Protective Effect Of Oxytocin Via VTA-NAc Dopamine Signaling Pathway On Stress-induced Gastric Ulcers In Rats And Its Potential Mechanism

Objective: Oxytocin(OT)is one of the important brain gut peptides secreted by hypothalamus.In recent years,it has been found that it plays an important role in gastrointestinal function in addition to energy metabolism and feeding behavior.Studies showed that enteric OT/OTR signaling protects the intestinal tract and regulates of gastrointestinal(GI)motility,intestinal permeability,and mucosal maintenance to reduce intestinal inflammation[1].OT can also inhibit gastric emptying by stimulating the release of cholecystokinin(CCK)[2,3].The purpose of this study was to investigate the regulatory and protective effects of OT on the formation and development of stress gastric ulcer in rats,and whether the ventral tegmental area(VTA)-nucleus accumbens(NAc)dopamine(DA)signaling pathway is involved in this regulatory process.Methods: 1.Healthy male Wistar rats were selected.The stress gastric ulcer model of rats was established by water immersion restraint stress;2.Observation of nerve fiber projection between VTA-NAc and the distribution of oxytocin receptor(OTR)and dopamine(DA)immunoreactive neurons in VTA by fluorescence gold retrograde tracing and immunohistochemical staining;3.The effects of OT and its receptor antagonist,atosiban,on the discharge of VTA DA neurons were observed by extracellular recording;4.The brain nuclei were catheterized and microinjected with drugs,to observe the protective effect and potential mechanism of VTA microinjection of OT on gastric mucosal cells in rats with stress gastric ulcer;5.The effect of OT on the content of hexose in gastric mucosa and the histological analysis of gastric mucosa were observed by Periodic acid Schiff and Alcian blue stain(AB-PAS)staining;6.Western blots and RT-PCR were used to detect the expression of dad2 r protein and m RNA in NAc;7.The effect of OT on DA and its metabolites in NAc was observed by highperformance liquid chromatography-electrochemical detector(HPLC-ECD).Results:(1)The effects of OT administered into the VTA on gastric ulcer index,p H of gastric content and mucus secretion in gastric ulcer model rats: 0.15 ?g OT,0.3 ?g OT,0.6 ?g OT,or 1.2 ?g OT administration into the VTA caused a significant decrease in gastric ulcer index(P<0.05),while PH of gastric content(P<0.05)and mucus secretion significantly increased(P<0.05),all in a dose-dependent manner.Therefore,OT was determined to have an ED50 value of 0.3 ?g,which was used for subsequent experiments.OT administered into the VTA significantly decreased gastric ulcer index(P<0.05),while p H of gastric content(P<0.05)and mucus secretion was significantly increased(P<0.05)compared to NS group,illustrating the protective effect of OT on the gastric mucosa.Atosiban administration in the atosiban-VTA group significantly increased gastric ulcer index(P<0.05),while p H of gastric content(P<0.05)and mucus secretion(P<0.05)significantly decreased compared to the OTVTA group.While OT administered into the VTA following atosiban,the protective effect of OT on gastric mucosa was significantly weakened(P<0.05).It is suggested that endogenous OT may participate in the protective effect of stress gastric ulcer,and the protective effect of OT on gastric ulcer mucosa may be related to the activation of OTR signaling pathway.Raclopride administration to NAc reduced the protective effect of VTA OT on the gastric mucosa of rats,compared with the OTVTA + NSNAc group(P<0.05~0.001).This result implicates VTA-NAc DA signaling pathway may be involved in the protective regulation of OT on stress-induced gastric ulcers.(2)The effect of VTA microinjection of OT on the content of hexosamine and neutral mucus in gastric mucosa of rats with gastric ulcer model: OT administered into the VTA significantly increased hexosamine levels(P<0.05)and PAS-stained neutral mucus substance(P<0.05)in the gastric mucosa compared with the NSVTA group.However,preinjection of atosiban blocked the effect of OT on promoting the biosynthesis and accumulation of hexosamine and promoting the secretion of gastric mucus substance in the gastric mucosa.It is suggested that OT may play a protective role in gastric mucosa by promoting the synthesis of hexosamine or increasing the secretion of neutral mucus.(3)The effect of microinjection of OT into VTA on the discharge activity of VTA DA neurons in rats: The electrophysiological experiment showed that 44.4 % of the 54 DA neurons increased significantly(P<0.01),identified as OT-E neurons;35.19 % of the neurons were inhibited(P<0.01),identified as OT-I neurons;20.40 % of the neurons did not change significantly(P>0.01);The effect of OT on discharges of DA neurons could be blocked by pre-administration of atosiban into the VTA(P<0.01).It is suggested that OT may be involved in the excitatory regulation of DA neurons by acting on OTR.Administration of atosiban alone had no effect on discharges of DA neurons.These results illustrate that OT may be involved in the mucosal protection of stress gastric ulcer by regulating the excitability of DA neurons.(4)The effect of VTA microinjection of OT on the expression of OTR / TH immunoreactive neurons in rats: Fluorescence immunohistochemistry study showed that,microinjected the retrograde tracer FG into the NAc,and observed FG-labeled DA neurons in the VTA,as expected.Staining for the DA and OTR revealed DA-immunoreactive(DA-IR)and OTRimmunoreactive(OTR-IR)neurons in the VTA.Some DA-IR neurons were also OTR-IR,and some FG-positive VTA neurons were both OTR-IR and DA-IR,indicating that some VTA DA neurons express the OTR.In addition,we microinjected the OT into the VTA,and observed TH-IR neurons which co-located with DA neurons were increased in the VTA(P<0.05).It suggests that OT can activate DA neurons in the VTA,this provides anatomical and functional basis for the follow-up study.The results of Western blot and RT-PCR showed that there were protein and m RNA expression of target protein DA D2 R in NAc.(5)The effect of VTA microinjection of OT on the content of nacda and its metabolite DOPAC: OT administered into the VTA caused an increase in DA and its major metabolite DOPAC in the NAc(P<0.05).The promote effect of OT on content of DA / DOPAC could be blocked by pre-administration of atosiban into the VTA(P<0.05).This result suggests that OT enhances VTA neuronal firing and subsequent DA release in the NAc,and that DA may play a role in OT's regulation of stress-induced gastric ulcers.(6)The effect of VTA microinjection of OT on gastric ulcer index and PH of gastric content in Oxtr-/-mice: OT administered into the VTA caused gastric ulcer index(P<0.05)significantly increased and p H of gastric content(P<0.05)significantly decreased in the NSOxtr-/-group,compared to the NSWT group,indicating that endogenous OT,acting through OTR,likely protects the gastric mucosa of mice.Further,there was no change in gastric ulcer index or p H of gastric content after OT administered in the VTA of Oxtr-/-mice(P>0.05)compared with the NSOxtr-/-group.Importantly,OTOxtr-/-mice showed increased gastric ulcer index(P<0.05),and decreased p H of gastric content(P<0.05)compared with the OTWT group.These results further implicate OTRs in the regulation stress-induced gastric ulcer,and corroborate our pharmacological findings with the OTR antagonist in rats.(7)The motor dorsal nucleus(DMV)of vagus nerve was damaged by electricity,to observe the effect of VTA microinjection of OT on gastric ulcer index,PH of gastric content and mucus secretion in gastric ulcer model rats: OT administration to the VTA of the shamlesion-DMV group showed decreased gastric ulcer index(P<0.05),and increased p H of gastric content(P<0.05)and mucus secretion(P<0.05)compared to the sham-lesionDMV+NSVTA group;OT administered into the VTA of DMV-lesion rats caused a significant increase in gastric ulcer index(P<0.05),while p H of gastric content(P<0.05)and mucus secretion was significantly decreased(P<0.05)compared to the sham-lesion-DMV+OTVTA group.These results indicate that the DMV may play a role in stress-induced gastric ulcers and the protective effect of OT on gastric mucosa.Conclusion: There was co-expression of OTR and th immunoreactive neurons in VTA.VTA microinjection of OT can significantly reduce the gastric ulcer index,increase the PH of gastric content and mucus secretion in gastric ulcer model rats.VTA pre-injection of atosiban can block the protective effect of OT on gastric mucosa,suggesting that the protective effect of OT on gastric ulcer mucosa may be related to the activation of OTR signaling pathway;Raclopride administration to the NAc can partially block the protective effect of OT on gastric mucosa in gastric ulcer model rats,suggesting that this regulatory mechanism involves the activation of VTA-NAc DA signaling pathway.This study is of great significance to explore the pharmacological strategies for the treatment of gastric ulcer or other diseases of the digestive system.