Effect Of Berberine Hydrochloride On Nerve Conduction Of Colonic Smooth Muscle In IBS-D Rats

Objective(1)To study the pharmacological effects of berberine hydrochloride on chronic restrain stress model of IBS-D rats,and to research its mechanism of action;(2)To study the effect of berberine hydrochloride on the contractile tension of isolated colonic longitudinal muscle IBS-D rats,and to study the effect of berberine hydrochloride on the cholinergic,nitrogen and purine nerve conduction pathways.Methods1)Study on the pharmacodynamics and mechanism of berberine hydrochloride on IBS-D rats:chronic restrain stress model was used to induce the establishment of IBS-D rat model.Sixty rats were randomly divided into control group(10 rats)and model group(50 rats).The control group received no treatment,while the model group received bound stimulation to establish the model,2h per day for 14 days.The 50 rats were randomly divided into the model group,the positive medicine group(piviprium bromide group)and the low,medium and high dose groups of berberine hydrochloride,and were given gastric gavage.The control group and the model group were given gastric normal saline once a day for 14 consecutive days.DO,D7,D14,D21 and D28 days,the abdominal withdrawal reflex score(AWR)of rats in each group were measured to evaluate the sensitivity,and the defecation area was collected and counted.The contents and protein expressions of colonic 5-HT3AR,nNOS,Myosin Va,LC8 and SLC17A9 mRNA were measured by qPCR.(2)Regulation of berberine hydrochloride on nerve conduction of isolated colonic smooth muscle of IBS-D rats:we studied on isolated colonic smooth muscle of rats,to research the effects of berberine hydrochloride on spontaneous contraction,cholinergic,nitrogen and purine energy of smooth muscle.The colonic smooth muscle were obtained from the aforementioned IBS-D rats.Intercepting 1 cm colon segment of SD rats asphyxiated by CO2,filled its with 37oC Kreb 's solution of eight cavity perfusion system organ bath slot,and hung on fixing device with a sensor connected,continuous ventilation with mixture(95%CO2+5%O2)LabChart software was used to record the effect of berberine hydrochloride cumulative concentration(1.625,3.125,1.625,3.125,25,50,100 and 200 ?mol/L)on colonic longitudinal muscle fasciculation tension of rats,and half maximal effective concentration(EC50)was calculated.After wash and balance for lh,add P2 receptor(purine energy)agonist ?-?pmeATP(100 ?mol/L),P2Y1 receptor(purine energy)antagonist MRS2500(1 ?mol/L),nitric oxide synthase(NOS,nitrogen energy)inhibitor L-NAME(10 ?mol/L),choline receptor agonist CCh(10-9 ?mol/L?10-5 ?mol/L),TTX(tetrodotoxin,2?mol/L)in turn,with proper incubation time.Electric field stimulation(EFS,40 V,2?30 Hz,0.5 ms pulse duration,and 10 s)was given except for ?-?meATP and CCh to observe the changes in colonic muscle tension of IBS-D rats and record their motion state.After the preincubation of berberine hydrochloride(40 ?mol/L),the changes in muscle strip tension were observed and recorded,and compared with the previous experiment,to evaluate the effect of berberine hydrochloride.Results(1)Pharmacodynamics of berberine hydrochloride on IBS?D rats:Compared with the model group,the AWR and fecal area decreased in the berberine hydrochloride medium and high dose groups(P<0.05).The efficacy of the high-dose group was the most obvious,which was significantly different from that of the positive group(P<0.05).Compared with the colon of normal group,the mRNA levels of 5-HT3AR,Myosin Va and nNOS in the IBS-D model group were all abnormal.The mRNA expressions of 5-HT3AR and Myosin Va in the colon of IBS-D group were increased,while the expression of nNOS was decreased.LC8 and SLC17A9 showed no significant difference,indicating that the nitrogen-energy nerve conduction in the colon of IBS-D rats with chronic binding stress model established in this study may be abnormally changed,and other neurotransmission mode may be changed.The mRNA levels of 5-HT3AR and Myosin Va in the group which had been given BBH were significantly lower,indicating that BBH might improve the abnormal changes in the chronic restrain stress model.(2)The regulation of berberine hydrochloride on nerve conduction of isolated colonic smooth muscle in IBS-D rats:berberine hydrochloride inhibited the spontaneous contraction of colonic smooth muscle in a dose-dependent manner,with an EC50 of 25.17?mol/L.Secondly,choline receptor agonist carbacholine(CCh)can increase the tension of smooth muscle,and the tension in the stress model group was significantly higher than that in the control group(P<0.05).Berberine hydrochloride can antagonize the effect of choline receptor agonist carbacholine(CCh)on smooth muscle,and can significantly reduce the tension of smooth muscle(P<0.05).In addition,L-NAME can increase colonic smooth muscle tension,causing contraction,and the tension of colonic smooth muscle in stress model rats was significantly higher than that in the control group(P<0.05).However,by incubating berberine hydrochloride in advance,it can antagonize the contraction effect of L-NAME and reduce the tension to a diastolic state.Furthermore,?-?meATP could relax the smooth muscle,and the tension of the control group was significantly lower than that of the model group(P<0.05),while the preincubation of berberine hydrochloride could increase the tension of the smooth muscle and antagonize the effect of ?-? meATP.MRS2500 can increase the smooth muscle tension,while the preincubation of berberine hydrochloride can reduce the smooth muscle tension,but there is a significant difference only in the model group(P<0.05).ContractionBerberine is the main active ingredient of Rhizoma Coptidis in the treatment of IBS-D,which can significantly improve the visceral hypersensitivity and diarrhea symptoms of IBS-D rats.This effect is achieved through the regulation of intestinal nerve conduction.At the IBS-D colon smooth muscle tissue,BBH may increase the level of NO release,increase the activity of NOS,increase the nNOS mRNA expression level,In addition,it may competitively antagonize cholinergic receptors and regulate the expression of colonic neurotransmitter 5-HT.However,the effects of berberine on P2 receptor antagonist and agonist are different,so its effect on purinergic neural pathway is unknown,and further research is needed.This multi-target regulation of berberine is finally manifested as an inhibitory effect on the excessive contraction of the colon,which is an important pharmacological mechanism to alleviate IBS-D diarrhea symptoms.