大黄素对脂多糖诱导肠上皮细胞缺氧诱导因子-1α/环氧化酶-2通路的干预

The effects of Rheum emodin on LPS-induced HIF-1α and COX-2 expression in intestinal epithelial cells

目的 观察脂多糖（LPS）处理肠上皮细胞对缺氧诱导因子-1α （HIF-1α）及下游炎症相关靶基因环氧化酶-2 （COX-2）表达的影响,探讨大黄素干预的可能作用靶点.方法 建立LPS处理人肠上皮细胞的体外实验模型.①用Western blot检测LPS不同剂量和不同时间处理组的HIF-1α和COX-2蛋白表达的变化趋势;检测LPS和不同剂量大黄素共同干预组的HIF-1α、COX-2、Phospho-IκB-α和Phospho-NF-κB p65蛋白表达的变化趋势.②用PCR检测LPS处理组与LPS和大黄素共同干预组HIF-1α的mRNA水平.③用MTT法检测大黄素对肠上皮细胞增殖情况的影响.数据采用单因素方差分析,以P＜0.05为差异具有统计学意义.结果 ①LPS可引起HIF-1α表达升高,且有时间和剂量效应关系.随LPS质量浓度的增加,HIF-1α的表达量先增高后降低,在质量浓度为10-3 mg/mL时HIF-1α表达量最高（P＜0.05）;HIF-1α表达首先在0.5h达峰值,而后到4h下降至最低,最后又增高（P＜0.05）.COX-2蛋白表达变化趋势与HIF-1α基本一致（P＜0.05）.大黄素可抑制LPS诱导的HIF-1α、COX-2、Phospho-IκB-α和Phospho-NF-κB p65的表达,并有明显量效关系（P＜0.05）.②PCR检测LPS刺激后HIF-1α的mRNA水平增高,而大黄素抑制其增高.③MTT法检测不同浓度的大黄素（0μmol/L、20 μmol/L、40μmol/L、60 μmol/L、80 μmol/L）对细胞增殖无明显影响（0.95 ＆#177;0.02、0.89＆#177;0.03、0.88 ＆#177;0.04、0.91 ＆#177;0.03、0.83＆#177;0.03,P＞0.05）.虽然大黄素在此浓度范围产生了生物学效应,但对肠细胞无药物毒性.结论 LPS激活肠上皮细胞HIF-1α/COX-2信号通路,且具有时间和剂量依赖性.大黄素可能通过阻断LPS/HIF-1 α/COX-2的缺氧通路和LPS/IκB-α/NF-κB/COX-2的炎症通路而对肠上皮细胞起到保护作用.

Objective To observe the level of hypoxia-inducible factor-1 alpha （HIF-1α） and its downstream target gene cyclooxygenase-2 （COX-2） in LPS-treated intestinal epithelial cells,and to explore the possible intervention targets of Rheum emodin.Methods Human intestinal epithelial cells were cultured in vitro treated with LPS to establish the experimental model.The protein level trends of HIF-1α and COX-2 were measured by Western blot in LPS dose-dependent and time-dependent manners.The protein level trends of HIF-1α,COX-2,Phospho-IκB-α and Phospho-NF-κB p65 were measured in LPS plus various concentrations of Rheum emodin treated groups.The expression of HIF-1α mRNA were detected by PCR after cells treated with LPS or LPS plus Rheum emodin,respectively.The effect of Rheum emodin on the proliferation of intestinal epithelial cells was measured by MTT assay in each group.Data were analyzed with ANOVA,and P 〈0.05 was considered significant.Results LPS induced the protein level of HIF-1α in a dose-dependent and a time-dependent manners.With increasing concentrations of LPS,the protein level of HIF-1α increased to the peak when cells were treated with LPS at 10-30mg/mL,and then gradually decreased （P 〈0.05）.Firstly the protein level of HIF-1α reached the peak at 0.5 h after treatment,and then decreased to the lowest level at 4 h,and finally returned to a high level （P〈0.05）.The protein level trend of COX-2 went a similar way to that of HIF-1α （P 〈0.05）.Rheum emodin inhibited the protein levels of LPS-induced HIF-1α,COX-2,Phospho-IκB-α and Phospho-NF-κB p65 with a significant dose-effect relationship （P 〈 0.05）.The PCR showed Rheum emodin inhibited LPS-induced increasing expression of HIF-1α mRNA.MTT assay showed different concentrations of Rheum emodin （0 μmol/L,20 μmol/L,40 μmol/L,60 μmol/L,80 μmol/L） had no significant effect on cell proliferation （0.95 ＆#177; 0.02,0.89 ＆#177; 0.03,0.88 ＆#177; 0.04,0.91 ＆#177; 0.03,0.83 ＆#177; 0.03,P 〉 0.05）.Although Rheum emodin produced biological effect at this concentration range,and it had no toxicity to intestinal cells.Conclusions LPS induces HIF-1α/COX-2 signaling pathway in a time-dependent and a dose-dependent manners in intestinal epithelial cells.Rheum emodin blocks the hypoxia pathway of LPS/HIF-1α/COX-2 and the inflammatory pathway of LPS/IκB-α/NF-κB/COX-2,which may play a protective effect on intestinal epithelial cells.