基于NLRP3/Caspase-1通路探讨薏苡仁多糖对溃疡性结肠炎体外炎症模型的干预作用及机制

Intervention effect of coix seed polysaccharides on an in vitro inflammation model of ulcerative colitis and its mechanism:a study based on the NLRP3/caspase-1 pathway

目的:从炎症理论角度出发,探讨薏苡仁多糖(Coixan)对溃疡性结肠炎(ulcerative colitis,UC)体外炎症模型的干预作用及其机制。方法:体外培养NCM460细胞,采用不同剂量(0、2.5、5、10、20和40μg/mL)脂多糖(lipopolysaccharide,LPS)诱导NCM460细胞,筛选出最佳诱导剂量(20μg/mL);用不同浓度(0、5、10、20、40和80μg/mL)的Coixan诱导NCM460,筛选出最佳促增殖浓度(10、20、40μg/mL)。实验随机设为空白组、模型组(LPS诱导)、LPS+Coixan低、中、高剂量(10、20、40μmol/L)组,LPS+柳氮磺吡啶(sulfasalazine,SASP)(200μg/mL)组,除空白组外用LPS(20μg/mL)刺激48 h建立UC体外细胞炎症模型后,分别以薏苡仁多糖及SASP进行48 h干预治疗后,ELISA法检测细胞上清液中炎症因子白细胞介素-1β(interleukin-1β,IL-1β)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)水平;免疫荧光检测焦亡相关蛋白Caspase-1荧光的表达量;采用qRT-PCR技术检测各组NCM460中NLRP3/Caspase-1通路中焦亡相关NLRP3、Caspase-1、GSDMD-N、IL-1β基因表达水平的影响;Western blot技术检测各组焦亡相关NLRP3、Caspase-1、IL-1β蛋白表达水平的影响。结果:与0μg/mL比较(20μg/mL)LPS诱导48 h对NCM460细胞生长抑制较为适中(P<0.01),同时,干预48 h时,炎症因子IL-1β、TNF-α含量明显升高(P<0.01),因此,选择(20μg/mL)LPS诱导48 h作为造模方式。与0μmol/L的Coixan相比,当Coixan浓度为10、20,40μmol/L时,细胞存活率增高,特别是Coixan浓度为10、20μmol/L(P<0.05)。与正常对照组比较,模型组中炎症因子IL-1β、TNF-α的含量,NLRP3、Caspase-1β、IL-1β蛋白和NLRP3、Caspase-1、GSDMD-N、IL-1β基因表达均显著升高(P<0.01);与模型组比较,药物干预组中,中、高剂量组和柳氮磺吡啶组中细胞IL-1β、TNF-α分泌量减少,差异有统计学意义(P<0.01);与模型组比较,药物干预组中,高剂量组和柳氮磺吡啶组中焦亡相关蛋白Caspase-1表达水平明显下调(P<0.01);与模型组比较,药物干预组中,低、中、高剂量组和柳氮磺吡啶组中NLRP3、Caspase-1蛋白和基因表达明显降低(P<0.05或P<0.01),药物干预组中,高剂量组、柳氮磺吡啶组中GSDMD-N、IL-1β基因表达均显著降低(P<0.01);与模型组比较,药物干预组中,中、高剂量组和柳氮磺吡啶组中焦亡相关蛋白(NLRP3,Caspase-1,IL-1β)表达水平明显下调(P<0.01)。结论:薏苡仁多糖可能通过下调焦亡相关因子NLRP3、Caspase-1、GSDMD-N、IL-1β的表达水平,从而抑制人结肠上皮细胞的细胞焦亡,发挥人结肠上皮细胞保护作用。

Objective:To investigate the intervention effect of coix seed polysaccharides(Coixan)on an in vitro inflammation model of ulcerative colitis(UC)and its mechanism from the perspective of in⁃flammation theory.Methods:NCM460 cells were cultured in vitro and were induced by different doses(0,2.5,5,10,20,and 40μg/mL)of lipopolysaccharide(LPS)to obtain the optimal induction dose of 20μg/mL,and NCM460 cells were induced by different concentra⁃tions(0,5,10,20,40,and 80μmol/L)of Coixan to obtain the optimal concentrations for promoting proliferation(10,20,and 40μmol/L).The cells were randomly divided into blank group,model group(LPS induction),LPS+low-,middle-,and high-dose Coixan(10,20,and 40μmol/L)groups,and LPS+salicylazosulfapyridine(SASP)(200μg/mL)group,and after all cells except those in the blank group were stimulated with LPS(20μg/mL)for 48 hours to establish an in vitro cell inflammation model of UC,Coixan and SASP were used for 48 hours of intervention.ELISA was used to measure the levels of the inflammatory factors interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)in supernatant;immunofluorescence assay was used to measure the expression level of the pyroptosis-related protein caspase-1;qRT-PCR was used to measure the gene expression levels of NLRP3,caspase-1,GSDMD-N,and IL-1βassociated with pyroptosis in the NLRP3/caspase-1 pathway;Western blot was used to measure the protein expression levels of NLRP3,caspase-1,and IL-1βassociated with pyroptosis in each group.Results:Compared with 0μg/mL LPS,20μg/mL LPS for 48 hours of induction moderately inhibited the growth of NCM460 cells(P<0.01),and meanwhile,there was a significant increase in the content of the inflammatory factors IL-1βand TNF-αat 48 hours of intervention(P<0.01);therefore,20 ug/mL LPS induction for 48 hours was selected for modeling.Compared with 0μmol/L Coixan,there was an increase in cell viability after intervention with Coixan at concentrations of 10,20,and 40μmol/L,especially Coixan at concentrations of 10 and 20μmol/L(P<0.05).Compared with the normal control group,the model group had significant increases in the levels of the inflammatory factors IL-1βand TNF-α,the protein expression levels of NLRP3,caspase-1,and IL-1β,and the gene expression levels of NLRP3,caspase-1,GSDMD-N,and IL-1β(P<0.01);compared with the model group,the middle-and highdose groups and the SASP group had significant reductions in the secretion of IL-1βand TNF-α(P<0.01).Compared with the model group,the middle-and high-dose groups and the SASP group had a significant reduction in the expression level of the pyroptosisrelated protein caspase-1(P<0.01);compared with the model group,the low-,middle-,and high-dose groups and the SASP group had significant reductions in the protein and gene expression levels of NLRP3 and caspase-1(P<0.05 or P<0.01),and the middle and high-dose groups and the SASP group had significant reductions in the gene expression levels of GSDMD-N and IL-1β(P<0.01);compared with the model group,the middle-and high-dose groups and the SASP group had significant reductions in the expression levels of the pyroptosis-related proteins NLRP3,caspase-1,and IL-1β(P<0.01).Conclusions:Coixan may exert a protective effect on human intestinal epithelial cells by downregulating the expression levels of the pyroptosis-related factors NLRP3,caspase-1,GSDMD-N,and IL-1βand inhibiting the pyroptosis of intestinal epithelial cells.