摘要
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、1 μg·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0.50 μg·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.
Abstract
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 py-roptosis 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 1 μg·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,lac-tate dehydrogenase(LDH),interleukin(IL)-18,and tumor necrosis factor(TNF)-α release.Then,J774A.1 macrophages were ran-domly 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 re-sults showed that the stimulation condition of 0.50 μg·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-18,and TNF-α.FTRAs were able to signifi-cantly inhibit the activation of GSDMD proteins and significantly down-regulate the protein expression of the pyroptosis pathway signa-ture 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 clas-sical and non-classical pyroptosis signaling pathways and reducing the production of inflammatory cytokines.
基金项目
国家自然科学基金(81873067)
泸州市科技局项目(2022-JYJ-124)
西南医科大学校级自然科学基金(2020ZRQNB006)
NETL
NSTL
万方数据