松果菊苷调控SIRT1/STAT3信号通路改善CLP大鼠肝损伤及糖代谢紊乱的研究

Echinacoside regulates SIRT1/STAT3 signaling pathway to improve liver injury and glucose metabolism disorder in CLP rats

目的观察松果菊苷(ECH)对盲肠结扎穿刺(CLP)所致脓毒症大鼠肝损伤及糖代谢紊乱的治疗作用,并探讨其可能机制。方法48只雄性SD大鼠随机分为4组,分别为假手术组(Sham)、模型组(CLP)、治疗组(CLP+ECH)及抑制剂组(CLP+ECH+EX527)。假手术组,仅接受剖腹手术;模型组,进行盲肠结扎和穿刺;治疗组,CLP建模后每天灌胃松果菊苷(30 mg/kg),持续7 d;抑制剂组,CLP前1 h注射SIRT1抑制剂EX527(5 mg/kg),后同治疗组。检测各组大鼠空腹血糖、胰岛素及血清生化指标水平;ELISA法检测血清IL-1β、IL-6和TNF-α水平;DCFH-DA染色观察各组大鼠肝脏组织中活性氧(ROS)生成情况;HE染色观察各组大鼠肝组织病理改变;Western blot检测各组大鼠肝组织中沉默信息调节因子1(SIRT1)、葡萄糖6-磷酸酶(G6Pase)、磷酸烯醇式丙酮酸羧化激酶(PEPCK)、磷酸化信号转导和转录激活因子3(p-STAT3)和磷酸化蛋白激酶B(p-AKT)表达。结果与假手术组比较,模型组大鼠血糖浓度、血清胰岛素、ALT、AST、ROS、IL-1β、IL-6、TNF-α水平上升,而肝糖原、生存率下降(均P<0.05)。经松果菊苷治疗后,CLP大鼠血糖浓度、血清胰岛素、ALT、AST、ROS、IL-1β、IL-6、TNF-α水平下降,肝糖原、生存率上升(均P<0.05);SIRT1抑制剂干预后,抑制剂组的血清胰岛素、ALT、AST、IL-6、ROS水平升高(P<0.05)。HE染色发现,模型组大鼠肝脏组织存在炎症细胞的浸润及肝细胞坏死,而松果菊苷可减轻局灶性和块状坏死;Western blot检测发现:与假手术组比较,模型组大鼠肝组织中SIRT1、p-STAT3和p-AKT蛋白表达水平下降,而G6Pase、PEPCK蛋白表达水平升高(P<0.05),而松果菊苷治疗后SIRT1、p-STAT3和p-AKT蛋白表达水平升高,G6Pase、PEPCK蛋白表达水平下降(P<0.05)。SIRT1抑制剂干预后,抑制剂组SIRT1、p-STAT3、p-AKT蛋白表达降低,G6Pase、PEPCK蛋白表达升高(P<0.05)。结论松果菊苷是脓毒症相关肝损伤及糖代谢紊乱的潜在治疗剂,其机制可能通过SIRT1介导激活肝脏中STAT3、AKT而起作用。

Objective To observe the therapeutic effect of echinacoside(ECH)on liver injury and glucose metabolism disorder in sepsis rats induced by cecal ligation and puncture(CLP),and to explore its possible mechanism.Methods Forty eight male Sprague Dawley(SD)rats were randomly divided into four groups:sham group(sham),model group(CLP),treatment group(CLP+ECH)and inhibitor group(CLP+ECH+EX527).The sham group only received laparotomy,and the model group underwent CLP.The treatment group was intragastric administration of echinacea(30 mg/kg)every day after CLP modeling.The inhibitor group was injected with silence information regulator 1(SIRT1)inhibitor EX527(5 mg/kg)one hour before CLP,and then treated the same as the treatment group.Fasting blood glucose,insulin and serum biochemical indexes were detected in virous groups.The serum levels of interleukin(IL)-1β,IL-6 and tumor necrosis factor-α(TNF-α)were detected by enzyme-linked immunosorbent assay(ELISA).2′,7′-dichlorofluorescein diacetate(DCFH-DA)staining was used to observe the production of reactive oxygen species(ROS)in liver tissue of rats in each group;hematoxylin-eosin(HE)staining was used to observe the pathological changes of liver tissue in each group;The expressions of SIRT1,glucose-6-phosphatase(G6Pase),phosphoenolpyruvate carboxykinase(PEPCK),phosphorylated signal transducers and activators of transcription 3(p-STAT3)and phosphorylated protein ki-nase B(p-AKT)were detected by Western blot.Results Compared with sham group,the levels of serum glucose,serum insulin,alanine aminotransferase(ALT),aspartate aminotransferase(AST),ROS,IL-1β,IL-6 and TNF-αin model group increased,while the liver glycogen and survival rate decreased(all P<0.05).After echinacoside treatment,the serum glucose,serum insulin,ALT,AST,ROS,IL-1β,IL-6 and TNF-αlevels decreased,and the liver glycogen and survival rate increased(all P<0.05);After SIRT1 inhibitor intervention,the levels of serum insulin,ALT,AST,IL-6 and ROS in the inhibitor group increased(P<0.05).HE staining showed that there were infiltration and necrosis of inflammatory cells in the liver tissue of model group,and echinacoside could significantly reduce the focal and massive necrosis;Western blot showed that compared with the sham group,the expression levels of SIRT1,p-STAT3 and p-AKT protein in the model group decreased,while the expression levels of G6Pase and PEPCK protein increased(P<0.05);After echinacoside treatment,the expression levels of SIRT1,p-STAT3 and p-AKT increased,while the expression levels of G6Pase and PEPCK decreased(P<0.05).After SIRT1 inhibitor intervention,the expression of SIRT1,p-STAT3 and p-AKT protein decreased,and the expression of G6Pase and PEPCK protein increased in the inhibitor group(P<0.05).Conclusions Echinacoside is a potential therapeutic agent for sepsis associated liver injury and glucose metabolism disorders,which may play a role by targeting SIRT1 to activate STAT3 and AKT in the liver.