基于HGF/PI3K/Akt信号轴研究赤芍-附子药对促进慢加急性肝衰竭肝细胞再生的作用机制

Mechanism of Paeoniae Radix Rubra and Aconiti Lateralis Radix Praeparata herbal pair in promoting hepatocellular regeneration in chronic acute liver failure based on HGF/PI3K/Akt signaling axis

基于肝细胞生长因子(hepatocyte growth factor,HGF)/磷脂酰肌醇3-激酶(phosphoinositide 3-kinase,PI3K)/蛋白激酶B(protein kinase B,Akt)信号轴研究赤芍-附子药对对慢加急性肝衰竭(acute-on-chronic liver failure,ACLF)大鼠的治疗作用及对肝细胞再生的影响。采用牛血清白蛋白皮下及尾静脉注射联合脂多糖(lipopolysaccharides,LPS)+D-氨基半乳糖(D-galactosamine,D-GalN)腹腔注射构建ACLF大鼠模型,实验设置正常对照(NC)组、模型(vehicle)组、赤芍-附子药对(SFYD,5.85 g·kg^(-1))组、促肝细胞生长颗粒(HGFG,4.05 g·kg^(-1))组。苏木素-伊红(hematoxylin-eosin staining,HE)染色观察大鼠肝组织病理变化。全自动生化分析仪检测血清谷丙转氨酶(alanineamino transferase,ALT)、谷草转氨酶(aspartate transa-minase,AST)、总胆红素(total bilirubin,TBIL)。ELISA法检测白细胞介素-1β(interleukin-1β,IL-1β)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)炎症因子水平。免疫荧光染色检测增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、细胞增殖相关抗原(antigen identified by monoclonal antibody,Ki67)及细胞周期蛋白(cell cycle protein B1,CyclinB1)阳性表达。实时荧光定量聚合酶链式反应(RT-qPCR)法、蛋白免疫印迹(Western blot)法检测HGF、结合蛋白Gab1(grb2 associated binding protein 1,Gab1)、PI3K、Akt、磷酸化PI3K(phosphorylation PI3K,p-PI3K)、磷酸化Akt(phosphorylation Akt,p-Akt)mRNA及蛋白表达水平。结果显示,与vehicle组比较,SFYD组大鼠肝组织病理评分降低,肝功能及炎症反应改善,PCNA、Ki67、CyclinB1荧光表达增强。同时与模型组相比,SFYD组HGF、Gab1蛋白表达上调,PI3K、Akt磷酸化激活。以上研究表明赤芍-附子药对通过减轻肝细胞炎症损伤以及促进肝细胞再生来达到抗肝衰竭的效应,其具体调控机制可能与激活HGF/PI3K/Akt信号通路有关。

Based on the hepatocyte growth factor(HGF)/phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)signaling axis,this study investigated the therapeutic effect of Paeoniae Radix Rubra and Aconiti Lateralis Radix Praeparata(PRR-ALRP)her-bal pair on acute-on-chronic liver failure(ACLF)rats and its impact on hepatocellular regeneration.The rat model of ACLF was constructed by subcutaneous and tail vein injection of bovine serum albumin combined with intraperitoneal injection of lipopolysaccharides(LPS)+D-galactosamine(D-GalN).The rats were divided into normal control(NC)group,model(vehicle)group,PRR-ALRP(5.85 g·kg^(-1))group,and hepatocyte growth factor granules(HGFG,4.05 g·kg^(-1))group.Hematoxylin-eosin(HE)staining was used to observe pathological changes in rat liver tissues.Serum alanine aminotransferase(ALT),aspartate transaminase(AST),and total bilirubin(TBIL)were detected using an automatic biochemical analyzer.The levels of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)inflammatory factors were detected by ELISA.Immunofluorescence staining was used to detect the positive expression of proliferating cell nuclear antigen(PCNA),antigen identified by monoclonal antibody(Ki67),and cell cycle protein B1(CyclinB1).Real-time fluorescence-based quantitative polymerase chain reaction(RT-qPCR)and Western blot were used to detect the mRNA and protein expression levels of HGF,growth factor receptor-bound protein 1(Gab1),PI3K,Akt,phosphorylated PI3K(p-PI3K),and phosphorylated Akt(p-Akt).The results showed that compared with the vehicle group,the PRR-ALRP group had reduced liver tissue pathological scores,improved liver function,and reduced inflammatory response,with enhanced PCNA,Ki67,and CyclinB1 fluorescence expression.Furthermore,compared with the model group,the PRR-ALRP group showed upregulated expression of HGF and Gab1 proteins,as well as activation of PI3K and Akt phosphorylation.These findings suggest that PRR-ALRP herbal pair exerts anti-liver failure effects by alleviating hepatocyte inflammatory damage and promoting hepatocellular regeneration,and its specific regulatory mechanism may be related to the activation of the HGF/PI3K/Akt signaling pathway.