芍药苷通过STAT3抑制IL-13诱导的BEAS-2B细胞氧化应激和自噬

Paeoniflorin inhibits IL-13-induced oxidative stress and autophagy in BEAS-2B cells via STAT3

目的:评估芍药苷通过信号转导与转录激活子3(signal transducer and activator of transcriptions 3,STAT3)对白细胞介素-13(interleukin-13,IL-13)诱导的BEAS-2B哮喘细胞模型的炎症、氧化应激和自噬的改善作用。方法:用1、10和30μmol·L^(-1)的芍药苷干预IL-13诱导前后的BEAS-2B细胞,CCK-8检测细胞增殖活力,筛选芍药苷最佳作用条件。然后利用芍药苷和STAT3激活剂干预IL-13诱导的细胞模型,将细胞分为对照组、模型组、STAT3激活剂组、芍药苷组和STAT3激活剂+芍药苷共处理组。CCK-8检测各组细胞增殖活力,ELISA试剂盒检测各组细胞上清中IL-4、IFN-γ的含量,及细胞中MDA、CAT、SOD水平,Western blot检测各组细胞LC3Ⅱ、LC3Ⅰ、P62、p-STAT3和STAT3的蛋白表达。结果:CCK-8检测结果显示,10μmol·L^(-1)的芍药苷作用24 h为最佳干预条件。与对照组相比,模型组细胞增殖能力下降(P<0.05),IFN-γ、SOD和CAT含量下降(P<0.05),IL-4和MDA含量增加(P<0.05),LC3Ⅱ/Ⅰ蛋白表达比值和p-STAT3/STAT3蛋白表达比值增加(P<0.05),P62蛋白表达下降(P<0.05)。与模型组相比,STAT3激动剂组细胞增殖能力下降(P<0.05),IFN-γ、SOD和CAT含量下降(P<0.05),IL-4和MDA含量增加(P<0.05),LC3Ⅱ/Ⅰ蛋白表达比值和p-STAT3/STAT3蛋白表达比值增加(P<0.05),P62蛋白表达下降(P<0.05);芍药苷组细胞增殖能力增加(P<0.05),IFN-γ、SOD和CAT含量增加(P<0.05),IL-4和MDA含量下降(P<0.05),LC3Ⅱ/Ⅰ蛋白表达比值和p-STAT3/STAT3蛋白表达比值下降(P<0.05),P62蛋白表达增加(P<0.05)。与STAT3激动剂组相比,STAT3激动剂+芍药苷组共处理逆转了STAT3激动剂的作用。结论:芍药苷通过抑制STAT3蛋白磷酸化,抑制炎症反应,从而改善哮喘中细胞氧化应激和自噬。

OBJECTIVE To evaluate the effects of paeoniflorin on interleukin-13(IL-13)-induced inflammation,oxidative stress and autophagy in BEAS-2B asthma cell model via signal transducer and activator of transcriptions 3(STAT3).METHODS BEAS-2B cells were intervened with paeoniflorin(1,10,30μmol·L^(-1))before and after IL-13 induction.Cell proliferation activity was detected by CCK-8 and optimal conditions of paeoniflorin were screened.Then paeoniflorin and STAT3 activators were utilized for intervening IL-13-induced cell models.The cells were assigned into five groups of control,model,STAT3 activator,paeoniflorin and STAT3 activator+paeoniflorin co-treatment.Cell proliferation activity was detected by CCK-8;IL-4 and IFN-γcontents in supernatant of each group were detected by enzyme-linked immunosorbent assay(ELISA);MDA,CAT and SOD levels were detected by ELISA.And the protein expressions of LC3Ⅱ,LC3Ⅰ,P62,p-STAT3 and STAT3 in each group were detected by Western blot.RESULTS The results of CCK-8 indicated that 10μmol·L^(-1)of paeoniflorin for 24 h was the optimal intervention.As compared with control group,cell proliferation capacity dropped(P<0.01),the contents of IFN-γ,SOD and CAT declined(P<0.01),the contents of IL-4 and MDA spiked(P<0.01),the ratio of LC3Ⅱ/Ⅰ protein expression and the ratio of p-STAT3/STAT3 protein expression rose(P<0.05),and the expression of P62 protein decreased in model group(P<0.01).Compared with model group,proliferation capability of cells(P<0.01)and contents of IFN-γ,SOD and CAT decreased(P<0.01)while the contents of IL-4 and MDA spiked in STAT3 agonist group(P<0.01).LC3II/I and p-STAT3/STAT3 protein expression ratios rose(P<0.05)while there was a down-regulation of P62 protein expression(P<0.01).In paeoniflorin group,cell proliferation capacity spiked(P<0.01),IFN-γ,SOD and CAT contents jumped(P<0.01),IL-4 and MDA contents dropped(P<0.01)and LC3Ⅱ/Ⅰ and p-STAT3/STAT3 protein expression ratios decreased(P<0.01),and the expression of P62 protein became up-regulated(P<0.01).As compared with STAT3 agonist group,the effect of STAT3 agonist was reversed after a co-treatment of STAT3 agonist and paeoniflorin.CONCLUSION Paeoniflorin may suppress inflammatory responses through blunting the phosphorylation of STAT3 protein and improve oxidative stress and autophagy of asthma cell.