归芪益元膏联合顺铂通过内质网应激途径和线粒体凋亡途径对Lewis肺癌小鼠的影响

Effect of Guiqi Yiyuan Ointment Combined with Cisplatin on Mice with Lewis Lung Cancer Through Endoplasmic Reticulum Stress Pathway and Mitochondrial Apoptosis Pathway

目的:探讨归芪益元膏联合顺铂通过内质网应激途径和线粒体凋亡途径对Lewis肺癌小鼠的影响。方法:将50只SPF级雄性C57BL/6小鼠,随机分为模型组、顺铂组(0.005 g·kg^(-1))、归芪益元膏联合顺铂(联合)低、中、高剂量组(0.005+1.6 g·kg^(-1)、0.005+3.3 g·kg^(-1)、0.005+6.6 g·kg^(-1))。各组小鼠腋下接种Lewis细胞悬液,构建Lewis肺癌移植瘤小鼠模型。连续给药14 d后取材,称量小鼠体质量,剥离瘤体称量瘤体质量,计算脏器指数和抑瘤率;苏木素-伊红(HE)染色观察肿瘤组织病理形态变化;流式细胞仪检测肿瘤细胞凋亡率和活性氧(ROS)比率;蛋白免疫印迹法(Western blot)检测内质网应激途径中葡萄糖调节蛋白78(GRP78)、磷酸化蛋白激酶R样内质网激酶(p-PERK)、活化转录因子4(ATF4)、凋亡蛋白C/EBP同源蛋白(CHOP)和线粒体凋亡途径中胱天蛋白酶-9(Caspase-9)、B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)蛋白表达情况。结果:与模型组比较,归芪益元膏联合顺铂组小鼠毛色较光泽,精神反应状态较好;各给药组肿瘤质量均降低(P<0.05),抑瘤率均升高(P<0.05);联合组胸腺、脾脏指数均升高(P<0.05);各给药组肿瘤组织病理形态改变明显,异型性逐渐降低,联合高剂量组肿瘤组织被大量破坏;各给药组肿瘤细胞凋亡率及ROS生成率均升高(P<0.05);各给药组肿瘤组织中Bcl-2蛋白表达水平逐渐降低(P<0.05),而GRP78、p-PERK、ATF4、CHOP、Bax、Caspase-9蛋白表达水平明显升高(P<0.05)。与顺铂组比较,联合组肿瘤质量均降低(P<0.05),联合中、高剂量组抑瘤率均升高(P<0.05);联合组胸腺指数和脾脏指数、肿瘤细胞凋亡率、ROS比率明显升高(P<0.05);联合中、高剂量组GRP78、p-PERK、Bax、Caspase-9蛋白表达水平明显升高(P<0.05);联合组ATF4、CHOP蛋白表达水平升高(P<0.05),Bcl-2蛋白表达水平逐渐降低(P<0.05)。结论:归芪益元膏联合顺铂可对Lewis肺癌小鼠产生抑瘤作用,降低肿瘤质量,提高抑瘤率,诱导肺癌细胞凋亡,其机制可能与调节内质网应激途径和线粒体凋亡途径相关。

Objective:To explore the effects of Guiqi Yiyuan ointment combined with cisplatin on mice with Lewis lung cancer through the endoplasmic reticulum stress pathway and mitochondrial apoptosis pathway.Method:Fifty SPF male C57BL/6 mice were randomly divided into the model group,cisplatin group(0.005 g·kg^(-1)),and low,medium,and high dose groups of Guiqi Yiyuan ointment combined with cisplatin(0.005+1.6 g·kg^(-1),0.005+3.3 g·kg^(-1),and 0.005+6.6 g·kg^(-1)).Lewis cell suspension was inoculated under the axilla of mice in each group to construct the Lewis lung cancer xenograft mouse model.After continuous administration for 14 days,the mice were sacrificed.The body weight of the mice was measured,and the tumor weight was measured after the tumors were removed.The organ index and tumor inhibition rate were calculated.Hematoxylin-eosin(HE)staining was used to observe the pathological changes in tumor tissue.Flow cytometry was used to detect the apoptosis rate of tumor cells and the ratio of reactive oxygen species(ROS).Western blot was used to detect the expression of glucose-regulated protein 78(GRP78),phosphorylated activated protein kinase R-like endoplasmic reticulum kinase(p-PERK),activated transcription factor 4(ATF4),and apoptosis protein C/EBP homologous protein(CHOP)in the endoplasmic reticulum stress pathway,as well as cysteine aspartate protease-9(Caspase-9),B-cell lymphoma-2(Bcl-2),and Bcl-2 associated X protein(Bax)in the mitochondrial apoptosis pathway.Result:Compared with those in the model group,the mice in the groups of Guiqi Yiyuan Ointment combined with cisplatin had shinier fur and better mental response status.Tumor mass was reduced in all treatment groups(P<0.05),and tumor inhibition rate was increased in all treatment groups(P<0.05).The thymus and spleen indices of the combined group were increased(P<0.05),and obvious pathological changes were observed in the tumor tissue of all treatment groups,with a gradual decrease in heteromorphism.Destruction of massive tumor tissue was observed in the high-dose combined group,and the apoptosis rate and ROS generation rate of tumor cells were increased in all treatment groups(P<0.05).The protein expression level of Bcl-2 in the tumor tissue gradually decreased(P<0.05),while the protein expression levels of GRP78,p-PERK,ATF4,CHOP,Bax,and Caspase-9 were significantly increased(P<0.05).Compared with the cisplatin group,tumor mass was reduced in the combined group(P<0.05),and tumor inhibition rates in the low and high-dose combined groups were increased(P<0.05).The thymus index,spleen index,apoptosis rate of tumor cells,and ROS ratio in the combined group were significantly increased(P<0.05),while the protein expression levels of GRP78,p-PERK,Bax,and Caspase-9 were increased in the low and high-dose combined groups(P<0.05).The protein expression levels of ATF4 and CHOP were increased in the combined group(P<0.05),while the expression level of Bcl-2 protein gradually decreased(P<0.05).Conclusion:Guiqi Yiyuan ointment combined with cisplatin can exert anti-tumor effects in mice with Lewis lung cancer,reduce tumor mass,increase tumor inhibition rate,and induce apoptosis of lung cancer cells.Its mechanism may be related to the regulation of the endoplasmic reticulum stress pathway and mitochondrial apoptosis pathway.