小檗碱对内毒素刺激致Caco-2细胞间高通透性的保护作用*

doi:10.3969/j.issn.1672-5069.2018.01.008

实用肝脏病杂志 ›› 2018, Vol. 21 ›› Issue (1): 34-37.doi: 10.3969/j.issn.1672-5069.2018.01.008

小檗碱对内毒素刺激致Caco-2细胞间高通透性的保护作用^*

李鑫, 王洪岩, 迟程, 徐有青

100050 北京市首都医科大学附属北京天坛医院消化内科（李鑫,迟程,徐有青）;
哈尔滨医科大学附属第二医院体检中心（王洪岩）

收稿日期:2017-03-31 出版日期:2018-01-10 发布日期:2018-01-29
通讯作者: 徐有青,E-mail:youqingxuttyy@163.com
作者简介:李鑫,女,35岁,医学博士,主治医师。主要从事酒精性肝病的防治研究。E-mail: lixinqingzhou@163.com
基金资助:
*国家自然科学基金资助项目（编号：81570536）; 北京市自然科学基金资助项目(编号：7154194)

Protective effect of berberine on endotoxin-induced intercellular hyperpermeability in Caco-2 cells in vitro

Li Xin, Wang Hongyan, Chi Cheng, et al

Department of Gastroenterology,Tiantan Hospital,Capital Medical University,Beijing 100050,China

Received:2017-03-31 Online:2018-01-10 Published:2018-01-29

摘要/Abstract

摘要： 目的探讨小檗碱（BBR）对脂多糖（LPS）刺激致Caco-2细胞间高通透性的保护作用。方法体外培养肠上皮细胞Caco-2,分为四组：①对照组（培养基）;②LPS组（培养基+LPS 2 μg/ml）;③BBR组（培养基+BBR 10 μM）;④BBR/LPS组（培养基+LPS 2 μg/ml+BBR 10μM）。在倒置显微镜下直接观察细胞生长形态,采用荧光黄透过率检测细胞间通透性,采用Western-blot法检测细胞间紧密连接主要结构蛋白occludin、细胞膜Toll样受体-4（TLR4）表达,采用免疫荧光法检测细胞TLR4下游分子MyD88表达。结果在倒置显微镜下可见对照组和BBR组细胞连接完整,LPS组细胞间出现锯齿样断裂,但在BBR/LPS组细胞间连接毛糙,但无明显片状中断;对照组和BBR组荧光黄透过浓度分别为（683.3±98.9）μg/ml和（849.0±89.7）μg/ml)以及细胞occludin、TLR4和MyD88蛋白表达差异均无统计学意义（P>0.05）;与对照组比,LPS组透过荧光黄浓度（1886±176.0 μg/ml）显著增加,TLR4蛋白表达增加,occludin表达减少,胞浆MyD88荧光信号增强;与LPS组比,BBR/LPS组透过荧光黄浓度（1071.0±65.9 μg/ml）显著下降,TLR4蛋白表达减弱,occludin表达增加,胞浆MyD88荧光信号减弱。结论 BBR通过减弱TLR4/MyD88活性,增加occludin表达,进而减轻LPS刺激致Caco-2细胞间高通透性。

关键词: Caco-2细胞, 小檗碱, 内毒素, 紧密连接

Abstract: Objective The aims of this study is to determine whether berberine（BBR） has a protective effect on lipopolysaccharide（LPS）-induced intercellular high permeability in Caco-2 cells in vitro. Methods Caco-2 cells was incubated and divided into four groups,e.g. control（cultured in Dulbecco's modified eagle medium）,LPS（co-incubated with medium and 2 μg/ml of LPS,BBR （co-incubated with medium and 10μM BBR） and BBR/LPS group （co-incubated with medium,2μg/ml of LPS and 10μM BBR）. The morphological features of cell growth were observed under inverted microscope. Paracellular permeability was assessed by measuring transepithelial fluorescein permeability. Tight junction protein occludin and Toll-like receptors-4（TLR4） were detected by Western blotting. The expression of downstream signal molecule,MyD88,was detected by immunofluorescence. Results We observed that the intercellular junction were long and thin in control and BBR group, while a saw tooth fracture in intercellular junctions was found in LPS group and a coarse margin but without interruption was observed in BBR/LPS group;there were no significantly differences between control and BBR group as respect to fluorescein permeability （683.3±; 98.9 μg/ml vs. 849.0±; 89.7 μg/ml,respectively）,and the expression of occludin,TLR4 and MyD88 （P>; 0.05）;the fluorescein penetrating concentration （1886±; 176.0 μg/ml） was much higher,TLR4 expression increased,occludin expression decreased,and enhanced MyD88 fluorescence signal in cytoplasm was observed in LPS group as compared with in control group（P<; 0.05）;the fluorescein penetrating concentration（1071.0±; 65.9 μg/ml） and TLR4 expression decreased,occludin expression increased, and weakened MyD88 fluorescence signal in cytoplasm was observed in BBR/LPS group as compared with LPS group （P<; 0.05）. Conclusion Our findings suggest that BBR alleviates LPS-induced intercellular high permeability by weakening TLR4/MyD88 activity and increasing the expression of occludin.

Key words: Caco-2 cells, Berberine, Lipopolysaccharide, Occludin, Tight junction

李鑫, 王洪岩, 迟程, 徐有青. 小檗碱对内毒素刺激致Caco-2细胞间高通透性的保护作用^*[J]. 实用肝脏病杂志, 2018, 21(1): 34-37.

Li Xin, Wang Hongyan, Chi Cheng, et al. Protective effect of berberine on endotoxin-induced intercellular hyperpermeability in Caco-2 cells in vitro[J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2018, 21(1): 34-37.

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小檗碱对内毒素刺激致Caco-2细胞间高通透性的保护作用^*

Protective effect of berberine on endotoxin-induced intercellular hyperpermeability in Caco-2 cells in vitro

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