create a model and Qihuang Siwu Decoction was used to intervene.It was found that compared with miRNA-NC mimics group,miR-21-5p mimics inhibited SMAD7 protein(P<0.01)and gene(P< 0.05)expression,and reduced the protein expression of E-cadherin(P<0.01),increased the protein expression of a-SMA(P<0.01),and also promoted the migration ability of HKC(P<0.01).Compared with miRNA-NC inhibitor group,miR-21-5p inhibitor promoted gene and protein expression of SMAD7(P<0.05),increased E-cadherin protein expression(P<0.01),and inhibited a-SMA protein expression(P<0.01),and partially inhibited the migration ability of HKC(P<0.01).Conclusion: 1.Clinical studies have shown that Qihuang Siwu Decoction can effectively improve the clinical symptoms of patients with chronic kidney disease in stages3-5,protect kidney function,reduce urine protein,relieve complications such as anemia and calcium and phosphorus metabolism disorders,and reduce fibrosis,oxidative stress and micro-inflammatory state,and is safe and effective.It is clinically worthy of promotion and application.2.Further studies in vivo showed that Qihuang Siwu Decoction could regulate the disordered miRNA-mRNA co-expression network in CsA induced renal fibrosis mouse model,by improving the interaction with EMT and ECM receptors,TGF-? signal regulation,endoplasmic reticulum stress,fatty acid metabolism and oxidative stress in the co expression network.In turn,it can improve kidney pathological damage,apoptosis and ECM deposition with multiple targets,reduce micro-inflammation and oxidative stress,protect renal function,and improve renal fibrosis.3.Further in vitro studies have shown that in the CsA-induced HKC injury model,Qihuang Siwu Decoction can inhibit EMT by promoting the proliferation of HKC,inhibiting its apoptosis and migration,and improving the expression of EMT-related phenotypic genes.Among them,in the miRNA-mRNA co-expression network,Qihuang Siwu Decoction reduces the expression of its target gene SMAD7 by increasing miR-21-5p,which may be an important mechanism for inhibiting EMT and improving renal fibrosis. |
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