大黄游离蒽醌对J774A.1巨噬细胞焦亡的影响及机制研究

Effect and mechanism of free total rhubarb anthraquinones on pyroptosis of J774A.1 macrophages

该研究以脂多糖(LPS)+三磷酸腺苷(ATP)刺激的J774A.1巨噬细胞建立细胞焦亡体外模型,探讨大黄游离蒽醌(free total rhubarb anthraquinones,FTRAs)对细胞焦亡的干预机制。体外培养J774A.1巨噬细胞,实验分组为空白对照组、不同质量浓度脂多糖(LPS,0.25、0.5、1μg·mL^(-1))+ATP(1.25、2.5、5 mmol·L^(-1))组,通过CCK-8法检测细胞活力、碘化丙锭(PI)凋亡细胞染色法、乳酸脱氢酶(LDH)及白细胞介素(IL)-18和肿瘤坏死因子(TNF)-α释放,建立巨噬细胞焦亡体外模型。之后将J774A.1巨噬细胞随机分为6组:空白对照组,LPS+ATP组,FTRAs高剂量单独作用组,FTRAs低、中、高剂量预保护组。检测上述细胞焦亡的表型特征和关键指标作为评判FTRAs对LPS+ATP诱导的细胞焦亡的影响依据。Western blot法和RT-PCR法检测半胱氨酸蛋白酶(caspase)-1/11细胞焦亡通路相关蛋白和mRNA表达水平,阐明其抗焦亡效应的分子机制。结果显示巨噬细胞焦亡体外模型以0.50μg·mL^(-1)LPS+5.00 mmol·L^(-1)ATP的刺激条件效果最佳。FTRAs预保护细胞24 h,能够提高焦亡条件下细胞的活力、减轻细胞的受损程度、降低PI染色阳性率并减少LDH、IL-18和TNF-α的释放。FTRAs能够明显抑制GSDMD蛋白的活化,并且显著下调焦亡通路特征分子TLR4、NLRP3、cleaved-caspase-1、cleaved-caspase-11的蛋白表达,但是对ASC蛋白无明显影响。FTRAs还能够明显抑制caspase-1、caspase-11和GSDMD的mRNA表达。这些研究结果表明,FTRAs对LPS+ATP诱导的细胞焦亡模型具有抑制作用,且FTRAs通过调控经典和非经典细胞焦亡信号通路,减少炎性炎症因子的产生,发挥抗焦亡效应。

In this study,J774A.1 macrophages stimulated by lipopolysaccharide(LPS)and adenosine triphosphate(ATP)were used to establish an in vitro model of pyroptosis,and the intervention mechanism of free total rhubarb anthraquinones(FTRAs)on pyroptosis was investigated.J774A.1 macrophages were cultured in vitro,and the experiment was assigned to the control group and groups with different concentrations of LPS(0.25,0.5,and 1μg·mL^(-1))and ATP(1.25,2.5,and 5 mmol·L^(-1)).An in vitro model of macrophage pyroptosis was established by detecting cell viability through CCK-8,propidium iodide(PI)apoptotic cell staining,lactate dehydrogenase(LDH),interleukin(IL)-18,and tumor necrosis factor(TNF)-αrelease.Then,J774A.1 macrophages were randomly divided into six groups:blank control group,LPS+ATP group,high-dose FTRA group,and low,medium,and high-dose FTRA pre-protection group.The phenotypic characteristics and key indicators of pyroptosis were detected as the basis for evaluating the effect of FTRAs on pyroptosis induced by LPS and ATP.Western blot and RT-PCR were used to detect the expression levels of protein and mRNA related to the pyroptosis pathway in caspase-1/11 and elucidate the molecular mechanism of the anti-pyroptosis effect.The results showed that the stimulation condition of 0.50μg·mL^(-1) LPS+5.00 mmol·L^(-1) ATP was the most effective in the in vitro model of macrophage pyroptosis.FTRAs pre-protected cells for 24 h and then can increase cell viability under pyroptosis conditions,alleviate cell damage,lower the positive rate of PI staining,and reduce the release of LDH,IL^(-1)8,and TNF-α.FTRAs were able to significantly inhibit the activation of GSDMD proteins and significantly down-regulate the protein expression of the pyroptosis pathway signature molecules,TLR4,NLRP3,cleaved-caspase-1,and cleaved-caspase-11,but they had no significant effect on ASC proteins.FTRAs were also able to significantly inhibit the mRNA expression of caspase-1,caspase-11,and GSDMD.These results indicate that FTRAs have an inhibitory effect on the pyroptosis model induced by LPS and ATP and play an anti-pyroptosis effect by regulating classical and non-classical pyroptosis signaling pathways and reducing the production of inflammatory cytokines.