Study Of The Mechanism Of Lianpo Yin In Treatment Of Functional Dyspepsia By Brain-gut Co-regulated Pathways

Objective 1 By studying status of treatment for functional dyspepsia in traditional Chinese Medicine and western medicine to broaden the scope of application of lianpiao Yin(LPY),a classical pres cription in warm disease.2 To investigate the mechanism of Lianpo Yin(LPY)in the t reatment of Functional dyspepsia(FD)with multiple-pathway an d multiple-target.(1)To study the effect of LPY on the contraction activity o f SD rats' circular smooth muscle strips and to explore the pos sible mechanism of its action on circular smooth muscle strips in gastric antrum.(2)To study the effects of LPY on body weight,gastric empt ying and gastric antral muscle contraction in FD rats and to ex plore its mechanism of regulating gastric motility in FD rats.(3)To study the effect of LPY on behavior and “brain-gut” interaction of FD rats.Methods 1 Theoretical research:Through the documentation of carding and analysis,we integrated researched the basic theory?clinical and experimental research on treatment of functional dyspep sia with LPY.2 Experimental research:(1)(1)The preparations of isolated circular antral strips were suspended in the organ bath and the spontaneous contractile activity was noted.Consecutive concent rations of Serum with LPY(10?L?20?L?40?L?60?L)(n=8)or equal volumes of blank serum(control,n=8)were administrated i nto the bath to note the responses of strips;The cumulative con centrations of Acetycholine(10-8-10-4mol/L)were administrated i nto the organ bath to note the responses of strips(n=8)and com pared with Serum with LPY.The contractions induced by Serum wit h LPY were characterized by using antagonists,such as non-selec tive muscarinic(M)receptor antagonist Atropine(10-5mol/L,n=8),exogenous Nitricoxide(NO)donor L-arginine(10-5mol/L,n=8)and nicotinic(N)receptor antagonist Hexamethonium(10-5mol/L,n=8)on contractile activity of strips induced by Serum with LPY.(2)The preparations of isolated circular antral strips were suspended in the organ bath and the spontaneous contractile act ivity was noted.Consecutive concentrations of Krebs with LPY(400?L?800?L?1200?L?1600?L)(n=8)or equal volumes of Krebs(c ontrol,n=8)were administrated into the bath to note the respon ses of strips;The contractions induced by Krebs with LPY werech aracterized by using agonists,such as Acetycholine(10-5mol/L,n=8)and cholinergic receptor agonist Neostigmine(10-5mol/L,n=8)on contractile activity of strips induced by Krebs with LPY.(2)Neonatal SD rat of 8-day-old were randomly divided into normal control group(NC,n=10),functional dyspepsia group(FD,n=8),functional dyspepsia rats treated with domperidone group(DPLT+FD,n=8),and functional dyspepsia rats treated with LPY group(LPY+FD,n=8),adaptiving feeding for 2 days.At 10-day-old,the rats were stimulated by iodoacetamide: NC group(2% sucros e 0.2ml per day,gavage);FD group,DPLT+FD group and LPY+FD gro up(0.1% iodoacetamide(dissolved in 2% sucrose solution)0.2ml per day,gavage)for 7days.The body weight was recorded per we ek.At seven-week,HC group and FD group were gavaged with saline,DPLT+FD group(Domperidone 20mg/kg per day,gavage)and LPY+FD group(LPY,0.920g/kg per day,gavage)for a week.At eight-week,the food intake and gastric emptying was tested.The rats in ea ch group were sacrificed with cervical dislocation,the gastric antrum was isolated,and the circular smooth muscle strips wer e prepared.Record the responses of strips to accumulative conce ntrations of Acetylcholine(10-8-10-4mol/L)and 5-HT(10-5mmol/L).(3)Neonatal SD rat of 8-day-old were randomly divided into normal control group(NC,n=10),functional dyspepsia group(FD,n=8),functional dyspepsia rats treated with fluoxetine group(F luoxetine+FD,n=8),and functional dyspepsia rats treated with LP Y group(LPY+FD,n=8),adaptiving feeding for 2 days.At 10-day-old,the rats were stimulated by iodoacetamide:NC group(2% sucrose 0.2ml per day,gavage);FD group,Fluoxetine+FD group and LPY+F D group(0.1% iodoacetamide(dissolved in 2% sucrose solution)0.2ml per day,gavage)for 7 days.The body weight was recorded p er week.At seven-week,HC group and FD group were gavaged with saline,Fluoxetine +FD group(Fluoxetine,2mg/kg per day,gavage)and LPY+FD group(LPY,0.920g/kg per day,gavage)for a week.At eight-week,the Sucrose preference test was determined.Then,r ats in each group were sacrificed with cervical dislocation,and bilateral hippocampal tissues were taken and stored at-80?.The content of the monoamine neurotransmitters 5-hydroxytryptam ine(5-HT),norepinephrine(NE)and dopamine(DA)in the hippocampa l tissues of rats in each group were determined by high perform ance liquid chromatography(HPLC).Results 1 Theoretical research: In clinical,refering to the princi ple of “combination of disease and syndrome”,the disease?syn drome and symptom differentiation will be supposed to combined in tradition Chinese and western medicine.Meanwhile,following the principle of treating different diseases with the same the rapy,we shoud focus on pathogenesis and disease position,regar dless of symptom.When it show retention of damp-heat in spleen and stomach,we can apply LPY to treatment of diseses in all s ystems,especially the digestive system diseases.2 Experimental research:(1)(1)Serum with LPY dose-dependent increased the contractions of antral circular smooth muscle,an d its induced contractility was significantly increased compari ng with control group(Blank serum)(P<0.01 for all).Accumulati ve concentrations of Acetylcholine dose-dependently elicited co ntractions of antral smooth muscle strips,and the effect of Ace tylcholine(10-5mol/L)comparing with the maximal effect(60?L)of serum with LPY was not significantly different(P>0.05).The contractions of antral circular strips induced by Atropine + S erum with LPY,L-arginine + Serum with LPY,Hexamethonium + Ser um were significantly decreased compared with Serum with LPY(P<0.01 for all).The contractions induced by Serum with LPY were completely suppressed by Atropine,however,L-arginine and Hex amethonium partly inhibited the contractions of strips induced by Serum with LPY.(2)Krebs with LPY dose-dependently inhibited the contraction s of antral smooth muscle strips,with a significant difference compared with control group(Krebs)(P<0.01).Cumulative dose o f Krebs with LPY significantly inhibited contractions of antral smooth muscle strips induced by Acetycholine(10-5mol/L)(P<0.01).Maximal inhibitory effect of Krebs with LPY(1600?L)signi ficantly inhibited the contractions of antral smooth muscle str ips induced by Neostigmine(10-5mol/L)(P<0.05).(2)After making model,body-weight and the growth rate of b ody-weight in each FD model group were significantly reduced co mpared with NC group(P<0.05),which were significantly increa sed after treatment of doperidone and LPY(P<0.01 for both).In FD group,the food intake and gastric emptying of rats were si gnificantly decreased than NC group,which were significantly i ncreased after treatment after LPY and doperidone(P<0.01 for both).It was no significant difference between DPLT+FD group an d LPY+FD group(P>0.05).In basal state,frequency of antral st rips' contractions in FD rats was significantly decreased compa red with normal rats(P<0.01 for all),yet,the frequency was si gnificantly increased after intervention with LPY(P<0.01).It had no significant difference between LPY+FD group with DPLT+F D group(P>0.05).There was no significant difference in the am plitude of antral strips' contractions in each group(P>0.05 f or all).Acetylcholine-induced contractions of antral circular s trips in FD rats were significantly decreased(P<0.01 vs contro l for all).Treatment with LPY makedly increased Acetylcholine-induced circular strips contractions of FD rats(P<0.01 vs FD fo r all).EC50 in FD group higher than FD+DPLT group and FD+LPY gr oup(P<0.01),and it was no significant difference between eff ect of LPY and DPLT(P>0.05).The maximal contractions effect w as shown at the highest cumulative amount of acetylcholine(10-4mol/L)in each group,and there was no significant difference a mong all groups(P>0.05).Compared with normal,the reaction of antral circular strips in FD rats to 5-HT(10-5mol/L)was signi ficantly reduced(P<0.05).After treatment of LPY,response of antral circular strips to 5-HT shown a increase(P<0.05).(3)Compared with normal rats,the sucrose preference of FD rats decreased significantly(P<0.01),and the sucrose prefere nce of FD rats increased significantly after treatment of LPY(P<0.05).The content of monoamine transmitters 5-HT and NE in hi ppocampus of FD rats was significantly reduced by comparison wi th normal rats(P<0.01),while the content of DA was not signif icant changed(P>0.05).After treatment of LPY,content of 5-HT and NE in hippocampus of FD rats were significantly increased(P<0.01),which had no significant difference compared with ef fect of fluoxetine(P>0.05).Conclusions 1 Theoretical research: We can expand the clinical applicat ion scope of LPY and treat it as the primary prescription for the damp-heat syndrome of functional dyspepsia.2 Experimental research:(1)(1)Serum with LPY promote contrac tions of antral circular smooth muscle in normal rats by dose-d ependently.The effect of serum with LPY on antral strips is mai nly through the activation of the muscarine receptor,meanwhile,partly through activation of N receptor and inhibition of NO release to cause contraction of antral circular smooth muscle in normal rats.(2)Krebs with LPY inhibit the contractions of antral circular smooth muscle in normal rats by dose-dependently.The mechanism may be related to blocking the Acetylcholine pathway and exer ting the anticholinergic effect.(2)The gastric emptying of FD rats is delayed with attenuated contractile activity of antral strips.LPY can significantly improve the gastric emptying and contractile activity of antral circular strips in FD rats,improving the reactivity of antral strips to Acetylcholine and 5-HT.(3)FD rats exhibit anxiety-like or/and depression-like behavior,and LPY can improve the anxiety-like or/and depression-like behavior of FD rats.LPY upregulate the levels of 5-HT and NE in hippocampus of FD rats,which maybe involved in regulate the disorder of“brain-gut”interaction and exert the therapeutic effect on FD rats through pathway of“brain-gut”axis.3 The study explained the mechanism of Lianpo Yin in treatment of functional dyspepsia by brain-gut co-regulated pathways.It reflects holism concept of traditional chinese medicine,and fits the definition of FD as“disorders of brain-gut interacti on”in Rome IV,which provides a new reference for multi-dimensi onal diagnosis and treatment of FD.