Rock2负性调控pVHL稳定HIF-1α表达在脓毒症致肝损伤的作用及机制研究

Sepsis is characterized by a severe inflammatory response to infection, and its complications, including acute liver injury, can be fatal. Rho-associated kinases Rock2 is a key regulator of actin cytoskeleton dynamics downstream of Rho GTPases that participate in the control of inflammation. HIF-1α has emerged to play central roles in response to several microenvironmental stresses in addition to hypoxia, including exposure to infectious pathogens, inflammatory cytokines. But the role of Rock2 and HIF-1α in sepsis-induced hepatic injure has not been fully elucidated. Our previous results found that both Rock2 and HIF-1α were significantly elevated , and the expression of Rock2 was positively correlated with HIF-1α expression in sepsis-induced hepatic injure. Knockdown of Rock2 could decrease the HIF-1α expression level, increase the pVHL expression and inhibit both the production of inflammatory mediators and the apoptosis of hepatocyte. Therefore, we speculated that Rock2 damage in sepsis-induced hepatic injure by regulating HIF-1α dependent pVHL. To test this hypothesis and investigate the mechanism of this action, we will collection clinical significance of Rock2 and HIF-1α expression in sepsis patients, and observe the effect of Rock2 regulating HIF-1α expression dependent pVHL by gene transfection and RNA interference technology. Then, we will further clarify the mechanism through Rock2 regulates HIF-1α dependent pVHL by coimmunoprecipitation and confocal laser scanning microscope. This study will provide the new theoretical for clarifying the mechanism of sepsis-induced hepatic injure.

脓毒症引发失控性炎症反应可致肝损伤，合并肝功能异常的脓毒症患者预后差。Rho相关激酶Rock2与缺氧诱导因子HIF-1α均可促进炎症反应，但二者在脓毒症致肝损伤中相互关系及促炎机制不明。我们前期发现脂多糖致脓毒症肝脏中Rock2和HIF-1α均过表达。干扰Rock2可致HIF-1α降解E3连接酶pVHL表达增高，HIF-1α蛋白表达下调，细胞凋亡受抑。据此推测Rock2通过负性调控pVHL抑制HIF-1α泛素化降解，从而在脓毒症致肝损伤中发挥作用。本课题拟收集脓毒症患者标本，确定Rock2和HIF-1α表达相关性及相关作用，采用点突变、免疫共沉淀、激光共聚焦等方法研究Rock2负性调控pVHL稳定HIF-1α表达的途径，并利用小鼠脓毒症模型进一步验证Rock2调控HIF-1α影响肝损伤的生物学特征。本研究有望为脓毒症治疗提供新的理论依据。

脓毒症发病率高，病情凶险且治疗昂贵，是重症监护病房病人死亡的第一大原因，总体死亡率大约为30%~50%，伴随有基础疾病的患者死亡率更是超过70%。脓毒症发病机制未明，革兰阴性菌具有脂多糖(lipopolysaccharide，LPS)结构，LPS是脓毒症最重要的启动因子，对肝脏有直接损害作用。肝脏既是清除内毒素的场所，又是炎症攻击最为严重的器官之一，肝细胞功能丧失可诱导多器官功能障碍的发生。减轻肝损伤，恢复肝功能可明显降低脓毒症的致死率，但迄今为止，脓毒症致肝损伤机制尚未得到明确阐述，亦无有效防治措施。寻找切实有效的保护策略改善脓毒症致肝损伤，对降低患者病死率，减少住院费用至关重要。通过我们的研究成功建立Rock2基因敲除小鼠，并在基因敲除鼠上成功构建脓毒症动物模型，发现脓毒症肝脏肝功能和炎症因子均有明显升高，Rock2、HIF-1α在脓毒症肝脏中也明显的高表达，在Rock2敲除鼠中发现肝损伤得到明显改善。在细胞实验中，我们也发现沉默或者过表达Rock2表达，LPS诱导肝窦内皮细胞会呈现程度明显不同的炎症反应。沉默或者过表达Rock2表达也影响了HIF-1α的表达，而且二者存在明显正相关关系。在沉默或过表达Rock2的细胞中回复或增加HIF-1α，发现炎症因子表达出现相应的改变。加入泛素-蛋白体酶抑制剂MG132和放线菌酮CHX，可以明确Rock２通过影响HIF-1α的泛素化降解干扰HIF-1α表达。深入研究发现Rock2是通过负性调控pVHL抑制HIF-1α泛素化降解。通过本课题明确了Rock2通过影响HIF-1α在脓毒症对肝损害的作用；阐明了Rock2通过负调控pVHL稳定HIF-1α表达的分子机制。本课题研究完善Rock2蛋白的功能，为患者提供一种很有前景的治疗策略。

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