摘要目的 评价RhoA在富氢液减轻脂多糖(LPS)诱发人肠屏障功能障碍中的作用.方法 将Caco-2细胞以2×105个/ml的密度接种于transwell小室,每孔400μl,采用随机数字表法,将其分为5组(n=50):对照组(C组)、富氢液组(H2组)、LPS组、LPS+富氢液组(LPS+HRS)组和LPS+ RhoA抑制剂C3胞外酶组(LPS+C3组).H2组和LPS+HRS组用含0.6 mmol/L富氢液的培养基培养,LPS组、LPS+HRS组和LPS+C3组加入LPS 100 μg/ml;于加入LPS前1h时,LPS+C3组加入C3胞外酶2.5 μg/ml.每组分别于LPS孵育前即刻、3、6、12、24、36、48 h时取5孔,测定上皮细胞电阻(TEER)和渗透系数(PE),每组于LPS孵育24 h时取5孔,采用Western blot法和免疫荧光法测定occludin与E-cadherin的表达,采用GST pull-down法测定RhoA活性.结果 与C组比较,LPS组、LPS+HRS组和LPS+C3组LPS孵育6-48 h时TEER降低,PE和RhoA活性升高,occludin和E-cadherin的表达下调(P＜0.05);H2组上述指标差异无统计学意义(P＞0.05);与LPS组比较,LPS+ HRS组和LPS+C3组LPS孵育6-48 h时TEER升高,PE和RhoA活性降低,occludin和E-cadherin的表达上调(P＜0.05);与LPS组比较,LPS+HRS组和LPS+C3组occludin和E-cadherin在细胞膜处分布部分增多,在细胞质中分布减少,“铁丝网”样形态的连续性与完整性部分恢复.结论 富氢液可减轻LPS诱发人肠屏障功能障碍,其机制与抑制RhoA激活,从而上调occludin和E-cadherin的表达及抑制其分布紊乱有关.

abstractsObjective To evaluate the role of RhoA in amelioration of lipopolysaccharide (LPS)-induced human intestinal barrier dysfunction by hydrogen-rich saline.Methods Caco-2 cells were plated on transwells at a density of 2× 105/ml (400 μl/well),and randomly divided into 5 groups (n =50 each) using a random number table:control group (group C),hydrogen-rich saline group (HRS group),LPS group,LPS + hydrogen-rich saline group (group LPS+HRS),and LPS + RhoA inhibitor C3 exoenzyme group (group LPS+C3).Cells were cultured in the medium containing 0.6 mmol/L hydrogen-rich saline in HRS and LPS+HRS groups,or in the medium containing 100 μg/ml LPS in LPS,LPS+HRS and LPS+C3 groups.In group LPS+C3,C3 exoenzyme 2.5 μg/ml was added at 1 h before addition of LPS.Five wells were selected at 0,3,6,12,24,36 and 48 h of incubation with LPS,and the transepithelial electrical resistance (TEER) and permeability coefficient (PE) were measured.Five wells were selected at 24 h of incubation with LPS for determination of occludin and E-cadherin expression (by Western blot and immunofluorescence) and RhoA activity (using GST pull-down assay).Results Compared with group C,TEER was significantly decreased,PE and RhoA activity were increased,and the expression of occludin and Ecadherin was down-regulated at 6-48 h of incubation with LPS in LPS,LPS+ HRS and LPS+C3 groups (P＜ 0.05),and no significant change was found in the variables mentioned above in group HRS (P＞0.05).Compared with group LPS,TEER was significantly increased,PE and RhoA activity were decreased,and the expression of occludin and E-cadherin was up-regulated at 6-48 h of incubation with LPS in LPS+HRS and LPS+C3 groups (P＜0.05).Compared with group LPS,the distribution of occluding and E-cadherin in the cell membrane was significantly increased,the distribution in the cytoplasm was decreased,and the structures of occludin and E-cadherin partly recovered in LPS+HRS and LPS+C3 groups.Conclusion Hydrogen-rich saline can alleviate LPS-induced intestinal barrier dysfunction via inhibiting RhoA activation,thus up-regulating occludin and E-cadherin expression and inhibiting the disturbance of the distribution.