罗格列酮抑制环孢素诱导的大鼠肾脏成纤维细胞基质金属蛋白酶9和金属蛋白酶1组织抑制剂表达

目的 观察罗格列酮(RGZ)对环孢素A(CsA)作用下大鼠肾脏成纤维细胞(NRK)过氧化物酶体增生物激活受体γ(PPARγ)和基质金属蛋白酶-9(MMP-9)表达的影响,探讨罗格列酮对环孢素A肾毒性保护作用的分子机制.方法 构建、筛选和扩增PPARγ基因的siRNA载体,并将载体转染至体外培养的NRK细胞.体外培养NRK细胞,随机分组.(1)对照组:不加处理；(2)RGZ组:加RGZ( 10 μmol/L)；(3)CsA组:加CsA( 1.0 mg/L)；(4)CsA+RGZ 组:同时加CsA( 1.0 mg/L)及RGZ(10 μmol/L)；(5)CsA+RGZ+siRNA组:质粒pRNAT- U6.2/LentiPPARγ-236转染NRK细胞,然后同时加入CsA( 1.0 mg/L)及RGZ(10 μmol/L).培养24 h后,用实时荧光定量和RT-PCR法检测各组细胞中PPARγ、MMP-9、金属蛋白酶1组织抑制剂(TIMP-1) mRNA表达,用Western印迹法检测纤连蛋白(FN)的蛋白表达.结果 CsA明显上调PPARγ、MMP-9和TIMP-1 mRNA的表达(均P＜0.05);RGZ与CsA合用后,PPARγ、MMP-9和TIMP-1 mRNA表达下降(均P＜0.05)；应用PPARγ siRNA后,与RGZ+ CsA组相比,PPARγ mRNA表达显著下降(P＜0.05),MMP-9 mRNA和TIMP-1 mRNA表达有所增加(均P＜ 0.05).CsA明显上调FN蛋白表达(P＜0.05)；RGZ与CsA合用后,FN蛋白表达下降(P＜0.05)；应用含PPARγ siRNA后,FN蛋白表达有所增加(P＜0.05).结论 罗格列酮可以显著减轻CsA诱导NRK细胞分泌的FN蛋白及MMP-9、TIMP-1 mRNA的表达,构建的siRNA质粒转染NRK细胞,能够有效地阻断NRK中PPARγ mRNA的表达,部分阻断罗格列酮对CsA毒性的改善作用.

Effects of rosiglitazone on the expression of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in renal interstitial fibroblasts induced by cyclosporine A

Objective To investigate the effects of rosiglitazone on the expression of peroxisome proliferator-activated receptor-γ(PPARγ) and matrix metalloproteinase-9(MMP-9) in renal interstitial fibroblasts induced by cyclosporine A, and discuss renal protective effect of rosiglitazone on renal toxicity of cyclosporine A. Methods Construction, screening and amplification of the target siRNA vector for PPARγ were carried out. The inhibitory effect of siRNA on the expression of PPARγ in normal rat kidney(NRK) cells was evaluated. NRK cells were cultured in the routine way. Experimental groups: (1)control group: single NRK cells without treatment; (2)RGZ group: NRK cells with RGZ (10 μmol/L); (3)CsA group: NRK cells with CsA (1.0 mg/L); (4)CsA+RGZ group: NRK cells with CsA (1.0 mg/L) plus RGZ (10 μmol/L); (5)CsA+RGZ+siRNA group: pRNAT-U6.2/Lenti-PPARγ-236 plasmid transfected into NRK cells, then CsA (1.0 mg/L) plus RGZ (10 μmol/L). The mRNA expression of PPARγ was detected by real-time RCR. The mRNA expressions of MMP-9 and TIMP-1 were detected by RT-RCR. The protein expression of FN was detected by Western blotting. Results CsA up-regulated the mRNA level of PPARγ, MMP-9 and TIMP-1 (P<0.05), and the up-regulation was inhibited by RGZ significantly (P<0.05). The application of PPARγ siRNA resulted in the decreasing of PPARγ mRNA (P<0.05), and partly reversed the inhibition effect of RGZ on MMP-9 and TIMP-1 mRNA (all P<0.05). CsA up-regulated the protein level of FN (P<0.05), and this effect was significantly inhibited by RGZ (P<0.05). The application of PPARγ siRNA could reverse the inhibition effect of RGZ on FN protein expression (P<0.05). Conclusion RGZ can inhibit the expressions of FN, MMP-9 and TIMP-1 induced by CsA which may be the mechanism of the protective effect of RGZ on renal interstitial fibrosis induced by CsA.