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

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,瘤体形态规则,联合组小鼠肿瘤质量下降(P<0.05);参芪抑瘤方组ALT和AST水平下降(P<0.05);参芪抑瘤方组和联合组IL-1β和IL-18均下降(P<0.05,P<0.01),以联合组最为明显(P<0.01);免疫组化结果显示,联合组小鼠肿瘤组织中GSDMD蛋白表达显著降低(P<0.01);免疫荧光检测发现参芪抑瘤方组NLRP3在肿瘤组织中表达显著降低(P<0.01);Western blot结果显示参芪抑瘤方组及联合组NLRP3蛋白表达水平下降显著(P<0.01),联合组Caspase-1蛋白表达水平下降显著(P<0.01);GSDMD蛋白表达下降不明显,差异无统计学意义。结论:参芪抑瘤方联合顺铂具有明显抑瘤作用,其抗肿瘤作用可能是通过下调NLRP3/Caspase-1/GSDMD炎性焦亡途径抑制细胞焦亡,缓解肝癌小鼠的炎症反应,达到抗肿瘤的作用。

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 and morphological lesions of the tumor tissues in the model group were significant,while those in all groups with drug intervention were improved to a certain extent.The karyolysis and nuclear rupture in the Shenqi Yiliu prescription group and the combination group were more significant.In the liver function test,the serum ALT and AST levels of mice in the Shenqi Yiliu prescription group and the combination group decreased(P<0.01),and the inflammatory factors IL-1βand IL-18 in each group with drug intervention decreased(P<0.05,P<0.01).Among them,the declining trend of IL-1βand IL-18 in the Shenqi Yiliu prescription group was the most significant(P<0.01).TUNEL staining showed that the positive TUNEL staining in each group with drug intervention decreased after intervention(P<0.05,P<0.01),especially the cisplatin group and Shenqi Yiliu prescription group(P<0.01).Western blot,IHC,and IF found that the protein expression levels of NLRP3,Caspase-1,and GSDMD in each group with drug intervention decreased(P<0.05,P<0.01).Compared with the mice in the cisplatin group,those in the Shenqi Yiliu prescription group and the combination group had better mental state and regular tumor morphology,and the tumor weight of the mice in the combination group decreased(P<0.05).The levels of ALT and AST in the Shenqi Yiliu prescription group decreased(P<0.05),and the levels of IL-1βand IL-18 in the Shenqi Yiliu prescription group and the combination group decreased(P<0.05,P<0.01),especially in the combination group(P<0.01).The results of IHC showed that the expression of GSDMD protein in the tumor tissues of mice in the combination group was reduced(P<0.01).IF detection showed that the expression of NLRP3 in the tumor tissues of the Shenqi Yiliu prescription group was reduced(P<0.01).The results of Western blot showed that the expression level of NLRP3 protein in the Shenqi Yiliu prescription group and the combination group decreased(P<0.01),and the expression level of Caspase-1 protein in the combination group decreased(P<0.01).The decrease in GSDMD protein expression was not significant,and the difference was not statistically significant.Conclusion:Shenqi Yiliu prescription combined with cisplatin has an obvious anti-tumor effect,which may be achieved by down-regulating the NLRP3/Caspase-1/GSDMD inflammatory pyroptosis pathway to inhibit cell pyroptosis,and relieve the inflammatory response in mice with liver cancer.