RIP3相关蛋白抑制肝细胞necroptosis防治急性肝衰竭的分子机制及三黄茵赤汤的干预作用

Acute liver failure (ALF) is a severe disease with high mortality. Previous clinical and experimental studies preliminarily confirmed SHYCT with function of prevention and treatment of ALF, meanwhile, found hepatocytes apoptosis and programmed necrosis (necroptosis) pathological features, and significantly higher protein RIP3. Therefore, we surmise that ALF happened necroptosis of RIP3 dependent. Based on the above assumptions, We established Acute liver failure(ALF) animal model by RIP3 knockout mice and normal mice, and cell model by CRISPR / Cas9 system overexpression and silence of RIP3 gene in normal liver cell line. From animal cells on two levels to verify role RIP3 and network proteins in liver cell necroptosis with both positive and negative angles. Using Luminex, Immunofluorescence, qPCR,etc. techniques to analyze the expression of Caspase8- RIP3-MLKL pathway network protein and mRNA. Reveal SHYCT decoction regulate RIP3 network protein inhibits liver cells necroptosis molecular mechanisms to find new targets for prevention and treatment of ALF. At the same time, help to further interpretation of that decoction multi-way, multi-target prevention and treatment of ALF, the scientific connotation, provide sufficient theoretical basis for clinical application.

急性肝衰竭是病死率高的一种危重疾病。课题组前期研究初步证实三黄茵赤汤具有防治急性肝衰竭的作用，同时发现肝细胞具有凋亡和程序性坏死（necroptosis）的病理特征，且RIP3显著升高。因此，我们推测急性肝衰竭发生了RIP3依赖的necroptosis。基于上述假设，应用正常和RIP3基因敲除小鼠建立急性肝衰竭动物模型，通过CRISPR/Cas9系统技术过表达和沉默RIP3基因建立细胞模型。从动物、细胞两个层面正反两个角度验证RIP3及相关蛋白在肝细胞necroptosis中的作用。采用液相芯片、免疫荧光和qPCR等技术，分析Caspase8- RIP3-MLKL通路相关蛋白及mRNA的表达。阐明三黄茵赤汤调节RIP3相关蛋白抑制肝细胞necroptosis的分子机制，寻找防治急性肝衰竭的新靶点。同时，有助于进一步诠释该方多途径、多靶点防治急性肝衰竭的科学内涵，为临床应用提供理论依据。

目的：研究RIP3相关蛋白抑制肝细胞necroptosis防治急性肝衰竭的分子机制及三黄茵赤汤的干预作用。方法：正常和RIP3基因敲除小鼠通过D-氨基半乳糖联合LPS建立急性肝衰竭动物模型，并给予三黄茵赤汤干预；正常和RIP3基因沉默LO2肝细胞通过D-氨基半乳糖建立急性肝细胞损伤模型，并给予三黄茵赤汤粉末干预。检测小鼠肝功能和凝血功能；HE染色和电镜分析肝细胞结构和亚细胞结构的变化；免疫印迹、免疫组化和qPCR等技术分析程序性坏死通路相关蛋白及mRNA的表达。结果：与模型组比较，三黄茵赤汤高、中、低剂量减少血清ALT, AST的表达水平，缩短INR 时间，提高小鼠是生存率，改善肝脏病理损伤。三黄茵赤汤高、中、低剂量组均能减少HMGB1、TLR4、Caspase3、Caspase-1、NLRP3、RIP3、RIP1、MLKL的表达，升高Bcl-2/Bax的比值。结论：三黄茵赤汤高、中、低剂量具有防治急性肝衰竭的作用，并成剂量依赖。其可能机制是通过下调程序性关键蛋白RIP3并调控程序性相关蛋白，抑制肝细胞坏死，从而防治急性肝衰竭。