青蒿素对人皮肤瘢痕疙瘩成纤维细胞的抑制作用及相关机制初探
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上海市同仁医院院级课题(TRYJ201609);上海市长宁区科委课题(CNKW2018Y08);上海市长宁区卫计委课题(20174Y003);上海交通大学医工交叉基金(YG2017QN59)


Preliminary study on the inhibitory effects of artemisinin on human skin keloid fibroblasts and its related mechanism
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    摘要:

    目的:探讨青蒿素对人皮肤瘢痕疙瘩成纤维细胞的抑制作用及相关机制。方法:进行人瘢痕疙瘩成纤维细胞的原代分离、培养和传代。不同浓度青蒿素分别预处理人瘢痕疙瘩成纤维细胞,每隔24 h用CCK-8法测定细胞增殖情况,连续测定5 d,观察药物处理的最佳浓度。后续实验分为空白组、青蒿素组、青蒿素+IL-6组、青蒿素+AG490组,流式细胞术检测各组早期细胞凋亡率,real-time PCR法检测各组基质金属蛋白酶(matrix metalloproteinase,MMP)2、MMP9、STAT3 mRNA的表达水平,Western blot法检测各组MMP2、MMP9、STAT3、p-STAT3蛋白的表达水平。结果:根据CCK-8实验结果选择150 μg/mL青蒿素作用48 h的细胞为后续实验干预浓度和时间。流式细胞术结果显示,青蒿素组、青蒿素+IL-6组、青蒿素+AG490组早期凋亡细胞百分比明显高于空白组,青蒿素+IL-6组的早期凋亡细胞百分比低于青蒿素组和青蒿素+AG490组(P < 0.01)。real-time PCR检测结果显示,与空白组比较,青蒿素组、青蒿素+IL-6组、青蒿素+AG490组中MMP2、MMP9 mRNA的表达均显著降低(P < 0.01);MMP2、MMP9 mRNA在青蒿素组、青蒿素+AG490组的表达较青蒿素+IL-6组显著降低(P < 0.01)。Western blot检测结果显示,与空白组比较,青蒿素组、青蒿素+IL-6组、青蒿素+AG490组中STAT3、p-STAT3、MMP2、MMP9蛋白的表达均明显降低(P < 0.01),p-STAT3、MMP2、MMP9蛋白在青蒿素组、青蒿素+AG490组中的表达低于在青蒿素+IL-6组中的表达(P < 0.01)。结论:青蒿素对瘢痕疙瘩起抑制作用,其机制可能通过抑制p-STAT3的过度激活来抑制MMP2、MMP9的表达。

    Abstract:

    Objective:To investigate the inhibitory effect of artemisinin on human skin keloid fibroblasts and its related mechanism. Methods:Primary isolation,culture and passage of human keloid fibroblasts were performed. Human keloid fibroblasts were pretreated with different concentrations of artemisinin,and cell proliferation was measured by CCK-8 method every 24 hours for 5 consecutive days to observe the optimal concentration of drug treatment. The following experiments were divided into four groups:blank group,artemisinin group,artemisinin+IL-6 group and artemisinin+AG490 group. Flow cytometry was used to detect the apoptosis rate in the early stage of each group,real-time PCR was used to detect the mRNA expression levels of matrix metalloproteinase(MMP)2,MMP9 and STAT3 in each group,and Western blot was used to detect the protein expression levels of MMP2,MMP9,STAT3 and p-STAT3 in each group. Results:According to the results of CCK-8,150 μg/mL artemisinin cells for 48 hours were selected as the concentration and time of follow-up intervention. Flow cytometry results showed that the percentage of early apoptotic cells in artemisinin group,artemisinin+IL-6 group and artemisinin+AG490 group was significantly higher than that in the blank group,and the percentage of early apoptotic cells in artemisinin+IL-6 group was lower than that in artemisinin group and artemisinin+AG490 group(P < 0.01). Real - time PCR showed that compared with the blank group,the expression of MMP2 and MMP9 mRNA in artemisinin,artemisinin+IL-6 groups,artemisinin+AG490 group were significantly lower(P < 0.01). The expressions of MMP2,MMP9 mRNA in artemisinin,artemisinin+AG490 group were lower than in artemisinin+IL-6 group(P < 0.01). Western blot showed that compared with the blank group,the expressions of STAT3,p-STAT3,MMP2,MMP9 protein in artemisinin,artemisinin+IL-6 groups,artemisinin+ AG490 group were significantly lower(P < 0.01). The expressions of p-STAT3,MMP2,MMP9 protein in artemisinin,artemisinin+AG490 group were lower than in artemisinin+IL-6 group(P < 0.01). Conclusion:Artemisinin has an inhibitory effect on keloid,and its mechanism may inhibit the experession of MMP2,MMP9 by inhibiting the excessive activation of p-STAT3.

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殷 芳,许 瑾,张蓓蓓,权 哲,王婷婷,钱 苗,刘 玮,杜霁儒,支媛婷,宋宁静.青蒿素对人皮肤瘢痕疙瘩成纤维细胞的抑制作用及相关机制初探[J].南京医科大学学报(自然科学版),2020,(8):1119-1124

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  • 收稿日期:2019-10-18
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  • 在线发布日期: 2020-08-21
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