基于NLRP3/Caspase-1通路探讨银屑平丸含药血清对TNF-α诱导的人表皮角质形成细胞焦亡的影响

Effects of serum containing Yinxieping Pill on TNF-α-induced pyroptosis of human epidermal keratinocytes based on the NLRP3/Caspase-1 pathway

目的探讨银屑平丸含药血清通过调控NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor thermal protein domain associated protein 3,NLRP3)/胱天蛋白酶-1(cysteine aspartate-specific proteinase-1,Caspase-1)信号通路改善肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)诱导的人表皮角质形成细胞焦亡的作用机制。方法制备银屑平丸含药血清、阿维A含药血清及空白血清,采用CCK-8试剂检测空白血清对人表皮角质形成细胞存活率的影响;体外常规培养人表皮角质形成细胞,使用TNF-α诱导人表皮角质形成细胞建立体外银屑病损伤模型,设置正常组、模型组、空白血清组、银屑平丸含药血清组、阿维A含药血清组;ELISA法检测各组细胞上清液中白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-18(interleukin-18,IL-18)水平;RT-PCR检测各组细胞NLRP3,功能蛋白D(gasdermin D,GSDMD)、Caspase-1 mRNA表达;Western blot法检测各组细胞NLRP3、GSDMD、Caspase-1蛋白的表达。结果与正常组比较,空白血清干预后细胞存活率无明显改变。与正常组比较,模型组和空白血清组细胞上清中IL-1β、IL-18水平显著升高(P<0.01),且NLRP3、GSDMD、Caspase-1蛋白及mRNA表达水平显著升高(P<0.05);与模型组比较,银屑平丸含药血清组及阿维A含药血清组细胞上清IL-1β、IL-18蛋白及mRNA水平明显下降(P<0.01),NLRP3、GSDMD、Caspase-1蛋白及mRNA表达显著下降(P<0.01)。结论银屑平丸含药血清可下调TNF-α诱导的人表皮角质形成细胞NLRP3、GSDMD、Caspase-1蛋白及mRNA表达,降低炎症因子表达水平,其作用机制可能与调控NLRP3/Caspase-1通路有关。

Objective To investigate the mechanism of action of serum containing Yinxieping Pill(SCYXPP)in improving pyroptosis of human epidermal keratinocytes induced by tumor necrosis factor-α(TNF-α)through regulating the NOD-like receptor thermal protein domain associated protein 3(NLRP3)/cysteine aspartate-specific proteinase-1(Caspase-1)signaling pathway.Methods SCYXPP,serum containing Acitretin(SCA),and blank serum were prepared.CCK-8 reagent was used to detect the effect of blank serum on cell viability of the human epidermal keratinocytes.Human epidermal keratinocytes were cultured in vitro under conventional conditions and TNF-α was applied to them to establish an in vitro psoriasis injury model.Then normal,model,blank serum,SCYXPP,and SCA groups were set up.The levels of interleukin-1β(IL-1β)and interleukin-18(IL-18)in the cell supernatant of each group were determined by ELISA,the mRNA expressions of NLRP3,gasdermin D(GSDMD),and Caspase-1 in the cells of each group were examined by RT-PCR,and the protein expressions of NLRP3,GSDMD,and Caspase-1 were checked by Western blot.Results Compared with the normal group,there was no significant change in cell viability in the blank serum group.Compared with the normal group,the levels of IL-1β and IL-18 in the cell supernatant of the model and blank serum groups were significantly elevated(P<0.01),and the mRNA and protein expressions of NLRP3,GSDMD,and Caspase-1 were significantly higher(P<0.05).Compared with the model group,the IL-1β and IL-18 levels in the cellular supernatant of SCYXPP and SCA groups significantly decreased(P<0.01),and the mRNA and protein expressions of NLRP3,GSDMD,and Caspase-1 were significantly reduced(P<0.01).Conclusion SCYXPP can down-regulate the mRNA and protein expressions of NLRP3,GSDMD,and Caspase-1 in human epidermal keratinocytes induced by TNF-α,and reduce the expression levels of inflammatory factors.Its mechanism of action may be related to the regulation of the NLRP3/Caspase-1 pathway.