Effects Of MCHR1 Antagonist Via VTA-DMV Pathway On The Stress-induced Gastric Mucositis In Rats And Its Underlying Mechanism

Objective:Anxiety is a common disease symptom,which can cause autonomic nervous dysfunction in a period of time.As one of the most important factors affecting human health,its specific effects and potential mechanisms have attracted wide attention of researchers.Ventral tegmental area(VTA)is one of the central nervous nuclei involved in emotion,which is closely related to the regulation of anxiety behavior and its influence.Melanin concentrating hormone(MCH)and related receptors are closely related to the regulation of emotion and stress response.This study will explore whether SNAP94847,an antagonist of MCH receptor,is involved in the regulation of the neural pathway from ventral tegmental area to dorsal vagal nucleus(VTA-DMV),and then participate in the protection of gastric mucosal stress injury caused by anxiety,and its potential mechanism.Methods:1.The MCH receptor 1 immunoreactive neurons in VTA and the existence of signal projection pathway between VTA and DMV were observed in 10 rats randomly.2.Using single cell discharge recording method,30 normal rats and 30 anxiety model rats were randomly selected to observe the effects of NS,MCH or MCH receptor antagonist SNAP94847 microinjection on the firing activity of DA neuronsin the VTA.3.The elevated cross maze test(EPM)was used to observe the changes of anxiety like behavior after microinjection of ns or SNAP94847 into VTA.Six normal rats and twelve anxiety rats were randomly selected and divided into three groups(n=6):(1)VTA microinjection of 0.5μL NS in normal rats,(2)0.5μL NS in VTA of anxiety rats,and(3)6μg/0.5μL SNAP94847 was microinjected into VTA of anxiety rats.4.The gastric mucosal injury and related inflammatory indexes were observed by microinjection of ns or MCH receptor antagonist SNAP94847 into VTA of rats.Six normal rats and twelve anxiety rats were randomly divided into 3 groups(n=6):(1) VTA microinjection 0.5μL NS in normal rats,(2)0.5μL NS in VTA of anxiety rats, and(3)microinjection of 6μg/0.5μL SNAP94847 into VTA of anxiety rats,The degree of gastric mucosal injury was observed,and the area percentage of PAS stained mucosal material in gastric mucosa was observed under microscope.The activities of catalase(CAT),glutathione peroxidase(GSH PX),myeloperoxidase(MPO), malondialdehyde(MDA),tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6) were observed;5.After lesion of gastric mucosa,rats were injected with SNAP94847.Twenty four anxiety rats were randomly selected and divided into four groups(n=6).SNAP94847 was microinjected into VTA in each group:(1)sham lesion+NS group;(2)sham lesion+SNAP94847 group;(3)electrical lesion+NS group;(4)electrical lesion+ SNAP94847 group.The inflammatory markers CAT,GSH Px,MPO,MDA,TNF-α and IL-6 were detected;Results:1.Retrograde tracing combined with fluorescence immunohistochemical staining showed that FG labeled neurons were observed in the ipsilateral VTA seven days after the microinjection of FG into one side of DMV.MCHR1 immunofluorescence staining was performed with the same slice,and MCHR1 were co-labeled with FG in the VTA.2.The results of electrophysiological experiments showed that 42 DA neurons were identified in VTA of 30 normal rats.After microinjection of MCH,the firing frequency of 17 DA neurons increased by(62.5±4.3)%(P<0.01),and the firing frequency of 25 DA neurons decreased by(51.9±4.1)%.40 DA neurons were identified in VTA of 30 anxiety rats.After microinjection of MCH,the firing frequency of 18 DA neurons increased by(92.4±20.8)%(P<0.01),and the firing frequency of 22 DA neurons decreased by(66.1±13.9)%(P<0.01).The excitatory or inhibitory effects of MCH on the DA neurons could be significantly inhibited by the injection of SNAP94847 into the VTA(P<0.01).Compared with normal rats ~1MCH group,DA neurons in VTA in ~1MCH group of anxiety rats were more activated or inhibited by MCH significantly(P<0.05).3.EPM results showed that compared with normal+NS group,the number and time percentage of entering the open arm in anxiety+NS group were significantly decreased(P<0.05),while compared with anxiety+NS group,the number and time percentage of rats in anxiety+SNAP94847 group were significantly increased(P<0.05).4.To observe the effects of SNAP94847 on gastric injury in the normal and anxiety rats. The results showed that compared with the normal+NS rats,gastric mucosal bleeding lesions were observed in the anxiety+NS group,and the secretion of neutral mucus in gastric mucosa was significantly decreased(P<0.05).Compared with the anxiety+ns rats,bleeding ulcer lesions in the anxiety+SNAP94847 group were alleviated,and the secretion of neutral mucus in gastric mucosa was significantly increased(P<0.05).5.The effects of MCHR1 signaling pathway on gastric inflammation in normal and anxiety rats were observed.Compared with the control group,the activity of CAT and the content of GSH Px was significantly decreased(P<0.05),while the activity of MPO was significantly increased(P<0.05);the contents of MDA,TNF-αand IL-6 were also significantly increased(P<0.05);compared with anxiety+NS rats,CAT activity and GSH Px of content of anxiety+SNAP94847 group was significantly higher than that of anxiety+NS rats(P<0.05);while MPO decreased significantly(P<0.05);MDA,TNF-αand IL-6 were also significantly decreased(P<0.05).6.Compared with sham lesion+NS group,the CAT activity in sham lesion+ SNAP94847 group was significantly increased(P<0.05),GSH Px content was significantly increased(P<0.05),MPO activity was significantly decreased(P<0.05),MDA,TNF-αand IL-6 contents were significantly decreased(P<0.05);compared with sham lesion+SNAP94847 group,CAT activity of sham+ SNAP94847 group was significantly decreased(P<0.05).The content of GSH Px decreased significantly(P<0.05),while the activity of MPO increased significantly(P<0.05),and the contents of MDA,TNF-αand IL-6 increased significantly(P<0.05).Conclusions:There was a MCHR1 expression pathway between VTA and DMV,and DA neurons in VTA were regulated by MCHergic signaling pathway.Moreover,microinjection of SNAP94847 into VTA can regulate the gastric injury induced by anxiety and anxiety like behavior in rats.Moreover,MCH receptor signaling pathway and DMV nucleus are closely related to this effect.