益生菌VSL#3对实验性结肠炎大鼠结肠黏膜Claudins蛋白表达的影响

目的 观察益生菌VSL#3对2，4，6-三硝基苯磺酸(TNBS)诱导的大鼠急性结肠炎的保护作用，以及对紧密连接蛋白Claudins（Claudin-1，-2，-3）在结肠黏膜上表达的影响。方法 24只健康雌性SD大鼠采用TNBS一次性灌肠法建立大鼠结肠炎模型，造模完成后第2天开始，8只大鼠每日给予2ml0.9％ NaC1溶液灌胃（TNBS组），8只给予100 mg VSL#3（VSL#3组），另8只给予200 mg 5-氨基水杨酸(5-ASA)灌胃（5-ASA组）。5只同级别未造模大鼠作为对照组每日给予0.9％ NaCl溶液灌胃，均持续1周并处死。处死时留取粪便标本并做细菌培养以检测肠道菌群的变化。通过观察腹泻、便血和体重情况计算疾病活动指数(DAI)。取结肠组织做HE染色，观察病理学改变；酶联免疫吸附试验(ELISA)检测肠组织髓过氧化物酶(MPO)的变化；量子点免疫荧光标记法检测Claudins在结肠黏膜的表达和分布。结果 与TNBS组比较，益生菌VSL#3和5-ASA治疗均能降低结肠炎大鼠明显升高的DAI和肠组织MPO水平，减轻结肠炎性反应评分。与对照组比较，结肠炎大鼠的肠道细菌计数发生改变，Claudin-1，-2，-3蛋白表达均明显增加（平均荧光强度分别为66.200±5.737、71.780±6.670、61:300土5.199,t值分别＝17.237、27.909和21.788;P值均＜0.01）；而与TNBS组和5-ASA组比较，VSL#3可调节肠道菌群平衡，增加Claudin-1，-3的表达（Claudin-1:75.550±8.717比66.200±5.737和67.080±5.401；t＝9.348，8.469；P值均＜0.05；Claudin-3:68.820±7.443比61.300土5.199和59.830±5.930；t＝7.519,8.988，P值均＜0.05)，降低Claudin-2的表达(58.740±6.457比71.780±6.670和66.870±5.791；t＝13.033，P＜0.01；t＝8.123，P＜0.05)。结论 益生菌VSL#3可能通过改变Claudins蛋白的表达，增强肠黏膜屏障功能，从而发挥缓解急性结肠炎性反应的作用。

The effects of probiotics VSL # 3 on the expression of claudins in the colon mucosa of experimental colitis rats

Objective To observe the protective effect of probiotics VSL # 3 on the rats of trinitrobenzene sulphonic acid (TNBS) induced acute colitis, and the effect on the expression of tight junction proteins claudins (claudinl, 2, and 3) in colon mucosa. Methods Rat of colitis model was induced by TNBS one time enema. Since the second day after the model established, 2ml 0.9％ sodium chloride was intragastric administrated into 8 rats each day (TNBS group), 8 rats with 100 mg VSL # 3 (VSL # 3 group ), another 8 rats with 5-aminosalicylic acid (5-ASA) intragastric administration (5-ASA group), and 0.9％ sodium chloride was intragastric administrated daily into 5 no modeling rats as control group. All were treated for 7 days and then sacrificed. Fecal samples were collected for bacterial culturing and detecting the changes of colon bacteria colonies. Disease activity index (DAI was calculated according to diarrhea, hemafecia and weight. The colon tissue was taken for HE staining and the pathological changes were observed under microscope.The changes of myeloperoxidase (MPO) level in colon tissue were examined by enzyme-linked immunosorbent assay (ELISA). The expression and distribution of claudins 1, 2, and 3 in colon tissue was detected by a novel quantum dots immunofluorescence labeling technique. Results Compared with TNBS group,both VSL # 3 and 5-ASA treatment could improve colitis, as well as decreas DAI and colonic MPO level, and lower the inflammatory score of colitis. Compared with control group, the bacteria counting in the colon of colitis rats changed, the expression of Claudins 1, 2, and 3 expression was significantly increased (average density: 66.200±5.737, 71.780±6.670 and 61.300±5. 199,t＝17.237, 27.909and 21.788, all P＜0.01). Compared with TNBS group or 5-ASA group, VSL# 3 could adjust the balance of intestinal bacteria and increase the expression of Claudin 1 and 3 (Claudin 1 : 75. 550 ±8.717, 66.200±5.737 and 67.080±5.401;t＝9.348, 8. 469; all P＜0.05; Claudin-3: 68. 820±7.443, 61.300±5. 199 and 59. 830±5. 930;t＝7. 519, 8. 988, all P＜0.05), however claudin 2 expression was down-regulated (58. 740±6. 457, 71. 780±6. 670 and 66. 870±5. 791; t＝ 13. 033,P＜0.01, t＝8. 123, P＜0. 05). Conclusion Probiotics VSL# 3 may enhance the intestinal mucosal barrier function through changing the claudins expression, and play a role in the relief of inflammatory reaction in acute colitis.