大建中汤对肠易激综合征大鼠内脏痛的影响

Effect of Da Jianzhongtang on Visceral Pain in Rats with Irritable Bowel Syndrome

目的:研究大建中汤对肠易激综合征(IBS)内脏痛大鼠P物质(SP),肥大细胞(MC),MC模上Toll样受体2(TLR2),Toll样受体4(TLR4)和核转录因子-κB p65(NF-κB p65)影响,并探讨其对IBS内脏痛的作用机制。方法:将SD 3日龄新生大鼠48只,随机分为正常组,IBS模型组,酮替芬组(0.18 mg·kg^(-1)),大建中汤低、中、高剂量组(2.16,1.08,0.54 g·kg^(-1))。每组8只,连续灌胃给药2周。采用母婴分离法制备IBS内脏痛大鼠模型,利用腹壁撤退反应(AWR)评估大鼠内脏敏感性;蛋白免疫印迹法(Western blot)检测结肠组织SP,NF-κB p65蛋白;免疫荧光染色法检测MC膜上TLR2,TLR4蛋白;甲苯胺蓝染色法检测结肠组织MC脱颗粒率。结果:与正常组大鼠比较,模型大鼠中60,40,20 mmHg压力下AWR评分明显升高(P<0.05,P<0.01),结肠组织中MC脱颗粒率,SP蛋白表达显著上升(P<0.01),MC模上TLR2,TLR4及核内NF-κB p65蛋白表达显著升高(P<0.01);与模型组大鼠比较,大建中汤高剂量组(压力40,20 mmHg,1 mmHg≈0.133 kPa),大建中汤中剂量组(压力60,40,20 mmHg)的AWR评分,结肠MC脱颗粒率,SP,TLR2,TLR4和NF-κB p65表达明显下降(P<0.05,P<0.01)。结论:大建中汤能够降低IBS模型大鼠内脏痛,其下调SD大鼠结肠组织中SP,进而影响MC活性可能是机制之一。

Objective:To investigate the effects of Da Jianzhongtang on substance P(SP),mast cells(MC),Toll like receptor 2(TLR2),TLR4 on MC model and nuclear transcription factor(NF)-κB p65 in visceral pain rats with irritable bowel syndrome(IBS),and explore its mechanism of action on IBS visceral pain.Method:Forty-eight 3-day-old SD rats were randomly divided into 6 groups:the control group(control),irritable bowel syndrome group(IBS),ketotifen group(Ketotifen,0.18 mg·kg^(-1)),Da Jianzhongtang low,medium and high dose groups(DJZT-L,DJZT-M,DJZT-H,2.16,1.08,0.54 g·kg^(-1)),with 8 rats in each group.Intragastric administration lasted for 2 weeks.Maternal separation method was used to establish the IBS visceral pain model in rats.The visceral sensitivity of rats was evaluated at 60,40 and 20 mmHg(1 mmHg≈0.133 kPa)with Abdominal wall withdrawal response(AWR)scale.SP and NF-κB p65 protein expression levels in colon tissue were detected with Western blotting technique.TLR2 and TLR4 proteins on mast cell membrane were detected by immunofluorescence staining.The degranulation rate of mast cells in colon tissue was detected by toluidine blue staining.Result:Compared with normal rats,AWR scores of model rats significantly increased at60,40,and 20 mmHg pressure(P<0.05,P<0.01),the degranulation rate of mast cells in colon tissue and SP protein expression in colon tissue significantly increased(P<0.01),TLR2,TLR4,and nuclear NF-κB p65 expression on mast cell membrane significantly increased(P<0.01).Compared with model rats,the AWR scores of DJZT-H group(pressure of 40,20 mmHg)and DJZT-M group(pressure of 60,40,20 mmHg)significantly decreased.Meanwhile,the degranulation rate of colon mast cells,and the SP,TLR2,TLR4,and NF-κB p65 expression also significantly decreased(P<0.05,P<0.01).Conclusion:Da Jianzhongtang can affect mast cell activity and finally decrease visceral pain of IBS rats by down-regulating SP in colon tissue.