扁蒴藤素调节CCL2-CCR2信号轴对LPS诱导的肺泡上皮细胞损伤的影响

Effect of pristimerin on LPS-induced alveolar epithelial cell injury by regulating the CCL2-CCR2 signaling axis

目的 探究扁蒴藤素(PT)调节CC趋化因子配体2(CCL2)-CC趋化因子受体2(CCR2)信号轴对脂多糖(LPS)诱导的肺泡上皮细胞损伤的影响。方法 常规培养肺泡上皮细胞A549,将其随机分为Control组(正常培养)、LPS组(10μg/mL LPS处理)、PT-L组(1μmol/L PT)、PT-H组(2μmol/L PT)和PT-H+RS504393组(2μmol/L PT+50 ng/mL CCL2抑制剂RS504393)。采用CCK-8法检测细胞活力;采用EdU实验测定细胞增殖情况;采用流式细胞术检测细胞凋亡情况;采用酶联免疫吸附试验(ELISA)检测细胞炎性因子肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、IL-6水平;采用Western Blot检测各组细胞中CCL2、CCR2和凋亡相关蛋白表达水平。结果 与Control组比较,LPS组A549细胞吸光度(A)450(48、72 h)、细胞增殖率、Bcl-2蛋白表达水平下降(P<0.05),细胞凋亡率、TNF-α、IL-1β、IL-6水平,CCL2、CCR2、Bax蛋白表达水平上升(P<0.05)。与LPS组比较,PT-L组、PT-H组A549细胞A450(48、72 h)、细胞增殖率、Bcl-2蛋白表达水平上升(P<0.05),细胞凋亡率、TNF-α、IL-1β、IL-6水平,CCL2、CCR2、Bax蛋白表达水平下降(P<0.05)。CCL2抑制剂RS504393促进了PT对LPS诱导的肺泡上皮细胞损伤的改善作用。结论 PT可能通过下调CCL2-CCR2信号轴对LPS诱导的肺泡上皮细胞损伤具有改善作用。

Objective To investigate the effect of pristimerin(PT)on lipopolysaccharide(LPS)induced alveolar epithelial cell injury by regulating the CC chemokine ligand 2(CCL2)-CC chemokine receptor 2(CCR2)signaling axis.Methods A549 alveolar epithelial cells were routinely cultured and randomly separated into control group(normally cultured),LPS group(treated with 10μg/mL LPS),PT-L group(1μmol/L PT),PT-H group(2μmol/L PT)and PT-H+RS504393 group(2μmol/L PT+50 ng/mL RS504393).CCK-8 method was applied to detect cell activity.EdU experiment was applied to measure cell proliferation.Flow cytometry was applied to detect cell apoptosis.Enzyme linked immunosorbent assay(ELISA)was applied to detect the levels of inflammatory factors such as tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and IL-6 in cells.Western Blot was applied to detect the expression levels of CCL2,CCR2 and apoptosis related proteins of cells in each group.Results Compared with the control group,the absorbance(A)450(48,72 h),cell proliferation rate,and Bcl-2 protein expression level of A549 cells in the LPS group were greatly reduced(P<0.05),and the apoptosis rate,TNF-α,IL-1β,IL-6 levels,CCL2,CCR2,and Bax protein expression levels were greatly increased(P<0.05).Compared with the LPS group,the A 450(48,72 h),cell proliferation rate,and Bcl-2 protein expression level of A549 cells in the PT-L and PT-H groups were greatly increased(P<0.05),and the apoptosis rate,TNF-α,IL-1β,IL-6 levels,CCL2,CCR2,and Bax protein expression levels were greatly decreased(P<0.05).The CCL2 inhibitor RS504393 promoted the improvement effect of PT on LPS induced alveolar epithelial cell injury.Conclusion PT may improve LPS-induced alveolar epithelial cell injury by down-regulating the CCL2-CCR2 signaling axis.