健脾清热活血类方药对溃疡性结肠炎肠黏膜功能障碍及claudin-2、claudin-5的影响

The mechanism of Jian Pi Qing Re Huo Xue decoction on improving colon mucosa dysfunction and regulating the expression of claudin-2 and claudin-5 in DSS-induced colitis

[目的]基于检测溃疡性结肠炎结肠黏膜组织形态、超微结构、上皮紧密连接,及紧密连接蛋白claduin-2、claudin-5表达的变化,探讨健脾清热活血方防治溃疡性结肠炎的可能机制。[方法]72只雄性Balb-c小鼠按体重随机分为6组,即正常组、模型组、对照组、治疗组(低、中、高剂量),每组12只。除正常组外,其余各组小鼠均采用自由饮用3%DSS 6周法制备溃疡性结肠炎模型,自第7周起分组给药,模型组予等剂量生理盐水,对照组予艾迪莎,治疗组予不同剂量健脾清热活血方,连续给药4周,第11周末处死全部小鼠。应用光镜、透射电镜及免疫组织化学观察结肠黏膜组织形态、超微结构及黏蛋白变化;应用荧光定量PCR技术检测紧密连接蛋白claudin-2、claduin-5表达变化。[结果]模型组光镜下见结肠黏膜不同程度缺损、溃疡形成,表面可见坏死组织,以及隐窝脓肿形成;电镜下见肠上皮细胞表面微绒毛稀疏,细胞连接间隙增宽,杯状细胞减少,线粒体肿胀,结构不清或呈空泡变,部分可见核固缩,并见凋亡小体;结肠黏蛋白分泌下降。治疗组光镜下见结肠黏膜充血肿胀、少量炎性细胞浸润,可见隐窝脓肿,未见明显溃疡;电镜下见肠上皮细胞表面绒毛排列尚整齐,可见线粒体肿胀,核固缩及凋亡小体;结肠黏蛋白分泌增加,与模型组比较,有统计学意义(P<0.05)。模型组claudin-2表达呈上升趋势、claudin-5表达呈下降趋势;治疗组claudin-2表达下降及claudin-5表达上升,经比较有统计学意义(P<0.05)。[结论]健脾清热活血方药可能通过降低claudin-2表达、上升claudin-5表达,促进黏膜修复,达到缓解溃疡性结肠炎的效用。

[Objective]We aim to explore the mechanism of Jian pi qing re huo xue decoction by observa- tion the colon mucosa morphology, ultrastructure and epithelial tight junctions which based on the changed expression of claduin-2,claudin-5 before and after the treatment of ulcerative colitis. [Methods] 72 Balb-c rats were randomly divided into the normal group, the blank group, the control group and the intervention group equally. Except normal group,other groups were administrated by drinking 3 % DSS for six weeks to making ulcerative colitis model. Since the seventh week, the blank group was given with the same doses of saline, the control group was treated with Editha, and the intervention group was given with different doses of Jianpiqingrehuoxue decoction for 4 weeks. All rats were killed at the eleventh week. The colon mucosa morphology, ultrastructure and the changed expression of mucin were detected by the light microscopy, the electron microscopy and the immuno histochemistry. The expression of tight junction protein including Claudin-2 ,claduin-5 were detected by the real-time PCR. [Results]There were different degrees of defected colonic mucosa,ulceration and crypt abscess with necrotic tissue on surface under light microscope in the blank group. Also there were microvilli sparse on the intestinal epithelial cell, the intercellular spaces, the decreased goblet cells,the swelling mitochondria, the unclear structure with vacuolar degeneration, the py- knosis nuclear and apoptotic bodies under the electron microscope. The colonic mucin secretion was de- creased. There was colonic mucosal hyperemia with swelling,inflammatory cell infiltration, and crypt ab- scess without obvious ulcer in the treatment group under light microscope. Also the intestinal epithelial cells were still neatly arranged with swelling mitochondrion, nuclear condensation and apoptotic bodies on the surface under electron microscope. The colonic mucin secretion was in the increasment. There was a statistical difference compared with the blank group（P〈0.05）. The expression of Claudin-2 in blank group was on a rising trend,and the expression of claudin-5 was decreased. The expression of Claudin-2 in inter vention group was on the decline, and the expression of claudin-5 was on the rise. There were statistical difference（P〈0. 05）. {-Conelusion]Jian pi qing re huo xue decoction can promote mucosal repair in order to remitting ulcerative colitis through regulating the expression of Claudin-2 and claudin-5.