前列腺素E2受体3亚型siRNA通过抑制MAPK信号通路减轻TGF-β1诱导的小鼠系膜细胞损伤

Prostaglandin E2 receptor subtype 3-siRNA reduces the mesangial cell damage induced by TGF -β1 through inhibiting MAPK pathway in mice

目的探讨前列腺素E2（PGE2）受体3亚型（EP3）对转化生长因子-B1（TGF—β1）诱导的小鼠肾小球系膜细胞的损伤及可能机制。方法体外培养原代野生型系膜细胞，采用脂质体Lipofectamine^TM 2000化学合成法将siRNA转染至系膜细胞中沉默EP3受体，筛选干扰效率最大的EP3-siRNA片段。实验分组：（1）正常对照组；（2）TGF-β1（10μg/L）组；（3）NC-siRNA＋TGF-β1（10μg／L）组；（4）EP3-siRNA组；（5）EP3-siRNA＋TGF—β1（10μg/L）组。CCK-8法检测TGF—β1对细胞增殖的影响；ELISA法检测细胞PGE2和cAMP的表达；实时荧光定量PCR法检测纤维连接蛋白（FN）、结缔组织生长因子（CTGF）、环氧化酶2（COX2）、膜结合型前列腺素E2合酶1（mPGES1）mRNA的表达；Western印迹检测FN、CTGF、COX2、mPGES1蛋白表达及ERK1／2、p38MAPK活性的变化。结果与正常对照组相比，TGF-β1组系膜细胞增殖明显增加（P〈0．05），PGE2和cAMP表达增加（均P〈0．05），FN、CTGF、COX2、mPGES1的mRNA和蛋白的表达均上调（均P〈0．05）；与TGF—β1组相比，EP3-siRNA＋TGF-β1组系膜细胞增殖减少（P〈0．05），FN、CTGF、COX2、mPGES1的mRNA和蛋白的表达均下调（均P〈0．05）。与正常组比较，TGF-β1组ERK1／2、p38MAPK磷酸化水平明显增强（均P〈0．05）；而EP3-siRNA＋TGF—β1组的ERK1／2、p38MAPK磷酸化水平较TGF—β1组明显下调（均P〈0．05）。结论EP3-siRNA可能通过增加cAMP的产生，抑制ERK1／2、p38MAPK磷酸化，反馈抑制COX2及PGE2，从而下调FN、CTGF的表达，减轻TGF-β1诱导的小鼠系膜细胞损伤。

Objective To investigate the effects and mechanisms of prostaglandin E2 receptor subtype 3 （EP3） on transforming growth factor β1 （TGF-β1）-induced mouse mesangial cells damage. Methods Primary mouse mesangial cells were separated and cultured. Three siRNAs were synthesized and transfected into mesangial cells for silencing EP3 by LipofectamineTM 2000 and the best one was chosen. MCs were grouped into： （1）control group; （2）TGF- β1 （10 μg/L） group; （3）NC- siRNA plus TGF-131 （10 μg/L） group; （4） EP3-siRNA group; （5）EP3-siRNA plus TGF-β1 （10 μg/L）. Then the proliferation of MCs was evaluated by CCK-8 assay. The expression of PGE2 and cAMP incell supernatant were detected by ELISA. The mRNA and protein expression of fibronectin （FN）, connective tissue growth factor （CTGF）, cyclooxygenase-2 （COX2）, membrane-bound prostaglandin E2 synthase 1 （mPGES1） were detected by real- time quantitative PCR and Western blotting. The phosphorylation of p38 MAPK and ERK1/2 was detected by Western blotting. Results Compared with control group, the cell proliferation induced by TGF-β1 was increased （P 〈 0.05）, the expression of PGE2 and cAMP were improved, mRNA and protein expression of FN, CTGF, COX2 and mPGES1 were up-regulated （all P 〈 0.05）. Compared with TGF-β1 group, the cell proliferation in EP3- siRNA plus TGF-151 group was reduced, the expression of FN, CTGF, COX2 and mPGES1 mRNA and protein were downregulated （all P 〈 0.05）, the phosphorylation of ERK1/2, p38 MAPK were also declined （P 〈 0.05）. Conchmion EP3-siRNA may reduce TGF-151-induced cell damage through upregulating the expression of cAMP, repressing the activity of ERK1/2 and p38 MAPK, inhibiting the expression of COX2 mPGES1 and PGE2 by feedback, then decreased the expression of FN and CTGF.