华支睾吸虫排泄/分泌产物对肝纤维化的影响

Effect of excretory/secretory products of Clonorchis sinensis on liver fibrosis

为了探讨华支睾吸虫排泄/分泌产物(ESPs)对大鼠肝星状细胞(HSCs)的影响,采用不同质量浓度的华支睾吸虫ESPs(CsESPs)处理HSC-T6细胞,利用CCK8法检测CsESPs对HSC-T6细胞活力的影响,并以MCC950预处理细胞作为对照,利用RT-qPCR和Western-blot分别检测细胞中NLRP3、Caspase-1、IL-1β、α-SMA、Col1α1和Col3α1的基因和蛋白表达水平。结果显示,与对照组相比,25、50和100μg/mL的CsESPs对细胞活力无明显影响;当CsESPs的质量浓度达到50μg/mL时,与对照组相比,NLRP3及其下游的Caspase-1、IL-1β基因的相对表达水平显著升高,HSC-T6细胞激活的标志基因α-SMA及纤维化相关的Col1α1基因的相对表达水平也显著升高;Western-blot结果显示,与对照组相比,50μg/mL的CsESPs可以使HSC-T6细胞中NLRP3、Caspase-1、IL-1β、α-SMA、Col1α1及Col3α1的蛋白表达水平显著升高,而在MCC950预处理细胞组,这6个指标的基因和蛋白表达水平均被明显抑制。结果表明,CsESPs可以使HSC-T6细胞活化,诱导细胞内NLRP3炎性小体活化及胶原表达,NLRP3炎性小体活化是华支睾吸虫感染所致肝纤维化的关键环节之一。

In order to investigate the effect of Clonorchis sinensis excretory/secretory products(ESPs)on rat hepatic stellate cell(HSCs),the HSC-T6 cells were treated with different concentrations of CsESPs,and the effects of CsESPs on cell viability were detected by CCK8 method,and at the same time,MCC950 pretreated cells were used as a control,and RT-qPCR and Western-blot were used to detect the gene and protein expression levels of NLRP3,Caspase-1,IL-1β,α-SMA,Col1α1 and Col3α1 in HSC-T6 cells.The results showed that,compared to the control group,25,50 and 100μg/mL CsESPs had no significant effect on cell viability.When the concentration of CsESPs reached 50μg/mL,the relative gene expression levels of NLRP3 and its downstream Caspase-1 and IL-1βwere significantly increased compared to the control group,and the mRNA expression levels ofα-SMA as the marker gene for HSC-T6 cells activation,and Col1α1 as the fibrosis gene,were also significantly higher compared to the control group.Western-blot results showed that,compared to the control group,50μg/mL CsESPs significantly increased the protein expression levels of NLRP3,Caspase-1,IL-1β,α-SMA,Col1α1 and Col3α1 in HSC-T6 cells.But at MCC950 pretreatment groups,the genes and protein expression levels of these six indicators were significantly inhibited.The results indicated that CsESPs can activate HSC-T6 cells and induce the activation of NLRP3 inflammasome and collagen expression in cells.The activation of NLRP3 inflammasome was one of the key links of liver fibrosis caused by Clonorchis sinensis infection.