| Objective:To investigate the effect and mechanism of Shu Gan Jian Pi(SGJP)formula in rats with Diarrhea-predominant pattern irritable bowel syndrome(D-IBS).Methods:This research was divided into three parts.The first part was for evaluation of efficiency of SGJP formula in D-IBS rats.The second part evaluated the effects of SGJP formula on the expression level of mast cells(MCs),5-hydroxytryptamine(5-HT),5-hydroxytryptamine receptor 3(5-HT3R)and 5-hydroxytryptamine receptor 4(5-HT4R)in rats with D-IBS.The third part discussed the effects of SGJP formula on the ion transport in colonic mucosa and submucosa of D-IBS rats.The specific methods were as follows:1-day-old male Sprague-Dawley(SD)rats were randomly divided into seven groups:normal control group,model group,SGJP formula high-dose group,SGJP formula middle-does group,SGJP formula low-dose group,Shen Ling Bai Zhu San(SLBZS)formula group and positive control group.D-IBS model rats were established by received neonatal maternal separation and restraint stress except the control group.The rat model of D-IBS was evaluated at the point of 60 days old.The weight change,defecation,behavioral Abdominal Withdrawal Reflex(AWR)scores and electromyographic(EMG)recordings of the obliquus externus abdominis responses to graded balloon distention were tested.After the success of molding,the SGJP high-,middle-and low-dose group were respectively received treatment with 4.66g/100g/d,2.33g/100g/d and 1.17g/100g/d SGJP formula,SLBZS group was received treatment with 1.17g/100g/d SLBZS formula and positive control group was given with 2.1mg/100g/d Pinaverium Bromide,while the normal control group and model group were received administration with equal amount of 0.9%sodium chloride solution.Each group was dosed with the method of gavage for 14 days.After treatment,waste water content,scores of AWR and EMG recordings of the obliquus externus abdominis responses to graded balloon distention were observed.The visceral sensitivity of rats was evaluated by AWR scores and EMG recordings.The optimal dose group of SGJP formula was selected for the mechanism discussion in next two parts.In the second and third part of research,the D-IBS model rats were randomly divided into three groups(model group,SGJP formula optimal dose-effect group and SLBZS formula group),and no handling rats served as the normal control group.The dose and methods of treatment were as above.After treatment,the number of MCs in colon tissue of rats was detected with method of toluidine blue stain.The expression level of 5-HT in colon and spinal cord tissue was detected with method of euzymelinked immunosorbent assay(ELISA).The expression of 5-HT4R in the colon tissue and 5-HT3R in the colon and spinal cord tissue were also evaluated with methods of Western blot and Real-time PCR.The effects of 5-HT,5-HT3R,5-HT4R on the ion transport in colonic mucosa and submucosa were observed with Short-circuit current(Isc)technology.Results:The weight of model rats was lower than the normal controls after neonatal maternal separation,but there was no significant difference between the model group and normal control group(P>0.05).The weight of model rats was significantly lower(P<0.05)than the normal controls after restraint stress.Compared with the normal control group,Significant increases(P<0.05)in waste water content(%),AWR scores and EMG activity of model rats were observed at balloon distention pressures of 40mmHg,60mmHg and 80 mmHg.Waste water content(%),AWR scores and EMG activity at balloon distention pressures of 40mmHg,60mmHg and 80mmHg in SGJP formula middle-,high-dose group and positive control group were significantly lower than those in model group(P<0.05)after administration.There was no significant difference between the SGJP formula middle-dose group and high-dose group(P>0.05),so SGJP formula with the middle dose can be considered as the optimal dose.The number of MCs,the content of 5-HT and the levels of protein and mRNA of 5-HT3AR,5-HT3BR in colon of model rats were significantly higher than those in the normal control rats(P<0.05),while the levels of protein and mRNA of 5-HT4R were lower than those in normal control rats(P<0.05).After administration with SGJP formula or SLBZS formula,the number of MCs,the content of 5-HT and the levels of protein and mRNA of 5-HT3AR,5-HT3BR in colon were significantly decreased(P<0.05)and the levels of protein and mRNAs of 5-HT4R in colons were significantly increased(P<0.05)compared with model group.And there was no significant difference with those in normal control group(P>0.05).The content of 5-HT and the level of 5-HT3AR in spinal cord of model rats were significantly higher than those in the normal control rats(P<0.05),and they were significantly decreased(P<0.05)compared with model group after administration with SGJP formula.However,the level of 5-HT3AR in SLBZS formula group was similar to that in model group(P>0.05).There was no significant difference in 5-HT3BR expression level in each groups(P>0.05).Rate in model group exhibited significantly lower(P<0.05)in short-circuit current changes(△Isc)induced by 5-HT compared with normal group,While 5-HT induced △Isc in SGJP formula group and SLBZS formula group were significantly higher(P<0.05)than that in model group.When the 5-HT3R or 5-HT4R was blocked,5-HT induced △Isc exhibited no significantly difference in each groups(P<0.05).The △Isc induced by 5-HT3R agonist was significant higher(P<0.05)in model group than that in normal group,SGJP formula group and SLBZS formula group.However,when neural function was blocked by Tetrodotoxin(TTX),the △Isc induced by 5-HT3R agonist did not significantly differ in four group(P>0.05).Conclusion:SGJP formula can effectively improve symptom of diarrhea and reduce the visceral sensitivity in D-IBS rats.And SGJP formula is superior to the SLBZS formula in reducing visceral sensitivity.Its mechanism may through(1)Lower MCs number,5-HT content and the level of 5-HT3R in colon,Lower 5-HT content and the level of 5-HT3R in spinal cord and higher the level of 5-HT4R in colon;(2)Affect the secretion of colonic mucosa by 5-HT,5-HT3R,5-HT4R in colon epithelial. |
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