葛根素对急性肝衰竭小鼠脾脏淋巴细胞亚型和肝脏炎症反应的影响

Effects of puerarin on splenic lymphocyte subtypes and hepatic inflammation in mice with acute liver failure

目的探究葛根素对急性肝衰竭(ALF)小鼠脾脏淋巴细胞亚型和肝脏炎症反应的影响。方法60只8~10周龄C57/BL6雄性小鼠按照随机数字表法分为对照组(正常小鼠,腹腔注射等量的0.9%氯化钠溶液),ALF组(ALF模型小鼠,腹腔注射等量的0.9%氯化钠溶液),葛根素治疗组(ALF模型小鼠,腹腔注射100 mg·kg^(-1)·d^(-1)剂量的葛根素),每组各20只,持续治疗10 d。脾脏和胸腺称重计算小鼠的脾脏和胸腺指数;通过稀释后的小鼠血清的吸光度值计算血清半数溶血值(HC50);MTT测定小鼠外周血中T淋巴细胞和B淋巴细胞的增殖;酶联免疫吸附试验(ELISA)测定小鼠血清细胞因子[γ干扰素(IFN-γ)、肿瘤坏死因子α(TNF-α)、白细胞介素(IL)-4、IL-17A和IL-10]水平;全自动Aeroset化学分析仪评估小鼠血清丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)和总胆红素(TBil)水平;蛋白质印迹法检测小鼠肝组织信号传导子和转录活化子1(STAT1)、STAT3和半胱氨酸蛋白酶-3(caspase-3)的蛋白表达。结果ALF组小鼠的脾脏指数和胸腺指数较对照组降低,差异均有统计学意义(P<0.05);葛根素治疗组脾脏指数和胸腺指数较ALF组升高,差异均有统计学意义(P<0.05)。ALF组小鼠HC50较对照组降低,差异有统计学意义(P<0.05);葛根素治疗组小鼠HC50较ALF组升高,差异有统计学意义(P<0.05)。ALF组T淋巴细胞和B淋巴细胞增殖较对照组降低,差异均有统计学意义(P<0.05);葛根素治疗组T淋巴细胞和B淋巴细胞增殖较ALF组升高,差异均有统计学意义(P<0.05)。ALF组小鼠血清IFN-γ、TNF-α、IL-4、IL-17A水平较对照组均升高,血清IL-10水平较对照组降低,差异均有统计学意义(P<0.05);葛根素治疗组血清IFN-γ、TNF-α、IL-4、IL-17A水平较ALF组均降低,IL-10水平较ALF组升高,差异均有统计学意义(P<0.05)。ALF组小鼠血清ALT、AST和TBil水平较对照组均升高,差异均有统计学意义(P<0.05);葛根素治疗组血清ALT、AST和TBil水平较ALF组均降低,差异均有统计学意义(P<0.05)。ALF组STAT1和caspase-3蛋白表达较对照组均升高,STAT3蛋白表达较对照组降低,差异均有统计学意义(P<0.05);葛根素治疗组STAT1和caspase-3蛋白表达较ALF组均降低,STAT3蛋白表达较ALF组升高,差异均有统计学意义(P<0.05)。结论葛根素可抑制ALF小鼠脾脏淋巴细胞亚型的促炎性免疫反应、减少肝细胞凋亡,并有助于重建STAT1和STAT3信号之间的平衡,从而对ALF的肝损伤发挥保护作用。

Objective To explore the effect of puerarin on splenic lymphocyte subtypes and liver inflammation in mice with acute liver failure.Methods Sixty 8-10 weeks old C57/BL6 male mice were divided into control group(normal mice,intraperitoneal injection of the same amount of normal saline),ALF group(ALF model mice,intraperitoneal injection of the same amount of normal saline)and puerarin treatment group(ALF model mice,intraperitoneal injection of puerarin at a dose of 100 mg·kg^(-1)·d^(-1))according to random number table method,20 mice in each group,continued treatment for 10 days,the spleen and thymus index of the mice were calculated by weighing the spleen and thymus.HC50 was calculated from the absorbance value of the diluted mouse serum.The proliferation of T lymphocytes and B lymphocytes in the peripheral blood of mice was measured by MTT.The serum cytokine[gamma interferon(IFN-γ),tumor necrosis factor alpha(TNF-α),interleukin(IL)-4,and IL-17A]levels of mice were determined by enzyme-linked immunosorbent assay(ELISA).The mouse serum alanine aminotransferase(ALT),aspartate aminotransferase(AST)and total bilirubin(TBil)levels were evaluated by the fully automated Aeroset chemical analyzer.The protein expression of signal transducers and transcription activators 1(STAT1),STAT3 and caspase-3 in mouse liver tissue was detected by Western blotting.Results The spleen index and thymus index of the ALF group were lower than those of the control group,the differences were statistically significant(P<0.05),and the spleen index and thymus index of the puerarin-treated group were higher than those of the ALF group,the differences were statistically significant(P<0.05).The HC50 of mice in the ALF group was lower than that in the control group,the difference was statistically significant(P<0.05),and the HC50 of the mice in the puerarin treatment group was higher than that in the ALF group,the difference was statistically significant(P<0.05).The proliferation of T and B lymphocytes in the ALF group were lower than that in the control group,the difference were statistically significant(P<0.05),and the proliferation of T and B lymphocytes in the puerarin treatment group were higher than that in the ALF group,the difference were statistically significant(P<0.05).The levels of serum IFN-γ,TNF-α,IL-4 and IL-17A in the ALF group were higher than those in the control group,the level of serum IL-10 was lower than that of the control group,the differences were statistically significant(P<0.05),and the levels of serum IFN-γ,TNF-α,IL-4 and IL-17A in the puerarin-treated group were lower than those in the ALF group,the level of serum IL-10 was higher than that in the ALF group,the differences were statistically significant(P<0.05).The serum ALT,AST and TBil levels of mice in the ALF group were higher than those in the control group,the differences were statistically significant(P<0.05),and the serum ALT,AST and TBil levels in the puerarin treatment group were lower than those in the ALF group,the differences were statistically significant(P<0.05).The expression of STAT1 and caspase-3 protein in the ALF group were higher than those in the control group,and the STAT3 protein expression in the ALF group was lower than that in the control group(P<0.05),the expression of STAT1 and caspase-3 protein in the puerarin treatment group were lower than those in the ALF group,the expression of STAT3 protein in the puerarin treatment group was higher than that in the ALF group,the differences were statistically significant(P<0.05).Conclusion Puerarin can inhibit pro-inflammatory immune responses of spleen lymphocyte subtypes in ALF mice,reduces hepatocyte apoptosis,and helps to restore the balance between STAT1 and STAT3 signaling,thereby exerting a protective effect on liver injury in ALF.