健脾消癌方对人结肠癌HCT116细胞焦亡的影响

Effect of Jianpi Xiaoai Prescription on Pyroptosis of Colorectal Cancer HCT116 Cells

目的 观察健脾消癌方含药血清对人结肠癌HCT116细胞焦亡的影响,探讨其可能作用机制。方法 10%、15%、20%健脾消癌方含药血清处理HCT116细胞;细胞毒性实验(CCK-8)检测健脾消癌方含药血清对HCT116细胞的抑制作用;乳酸脱氢酶(LDH)释放实验检测细胞上清LDH含量;酶联免疫吸附试验(ELISA)检测细胞上清中白介素1β(IL-1β)、白介素18(IL-18)释放量;荧光定量PCR(qRT-PCR)、蛋白免疫印迹法(Western blot)检测GSDMD、GSDME基因和蛋白表达。结果 健脾消癌方含药血清能够抑制HCT116细胞体外增殖(P<0.05);与空白鼠血清组比较,健脾消癌方含药血清干预HCT116细胞后细胞上清中的LDH、IL-1β、IL-18均有不同程度的上升(P<0.05);10%、15%、20%含药血清组的GSDME、GSDMD mRNA表达上调(P<0.05);10%、15%、20%含药血清组的GSDME蛋白表达下调(P<0.05),其中20%浓度较为明显,10%、20%含药血清组的GSDMD蛋白表达下调(P<0.01)。结论 健脾消癌方可诱导结肠癌HCT116细胞发生焦亡,其机制可能与GSDMD、GSDME蛋白的剪切有关。

Objective To observe the effect of Jianpi Xiaoai Prescription on pyroptosis of colorectal cancer cell HCT116;To discuss its possible mechanism of action for prevention and treatment of colorectal cancer. Methods By conducting CCK-8 experiments, the proliferation of colorectal cancer cell(HCT116) were detected;The LDH release test detects the LDH content of the cell supernatant;ELISA experiments detects the release of IL-1β and IL-18 in the cell supernatant;qRT-PCR and Western blot experiments were applied to verify the mRNA and protein expression level of GSDMD and GSDME. Results Jianpi Xiaoai Prescription showed in vitro inhibitory effect on the proliferation of HCT116(P<0.05);Compared with blank group, the release of of LDH、IL-1β、IL-18 in drug-containing groups were increased(P<0.05);The mRNA of GSDME and GSDMD were upregulated in 10%、15%、20% drug-containing groups(P<0.05);The protein expression levels of GSDME in 10%, 15% and 20% drug-containing groups were decreased(P<0.05), as well as the expression level of GSDMD in 10% and 20% drug-containing groups(P<0.01). Conclusion Jianpi Xiaoai Prescription can cause the pyroptosis of HCT116,whose mechanism may be related to the splicing of GSDMD and GSDME proteins.