基于内质网应激探讨扶正解毒方联合5-Fu对胃癌荷瘤小鼠术后复发及转移的影响  

Effect of Fuzheng Jiedu Prescription Combined with 5-Fu Against Postoperative Recurrence and Metastasis of Gastric Cancer in Mice:An Exploration Based on Endoplasmic Reticulum Stress

作　　者：冯颖[1,2] 吴喆 李杰 FENG Ying;WU Zhe;LI Jie(Guang'anmen Hospital Affiliated to China Academy of Chinese Medicine Sciences,Beijing 100053,China;The Affiliated Suzhou Hospital of Nanjing Medical University,Suzhou Municipal Hospital,Gusu school,Nanjing Medical University,Suzhou 215000,China)

机构地区：[1]中国中医科学院广安门医院,北京100053 [2]南京医科大学姑苏学院,南京医科大学附属苏州医院,苏州市立医院,苏州215000

出　　处：《中国实验方剂学杂志》2021年第16期75-83,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金面上项目(81774289,82074402)。

摘　　要：目的:研究扶正解毒方联合5-氟尿嘧啶(5-Fu)对胃癌荷瘤小鼠术后复发转移的抑制作用,并通过肿瘤微环境中CD4^(+)T细胞,CD8^(+)T细胞,调节性T(Treg)细胞含量的改变,内质网应激途径及磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)通路,探讨其可能的分子机制。方法:40只615小鼠随机分为模型组,扶正解毒方(25 g·kg^(-1))组,5-Fu(25 mg·kg^(-1))组,联合组(扶正解毒方25 g·kg^(-1)+5-Fu 25 mg·kg^(-1)),每组各10只,将小鼠前胃癌细胞(MFC细胞)接种于左后肢内侧爪垫下,通过手术切除移植瘤建立术后复发模型。苏木素-伊红(HE)染色法观察术后复发胃癌荷瘤小鼠肺转移病理形态的改变;流式细胞术检测复发瘤中CD4^(+)/CD8^(+)T值及脾脏中Treg细胞[CD4^(+),CD25^(+),叉头框蛋白P3(FOXP3)+细胞]的含量;蛋白免疫印迹法(Western blot)及免疫组化法(IHC)检测内质网应激相关蛋白[葡萄糖调节蛋白78(GRP78),肌醇需求酶1α(IRE1α),激活转录因子6(ATF6),蛋白激酶R样内质网激酶(PERK)]含量,以及PI3K/Akt/mTOR通路相关蛋白表达。结果:与模型组比较,联合组复发抑制率明显升高(P<0.05);各治疗组复发瘤重均明显降低(P<0.05);各治疗组肺转移数均降低,转移率均有所降低,联合组肺转移总数最少,转移率最低,但差异无统计学意义;扶正解毒方组、联合组CD4^(+)/CD8^(+)T值明显升高(P<0.05),Treg细胞含量明显降低(P<0.05);5-Fu组Treg细胞含量明显升高(P<0.05)。IHC结果显示,与模型组比较,各治疗组ATF6蛋白表达明显下降(P<0.05,P<0.01),IRE1α表达明显下降(P<0.05),Akt表达显著下降(P<0.01);5-Fu组及联合组mTOR表达明显下降(P<0.05)。Western blot结果显示,与模型组比较5-Fu组GRP78表达明显下降(P<0.05),5-Fu组及联合组PI3K,磷酸化Akt(p-Akt),mTOR表达明显下降(P<0.05)。结论:扶正解毒方联合5-Fu可以抑制荷瘤小鼠术后复发转移,机制可能与抑制PI3K/Akt/mTOR的信号通路,Objective:To investigate the inhibitory effect of Fuzheng Jiedu prescription(FZJDP combined with 5-fluorouracil(5-Fu)against postoperative recurrence and metastasis in gastric cancer-bearing mice and explore the possible mechanism based on changes in CD4^(+)T cells,CD8^(+)T cells,and regulatory T(Treg)cells of tumor microenvironment,endoplasmic reticulum stress(ERS)and phosphatidylinositol-3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR)pathway.Method:Forty 615 mice were randomly divided into the model group,FZJDP(25 g·kg^(-1))group,5-Fu(25 mg·kg^(-1))group,and combined(25 g·kg^(-1)FZJDP+25 mg·kg^(-1)5-Fu)group,with 10 mice in each group.Mouse forestomach carcinoma(MFC)cells were implanted into the inner side of footpad of the left hind paw and the transplanted tumor was then surgically excised to establish a postoperative recurrence model.Hematoxylin-eosin(HE)staining was conducted to observe the pathological changes in mice with gastric cancer recurrence and lung metastasis.The CD4^(+)/CD8^(+)T cell ratio in recurrent tumor and the percentages of Treg cells[CD4^(+),CD25^(+),and forkhead box protein P3(FOXP3)+cells]in spleen were detected by flow cytometry.The contents of ERS-related proteins[78-k Da glucose-regulated protein(GRP78),inositol-requiring enzyme 1 alpha(IRE1α),activating transcription factor 6(ATF6),and protein kinase R-like endoplasmic reticulum kinase(PERK)]and the expression of related proteins in the PI3K/Akt/mTOR signaling pathway were determined by Western blot and immunohistochemistry(IHC).Result:Compared with the model group,the combined group significantly increased the recurrent inhibition rate(P<0.05).The recurrent tumor weight was significantly decreased in each treatment group(P<0.05).The number of lung metastases and metastasis rate declined in each treatment group,and the lowest values were observed in the combined group,without any statistical significance.The CD4^(+)/CD8^(+)T cell ratios in the FZJDP group and combined group were significantly elevated(P

关 键 词：扶正解毒方 5-氟尿嘧啶 胃癌 内质网应激 肿瘤免疫微环境 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学]