Exploring The Mechanism Of The Active Components Of Pogostemonis Herba In Treatment Of IBS-D Rat Via Enteric Neurotransmission Regulation

Object（1）Modifying the rat model of Irritable bowel syndrome with predominant diarrhea（IBS-D）induced by chronic restrain stress,to observe the effects of single chronic restraint stress factor on diarrhea,visceral sensitivity and intestinal nerve conduction changes in rats,and to clarify the intestinal segments of abnormal nerve conduction.（2）To observation effects of Pogostemonis Herba formula granules and patchouli alcohol（PA）on defecation,visceral high sensitivity and the colonic abnormal nerve conduction changes of IBS-D rats.To discuss the drugs effects on the onset of IBS-D related intestinal neurotransmitters or receptors gene expression.（3）To observe the effects of PA on the contractile tension and frequency of isolated IBS-D rat colonic longitudinal smooth muscle,and to investigate its effects on cholinergic,nitrergic and purine nerve conduction pathways.METHODSThe changes of neurotransmission dysfunction of the modified rat model of IBS-D that induced by chronic restrain stress were studied.From multi-level and in-depth study on the regulation of colonic neurotransmission,the therapeutic mechanisms of PA in treatment of IBS-D were revealed.The study consists of four parts.（1）Improvement and Evaluation of Rat Model of IBS-D:Thirty specific-pathogen free Sprague Dawley rats were randomly divided into control group,one-hour model group and 2-hour model group,10 rats in each group and half being male.One-hour model group and 2-hour model group were given restrain stress one,2 hour（s）per day respectively,and the treatment lasted 14 days.On day 7,14,21,28 after modeling,the number of feces grains within 4 hours,area of the excrement and body mass were monitored.On modeling day 28,abdominal withdrawal reflex（AWR）scores for visceral sensitivity were calculated,the pathological features of gastric antrum,duodenal,ileac,colonic tissues were observed by hematoxylin-eosin staining,and the expression level of 5-hydroxytryptamine-3A receptor(5-HT_3AR)in rat colon tissue was detected by real time quantitative polymerase chain reaction（qPCR）.（2）Pharmacodynamics and the mechanism study of Pogostemonis Herba formula granules:The model of IBS-D rats was established by using the above modified method.Rats were randomly divided into 6 groups:control group,model group,the positive group（Pinaverium Bromide）,Pogostemonis Herba formula Granules of low,medium and high dose group,12 rats in each group.Visceral sensitivity and defecation of each rat were tested regularly.After a 14-day gavage administration,colonic mRNA expression levels of5-HT_3AR,5-hydroxytryptamine-4 receptor（5-HT₄R）,neurokinin 1 receptor（NK-1R）,neuronal nitric oxide synthase（nNOS）were also determinate.（3）Pharmacodynamics and the mechanism study of PA:Chronic restraint stress was adopted to induce IBS-D rat model.Rats were randomly divided into 6 groups:control group,model group,the positive group（Pinaverium Bromide）,PA of low,medium and high dose group,12 rats in each group.Visceral sensitivity and defecation of each rat were tested regularly.After a 14-day gavage administration,colonic mRNA expression levels of 5-HT_3AR,5-HT₄R,tachykinin precursor 1（Tac1）,NK-1R,vasoactive intestinal peptide（Vip）and nNOS were also determinate.（4）The regulation effect of PA on the nerve conduction of isolated IBS-D rat colonic smooth muscle:On the pharmacological platform of isolated colonic longitudinal smooth muscle in vitro,we investigated effects of PA on cholinergic,NO（nitrergic）and ATP（purines）nerve pathways.Samples of the colonic smooth muscle was obtained from the aforementioned IBS-D rats.After euthanasia by CO₂ asphyxiation,colons and jejuna were excised and placed in 37℃Kreb’s solution and the smooth muscle tension experiment was performed.Effect of cumulative PA concentrations(3×10^-7 mol/L to 1×10^-4 mol/L)on spontaneous contraction of the colonic longitudinal smooth muscle was displayed by a strip chart recorder LabChart software and half maximal effective concentration(EC₅₀)was also calculated.Electric field stimulation（40 V,2³0 Hz,0.5 ms pulse duration,and 10 s）was used to observe the nonadrenergic,noncholinergic neurotransmitter release in the IBS-D rat colon,and the ATP levels in samples were also determined.After the preincubation of PA（100μmol/L）,we observed and recorded the tension changes in the following conditions:1)cholinoceptor agonists carbachol(10^-9 mol/L to 10^-5 mol/L);2)NO synthase inhibitor L-NAME（10μmol/L）,the P2Y₁ receptor（purine）antagonist MRS 2500（1μmol/L）;3)P2-purinoceptor（purine）agonistα,β-me ATP（100μmol/L）;4)KCl（120mmol/L）.RESULTS（1）Improvement and Evaluation of Rat Model of IBS-D:Compared with control group,the number of feces grains in one-hour model group showed no obvious changes on day 7,14,21,28,but the area of the excrement was obviously increased and body mass was decreased on day 14（P<0.05 or P<0.01）;in2-hour model group,the number of feces grains and area of the excrement were increased on day 21,28,and body mass was decreased obviously on day 7,14,21,28（P<0.05 or P<0.01）.One-hour model group and 2-hour model group had higher AWR scores than the control group（P<0.05）,but the difference between the two model groups was insignificant（P>0.05）.The optical microscopy results showed that no various changes in gastric,duodenal,ileac,colonic tissues in the two model groups as compared with those in the control group.qPCR results showed that the expression level of 5-HT_3AR in rat colonic tissues of the two model groups was higher than that of the control group（P<0.05）,but the expression level of 5-HT_3AR in rat colonic tissues of model was not significantly different from that of the control group（P>0.05）.（2）Pharmacodynamics study of Pogostemonis Herba formula granules:Compared with control group,scores of abdominal withdrawal reflex and areas of defecation grains increased significantly（P<0.01）,colonic mRNA expression levels of5-HT_3AR,5-HT₄R,NK-1R increased significantly（P<0.05）,expression levels of nNOS decreased significantly（P<0.01）.Compared with model group,Pogostemonis Herba formula granules of low,medium and high dose groups of IBS-D rats showed obvious therapeutic effect.Compared with model group,colonic mRNA expression levels of Pogostemonis Herba formula granules groups showed significant decreases in 5-HT_3AR,5-HT₄R,NK-1R and significant increase in nNOS（P<0.05 or P<0.01）.（3）Pharmacodynamics study of PA:Compared with model group,PA of low,medium and high dose groups of IBS-D rats showed significantly decreased in scores of abdominal withdrawal reflex and areas of defecation grains（P<0.05）.Compared with model group,colonic mRNA expression levels of PA groups showed significant decreases in 5-HT_3AR,5-HT₄R,Tac1,NK-1R,Vip and significant increase in nNOS（P<0.05 or P<0.01）.（4）The regulation effect of PA on the nerve conduction of isolated IBS-D rat colonic smooth muscle:Firstly,PA exerted a concentration-dependent inhibitory effect on the spontaneous contraction of the colonic longitudinal smooth muscle,and the half maximal effective concentration(EC₅₀)was 41.9μmol/L.Secondly,PA exerted an antagonistic effect on the contraction of smooth muscle induced by KCl.（P<0.05）.Thirdly,PA also exerted an antagonistic effect on the contraction of smooth muscle induced by cholinergic receptor agonist carbachol（CCh）.Compared with CCh-treated IBS-D rat colon,the cholinergic contractile response of IBS-D rat colonic smooth muscle(EC₅₀=0.94μmol/L)was significantly decreased by PA(EC₅₀=37.43μmol/L)（P<0.05）.Fourthly,when the nitrergic pathway was blocked by L-NAME（10 mol/L）,the colonic smooth muscle appeared to increase in contractile tension.（P<0.05）.Pretreatment with PA resulted in inhibitory effect on L-NAME-induced contraction.Therefore,PA can inhibit the contraction of colonic smooth muscle caused by multiple neural pathways.However,ATP might not be the main neurotransmitter involved in inhibitory effects of PA in the colonic relaxation of stress-induced IBS-D rats.CONCLUSIONThis pharmacodynamic study revealed that patchouli alcohol is the main active ingredient of Pogostemonis Herba in the treatment of IBS-D and its efficacy is comparable to that of the single herbal drug.PA can effectively improve the diarrhea symptoms and visceral hypersensitivity of IBS-D rat.These results may contribute to the drug effect of PA to regulate intestinal neurotransmission.The underlying mechanisms may be:in colonic smooth muscle tissue of IBS-D rats,on the one hand,PA might increase nNOS mRNA expression level to raise the release level of nitric oxide.On the other hand,PA might have competitive antagonist effect to cholinergic receptors.In addition,PA might adjust expression of the colonic neurotransmitters 5–HT and so on.The above multitarget regulation effect might be the inhibitory effects of PA on the excessive contraction of the colon,to improve the symptoms of IBS-D.PA has the prospect of becoming a new drug or lead compound in the treatment of IBS-D.