基于NLRP3/Caspase-1/GSDMD炎性焦亡途径探讨参芪抑瘤方联合顺铂对H22肝癌荷瘤小鼠的作用机制  

Mechanism of Shenqi Yiliu Prescription Combined with Cisplatin on H22 Liver Cancer-bearing Mice Based on NLRP3/Caspase-1/GSDMD Pyroptosis Pathway

作　　者：杨梦莹 段永强[2,3] 贾育新 白敏 朱中博 李亚荣 马兰 张明雨 冯鑫 何兰兰 杨玉萍 YANG Mengying;DUAN Yongqiang;JIA Yuxin;BAI Min;ZHU Zhongbo;LI Yarong;MA Lan;ZHANG Mingyu;FENG Xin;HE Lanlan;YANG Yuping(Basic Medical College,Gansu University of Chinese Medicine,Lanzhou 730000,China;Key Laboratory of Ministry of Education of Traditional Chinese Medicine Prevention and Treatment of Highincidence Diseases in Ningxia,Yinchuan 750004,China;Key Laboratory of Study on Integrated Traditional Chinese and Western Medicine with High Incidence Diseases in Ningxia,Yinchuan 750004,China;Gansu Province Experimental Animal Industry Technology Center,Lanzhou 730000,China;General Hospital of Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]甘肃中医药大学基础医学院,兰州730000 [2]宁夏区域高发病中医药防治教育部重点实验室,银川750004 [3]宁夏区域高发病中西医结合研究重点实验室,银川750004 [4]甘肃省实验动物行业技术中心,兰州730000 [5]宁夏医科大学总医院,银川750004

出　　处：《中国实验方剂学杂志》2023年第21期114-122,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：甘肃省发改委第十一批建设项目(2305142201);甘肃中医药大学人才创新创业项目扶持基金项目(2015-RC-24);甘肃中医药大学校级研究生创新基金资助项目(2022CX32)。

摘　　要：目的:探讨参芪抑瘤方干预细胞焦亡在抗肿瘤效应中的作用机制。方法:随机抽取10只BALB/c小鼠(雄性)作为正常组,余40只建立肝癌小鼠异位移植瘤模型,建模5 d后随机分为模型组、顺铂组[2.5×10^(-3)g·kg^(-1)·(3 d)^(-1)]、参芪抑瘤方组(27 g·kg^(-1)·d^(-1))、联合组(参芪抑瘤方+顺铂)。正常组与模型组以等量生理盐水灌胃,连续干预10 d,观察各组小鼠一般情况,干预结束后,称量小鼠瘤体质量,计算抑瘤率,采用苏木素-伊红(HE)染色观察肿瘤组织病理变化;检测小鼠肝功能指标丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)水平;原位末端标记法(TUNEL)检测小鼠肿瘤组织细胞DNA损伤情况;免疫组化(IHC)、免疫荧光(IF)及蛋白免疫印迹法(Western blot)检测瘤组织中NOD样受体蛋白3(NLRP3)、胱天蛋白酶-1(Caspase-1)、消皮素D(GSDMD)蛋白表达水平;酶联免疫吸附测定法(ELISA)检测瘤组织中白细胞介素(IL)-1β和IL-18的含量。结果:与正常组比较,模型组小鼠精神状态差,倦卧懒动,毛色暗淡;肝功能检测血清ALT、AST水平显著升高(P<0.01)。与模型组比较,各给药组小鼠精神状态明显好转,且各给药组均可不同程度抑制瘤体生长,肿瘤质量均下降,其中联合组抑瘤率最强(P<0.01);HE染色示,模型组小鼠肿瘤组织病理形态学病变显著,各给药组均有一定程度改善,以参芪抑瘤方组及联合组细胞核,溶解破裂更为明显;肝功能检测中,参芪抑瘤方组及联合组小鼠血清ALT、AST水平均下降显著(P<0.01);各给药组炎症因子IL-1β和IL-18均下降(P<0.05,P<0.01);TUNEL染色示各给药组干预后TUNEL染色阳性降低(P<0.05,P<0.01),以顺铂组和参芪抑瘤方组降低显著(P<0.01);Western blot、免疫组化、免疫荧光在肿瘤组织中检测发现,各给药组NLRP3、Caspase-1、GSDMD蛋白表达水平均下降(P<0.05,P<0.01)。与顺铂组比较,参芪抑瘤方组和联合组小鼠精神状态佳,瘤体形态�Objective:To explore the anti-tumor effect and mechanism of Shenqi Yiliu prescription in the intervention of pyroptosis.Method:Ten male BALB/c mice were randomly selected and assigned to the blank group.The remaining 40 mice underwent the induction of the liver cancer xenograft model.After 5 days of modeling,40 surviving mice were randomly divided into model group,cisplatin group[2.5×10^(-3) g·kg^(-1)·(3 d)^(-1)],Shenqi Yiliu prescription group(27 g·kg^(-1)·d^(-1)),and a combination group(Shenqi Yiliu prescription group+cisplatin).The mice in the blank group and the model group were treated with an equal volume of normal saline for 10 days.The general conditions of mice in each group were observed.After the intervention,the tumor weight of the mice was weighed and the tumor inhibition rate was calculated.Hematoxylin-eosin(HE)staining was used to observe the pathological changes in tumor tissues.The levels of mouse liver function indicators,including alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were detected.The TdT-mediated dUTP-biotin nick end labeling(TUNEL)assay was used to detect DNA damage in mouse tumor tissue cells.Immunohistochemistry(IHC),immunofluorescence(IF),and Western blot were used to detect the protein expression levels of NOD-like receptor protein 3(NLRP3),cysteinyl aspartate-specific protease-1(Caspase-1),and gasdermin D(GSDMD)in tumor tissues.The levels of interleukin-1β(IL-1β)and interleukin-18(IL-18)in tumor tissues were detected by enzyme-linked immunosorbent assay(ELISA).Result:Compared with the mice in the blank group,those in the model group were in a poor mental state,sleepy,and lazy,and their fur color was dull,with increased levels of serum ALT and AST in liver function tests(P<0.01).Compared with the model group,the groups with drug intervention showed improved mental state,inhibited tumor growth to varying degrees,and decreased tumor weight,and the tumor inhibition rate in the combination group was the highest(P<0.01).HE staining showed that the pathological

关 键 词：NOD样受体蛋白3(NLRP3) 胱天蛋白酶-1(Caspase-1) 消皮素D(GSDMD) 参芪抑瘤方 原发性肝癌 

分 类 号：R2-0[医药卫生—中医学] R22R242R2-031R285.5R735.7