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目的探讨过氧化物酶增殖物激活受体(PPAR)γ1对血管紧张素Ⅱ(Ang Ⅱ)诱导的系膜细胞外基质积聚的抑制作用及其机制。方法脂质体转染质粒pIRES2-EGFP-PPARγ1／ WT(野生型)于系膜细胞,给予AngⅡ(10-7 mol／L)刺激48 h。采用RT-PCR检测TGF-β1、PAI-1、 c-fos、c-jun mRNA表达水平。采用ELISA法检测细胞上清液中TGF-β1和FN浓度。Western印迹观察胞浆中I-κB、NF-κB及胞核中NF-κB、pERK蛋白表达水平。利用PPARγ／与PPRE结合活性测定PPARγ1在系膜细胞中的活性。同时以PPARγ激动剂吡格列酮(6μmol／L)、不具有目的基因PPARγ1的空白质粒pIRES2-EGFP和表达功能缺陷-突变型(DN)PPARγ1的质粒pIRES2- EGFP-mPPARγ1／DN作为对照。结果 PPARγ1过表达能显著性抑制Ang Ⅱ诱导的系膜细胞 TGF-β1、PAI-1的mRNA高表达(P<0．05),同时下调c-fos和c-jun mRNA高表达(P<0．05)。转染PPARγ1／WT能显著降低AngⅡ 48 h刺激下细胞上清液中FN和TGF-β1浓度(P<0．05)。在AngⅡ作用下系膜细胞AT1表达增加(P<0．05),PPARγ1可显著性减少AT1的高表达(P< 0．05)。AngⅡ诱导的系膜细胞pERK表达明显升高,PPARγ1可显著性减少pERK表达(P<0．05)。转染PPARγ1／WT能上调AngⅡ刺激下系膜细胞胞浆中I-κB低表达,同时下调NF-κB由胞浆向细胞核的转移(P<0．05)。转染PPARγ1／DN并无上述作用。吡咯列酮具有与PPARγ1相同的显著性效应。PPARγ1／WT转染组PPARγ1的活性明显高于其他组(P<0．05)。结论 PPAR-γ1过表达能够抑制AngⅡ刺激下系膜细胞外基质的聚积,降低AT1受体蛋白表达,具有直接抗硬化的非代谢性效应,其机制可能是通过抑制ERK／AP-1及NF-κB等信号分子的传递。
Objective To investigate the inhibitory effect of peroxisome proliferator - activated receptor γ (PPARγ1) on the extracellular matrix accumulation induced by angiotensin Ⅱ (Ang Ⅱ) and its mechanism. Methods Plasmid pIRES2-EGFP-PPARγ1 / WT (wild-type) was transfected into mesangial cells and stimulated with AngⅡ (10-7 mol / L) for 48 h. The expression of TGF-β1, PAI-1, c-fos and c-jun mRNA were detected by RT-PCR. The concentration of TGF-β1 and FN in the cell supernatant was detected by ELISA. Western blotting was used to observe the expression of NF-κB and pERK in cytoplasm of I-κB, NF-κB and cytoplasm. The activity of PPARγ1 in mesangial cells was measured using PPARγ / PPRE binding activity. Plasmid pIRES2-EGFP-mPPARγ1 / DN expressing plasmid pIRES2-EGFP-mPPARγ1 / PPARγ1 (PPARγ agonist pioglitazone (6μmol / L), PPARγ1 lacking the target gene PPARγ1 and PPARγ1 expressing DNMT) were used as controls. Results PPARγ1 overexpression significantly inhibited the mRNA expression of TGF-β1 and PAI-1 in mesangial cells induced by AngⅡ (P <0.05), and down-regulated the expressions of c-fos and c-jun mRNA .05). Transfection of PPARγ1 / WT significantly reduced FN and TGF-β1 concentrations in the cell supernatant stimulated by AngⅡ for 48 h (P <0.05). The expression of AT1 in mesangial cells was increased by AngⅡ (P <0.05), while PPARγ1 significantly decreased the expression of AT1 (P <0.05). Ang Ⅱ induced a significant increase of pERK expression in mesangial cells, while PPARγ1 significantly decreased pERK expression (P <0.05). Transfection of PPARγ1 / WT could up-regulate the expression of I-κB in the cytoplasm of mesangial cells under the stimulation of AngⅡ, and at the same time down-regulated the translocation of NF-κB from cytoplasm to nucleus (P <0.05). Transfection of PPARγ1 / DN does not have the above effects. Pyrrolostone has the same significant effect as PPARγ1. The activity of PPARγ1 in PPARγ1 / WT group was significantly higher than that in other groups (P <0.05). Conclusion Overexpression of PPAR-γ1 can inhibit the accumulation of mesangial extracellular matrix and decrease the expression of AT1 receptor protein, which has the direct anti-sclerotic and non-metabolic effects. The mechanism may be that ERK / AP-1 and NF- κB signaling molecules such as the transfer.