健脾解毒汤联合5-氟尿嘧啶对胃癌大鼠p53/Hippo信号通路的调节作用

Regulatory effect of Jianpijiedu decoction combined with 5-fluorouracil on p53/Hippo signal pathway in rats with gastric cancer

目的研究健脾解毒汤联合5-氟尿嘧啶(5-FU)对胃癌大鼠p53/Hippo信号通路的调节作用。方法60只大鼠随机分为空白对照组(12只)及造模组(48只),采用N-甲基-N-硝基-N-亚硝基胍(MNNG)喂养法建立胃癌大鼠模型,建模完成后大鼠随机分为胃癌模型组、健脾解毒汤组、5-FU组、健脾解毒汤联合5-FU组,健脾解毒汤组按照13 g/kg(以生药含量计)灌胃给予大鼠健脾解毒汤,1次/d,5-FU组按照17.5 mg/kg隔日腹腔注射1次5-FU,健脾解毒汤联合5-FU组同时给予大鼠健脾解毒汤及5-FU,连续给药10周。采用酶联免疫吸附法(ELISA)测定大鼠血清胃动素(MTL)、胃蛋白酶原Ⅰ(PGⅠ)、胃泌素(GAS)水平及胃组织肿瘤坏死因子α(TNF-α)、B淋巴细胞瘤(Bcl-2)、Bcl-2相关的x蛋白(Bax)水平,苏木精-伊红(HE)染色法检查胃组织病理学变化,实时定量聚合酶链式反应(RT-qPCR)检测胃组织p53及YAP1 mRNA水平,免疫印迹法(Western blot)法检测大鼠胃组织p53及YAP1蛋白水平。结果与空白对照组比较,胃癌模型组大鼠血清MTL、PGⅠ及GAS水平,胃组织Bax水平显著降低(P<0.05),胃组织TNF-α、Bcl-2水平,胃组织p53、YAP1 mRNA及蛋白水平显著升高(P<0.05);与胃癌模型组大鼠比较,健脾解毒汤组、5-FU组及健脾解毒汤联合5-FU组大鼠血清MTL、PGⅠ及GAS水平,胃组织Bax水平显著升高(P<0.05),胃组织TNF-α、Bcl-2水平,胃组织p53、YAP1 mRNA及蛋白水平显著降低(P<0.05),大鼠胃组织病理学显著恢复,且与健脾解毒汤组及5-FU组比较,健脾解毒汤联合5-FU组上述指标改善更为显著。结论健脾解毒汤联合5-FU能够调节胃组织细胞凋亡相关蛋白水平,改善胃组织病理改变,其机制可能与调节p53/Hippo通路有关。

Objective To study the regulatory effect of Jianpijiedu decoction combined with 5-fluorouracil(5-FU)on p53/Hippo signal pathway in rats with gastric cancer. Methods 60 rats were randomly divided into the blank control group(n=12) and the model group(n=48). The rat model of gastric cancer was established by feeding N-methyl-N-nitro-N-nitrosoguanidine(MNNG). After modeling, the rats were randomly divided into gastric cancer model group, Jianpijiedu decoction group, 5-FU group, and Jianpijiedu decoction combined with 5-FU group, 12 rats in each group. The rats in Jianpijiedu decoction group were given Jianpijiedu decoction by gavage at the rate of 13 g/kg(based on the crude drug content), once a day. The rats in 5-FU group were given 5-FU intraperitoneally at the rate of 17.5 mg/kg every other day. The rats in Jianpijiedu decoction combined with 5-FU group were given Jianpijiedu decoction and 5-FU. All groups were treated for 10 weeks. The levels of serum motilin(MTL), pepsinogen(PG) I and gastrin(GAS) and the levels of tumor necrosis factor α(TNF)-α, Bcl-2 and Bcl-2 related x protein(Bax) in gastric tissues were measured by ELISA. Gastric histopathological changes were examined by HE staining. The levels of p53 and YAP1 mRNA in gastric tissues were detected by RT-qPCR, and the levels of p53 and YAP1 protein in gastric tissues were detected by western blotting. Results Compared with the blank control group, the levels of serum MTL, PGI and GAS and the level of Bax in gastric tissue were significantly decreased, while the levels of TNF-α, Bcl-2, p53, YAP1 mRNA and protein in gastric tissues were significantly increased in gastric cancer model group(P<0.05). Compared with the gastric cancer model group, the levels of serum MTL, PGI and GAS, and the level of Bax in gastric tissues in Jianpijiedu decoction group, 5-FU group and Jianpijiedu decoction combined with 5-FU group were significantly elevated, while the levels of TNF-αand Bcl-2 in gastric tissues, p53, YAP1 mRNA and protein in gastric tissues were significantly lowered. Meantime,the gastric histopathology of rats recovered obviously in the treated groups. Compared with Jianpijiedu decoction group and 5-FU group, Jianpijiedu decoction combined with 5-FU group improved the above indexes more significantly. Conclusion Jianpijiedu decoction combined with 5-FU can regulate the level of apoptosis related protein in gastric tissues and improve the pathological changes of gastric tissues. Its mechanism may be related to the regulation of p53/Hippo pathway.