中华急诊医学杂志
中華急診醫學雜誌
중화급진의학잡지
CHINESE JOURNAL OF EMERGENCY MEDICINE
2014年
4期
371-376
,共6页
祁蕾%傅强%崔乃强%张国倩
祁蕾%傅彊%崔迺彊%張國倩
기뢰%부강%최내강%장국천
脂多糖%缺氧诱导因子-1α%肠上皮细胞%大黄素%环氧化酶-2
脂多糖%缺氧誘導因子-1α%腸上皮細胞%大黃素%環氧化酶-2
지다당%결양유도인자-1α%장상피세포%대황소%배양화매-2
LPS%HIF-1α%Intestinal epithelial cell%Emodin%COX-2
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 ±0.02、0.89±0.03、0.88 ±0.04、0.91 ±0.03、0.83±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.
目的 觀察脂多糖(LPS)處理腸上皮細胞對缺氧誘導因子-1α (HIF-1α)及下遊炎癥相關靶基因環氧化酶-2 (COX-2)錶達的影響,探討大黃素榦預的可能作用靶點.方法 建立LPS處理人腸上皮細胞的體外實驗模型.①用Western blot檢測LPS不同劑量和不同時間處理組的HIF-1α和COX-2蛋白錶達的變化趨勢;檢測LPS和不同劑量大黃素共同榦預組的HIF-1α、COX-2、Phospho-IκB-α和Phospho-NF-κB p65蛋白錶達的變化趨勢.②用PCR檢測LPS處理組與LPS和大黃素共同榦預組HIF-1α的mRNA水平.③用MTT法檢測大黃素對腸上皮細胞增殖情況的影響.數據採用單因素方差分析,以P＜0.05為差異具有統計學意義.結果 ①LPS可引起HIF-1α錶達升高,且有時間和劑量效應關繫.隨LPS質量濃度的增加,HIF-1α的錶達量先增高後降低,在質量濃度為10-3 mg/mL時HIF-1α錶達量最高(P＜0.05);HIF-1α錶達首先在0.5h達峰值,而後到4h下降至最低,最後又增高(P＜0.05).COX-2蛋白錶達變化趨勢與HIF-1α基本一緻(P＜0.05).大黃素可抑製LPS誘導的HIF-1α、COX-2、Phospho-IκB-α和Phospho-NF-κB p65的錶達,併有明顯量效關繫(P＜0.05).②PCR檢測LPS刺激後HIF-1α的mRNA水平增高,而大黃素抑製其增高.③MTT法檢測不同濃度的大黃素(0μmol/L、20 μmol/L、40μmol/L、60 μmol/L、80 μmol/L)對細胞增殖無明顯影響(0.95 ±0.02、0.89±0.03、0.88 ±0.04、0.91 ±0.03、0.83±0.03,P＞0.05).雖然大黃素在此濃度範圍產生瞭生物學效應,但對腸細胞無藥物毒性.結論 LPS激活腸上皮細胞HIF-1α/COX-2信號通路,且具有時間和劑量依賴性.大黃素可能通過阻斷LPS/HIF-1 α/COX-2的缺氧通路和LPS/IκB-α/NF-κB/COX-2的炎癥通路而對腸上皮細胞起到保護作用.
목적 관찰지다당(LPS)처리장상피세포대결양유도인자-1α (HIF-1α)급하유염증상관파기인배양화매-2 (COX-2)표체적영향,탐토대황소간예적가능작용파점.방법 건립LPS처리인장상피세포적체외실험모형.①용Western blot검측LPS불동제량화불동시간처리조적HIF-1α화COX-2단백표체적변화추세;검측LPS화불동제량대황소공동간예조적HIF-1α、COX-2、Phospho-IκB-α화Phospho-NF-κB p65단백표체적변화추세.②용PCR검측LPS처리조여LPS화대황소공동간예조HIF-1α적mRNA수평.③용MTT법검측대황소대장상피세포증식정황적영향.수거채용단인소방차분석,이P＜0.05위차이구유통계학의의.결과 ①LPS가인기HIF-1α표체승고,차유시간화제량효응관계.수LPS질량농도적증가,HIF-1α적표체량선증고후강저,재질량농도위10-3 mg/mL시HIF-1α표체량최고(P＜0.05);HIF-1α표체수선재0.5h체봉치,이후도4h하강지최저,최후우증고(P＜0.05).COX-2단백표체변화추세여HIF-1α기본일치(P＜0.05).대황소가억제LPS유도적HIF-1α、COX-2、Phospho-IκB-α화Phospho-NF-κB p65적표체,병유명현량효관계(P＜0.05).②PCR검측LPS자격후HIF-1α적mRNA수평증고,이대황소억제기증고.③MTT법검측불동농도적대황소(0μmol/L、20 μmol/L、40μmol/L、60 μmol/L、80 μmol/L)대세포증식무명현영향(0.95 ±0.02、0.89±0.03、0.88 ±0.04、0.91 ±0.03、0.83±0.03,P＞0.05).수연대황소재차농도범위산생료생물학효응,단대장세포무약물독성.결론 LPS격활장상피세포HIF-1α/COX-2신호통로,차구유시간화제량의뢰성.대황소가능통과조단LPS/HIF-1 α/COX-2적결양통로화LPS/IκB-α/NF-κB/COX-2적염증통로이대장상피세포기도보호작용.
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 ± 0.02,0.89 ± 0.03,0.88 ± 0.04,0.91 ± 0.03,0.83 ± 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.
