痛泻要方对内脏高敏感大鼠肠道肥大细胞及细胞因子表达的影响

Effect of Tongxie Yaofang on Intestinal Mast Cells and Cytokine Expression of Rats with Visceral Hypersensitivity

目的观察痛泻要方对内脏高敏感大鼠肠道肥大细胞(mast cell,MC)及细胞因子表达的影响,探讨痛泻要方治疗内脏高敏感的作用及其机制。方法采用随机数字表法将30只成年雄性SD大鼠随机分成空白对照组、模型组及中药治疗组,每组10只,模型组及中药治疗组以结直肠扩张联合束缚刺激建立大鼠内脏高敏感模型,以腹部收缩反射(abdominal withdrawal reflex,AWR)评估大鼠内脏敏感性,模型构建成功后第2日中药治疗组予痛泻要方4 g/(kg·d)灌胃4周,模型组予以等量生理盐水灌胃4周,空白对照组大鼠不予任何刺激。4周后采用甲苯胺蓝染色法计数回盲部结肠黏膜MC,采用E LISA法检测血清及肠黏膜IL-4、IL-9表达,Western blot法检测肠黏膜蛋白酶激活受体-2(PAR-2)蛋白表达。结果与空白对照组大鼠比较,模型组大鼠内脏敏感性升高,回盲部MC增多,血清及肠黏膜中IL-4、IL-9及PAR-2蛋白表达水平升高(P<0.05);与模型组比较,中药治疗组大鼠内脏敏感性显著降低,MC数量减少,肠黏膜PAR-2蛋白表达下降(均P<0.05),大鼠肠黏膜IL-4及血清IL-9表达水平亦明显降低(P<0.05)。结论痛泻要方可能通过作用于MC相关细胞因子,减少MC脱颗粒,从而降低内脏高敏感。

Objective To observe the effect of Tongxie Yaofang（TY） on the number of mast cells（MCs） and the expression of cytokines in rats with visceral hypersensitivity,and to explore roles of TY in treating visceral hypersensitivity and its possible mechanism.Methods Totally 30 male adult Sprague Dawley（SD） rats were randomly divided into the blank control group,the model group,and the TY treatment group,10 in each group.The irritable bowel syndrome（IBS） rat model was established by combining colorectal distention with restraint stress in the TY treatment group and the model group.The visceral hypersensitivity was assessed by abdominal withdrawal reflex（AWR）.From the 2nd day of successful modeling,rats in the treatment group were admiministered with TY at the daily dose of 4 g/kg for4 successive weeks.Equal volume of normal saline was given to rats in the model group for 4 successive weeks.No treatment was given to rats in the blank control group.Four weeks later the number of MCs was counted by using toluidine blue staining.The expression of interleukin-4（IL-4） and interleukin-9（IL-9） both in colonic mucosa and serum were measured by enzyme linked immunosorbent assay（ELISA）,and the expression of protease-activated receptor type 2（PAR-2） was detected by Western blot.Results Compared with the blank control group,the visceral sensitivity was significantly elevated,the number of MCs in the ileocecal junction increased,and the expression of IL-4,IL-9,and PAR-2 in serum and the colonic mucosa significantly increased（P0.05）.Compared with the model group,the visceral sensitivity significantly decreased,the number of MCs reduced,and the expression of PAR-2 in the colonic mucosa significantly reduced（all P〈0.05）,and the expression of IL-4 in colonic mucosa and IL-9 in serum were obviously reduced in the TY treatment group（P〈0.05）.Conclusion TY might improve the visceral hypersensitivity by acting on MCs related cytokines and reducing degranulation of MCs.