硫化氢对外伤诱导急性肝损伤的调节作用及其分子机制探讨

Hydrogen sulfide(H2S) is an endogenously produced gaseous signaling molecule synthesized mainly by the trans-sulfuration enzymes cystathionine γ-lyase(CSE) and cystathionine β- synthase(CBS).Liver is one of few organs in which both CSE and CBS are simultaneously distributed in mammals. However, the roles of H2S and the related-mechanisms in the pathogenesis of acute liver injury have been unclear as well as whether or not there is regulation between CSE and CBS in the hepatic cells is elusive. In the current study, the changes of CSE-H2S and CBS-H2S systems such as expressions of CSE and CBS mRNA, proteins and H2S levels would be detected with real-time PCR, Western-blot and biochemical measurements during the aute liver injury induced by traumatic stress and the hepatic cell injury from lippolysaccharides (LPS).Whether or not there is regulation between CSE and CBS in the hepatic cells has been evaluated. With the help of multiple techniques such as siRNA method,flow cytometry,CSE and CBS inhibitors,the regulation of endogenous and exogenous H2S for the oxidative injury and apoptosis of hepatic cells resulted injury from LPS and signal transduction mechanisms underlying effects of H2S such as ERK1/2,Nrf 2,NF-κB as well as PKC-p44/42-STAT3 would be elucidated due to the facts that oxidative injury and apoptosis are key pathway to acute liver injury.

硫化氢（hydrogen sulfide,H2S）是一种内源性气体信使分子，主要由胱硫醚-γ-裂解酶（CSE）、胱硫醚-β-合酶（CBS）催化生成。肝脏是机体同时存在CSE、CBS的少数器官之一。H2S在急性肝损伤的作用及其机制尚未阐明。本课题首先用Real-time PCR、Western blot、生化检测等技术探讨外伤诱导急性肝损伤和脂多糖诱导肝细胞损伤过程中CSE-H2S、CBS-H2S体系基因、蛋白和H2S生成的规律性变化，并检测在该病理过程中CSE- H2S、CBS- H2S体系之间可能存在的相互调节关系；其次针对氧化损伤和细胞凋亡是肝细胞损伤的关键发病环节，用siRNA技术、流式细胞术和CSE、CBS抑制剂等多种方法和技术着力探讨内外源性H2S对肝细胞氧化损伤和细胞凋亡的调节作用及其相关的信号分子如ERK1/2、Nrf 2、NF-κB、PKC-p44/42-STAT3。

创伤应激能引起肝组织CSE-H2S体系下调、肝组织和血液H2S生成减少以及急性肝损伤；抑制内源性H2S生成或给予外源性H2S供体能分别加剧或减轻创伤应激引起的急性肝损伤，肝损伤与抗氧化能力降低有关。 转染分别或同时引起H2S生成酶CSE和CBS基因沉默、H2S生成减少导致静息的大鼠肝细胞凋亡增多，机制与激活线粒体凋亡途径有关；CSE和CBS分别基因沉默实验结果显示静息的大鼠肝细胞中CSE和CBS之间存在相互代偿效应。LPS能诱导大鼠肝细胞CSE-H2S和CBS-H2S体系上调、H2S生成呈时间依赖性增多，但其效应迥然不同，H2S生成少量增多能抑制细胞凋亡、进一步增多对细胞凋亡无明显影响，明显增多则介导LPS致细胞凋亡。其中，H2S生成明显增多介导LPS致细胞凋亡是通过激活线粒体凋亡和内质网应激介导凋亡途径实现的，并受PKC-ERK1/2-STAT3通路调控。外源性H2S对LPS诱导的大鼠肝细胞ERS及其介导的细胞凋亡有不同的作用，即存在早期和晚期预处理效应但无后处理作用，也就是说, H2S预处理能减轻LPS诱导的大鼠肝细胞ERS但后给予则加剧细胞ERS介导的细胞凋亡。H2S减轻LPS诱导的大鼠肝细胞ERS的主要作用机制有：开放ATP敏感的钾通道、抗氧化效应、上调ERS下游UPR信号通路的IREα和ATF6以及抑制PARP1、上调ERAD。其中，H2S抗氧化效应的机制有协同性上调LPS诱导大鼠肝细胞关键抗氧化信号通路Nrf 2-HO-1/Trx-1，Nrf 2 siRNA、HO-1 siRNA和Trx-1siRNA均能分别明显翻转H2S减轻LPS诱导的大鼠肝细胞ERS；减少了LPS诱导大鼠肝细胞活性氧（ROS）含量，其中细胞质膜NOX和线粒体是LPS诱导大鼠肝细胞ROS的主要来源，然而H2S减少ROS效应所作用细胞质膜NOX和线粒体的关键靶分子有待进一步研究。研究还发现，H2S通过抑制PARP1表达、上调ERAD通路而减轻LPS诱导的大鼠肝细胞ERS；高剂量的外源性H2S本身具有细胞毒效应，能引起非ERS依赖性细胞死亡增多。