养肝化瘀颗粒抗肝癌作用及其机制研究

Inhibitory Effect and Mechanism of Yanggan Huayu Granules on Hepatocellular Carcinoma

目的探讨养肝化瘀颗粒抗肝癌作用及其机制。方法建立小鼠H22肝癌皮下移植瘤模型,随机分为空白对照组,模型组,5-氟尿嘧啶(5-FU,10 mg·kg^(-1),ip)组,养肝化瘀颗粒高、中、低(6,3,1.5 g·kg^(-1),ig)剂量组,连续给药10 d。观察各组小鼠的肿瘤生长情况及血清甲胎蛋白(AFP)、肿瘤特异性生长因子(TSGF)水平;HE染色观察瘤组织的病理改变;Western Blot法检测β-连环蛋白(β-catenin)、活化型半胱天冬酶-3(Cleaved-Caspase3)蛋白表达,并进一步通过氯化锂(Li Cl)激动Wnt/β-catenin信号通路,研究其抑癌作用机制。结果养肝化瘀颗粒高、中、低剂量和阳性药5-FU作用后,抑瘤率分别为41.29%、50.03%、48.15%、52.83%;与模型组比较,各给药组血清肿瘤标志物AFP均有所下降(P<0.01,P<0.05),TSGF也有所下降,其中养肝化瘀颗粒中剂量组与5-FU组最为明显(P<0.01);瘤组织出现不同程度的坏死,与模型组比较,养肝化瘀颗粒中剂量组最为明显。养肝化瘀颗粒可以下调HepG2细胞、瘤组织中β-catenin蛋白表达水平,上调Cleaved-Caspase3蛋白表达水平。进一步通过LiCl激动Wnt/β-catenin信号通路,与空白对照组比较,LiCl组β-catenin蛋白表达显著上调(P<0.05),Cleaved-Caspase3蛋白表达则下调(P<0.05);与LiCl组比较,LiCl+养肝化瘀颗粒含药血清(10%、20%浓度)组β-catenin蛋白表达显著下调(P<0.01),Cleaved-Caspase3蛋白表达则显著上调(P<0.01)。结论养肝化瘀颗粒显著抑制小鼠H22皮下移植瘤的生长,可能是通过抑制β-catenin蛋白表达,下调Wnt/β-catenin信号通路,进而诱导肝癌细胞凋亡发挥抗肿瘤作用。

Objective To investigate the antitumor effect and the possible mechanism of the Yanggan Huayu granules（YGHY） on hepatocellular carcinoma（HCC）. Methods H22 xenograft mice model was established. The mice were randomly divided into six groups,blank control group,model group,5- fluorouracil（5- FU,10 mg·kg-1,ip） group,and YGHY groups of high, medium and low dose（6, 3, 1.5 g·kg-1, ig）. The medication lasted for 10 continuous days. After the last administration, the tumor growth of mice in each group was observed, and serum alpha- fetoprotein（AFP）and tumor specific growth factor（TSGF）were also examined. Tumor tissue pathological changes were observed after HE staining, and Western blot was used to detect the protein expression of β- catenin and semi- activated caspase- 3（Cleaved- Caspase3） in hepatocellular carcinoma cells and tumor tissues. The suppressor mechanism was further studied by observing Wnt/β- catenin signaling pathway activated by lithium chloride（Li Cl）.Results High, medium and low dosage of YGHY and 5- FU effectively inhibited the growth of H22 hepatocellular carcinoma cells with the inhibition rate of 41.29 %,52.09 %,48.15 %,52.83 %,respectively. Compared with the model group,YGHY and 5- FU effectively reduced the AFP（P0.01,P0.05）and TSGF in tumor- bearing mice,and the effect of medium- dose YGHY and 5- FU were strongest（P0.01）. Varying degrees of necrosis were shown in tumor tissue of the medication groups,and the necrosis was obvious in medium- dose YGHY group compared with the model group. Compared with the blank control group, the protein expression of β- catenin in Li Cl group was up- regulated and that of Cleaved- Caspase3 was down- regulated（P0.05）. The results of observation of Wnt signaling pathway activated by Li Cl showed that serum containing YGHY＋ Li Cl（10 %, 20 %） down- regulated β- catenin protein expression obviously and up- regulated Cleaved- Caspase3 protein expression（P〈0.01 compared with Li Cl group）. Conclusion YGHY can significantly inhibit tumor growth in the H22 xenograft mice, and the possible mechanism is through inhibiting β- catenin protein expression and then down- regulating the Wnt/β- catenin signaling pathway,thereby inducing the apoptosis of hepatocellular carcinoma cells.