肠润方对功能性便秘大鼠结肠黏膜AQP3、AQP9表达的影响

Experimental study on effect of Changrun Formula in regulating expression of AQP3 and AQP9 in colon mucosa of functional constipation rats

目的:观察肠润方对功能性便秘模型大鼠结肠黏膜AQP3、AQP9表达的影响,探究其调控AQP3、AQP9表达的分子机制。方法:60只SD大鼠随机分为空白组、模型组、阳性对照组(麻仁丸组)、肠润方组、肠润方联合P38MAPK抑制剂SB203580组。用复方地芬诺酯制造大鼠便秘模型,采用免疫组织化学及实时荧光定量PCR(q RT-PCR)检测大鼠便秘模型近端及远端结肠黏膜AQP3、AQP9的表达;Western Blot检测信号传导通路相关分子P38MAPK及P-P38MAPK的表达。结果:造模成功后,模型组近端结肠黏膜AQP3表达较空白组明显上升,而远端结肠黏膜AQP9表达较空白组明显下降(t值分别为3.148和7.069,P值均<0.01);经肠润方及麻仁丸治疗后,近端结肠黏膜AQP3表达下降,而远端结肠黏膜AQP9表达上升,与模型组比较,差异均有统计学意义(t值分别为3.175和9.31,P值均<0.05);各组间远端结肠黏膜AQP3表达及近端结肠黏膜AQP9表达比较,差异均无统计学意义(F值分别为1.639和2.544,P值均>0.05)。肠润方联合P38MAPK抑制剂SB203580治疗后,近端结肠黏膜AQP3 m RNA水平显著上升(t=5.922,P<0.01);而远端结肠黏膜AQP9 m RNA水平显著下降(t=4.038,P<0.01);P-P38/P38蛋白相对表达水平显著下降(t=19.419,P<0.01)。结论:肠润方对便秘模型大鼠的通便作用是通过抑制近端结肠AQP3表达及促进远端结肠AQP9表达来实现的,其调控机制可能与激活P38磷酸化,进而激活P38MAPK信号通路有关。

Objective： To observe the effect of Changrun Formula on regulating expression of AQP3 and AQP9 in colon mucosa of functional constipation rats, and investigate the molecular mechanism of expression of AQP3 and AQP9. Methods： 60 SD rats were randomly divided into 6 groups, including model group, blank control group, positive control group（Maziren Pill group）, Changrun Formula groups. The functional constipation rat models were established by using compound diphenoxylate. The expression of AQP3, AQP9, P38 MAPK, and AKT in the proximal and distal colonic mucosa of model rats was tested by using immunohistochemical and Western Blot. Results： The expression of AQP3 in the proximal colon mucosa of the model group was significantly higher than that of the blank group（P〈0.01）, while the expression of AQP9 in the distal colonic mucosa was significantly lower than that in the blank group（t=3.148 and 7.069, P〈0.01）; The expression of AQP3 in the proximal colon mucosa decreased and the expression of AQP9 in the distal colon mucosa increased compared with the model group（t=3.175 and 9.31, P〈0.05）. There was no significant difference in the expression of AQP3 between proximal colon mucosa and AQP3 in distal colonic mucosa（F values were 1.639 and 2.544, P〈0.05）. The level of AQP3 m RNA in proximal colonic mucosa was significantly increased（t=5.922, P〈0.01）, and AQP9 m RNA was significantly decreased in distal colonic mucosa（t=4.038, P〈0.01）; The relative expression of P-P38/P38 protein was significantly decreased（t=19.419, P〈0.01）. Conclusion： The therapeutic action ofChangrun Formula on functional constipation rat models might through inhibiting the expression of AQP3 in proximal colon, and accelerating on expression of AQP9 in distal colon, and the regulating mechanism might relate with the inhibition on P38 MAPK and AKT phosphorylation.