Effects Of Hydrogen Sulfide In The Hypothalamic Paraventricular Nucleus On Gastric Function In Rats

Hydrogen sulfide（H₂S）is a new type of gaseous neurotransmitter discovered in recent years,which can freely cross various biological membranes,has a wide range of targets and can exert its regulatory effects by influencing multiple signalling pathways,together with Nitric Oxide（NO）and Carbon monoxide（CO）,they form a unique network of gaseous neurotransmitters that regulate a variety of physiological activities as a neuromodulator.Early laboratory studies found that Na HS injections into the dorsal nucleus of the vagus,the nucleus accumbens and the nucleus tractus solitarius each had a significant effect on gastric function.The hypothalamus is the centre of endocrine and phytomotor regulation.The paraventricular nucleus（PVN）is one of the large nuclei of the hypothalamus and is located in the periventricular part of the hypothalamus.It contains thousands of neurons and is the site of the greatest concentration of neurons secreting oxytocin or pressin,and there are many peptides that excite or inhibit gastric motility and can be released through neurons The neurotransmitters dopamine,5-hydroxytryptamine and acetylcholine are important centres for the regulation of appetite,food intake and gastric function,and their action may be mediated through the paraventricular nucleus-vagal complex-vagal pathway involved in the regulation of gastric function.However,whether the functional neurons that synthesize H₂S in the PVN are activated under stress and what is the role of H₂S in the PVN in the regulation of gastric function have not been reported.Therefore,we ask the following questions:1.Whether functional neurons for H₂S synthesis in the PVN are activated during Restraint Water-Immersion Stress（RWIS）in rats.2.The effect of H₂S in the PVN on gastric motility and gastric acid secretion in rats.3.If H₂S in the PVN regulates gastric function,through which receptors or signalling pathways does it do so.To address these questions,a three-part experiment was designed for this study:Experimental study 1:Rats were randomly divided into three groups（n=6）according to different time periods（control RWIS 0 h and experimental RWIS 1 h and 3 h）,and the positive cystathionine beta-synthase（CBS）and c-Fos co-expression within the PVN of rats were observed by immunohistochemical fluorescent double-labeling technique The number of neurons and the changes in CBS protein expression within the PVN of rats after RWIS were detected by protein immunoblotting.The results showed that the number of positive neurons co-expressed with c-Fos and CBS was significantly increased in the RWIS 1 h and 3 h groups compared to the control RWIS 0 h group,and the number of positive neurons co-expressed with c-Fos and CBS was the highest after RWIS 1 h.The results showed that the number of positive neurons co-expressed with CBS was significantly increased in the RWIS 1 h and 3 h groups compared to the control RWIS 0 h group.The protein immunoblotting technique also revealed that the expression of CBS and c-Fos proteins in the PVN of the experimental group of RWIS 1 h and 3 h rats was significantly higher than that of the control group.Experimental study 2:Rats were randomly divided into a control group（PVN injected with0.1μL saline）,an experimental group（PVN injected with 1 nmol（0.1μL,0.01 mol/L）Na HS,2nmol（0.1μL,0.02 mol/L）Na HS,4 nmol（0.1μL,0.04 mol/L）Na HS,8 nmol（0.1μL,0.08mol/L）Na HS）（n=6）,and the effects on gastric motility were observed in rats after the PVN was injected with saline and different doses of Na HS,respectively.A balloon was placed at the pylorus of the rats and the changes in gastric motility were recorded by the BL-420Biofunctional Assay System.The results showed that gastric motility was significantly inhibited in rats after intra-PVN injection of different doses of Na HS compared to the saline control group,and the best inhibition was found at 2 nmol Na HS injection.The rats were randomly divided into a control group（PVN injected with 0.1μL saline）,an experimental group（PVN injected with 2nmol（0.1μL,0.02 mol/L）Na HS,4 nmol（0.1μL,0.04 mol/L）Na HS,8 nmol（0.1μL,0.08mol/L）Na HS）（n=6）,and a control group（PVN injected with 2 nmol（0.1μL,0.02 mol/L）Na HS,4 nmol（0.1μL,0.04 mol/L）Na HS,8 nmol（0.1μL,0.08 mol/L）Na HS）.The effects on gastric acid secretion in rats were observed after the injection of saline and different doses of Na HS,respectively.The results showed that the p H value of gastric juice was significantly lowered after intravitreal injection of different doses of Na HS compared with the saline control group and showed a dose-dependent trend.These results suggest that the rat PVN contains H₂S functional neurons that are involved in the regulation of gastric function and promote gastric acid secretion in rats.Experimental study 3:Rats were randomly divided into three groups:injected with 2 nmol（0.1μL,0.02 mol/L）Na HS,2 nmol（0.1μL,0.02 mol/L）D-AP5+2 nmol（0.1μL,0.02 mol/L）Na HS,2 nmol（0.1μL,0.02 mol/L）PDTC+2 nmol（0.1μL,0.02 mol/L）Na HS（n=6）,and the effects on gastric motility and gastric acid secretion were observed in rats with PVN injected with Na HS and NF-κB blocker PDTC followed by Na HS and NMDA receptor blocker D-AP5followed by Na HS,respectively.The results showed that the pre-injection of PDTC or D-AP5eliminated the inhibitory effect of Na HS on gastric motility and the facilitative effect on gastric acid secretion.Conclusion:It is shown that CBS neurons are involved in RWIS regulation,and intra-PVN injection of Na HS can significantly inhibit gastric motility and promote gastric acid secretion in rats.The regulatory effect of intra-PVN injection of Na HS on gastric function may be achieved through NMDA receptors and down-regulation of NF-κB signaling pathway,and this study provides an important experimental basis for the prevention and treatment of stress gastric ulcer in clinical practice.