Effect Of Atorvastatin On The HK-2Proliferation And The Secretion Of ECM Induced By IL-6

objective: To observe the effect of atorvastatin on the proliferationand the extracellular matrix secreted from Human proximal renal tubularepithelial cells (HK-2) induced by IL-6, and to investigate the mechanismof IL-6-induced kidney injury and the molecular mechanisms ofatorvastatin on anti-renal interstitial fibrosis.Methods:1.HK-2cells induced by different concentrations of IL-6(0ng/ml,0.5ng/ml,5ng/ml,50ng/ml) cultured for24h,48h,72h in vitro.MTT assay for analyzing the effect of IL-6on the proliferation of HK-2cells, Chemiluminescence immunoassay(CLIA) was used to detectsupernatant of cell culture including Fibronectin and collagen type Ⅳexploring the effect of the level of IL-6and the time induced by IL-6.2.Design was divided into3groups, blank control group, IL-6(5ng/ml)group and IL-6(5ng/ml) adding atorvastatin(10-5mol/l) group,cellscultured for48hours, then HK-2’s proliferation was detected byMTT,the secretions of Fibronectin and collagen typeⅣ were measuredby chemoluminescence immunoassay, CTGF was detected byImmunohistochemistry，the expression of CTGF and TGF-β1mRNAwere detected by PCR. western-blot was used to detect the expression ofCTGF and TGF-β1protein levels.Results:1.MTT Results show that HK-2proliferation was increasedsignificantly with the concentration of IL-6, and with the duration of action. HK-2proliferation was also significantly increased. Thesupernatant show that HK-2secrete FN and collagen type IV increasedwith the concentration of IL-6, HK-2secrete FN and type IV collagenalso increased with the time extension stimulating by IL-6.2. WhenHK-2were cultured for48hours, FN and collagen type IV inIL-6(5ng/ml) group were significantly increased compared with controlgroup(P<0.05), while in IL-6(5ng/ml) adding atorvastatin(10-5mol/l)group, FN and collagen type IV were significantly reduced comparedwith IL-6(5ng/ml) group (P<0.05).4. When HK-2cells were cultured for48hours, the results of Immunohistochemistry show that CTGFexpression of IL-6(5ng/ml) group was significantly increased comparedwith blank control group(P<0.05), while CTGF expression inIL-6(5ng/ml) adding atorvastatin(10-5mol/l) group was significantlyreduced compared with IL-6(5ng/ml) group(P<0.05).5. Results of PCRshow that CTGF’s mRNA expression were enhanced statistically in IL-6(5ng/ml) group than the control group (P<0.05),while CTGF mRNAexpression was decreased statistically in IL-6(5ng/ml) addingatorvastatin(10-5mol/l) group which compared with IL-6(5ng/ml) group(P<0.05).6. The Western blot results show that the protein of CTGF andTGF-β1statistically expressed more in IL-6(5ng/ml) group than those inthe control group(P<0.05), while the protein of CTGF and TGF-β1statistically expressed in IL-6(5ng/ml) adding atorvastatin (10-5mol/l)group weaker compared with IL-6(5ng/ml) group (P<0.05). Conclusion:1. IL-6could induce renal tubular epithelial cellproliferation and increase the secretion of extracellular matrix, by a timeand dose dependent manner.2. Atorvastatin could inhibite the effect ofIL-6-induced tubular epithelial cell proliferation and extracellular matrixsecretion.3. IL-6induce the tubular epithelial cells proliferation andECM secretion through upregulation of TGF-β1and CTGF cytokines.4.Atorvastatin intervente the renal fibrosis through inhibition expression ofCTGF and TGF-β1.