茯苓多糖调控NLRP3/细胞焦亡途径对HepG2肝癌细胞的抑制作用及机制

Inhibitory Effect and Its Mechanism of Pachymaran on HepG2 Hepatoma Cells by Regulating NLRP3/Pyrogen Pathway

目的通过观察茯苓多糖调控NLRP3介导细胞焦亡对HepG2肝癌细胞抑制作用,探讨茯苓多糖防治肝癌的作用机制。方法将HepG2肝癌细胞分为对照组、茯苓多糖组(800μg/mL)、MCC950组(1μmol/L)、MCC950+茯苓多糖组(800μg/mL+1μg/mL)。CCK-8法检测茯苓多糖对肝癌HepG2细胞的抑制率并筛选药物浓度;划痕实验检测细胞迁移能力;荧光实时定量PCR法检测HepG2细胞NLRP3、Caspase-1、GSDMD、IL-1βmRNA转录水平;Western Blot检测HepG2细胞NLRP3、Caspase-1、GSDMD、IL-1β、IL-18蛋白表达情况;免疫荧光检测各组细胞Caspase-1的表达情况。结果与对照组相比,茯苓多糖组对细胞有明显抑制作用,且800μg/mL茯苓多糖干预48 h后,细胞抑制效果最好;NLRP3、GSDMD、Caspase-1、IL-1βmRNA表达显著增高(P<0.05);NLRP3、GSDMD、Caspase-1、IL-1β、IL-18蛋白表达显著增高(P<0.05);且促进Caspase-1入核表达。与MCC950组相比,MCC950+茯苓多糖组对HepG2细胞有明显的抑制作用;NLRP3、GSDMD、Caspase-1、IL-1βmRNA表达显著增高(P<0.05);NLRP3、GSDMD、Caspase-1、IL-1β、IL-18蛋白表达显著增高(P<0.05);且促进Caspase-1入核表达。结论茯苓多糖可能通过抑制焦亡经典通路NLRP3/Caspase-1/GSDMD的表达含量发挥作用。

Objective To observe the inhibitory effect of pachymaran on HepG2 hepatoma cells mediated by NOD-like receptor protein 3(NLRP3)and explore the mechanism of pachymaran in the prevention and treatment of liver cancer.Methods HepG2 hepatoma cells were divided into control group,pachymaran group(800μg/mL),MCC950 group(1μg/mL),MCC950+pachymaran group(800μg/mL+1μg/mL).The inhibitory rate of pachymaran on HepG2 cells was detected by CCK-8 and the drug concentration was screened.The ability of cell migration was measured by scratch test.RT-qPCR was used to detect the transcription levels of NLRP3,Caspase-1,gasdermin D(GSDMD)and interleukin-1β(IL-1β)mRNA.Western Blot detected the expressions of cardiomyocyte NLRP3,Caspase-1,GSDMD,IL-1βand interleukin-18(IL-18)protein.The immunofluorescence methods were used to observe the HepG2 cells pyroptosis.Results Compared with those of the control group,the viability of pachymaran group was significantly reduced,and 800μg/mL pachymaran had the best inhibitory effect on cells after 48 h intervention,and the levels of NLRP3,GSDMD,Caspase-1 and IL-1βmRNA were increased(P<0.05),the expressions of NLRP3,GSDMD,Caspase-1,IL-1βand IL-18 protein were significantly increased(P<0.05),and the expression of Caspase-1 was promoted.Compared with the MCC950 group,the MCC950+pachymaran group had significant inhibitory effect on HepG2 cells,and the levels of NLRP3,GSDMD,Caspase-1 and IL-1βmRNA were increased(P<0.05),the expressions of NLRP3,GSDMD,Caspase-1,IL-1βand IL-18 protein were significantly increased(P<0.05),and the expression of Caspase-1 was promoted.Conclusion Pachymaran may play a role by inhibiting the expressions of NLRP3/Caspase-1/GSDMD.