基于“气血辨证”理论探讨益气和血方对CCL4诱导的肝硬化大鼠的治疗作用及其对TGF-β1表达的影响

Discussion on the therapeutic effect of Yiqi Hexue Formula on CCL4-induced cirrhosis and its effect on TGF-β1 expression based on the theory of “qi and blood identification”

关键词:  “气血辨证”理论  益气和血方  肝硬化  治疗作用  TGF-β1

Key words:  qi and blood identification theory  Yiqi Hexue Formula  liver cirrhosis  therapeutic effect  TGF-β1

摘要:

[目的] 探讨益气和血方对四氯化碳（CCL₄）诱导的肝硬化大鼠的治疗作用及其对转化生长因子-β1（TGF-β1）表达的影响。[方法] 将SPF级SD雄性大鼠46只随机分为空白组和造模组两组。连续造模6周后将模型成功的大鼠再随机分为模型组、益气和血方低剂量组、益气和血方中剂量组、益气和血方高剂量组、扶正化瘀组5组。第10周末，检测大鼠血清肝功能，对大鼠肝组织进行苏木精-伊红（HE）染色、Masson染色观察病理变化，采用蛋白免疫印迹法（Western Blot）检测TGF-β1在大鼠肝组织中的蛋白表达水平。[结果] 1）与模型组比较，益气和血方中高剂量组和扶正化瘀组丙氨酸转氨酶（ALT）、天门冬氨酸氨基转移酶（AST）、总胆红素（TBIL）、碱性磷酸酶（ALP）含量显著降低（P<0.01），白蛋白（ALB）和胆碱酯酶（CHE）含量显著升高（P<0.05，P<0.01）。与益气和血方低剂量组比较，益气和血方高剂量组ALT、AST、TBIL、ALP含量显著降低（P<0.01），ALB、CHE含量显著升高（P<0.01）；益气和血方中剂量组ALT、AST、TBIL含量显著降低（P<0.01），ALB含量显著升高（P<0.05）；扶正化瘀组ALT、AST、TBIL、ALP含量显著降低（P<0.05，P<0.01），CHE含量显著升高（P<0.01）。与扶正化瘀组比较，益气和血方高剂量组ALT、AST含量显著降低（P<0.05，P<0.01），ALB含量显著升高（P<0.01）。2）益气和血方中、高剂量组肝小叶结构基本正常，肝小叶内肝细胞索排列较为整齐，病变面积明显减少，有少量炎性细胞浸润，肝小叶周围纤维组织明显减少。益气和血方低剂量组肝小叶结构破坏程度较模型组相比减轻，肝细胞排列不整齐，可见部分细胞空泡或脂肪样变，部分肝细胞间仍可见一定程度的纤维组织增生。3）益气和血方各剂量组和扶正化瘀组TGF-β1蛋白水平显著低于模型组（P<0.01），扶正化瘀组、益气和血方中高剂量组蛋白水平显著低于益气和血方低剂量组（P<0.01），益气和血方高剂量组蛋白水平显著低于扶正化瘀组（P<0.01）。[结论] 益气和血方能够显著改善CCL₄诱导的肝硬化大鼠的肝功能，抑制肝硬化的进展，作用机制可能与其下调或抑制TGF-β1的蛋白表达有关。

Abstract:

[Objective] To investigate the therapeutic effect of Yiqi Hexue Formula on carbon tetrachloride（CCL₄）-induced cirrhosis in rats and its effect on transforming growth factor β1（TGF-β1） expression. [Methods] Forty-six SPF grade SD male rats were randomly divided into two groups：blank group and modeling group. After 6 consecutive weeks of modeling，the successful rats were randomly divided into the model group，the low-dose group of Yiqi Hexue Formula，the medium-dose group of Yiqi Hexue Formula，the high-dose group of Yiqi Hexue Formula，and the group of reinforcing healthy qi and transforming stasis. At the end of the 10th week，the serum liver function of rats was tested，the liver tissues of rats were stained with HE staining and Masson staining to observe the pathological changes，and the protein expression level of TGF-β1 in the liver tissues of rats was detected by protein immunoblotting（Western Blot）. [Results] 1）Compared with the model group，alanine aminotransferase（ALT），aspartate aminotransferase（AST），total bilirubin（TBIL），and alkaline phosphatase（ALP） contents were significantly decreased in the medium and high dose groups of Yiqi Hexue Formula and the reinforcing healthy qi and transforming stasis group（P<0.01），and albumin（ALB） and cholinesterase（CHE） contents were significantly higher（P<0.05，P<0.01）. Compared with the low-dose group of Yiqi Hexue Formula，the contents of ALT，AST，TBIL，and ALP in the high-dose group of Yiqi Hexue Formula were significantly lower（P<0.01），and the contents of ALB and CHE were substantially higher（P<0.01）；in the middle-dose group of Yiqi Hexue Formula，the contents of ALT，AST，and TBIL were significantly lower（P<0.01），and the contents of ALB were significantly higher（P<0.05）；ALT，AST，TBIL，and ALP contents were significantly decreased（P<0.05，P<0.01） and CHE contents were significantly increased（P<0.01） in the reinforcing healthy qi and transforming stasis group. Compared with the reinforcing healthy qi and transforming stasis group，the ALT and AST contents of the high-dose group of Yiqi Hexue Formula were significantly lower（P<0.05，P<0.01），and the ALB content was significantly higher（P<0.01）. 2）The structure of the liver lobules in the middle and high dose groups of Yiqi Hexue Formula was basically normal，the hepatocyte cords in the liver lobules were arranged more neatly，the lesion area was significantly reduced，there was a small amount of inflammatory cell infiltration，and the fibrous tissue around the liver lobules was significantly reduced. In the low-dose group of Yiqi Hexue Formula，the degree of structural damage to the liver lobules was reduced compared with that of the model group，and the arrangement of the hepatocytes was not neat，with some cell vacuoles or steatosis，and a certain degree of fibrotic hyperplasia could be seen between some hepatocytes. 3）The TGF-β1 protein level was significantly lower in all dose groups of Yiqi Hexue Formula and reinforcing healthy qi and transforming stasis group than that in the model group（P<0.01），and the protein level was significantly lower in the middle and high dose groups of reinforcing healthy qi and transforming stasis and Yiqi Hexue Formula than that in the low dose group of Yiqi Hexue Formula（P<0.01），and the protein level in the high dose group of Yiqi Hexue Formula was significantly lower in the high dose group of reinforcing healthy qi and transforming stasis group than the low dose group of reinforcing healthy qi and transforming stasis and Yiqi Hexue Formula（P<0.01）. [Conclusion] Yiqi Hexue Formula can significantly improve liver function and inhibit the progression of cirrhosis in CCL₄-induced cirrhotic rats，and the mechanism of action may be related to its down-regulation or inhibition of TGF-β1 protein expression.