氧化苦参碱下调ZNF580以抑制LTC-14细胞分泌细胞外基质的分子机制研究

目的 探讨氧化苦参碱（OM）对转化生长因子-β1（TGF-β1）诱导的大鼠胰腺星状细胞 LTC-14中细胞外 基质（ECM）分泌的影响及其分子机制。方法 取生长良好的 LTC-14细胞株，分为对照组（正常培养的 LTC-14细 胞），TGF-β1组（10 μg/L TGF-β1刺激 12 h），TGF-β1+OM组（1 g/L OM预处理 30 min，再加入 10 μg/L TGF-β1刺激 12 h），TGF-β1 + SiZNF850组（LTC-14细胞中瞬时转染 ShRNA-ZNF580质粒，24 h后加入 10 μg/L TGF-β1刺激 12 h）。实时定量 PCR、Western blot 检测细胞内 Smad2、Smad3、ZNF580、α-肌动蛋白（α-SMA）、基质金属蛋白酶-2 （MMP-2）、基质金属蛋白酶组织抑制剂1（TIMP1）的mRNA和蛋白表达情况，酶联免疫吸附试验（ELISA）检测ECM中 胶原蛋白-Ⅰ（Col-Ⅰ）、Col-Ⅲ、纤维连接蛋白（FN）、肿瘤坏死因子-α（TNF-α）和白细胞介素-1β（IL-1β）的分泌情 况。结果 TGF-β1诱导的 LTC-14细胞中 Smad2、Smad3、ZNF580和 α-SMA的 mRNA和蛋白表达水平均升高（P＜ 0.05），MMP-2/TIMP1比值下降（P＜0.05），且 Col-Ⅰ、Col-Ⅲ、FN、TNF-α和 IL-1β的分泌水平升高（P＜0.05）；而用 OM预处理或沉默 ZNF580基因后，LTC-14细胞中 TGF-β1/Smads通路 Smad2、Smad3、α-SMA和 ZNF580的 mRNA和 蛋白表达水平均下调（P＜0.05），MMP-2/TIMP1表达比值升高（P＜0.05），细胞外基质 Col-Ⅰ、Col-Ⅲ、FN、TNF-α和 IL-1β分泌水平均降低（P＜0.05）。结论 OM通过抑制胰腺星状细胞LTC-14中TGF-β1/Smads通路激活，下调ZNF580， 阻滞胰腺星状细胞的活化，使ECM分泌减少、降解增多，减轻胰腺纤维化。ZNF580可能是OM作用的分子靶点。

Oxymatrine suppressed extracellular matrix expression in LTC-14 cells by down-regulating ZNF580 expression

Objective To probe the molecular mechanism and effect of oxymatrine (OM) on the secretion of extracellular matrix (ECM) in rat pancreatic stellate cell line LTC-14 induced by TGF-β1. Methods LTC-14 cells were divided into control group (LTC-14 cells in normal culture), TGF-β1 group (LTC-14 cells stimulated by 10 μg/L TGF-β1 for 12 h), TGF-β1+OM group (LTC-14 cells pretreated with 1 g/L OM for 30 min before TGF-β1 stimulation) and TGF-β1+ SiZNF850 group (LTC-14 cells were transiently transfected with ShRNA-ZNF580 plasmids for 24 h before TGF - β1 stimulation). After cultivation, cell supernatant was collected and total RNA and total protein were extracted. The expressions of Smad2, Smad3, ZNF580, α - SMA, MMP2 and TIMP1 were detected by Real-time PCR and Western blot assay. The secretion of ECM components, Col-I, Col-III, FN, TNF-α and IL-1β were detected by ELISA. Results The mRNA and protein expressions of Smad2, Smad3, ZNF580 and α-SMA were increased (P＜0.05), the expression ratio of MMP2/TIMP1 was down-regulated (P＜0.05), and the secretion of ECM components was increased in LTC-14 cells induced by TGF-β1 (P＜0.05). However, after OM intervention or ZNF580 gene knockdown, the mRNA and protein expressions of Smad2 and Smad3 were suppressed (P＜0.05), the mRNA and protein expressions of ZNF580 decreased, the expression ratio of MMP-2/ TIMP1 was up-regulated (P＜0.05), and the secretion of ECM components was reduced (P＜0.05). Conclusion Oxymatrine can reduce ECM secretion, increase ECM degradation and alleviate pancreatic fibrosis by inhibiting TGF- β1/ Smads/ZNF580 signaling pathway and blocking the activation of pancreatic stellate cells. ZNF580 may be a molecular target of OM in reducing pancreatic fibrosis.