桃红四物汤对溃疡性结肠炎大鼠SCF/c-kit通路及Cajal间质细胞的影响

Effect of Taohongsiwutang on SCF/c-kit pathway and interstitial cells of Cajal in rats with ulcerative colitis

目的探讨桃红四物汤(THSWT)对溃疡性结肠炎(UC)大鼠干细胞因子(SCF)/酪氨酸蛋白激酶受体(c-kit)通路及Cajal间质细胞(ICC)的影响。方法将SD大鼠随机分为假手术组、模型组、THSWT低剂量组(50 mg/kg)、THSWT中剂量组(100 mg/kg)、THSWT高剂量组(200 mg/kg)、柳氮磺吡啶组(0.3 g/kg),每组10只。除假手术组外,其余各组大鼠均利用结肠炎诱导剂(TNBS)溶液建立UC模型,按照各组给药剂量给予相应药物处理后,采集大鼠腹腔主动脉血及结肠组织;以苏木精-伊红(HE)染色检测各组大鼠结肠组织病理变化并对大鼠进行病理学评分;用透射电镜观察大鼠结肠组织ICC超微结构;以酶联免疫吸附(ELISA)法检测大鼠腹腔主动脉血清SCF水平;免疫荧光法检测大鼠结肠组织c-kit阳性ICC的表达;以Western blot免疫印迹法检测大鼠结肠组织中SCF、c-kit蛋白表达。结果与假手术组比较,模型组大鼠出现血便、黄色稀便、精神萎靡、反应迟钝、蜷缩扎堆等症状,并伴有体质量增长缓慢、脱毛、毛色暗黄等;结肠组织出现明显充血、淤血,肌层、肠黏膜变薄,结肠组织损伤评分及疾病活动指数(DAI)评分显著升高,结肠组织ICC数量较少且结构模糊;腹主动脉血清中SCF含量显著降低,结肠组织c-kit阳性ICC的荧光强度显著减弱,SCF、c-kit蛋白表达显著降低(P<0.05)。与模型组比较,THSWT低、中、高剂量组,柳氮磺吡啶组大鼠结肠组织病理损伤、ICC结构、结肠组织损伤评分及DAI评分有不同程度的缓解,血清中SCF含量显著升高,结肠组织c-kit阳性ICC的荧光强度显著增强,SCF、c-kit蛋白表达显著升高(P<0.05),THSWT各组呈剂量依赖性。THSWT高剂量组与柳氮磺吡啶组比较,差异无统计学意义(P>0.05)。结论 THSWT可缓解UC大鼠的结肠组织损伤,可能是通过激活SCF/c-kit通路、保护ICC结构完整性实现的。

Objective To investigate the effect of Taohongsiwutang(THSWT) on stem cell factor(SCF)/receptor tyrosine kinase(c-kit) pathway and interstitial cells of Cajal(ICC) in rats with ulcerative colitis(UC). Methods SD rats were randomly divided into sham operation group, model group, THSWT low-dose group(50 mg/kg), medium-dose group(100 mg/kg), high-dose group(200 mg/kg) and sulfasalazine group(positive control group, 0.3 g/kg), with 10 in each group. Except for the sham operation group, the other groups were used to establish UC model with 2, 46-Trinitro benzens a lfonic acid solution(TNBS) solution, after the corresponding drugs were given according to the dosage of each group, the abdominal aorta blood and colon tissue were collected;the pathological changes of colon tissue were detected by hematoxylin eosin(HE) staining, and the pathological scores of rats were evaluated;the ultrastructure of ICC in colon tissue was observed by transmission electron microscope;the level of serum SCF of abdominal aorta was detected by enzyme-linked immunosorbent assay(ELISA);the expression of c-kit positive ICC was detected by immunofluorescence, and the expression of SCF and c-kit proteins was detected by Western blot. Results Compared with those in the sham operation group, the rats in the model group had bloody stools, yellow loose stools, listlessness, slow reaction, curling up, and other symptoms, accompanied by slow weight growth, depilation, dark yellow hair color and other characteristics;the colonic tissue showed obvious congestion and blood stasis, and the muscular layer and intestinal mucosa became thinner;the colonic tissue injury score and disease activity index(DAI) score were significantly higher;the number of ICC in colon tissue was less and the structure was vague;the content of SCF in abdominal aorta serum was significantly lower,the fluorescence intensity of c-kit positive ICC was significantly weaken,and the expression of SCF and c-kit protein was significantly lower( P<0.05). Compared with those in the model group,the pathological damage of colon tissue,ICC structure,colonic tissue injury score and DAI score in low,medium and high dose THSWT groups and sulfasalazine group were alleviated in varying degrees;the content of SCF in serum was significantly higher,and the fluorescence intensity of c-kit positive ICC in colon tissue was significantly stronger,the expression of SCF and c-kit protein was significantly higher( P<0.05) with dose-dependency in THSWT groups,while there was no significant difference between THSWT high-dose group and sulfasalazine group( P > 0. 05).Conclusion THSWT can alleviate the colon injury in UC rats,which may be achieved by activating SCF/c-kit pathway and protecting the integrity of ICC structure.