优化溃结方对溃疡性结肠炎气滞血瘀模型大鼠的影响及其作用机制研究

Effect and Mechanism of Optimized Kuijie Decoction in Ulcerative Colitis Rats with TCM Syndrome Differentiation of Qi Stagnation and Blood Stasis

背景经验方优化溃结方能有效提高溃疡性结肠炎(UC)患者生活质量,促进肠黏膜愈合和临床缓解,但其作用机制尚不完全明确。目的探讨优化溃结方对UC气滞血瘀模型大鼠的影响及其作用机制。方法2023年9—10月,选取SPF级雄性SD大鼠70只并采用随机数字表法将其随机分为正常组、模型组、柳氮磺胺吡啶组、低剂量组、标准剂量组、益气组、活血组,每组10只。采用三硝基苯磺酸(TNBS)/乙醇二次致炎法结合束缚法建立UC气滞血瘀模型。正常组大鼠在造模时使用0.9%氯化钠溶液灌肠,并与其他组大鼠进行同步抓取、固定,在造模成功后给予等体积水灌胃,1次/d,共灌胃14次;模型组大鼠在造模成功后给予等体积水灌胃,1次/d,共灌胃14次;柳氮磺胺吡啶组、低剂量组、标准剂量组、益气组、活血组大鼠分别在造模成功后给予柳氮磺胺吡啶药液0.54 g/kg灌胃、低剂量优化溃结方药液0.837 g/kg、标准剂量优化溃结方药液1.674 g/kg灌胃、黄芪药液1.8 g/kg、红花药液0.9 g/kg,均为1次/d,均灌胃14次。采用高精度透射电镜观察7组大鼠干预后结肠组织超微结构变化,并比较7组大鼠干预后结肠组织趋化因子受体4(CXCR4)、血管内皮生长因子A(VEGFA)、转化生长因子激酶1(TAK1)表达水平。结果低剂量组、标准剂量组大鼠干预后结肠组织超微结构趋于正常。模型组大鼠干预后结肠组织CXCR4、VEGFA、TAK1蛋白灰度比值、mRNA相对表达量高于正常;柳氮磺胺吡啶组、低剂量组、标准剂量组、益气组、活血组大鼠干预后结肠组织CXCR4、VEGFA、TAK1蛋白灰度比值、mRNA相对表达量低于模型组(P<0.05);标准剂量组大鼠干预后结肠组织CXCR4、VEGFA、TAK1蛋白灰度比值低于柳氮磺胺吡啶组,活血组大鼠干预后结肠组织VEGFA、TAK1 mRNA相对表达量低于柳氮磺胺吡啶组(P<0.05)。结论优化溃结方及其益气、活血组分可有效降低UC气滞血瘀模型大鼠结肠组织CXCR4、VEGFA、TAK1表达水平,其治疗作用可能是益气组分、活血组分通过协同调节结肠组织中CXCR4、VEGFA、TAK1表达水平而实现。

Background Experiential effective prescription optimized Kuijie decoction can effectively improve the quality of life of patients with ulcerative colitis(UC),promote the intestinal mucosal healing and clinical remission,but its mechanism remains unclear now.Objective To investigate the effect and mechanism of optimized Kuijie decoction in UC rats with TCM syndrome differentiation of Qi stagnation and blood stasis.Methods From September to October 2023,70 SPFgrade male SD rats were randomly divided into the blank-control,model,salicylazosulfapyridine,low-dose,standarddose,Qi-enhancing and Blood-activating groups,with 10 rats in each group.UC model with TCM syndrome differentiation of Qi stagnation and blood stasis was established by trinitrobenzenesulfonic acid(TNBS)/ethanol secondary inflammation combined with restraint method.Rats in the blank-control group were enucleated with 0.9%sodium chloride solution at the time of modeling and were grasped and fixed synchronously with the rats in the other groups,and were given an equal volume of water by gavage once per day after the establishing for 14 days;rats in the model group were given an equal volume of water by gavage once per day after the establishing for 14 days;rats in the salicylazosulfapyridine,low-dose,standard-dose,Qi-enhancing and Blood-activating groups were given intragastric administration with salicylazosulfapyridine suspension(0.54 g/kg),low-dose optimized Kuijie decoction(0.837 g/kg),standard-dose optimized Kuijie decoction(1.674 g/kg),Astragalus suspension(1.8 g/kg)and Safflower suspension(0.9 g/kg)once per day after the establishing for 14 days,respectively.High-precision transmission electron microscope was used to observe the ultrastructural changes in the colonic tissues of the seven groups of rats after intervention,the expression levels of CXCR4,VEGFA,and TAK1 were compared among the seven groups of rats after intervention.Results The ultrastructure of colonic tissue of rats in the low-dose and standard-dose groups tended to be normal after intervention.The gray ratios of proteins and relative mRNA expression quality of CXCR4,VEGFA and TAK1 in colonic tissues of rats in the model group were higher than the blank-control after intervention(P<0.05);the gray ratios of proteins and relative mRNA expression quality of CXCR4,VEGFA and TAK1 in the salicylazosulfapyridine,low-dose,standard-dose,Qi-enhancing and Blood-activating groups were significantly lower than those in the model group(P<0.05);the gray ratios of proteins of CXCR4,VEGFA and TAK1 in the standard-dose group were significantly lower than those in the salicylazosulfapyridine group,relative mRNA expression quality of VEGFA and TAK1 in the Blood-activating group was significantly lower than that in the salicylazosulfapyridine group,respectively(P<0.05).Conclusion Optimized Kuijie decoction and its Qi-enhancing and Blood-activating components can effectively reduce the expression of CXCR4,VEGFA and TAK1 in colonic tissue in UC rats with TCM syndrome differentiation of Qi stagnation and blood stasis,its therapeutic effect may act through the coordinate regulating of the expression of CXCR4,VEGFA and TAK1 in colonic tissue by Qi-enhancing component and blood-activating component.