Salvianolic Acid B Prevents TGF-β1-induced Hk-2Cell Epithelial-mesenchymal Transition And Changes Related MicroRNAs’ Expression

BackgroundEpithelial-mesenchymal transition (EMT) plays a key role in the developmentof renal interstitial fibrosis. Studies suggested that renal EMT can be prevented ifgiven intervention at its early stage, this has a significance on delaying renal fibrosisor protecting residual renal function. MicroRNAs (miRNAs) are endogenousnon-coding RNA molecules,20–22nucleotides in length, involving in manydiseases. Researches suggested that some miRNAs mediated the development ofrenal interstitial fibrosis.Salvianolic acid B (SA-B), one drug obtained from Salviae Miltiorrhizae, hasmultiple pharmacological activities such as antioxidative, anti-hepatic fibrosis,cardiovascular protective and so on. Recently reports suggested that SA-B had renalprotective effect. In our study we had confirmed that SA-B could improve renalinterstitial fibrosis. But now the study of SA-B on preventing renal fibrosis and thepossible regulatory mechanism is still little known.We speculated that SA-B viapreventing the tubular EMT occurence and changing relative microRNAs’expression played a protective role on renal disease. We observed the changes of cellmorphology, the expression of α-SMA and E-cadherin to verification the prevent roleof SA-B on TGF-β1induced HK-2EMT. Next we made microRNA chip, screenedthe relative microRNAs and did further study of their downstream gene, we hopethat our work can confirm the role that salvianolic acid B could prevent tubular EMTand provide new theoretical and experimental basis for preventing and treating renal interstitial fibrosis.Methods1. Observed the effect of SA-B prevented TGF-β1-induced HK-2cells EMT(1)Grouping:①the normal control group②TGF-β1-induced EMT group(5ng/ml)③TGF-β1(5ng/ml)+SA-B(0.1,1,10,100uM). Added TGF-β1(5ng/ml) orTGF-β1(5ng/ml) and SA-B to the cells, cultured for48h.(2)Effect of different concentrations of SA-B (0.1,1,10,100uM) on TGF-β1(5ng/ml) induced HK-2cell viability was determined by methylthiazoletetrazplium(MTT) assay;(3)Cell morphology of different concentrations of SA-B on TGF-β1inducedHK-2cell was observed by inverted microscope;(4)Effect of different concentrations of SA-B on mRNA of α-SMA andE-Cadherin by RT-PCR;(5)Effect of different concentrations of SA-B on protein of α-SMA andE-Cadherin by immunofluorescence;2. Made miRNA chip, filtered and verified the expression of micorRNAs anddetected the expression of TGF-βRⅡwhich was regulated by miR-106b.(1) Made miRNA chip, filtered miRNAs having significant expressions;(2) Expression of miR-93, miR-106b, miR-34a and miR-493*by RT-PCR;(3) Transfected miR-106b mimics with transient transfection method, theexperiment were invided into3groups:①normal control group②TGF-β1(5ng/ml)+negative control (80nM)group③T GF-β1(5ng/ml)+miR-106bmimic(80nM) group.(4) mRNA and protein of TGF-βR Ⅱw eredetected by RT-PCR and Westernblotting.Results 1. Salvianolic acid B prevented TGF-β1-induced HK-2cells EMT(1) MTT resultsCompared to normal control group, the cell viability had no significantdifference in normal group and the TGF-β1induced group with the differentconcentrations of SA-B, it’s even increased in SA-B100uM group.(2) Changes in cell morphologyNormal HK-2cells showed cobblestone or paving stone-like form, TGF-β1induced HK-2cells showed slender spindle, similar to the fibrosis appearance. Theprotective effect of SA-B on cells showed in a concentration-dependent manner, thehigher the concentration, the more obvious the protective effect.(3) RT-PCR resultCompared to the normal group, the mRNA of α-SMA was significantly increasedwith the E-Cadherin significantly reduced in TGF-β1induced group (p<0.001).Compared to TGF-β1induced group, the mRNA of α-SMA and E-Cadherin wereseparately increased and reduced in a concentration-dependent manner in SA-Bgroups (P<0.05).(4) Immunofluorescence resultsThe protein α-SMA was significantly increased with the E-cadherin significantlyreduced in TGF-β1induced group，in SA-B100uM group, our results revealed thatSA-B not only prevent the expression of the α-SMA but also prohibit the loss of theE-cadherin in HK-2cells, while this phenomenon ware not obvious in SA-B1uMgroup.2. Filtered miRNAs and verified their expression, detected the expression ofTGF-βRⅡregulated by miR-106b.(1) Filtered the significantly changed miRNAs.According to the fold change>2.0or <0.5, we screened51miRNAs which had significantly change in the process of SA-B prevented HK-2cell EMT, among them9were reduced in TGF-β1induced group but increased in SA-B group, while42miRNAs increased in TGF-β1induced group but reduced in SA-B group.(2) RT-PCR verified the selected miRNAs.MiR-93, miR-106b and miR-34a in SA-B group showed significantly increased(P<0.05) while in TGF-β1group they were significantly decreased. MiR-493*wassignificantly increased in TGF-β1group while decreased in SA-B group(P<0.05).The expressions of these4miRNAs were consistent with the miRNA array results.(3) The expression of TGF-βR Ⅱ by RT-PCR, Western blotting.Overexpression of miR-106b could downregulated the expression of bothmRNA and protein of TGF-βR Ⅱ.Compared to TGF-β1+N.C. group, the mRNA ofTGF-βRⅡ were significantly reducedin miR-106b mimic transfection group(P<0.05) and the results of protein by western blotting were consistent with themRNA result.ConclusionSA-B prevents TGF-β1-induced HK-2cell EMT accompanied by changes inmiRNAs and some miRNAs are closely associated with the preventive role of SA-Bon HK-2EMT.