麦冬皂苷B调节Rho A/ROCK1信号通路对胃荷瘤大鼠肿瘤生长的抑制作用

Inhibitory effect of ophiopogonin B on tumor growth in gastric tumor-bearing rats by regulating Rho A/ROCK1 signaling pathway

目的 探讨麦冬皂苷B(ophiopogonin B,OP-B)对胃荷瘤大鼠肿瘤生长的影响及其作用机制。方法 将66只体质量为180~200 g的6周龄SPF级SD大鼠(雌雄各半)按随机数字表法分为模型组,OP-B低、中、高剂量组,激活剂组,每组10只。采用原位移植Walker-256肿瘤组织建立胃荷瘤模型,OP-B低、中、高剂量组大鼠分别灌胃17.5、35、70 mg/kg的OP-B并腹腔注射等量生理盐水,激活剂组灌胃70 mg/kg的OP-B并腹腔注射1 mg/kg的Rho A/ROCK1信号通路激活剂溶血磷脂酸(lysophosphatidic acid, LPA),模型组灌胃和腹腔注射等量的生理盐水;记录移植瘤质量及体积,计算抑瘤率;苏木精-伊红(hematoxylin-eosin, HE)染色观察肿瘤组织形态;原位末端转移酶标记技术(TdT-mediated dUTP-biotin nick end labeling, TUNEL)染色检测肿瘤组织细胞凋亡;免疫组织化学染色检测肿瘤组织细胞核增殖抗原67(proliferating cell nuclear antigen, Ki-67)、切割型半胱氨酸天冬氨酸蛋白水解酶-3(cleaved cysteinyl aspartate specific proteinase-3,Cleaved Caspase-3)表达;实时荧光定量聚合酶链式反应(real-time quantitative polymerase chain reaction, RT-qPCR)法检测Ras同源基因家族蛋白A(Ras homologous gene family member A,Rho A)、Rho相关卷曲螺旋蛋白激酶1(Rho-associated coiled-coil forming protein kinase1,ROCK1)的表达;Western blot检测Rho A、ROCK1蛋白表达。结果 与模型组比较,OP-B低、中、高剂量组肿瘤质量和体积、Ki-67阳性率及Rho A、ROCK1 mRNA和蛋白表达显著降低,抑瘤率、肿瘤细胞凋亡率、Cleaved Caspase-3阳性率显著升高(P<0.05);与OP-B高剂量组比较,激活剂组肿瘤质量和体积、Ki-67阳性率及Rho A、ROCK1 mRNA和蛋白表达显著升高,抑瘤率、肿瘤细胞凋亡率、Cleaved Caspase-3阳性率显著降低(P<0.05)。结论 OP-B可能通过抑制Rho A/ROCK1信号通路的激活,抑制胃荷瘤大鼠肿瘤生长。

Objective To investigate the effect of ophiopogonin B(OP-B)on tumor growth in gastric tumor-bearing rats and its underlying mechanism.Methods Sixty-six SPF SD rats(half male and female,6 weeks old,180~200 g)were randomly divided into model group,low-,medium-and high-dose OP-B groups,and activator group,with 10 rats in each group.The gastric tumor-bearing model was established by orthotopic transplantation of Walker-256 tumor tissue.The rats in the 3 doses groups were given 17.5,35 and 70 mg/kg OP-B,respectively,by gavage and intraperitoneal injection of the same amount of normal saline,and the rats in the activator group were intragastrically administered with 70 mg/kg OP-B and intraperitoneally injected with 1 mg/kg Rho A/ROCK1 signaling pathway activator,lysophosphatidic acid(LPA).The weight and volume of transplanted tumor were recorded to calculate the tumor inhibitory rate.The morphology of tumor tissue was observed with HE staining.The apoptosis of tumor cells were detected by TdT-mediated dUTP-biotin nick end labeling(TUNEL)staining.The expression of proliferating cell nuclear antigen 67(Ki-67)and cleavage cysteine aspartic acid proteolytic enzyme-3(Cleaved Caspase-3)in tumor tissues were detected by immunohistochemical staining.RT-qPCR and Western blotting were utilized to measure the expression of Ras homologous gene family member A(Rho A)and Rho-associated coiled-coil forming protein kinase1(ROCK1)at mRNA and protein levels.Results Compared with the model group,the tumor weight and volume,mRNA and protein levels of Ki-67,Rho A and ROCK1 were significantly decreased,and the tumor inhibitory rate,tumor cell apoptotic rate and Cleaved Caspase-3 protein level were obviously increased in the low-,medium-and high-dose OP-B groups(P<0.05).The activator group had heavier tumor weight and larger volume,increased mRNA and protein expression levels of Ki-67,Rho A and ROCK1,and lower tumor inhibitory rate and apoptotic rate and reduced Cleaved Caspase-3 expression when compared with the high-dose OP-B group(P<0.05).Conclusion OP-B may suppress the tumor growth of gastric tumor-bearing rats by inhibiting the activation of Rho A/ROCK1 signaling pathway.