目的 观察泛素连接酶9(ubiquitin conjugating enzyme 9,UBC9)在表没食子儿茶素没食子酸酯(epigallocatechin-3-gallate,EGCG)作用的脂多糖(lipopolysaccharide,LPS)诱导A549细胞中的表达,探讨UBC9与EGCG抗炎性反应机制的相关性。方法 根据筛选结果随机分为对照组、EGCG组、LPS组、EGCG＋LPS组4个组,MTT检测不同浓度的EGCG(50～400 μg/ml)和LPS(10～50 μg/ml)对A549细胞增殖活性的影响,筛选最佳作用浓度,进一步通过免疫组化染色法、蛋白质印迹法和Realtime-PCR检测分析各处理组UBC9蛋白及其mRNA的相对表达量。结果 MTT显示,200 μg/ml的EGCG细胞增殖抑制作用最强(F=1618.18,P＜0.001),LPS 25μg/ml细胞增殖活性最明显(F=237.38,P＜0.001)。免疫组化与蛋白质印迹检测均显示,与对照组比较,EGCG下调A549细胞UBC9蛋白表达(P＜0.001),LPS则上调UBC9表达量(P＜0.001),与LPS组比较,EGCG＋LPS组UBC9蛋白表达明显降低(P＜0.001)。同样,Realtime-PCR显示,与对照组比较,EGCG组UBC9 mRNA表达下调(F=89.34,P＜0.001),LPS组UBC9 mRNA表达上调(F=225.00,P＜0.001),与LPS组比较,EGCG＋LPS组UBC9 mRNA的表达受到抑制,显著降低(F=625.00,P＜0.001)。结论 EGCG下调LPS刺激的炎性反应模型的UBC9的表达,其抗炎机制可能与抑制UBC9蛋白与mRNA表达有关。
Abstract
Objective To observe the effect of EGCG on the expression of UBC9 in the A549 inflammatory model, and investigate its correlation with the anti-inflammatory mechanism of EGCG.Methods A549 cells were randomly divided in vitro into four group: control group, EGCG group, LPS group, and EGCG plus LPS group. The proliferative activity of A549 cells, which were incubated with different concentrations of EGCG(50~400 μg/ml) and LPS(10 ~50 μg/ml), was detected by MTT method to select the optimum concentration for experiment. The relative expressions of UBC9 protein and mRNA in each group were determined with immunohistochemical staining, Western blot and real-time-PCR, and compared.Results MTT showed that 200 μg/ml EGCG had the strongest inhibitory effect on proliferation of A549 cells(F=16181.18, P<0.001), while 25 μg/ml LPS had the strongest promoting effect (F=237.38, P<0.001). As observed with immunohistochemistry and Western blot, EGCG down-regulated the expression of UBC9 protein(P<0.001) while LPS up-regulated the expression(P<0.001) compared to control group. Compared to LPS group, the expression of UBC9 protein in EGCG plus LPS group was reduced significantly(P<0.001). Similarly, Real-time-PCR revealed that the expression of UBC9 mRNA decreased in EGCG group(F=89.34, P<0.001), but increased in LPS group(F=225.00, P<0.001) compared with control group. Compared to LPS group, the expression of UBC9 mRNA in EGCG plus LPS group was inhibited and reduced significantly(F=625.00, P<0.001).Conclusions EGCG can down-regulate the expression of UBC9 in the inflammatory model induced by LPS.The anti-inflammatory mechanism of EGCG might be mediated by suppression of UBC9 protein and mRNA expression.
关键词
表没食子儿茶素没食子酸酯 /
脂多糖 /
泛素连接酶9 /
炎性反应
Key words
epigallocatechin gallate /
lipopolysaccharide /
ubiquitin-conjugating enzyme 9 /
inflammation
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基金
山东省自然科学基金资助课题 (ZR2012CM008)
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