赤芍-附片对慢加急性肝衰竭大鼠PI3K/AKT信号通路及相关炎症因子表达的影响

Effects of Chishao(Radix Paeoniae Rubra)and Fupian(Radix Aconiti Lateralis)on PI3K/AKT Signaling Pathway and Expression of Related Inflammatory Factors in Rats with Chronic Acute Liver Failure

目的:探讨赤芍-附片对慢加急性肝衰竭(ACLF)大鼠的保护作用及其对磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(AKT)信号通路的影响。方法:48只SD大鼠随机分为空白组(6只)和ACLF造模组(42只)。ACLF造模组大鼠用牛血清白蛋白皮下及尾静脉注射联合腹腔注射D-氨基半乳糖+脂多糖制备ACLF大鼠模型,将造模大鼠随机分为模型组、阳性组、赤芍组、附片组、赤芍-附片组,各组大鼠予相应药物干预1周。检测肝功能、凝血酶原时间(PT),ELISA法检测肝组织白介素-1β(IL-1β)、白介素-6(IL-6)、肿瘤坏死因子-α(TNF-α),HE染色观察大鼠肝组织病理改变,Western blotting法检测肝组织PI3K、p-PI3K、AKT、p-AKT(Ser473)蛋白表达水平,实时荧光定量PCR技术检测PI3K mRNA、AKT mRNA表达水平。结果:与空白组比较,模型组大鼠肝组织出现肝细胞形态变化,呈融合性坏死、点状或灶状坏死,纤维组织增生,汇管区见大量炎症细胞浸润;与模型组比较,阳性组、赤芍组、附片组、赤芍-附片组大鼠肝组织肝细胞排列相对整齐,坏死程度改善,纤维组织减少,炎症细胞浸润减少。模型组大鼠血清ALT、AST、TBIL、TBA、PT水平及肝组织IL-1β、IL-6、TNF-α水平均高于空白组(P<0.05),肝组织中p-PI3K、p-AKT蛋白相对表达量及PI3K mRNA、AKT mRNA相对表达量均低于空白组(P<0.05)。阳性组、赤芍组、附片组、赤芍-附片组大鼠血清ALT、AST、TBIL、TBA、PT水平及肝组织IL-1β、IL-6、TNF-α水平均低于模型组(P<0.05),肝组织p-PI3K、p-AKT蛋白相对表达量及PI3K mRNA、AKT mRNA相对表达量均高于模型组(P<0.05);且赤芍-附片组大鼠血清ALT、AST、TBIL、TBA、PT水平及肝组织IL-1β、IL-6、TNF-α水平均低于阳性组、赤芍组、附片组(P<0.05),肝组织p-PI3K、p-AKT蛋白相对表达量及PI3K mRNA、AKT mRNA相对表达量均高于阳性组、赤芍组、附片组(P<0.05);各组大鼠PI3K、AKT蛋白相对表达量比较,差异均无统计学意义(P>0.05)。结论:赤芍-附片可能通过调节PI3K/AKT信号通路,下调相关炎症因子的表达,以减轻ACLF大鼠免疫炎症反应,从而发挥对肝脏的保护作用。

Objective:To investigate the protective effect of Chishao(Radix Paeoniae Rubra)and Fupian(Radix Aconiti Lateralis)on chronic acute liver failure(ACLF)rats and its effect on phosphatidylinositol 3 kinase(PI3K)/protein kinase B(AKT)signaling pathway.Methods:Among 48 SD rats,6 were randomly selected as blank group,and the other rats were treated with subcutaneous and tail vein injection of bovine serum albumin combined with intraperitoneal injection of D-galactose aminoglyaccharide+lipopolysaccharide to prepare ACLF rat model.The rats successfully constructed were randomly divided into model group,positive control group,Chishao group,Fupian group,and Chishao-Fupian group.Each group was given corresponding drug intervention for a week.Liver function and prothrombin time(PT)were detected.Interleukin-1β(IL-1β),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)were detected by ELISA,and rat liver tissues of model group were observed by HE staining.The protein expression levels of PI3K,p-PI3K,AKT,p-AKT(Ser473)in liver tissue were detected by Western blotting.Real-time fluorescence quantitative PCR was used to detect the expression levels of PI3K mRNA and AKT mRNA.Results:Compared with the blank group,the liver cells in the model group showed morphological changes,such as fusion necrosis,punctate or focal necrosis,fibrous tissue hyperplasia,and a large number of inflammatory cells infiltration in the portal area.Compared with model group,liver cells in positive control group,Chishao group,Fupian group and Chishao-Fupian group were arranged relatively neatly,and necrosis degree was improved.Fibrous tissue was reduced and inflammatory cell infiltration was reduced.The levels of ALT,AST,TBIL,TBA and PT in serum and IL-1β,IL-6 and TNF-αin liver tissue in model group were higher than those in blank group(P<0.05).The relative expression levels of p-PI3K and p-AKT protein and the relative expression levels of PI3K mRNA and AKT mRNA in liver tissues of Chishao Fupian group model group were lower than those in blank group(P<0.05).The levels of ALT,AST,TBIL,TBA and PT in serum and the levels of IL-1β,IL-6 and TNF-αin liver tissue of rats in positive control group,Chishao group,Fupian group and Chishao-Fupian group were lower than those in model group(P<0.05).The relative expression levels of p-PI3K and p-AKT protein and PI3KmRNA and AKT mRNA in liver tissues of positive control group,Chishao group,Fupian group and Chishao Fupian group.were higher than those in model group(P<0.05).The levels of ALT,AST,TBIL,TBA and PT in serum and IL-1β,IL-6 and TNF-αin liver tissue of rats in Chishao-Fupian group were lower than those in positive control group,Chishao group and Fupian group(P<0.05).The relative expression levels of p-PI3K and p-AKT protein and the relative expression levels of PI3K mRNA and AKT mRNA in liver tissue of Chishao-Fupian group were higher than those in positive control group,Chishao group and Fupian group(P<0.05).There was no significant difference in the relative expression levels of PI3K and AKT among all groups(P>0.05).Conclusion:Chishao(Radix Paeoniae Rubra)and Fupian(Radix Aconiti Lateralis)may regulate the expression of related inflammatory factors by regulating the PI3K/AKT signaling pathway to reduce the immune inflammatory response in rats with chronic acute liver failure,thereby exerting a protective effect on the liver.