| Objective:In this study,we intend to investigate the regulatory effect and specific mechanism of Bushen Huoxue Sanjie Capsule on the exosomal Micro RNA of mesenchymal stem cells to slow down renal interstitial fibrosis.Methods:Mesenchymal stem cells(MSCs)were isolated and cultured in vitro,and P3-generation MSCs in good growth condition were co-cultured with Bushen Huoxue Sanjie Capsule,and the culture supernatant was collected,from which exosomes were isolated and extracted for morphological size and surface marker identification.Significantly different micro RNA(mi RNA)of exosomes were analyzed,and the levels of secreted mi RNA of MSC-derived exosomes(MSC-exosomes,MSC-Exo)in each group were detected by q RT-PCR.Thirty-six male SD rats were randomly divided into three groups of 12 rats each,and the groups were set up as sham-operated group,UUO-molded group and exosome-treated+molded group to establish a unilateral ureteral ligation interstitial fibrosis model in rats,and exosomes were injected into the rats in the tail vein 7days after modeling.The expression levels of TGF-β1,COL1,Smad2 and α-SMA in renal tissues were detected by Western blot and mi R-21-5p in renal tissues by q RT-PCR.Results:The results of exosome identification showed that MSC-Exo was consistent with the exosome profile.A total of 26 significantly differentially expressed mi RNAs were screened(P<0.05),among which 5 mi RNAs were significantly up-regulated,including hsa-mi R-1282,hsa-mi R-182,hsa-mi R-1273 a,hsa-mi R-222,hsa-mi R-21,and 2 mi RNAs were significantly down-regulated,including hsa-mi R-451 b,hsa-mi R-1273 e.q RT-PCR confirmed that Bushen Huoxue Sanjie Capsule significantly up-regulated the expression of mi R-21-5P in MSCs-Exo(P<0.05).MASSON staining showed that compared with the sham-operated group,the UUO-molded group had increased collagen deposition in the interstitium and increased fibrotic area with the prolongation of obstruction time.The degree of various lesions was significantly less in the exosome+staging group than in the UUO-staging group at all time observation points,and the exosome+staging group had an ameliorating effect on collagen deposition in the periglomerular and tubular interstitium.Western blot results showed that the expression levels of TGF-β1,COL1,Smad2,and α-SMA were decreased in the exosome+staging group compared with the UUO-staging group(P< 0.05).q RT-PCR for the expression level of mi R-21-5p in renal tissues revealed that the expression level of mi R-21-5p in the exosomes+staging group was increased compared with the UUO-staging group(P<0.05).Conclusion:(1)Bushen Huoxue Sanjie Capsule could promote MSCs to secrete MSCs-Exo and increase its mi R-21-5p expression level.(2)MSCs-Exo after the action of Bushen Huoxue Sanjie Capsule could reduce the degree of kidney tissue fibrosis in UUO model rats.(3)MSCs-Exo after the action of Bushen Huoxue Sanjie Capsule may slow down the process of renal interstitial fibrosis by overexpressing mi R-21-5p and downregulating TGF-β1,Smad2,COL1,α-SMA and other proteins. |
