人参归脾丸对脾不统血证大鼠肝损伤的保护作用及其机制*

目的:观察人参归脾丸对脾不统血证大鼠的治疗作用及其对肝脏的影响。方法:40只SPF级雄性SD大鼠随机分为正常对照组(n=10)和实验组(n=30),前42 d,实验组每日游泳30 min,同时饮食一天禁食两天(饮食失节),第43～72日,在游泳力竭加饮食失节的基础上,每日皮下注射低分子肝素钙诱导出血,构建脾不统血证大鼠模型;将成功建立模型的30只大鼠随机分为3组(n=10):模型对照组(MD)、自然复健组(NR)、归脾丸组(GP)。第73～102日,模型组继续施加处理因素(游泳力竭、饮食失节及注射低分子肝素钙溶液),自然复健组和归脾丸组停止施加处理因素,归脾丸组每日灌胃人参归脾丸溶液(0.2 g/ml ,10 ml/(kg·d)),自然复健组灌胃等量生理盐水;第103日后,取血和肝脏,检测血清中各组大鼠丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、总胆红素(TBil)、总蛋白(TP)水平,检测血中红细胞(RBC)数量、血红蛋白(HGB)含量和红细胞压积(HCT),ELISA试剂盒检测大鼠血清中 IL-6和TNF-α 含量,Western blot检测各组大鼠肝组织细胞凋亡相关蛋白Bcl-2、Bax、Caspase3蛋白水平,荧光定量PCR法检测大鼠肝组织细胞凋亡相关蛋白Bcl-2、Bax、Caspase3 mRNA表达水平。结果:与正常对照组相比,模型组大鼠血清ALT、AST和TBil水平均显著升高,TP水平明显降低(P＜ 0.01),血RBC数量、HGB含量和HCT均明显降低(P＜0.01),血清IL-6和TNF-α含量均显著升高(P＜0.05),肝组织Bcl-2 蛋白及mRNA水平均明显降低、Bax和Caspase3 蛋白及mRNA水平均显著升高(P＜0.01);与模型组相比,自然复健组和归脾丸组大鼠血清ALT、AST和TBil水平均明显降低,TP水平显著升高(P＜0.01),血RBC数量、HGB含量和HCT均明显升高(P＜0.01),血清IL-6和TNF-α含量均明显降低(P＜0.05),Bcl-2 蛋白及mRNA水平升高,Bax和Caspase3 蛋白及mRNA 水平降低(P＜0.05);与自然复健组相比,归脾丸组血清ALT、AST和TBil均明显降低,TP含量明显升高(P＜0.01),血RBC数量明显升高(P＜0.05),肝脏Bcl-2蛋白和mRNA水平显著升高(P＜ 0.05),Caspase3蛋白水平明显降低(P＜0.05),Bax mRNA表达显著降低(P＜0.05)。结论:人参归脾丸对脾不统血证大鼠肝损伤具有保护作用,其机制可能与其抑制肝脏细胞凋亡、促炎细胞因子释放有关。

Protective effect of Ginseng Guipi Pill on liver injury in rats with spleen failing to control blood syndrome and its mechanism

Objective: To investigate the therapeutic effect of Ginseng Guipi pill on rats with spleen failing to control blood syndrome and its effect on liver. Methods: Forty SPF male Sprague-Dawley rats were randomly divided into normal control group (n=10) and experimental group (n=30). For the first 42 days, the experimental group swam for 30 minutes every day, eating for one day and fasting for two days (diet disorder). On the 43rd to 72nd day, the rats were injected with low-molecular-weight heparin calcium to induce hemorrhage on the basis of exhaustion of swimming and diet disorder to construct a rat model of spleen failing to control blood syndrome. Those thirty rat that successfully established the model were randomly divided into 3 groups (n=10), model control group (MD), natural rehabilitation group (NR) and Guipi Pill group (GP). On the 73rd to 103rd day, the MD group continued to apply factors (exhausted swimming, eating disorder, and injection of low-molecular-weight heparin calcium), the NR group and the GP group stopped applying the factors , the GP group was daily administered with Ginseng Guipi Pill solution (0.2 g/ml, 10 ml/(kg·d)) , and the NR group was given an equal dose of saline. After the 103 days, blood and liver samples were collected, and the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil) and total protein (TP) content were detected, the number of red blood cells (RBC), hemoglobin (HGB) content and hematocrit (HCT) were detected, the expressions of IL-6 and TNF-α in serum were determined by ELISA kit, the levels of apoptosis-related proteins Bcl-2, Bax and Caspase3 in liver tissue were detected by Western blot. The expression levels of apoptosis-related proteins Bcl-2, Bax and Caspase3 mRNA in liver tissue were detected by real-time PCR. Results: Compared with the control group, the serum levels of ALT, AST and TBil in the model group were increased significantly, while the serum level of TP was reduced significantly (P＜0.01), the number of RBC, HGB content and HCT were decreased significantly (P＜0.01), the expressions of IL-6 and TNF-α in serum were increased (P＜0.05), the Bcl-2 protein and mRNA levels were reduced, Bax and Caspase3 protein and mRNA levels were increased significantly (P＜0.01); Compared with the model group, the serum levels of ALT, AST and TBil were significantly lower in the natural rehabilitation group and the Guipi Pill group, the level of TP was increased significantly (P＜0.01), the blood RBC, HGB and HCT were increased significantly (P＜ 0.01), the expressions of IL-6 and TNF-α were decreased (P＜0.05), the levels of Bcl-2 protein and mRNA were increased significantly, the Bax, Caspase3 protein and mRNA levels were decreased (P＜0.05); Compared with the natural rehabilitation group, the serum levels of ALT, AST and TBil were reduced , and the TP content was increased significantly in the Guipi Pill group (P＜ 0.01), the number of RBC was increased significantly in the Guipi Pill group (P＜0.05), the levels of Bcl-2 protein and mRNA were increased (P＜0.05),the level of Caspase3 protein was decreased (P＜0.05), the expression of Bax mRNA was reduced significantly (P＜0.05). Conclusion: Ginseng Guipi Pill has protective effect on liver injury in rats with spleen failing to control blood syndrome. The mechanism may be related to the inhibition the liver cell apoptosis and the release of pro-inflammatory cytokines.