EMMPRIN的巯基亚硝基化在肝纤维化中调控基质金属蛋白酶表达水平的机制研究

EMMPRIN promotes the remodeling of extracellular matrix (ECM) by inducing extracellular metalloproteinases (MMPs) in liver fibrosis, but this mechanism is not clear. Our previous studies have shown that EMMPRIN is expressed on the surface of hepatocytes and liver endothelial cells (LSEC), and its s-nitrosylation sites are mutated to affect the expression of MMP-2 in hepatic stellate cells (HSCs). It was concluded that s-nitrosylated EMMPRIN was a key factor in the regulation of MMPs expression in liver fibrosis and may be regulated by nitric oxide synthase (NOSs). We first determine whether s-nitrosylated EMMPRIN exists during liver fibrosis by establishing the rat liver cirrhosis model. Then, we explore if EMMPRIN will be s-nitrosylated under the regulation of NOSs in hepatocytes and LSECs and as a result, the MMPs expression level of HSCs will be regulated by s-nitrosylated EMMPRIN. Finally, we observed the mechanism how exogenous s-nitrosylated EMMPRIN regulate MMPs in HSCs by NOSs. This new mechanism will further clarify the role of EMMPRIN in ECM remodeling in liver fibrosis.

研究证实EMMPRIN 在肝纤维化进程通过诱导细胞外金属蛋白酶（MMPs），促进细胞外基质（ECM）的重构。我们前期发现EMMPRIN在肝细胞和肝内皮细胞（LSEC）膜表面表达，将其巯基亚硝基化位点突变，影响肝星状细胞（HSCs）中MMP-2的表达。我们据此提出EMMPRIN蛋白的巯基亚硝基化是肝纤维化中调控MMPs表达的关键因素。我们采用建立肝纤维化模型体内观察EMMPRIN的巯基亚硝基化改变和分离原代肝脏细胞体外进行功能研究的方法，进行EMMPRIN蛋白的巯基亚硝基化改变和MMPs表达之间联系以及与一氧化碳合成酶（NOS）调控关系的研究，揭示NOS-EMMPRIN-MMPs之间相互调控的关系，阐明巯基亚硝基化的EMMPRIN参与肝纤维化ECM重构的机制，为以后肝纤维化的干预提供新的靶点。

背景与目的:研究EMMPRIN 巯基亚硝基化对肝纤维化过程中基质金属蛋白酶调节的作用。方法:建立CCl4诱导大鼠肝纤维化模型。我们检测并比较了正常和纤维化肝脏中EMMPRIN表达水平和巯基亚硝基化水平。我们随后在体外观察EMMPRIN的巯基亚硝基化水平变化。为此，我们设计合成了EMMPRIN巯基亚硝基化位点突变质粒和野生型质粒，并将其转染细胞。我们在检测了细胞中的巯基亚硝基化水平、迁移和MMPs活性后，根据EMMPRIN 巯基亚硝基化与MMPs活性的关系，论证了EMMPRIN 巯基亚硝基化在肝纤维化中的潜在作用。结果:CCl4组EMMPRIN mRNA和蛋白水平低于对照组，而免疫组化和免疫荧光染色结果显示CCl4组EMMPRIN 巯基亚硝基化水平高于对照组。原发性EMMPRIN不是来自静止的造血干细胞，而是来自附近的肝细胞和LSECs。将合成的质粒转染293细胞，收集上清液，将上清液转移到LX2细胞，发现EMMPRIN为分泌蛋白，可被肝星状细胞吸收。在肝星状细胞中摄取含有巯基亚硝基化位点突变(阻断巯基亚硝基化)的EMMPRIN使MMP2和MMP9活性减弱，降低细胞的迁移，提示巯基亚硝基化的EMMPRIN通过下调MMPs活性降低了肝星状细胞的迁移。结论:EMMPRIN可在肝纤维化过程中通过巯基亚硝基化使MMPs活性下降。这可能是MMPs在ECM沉积的可能调控机制之一