| Restraint Water-Immersion Stress(RWIS)model is a compound stress model,which can cause gastrointestinal dysfunction in a short time.It is widely used to study the pathogenesis and treatment of stress gastric mucosa injury.Our laboratory has long been engaged in the study of gastric mucosal injury induced by confining immersion stress in rats.It was found that neurons and astrocytes were activated in the spinal cord,nucleus tractus solitarius of brainstem,dorsal nucleus of vagus nerve,paraventricular nucleus of hypothalamus,supraoptic nucleus,parbrachial nucleus and other nuclear groups in rats during the process of restraint flooding stress,and were involved in the process of regulating the stress-induced gastric mucosa injury.However,previous studies mainly focused on the brain stem,hypothalamus and other lower centers,and the role of the higher central cortex in stress gastric mucosal injury was less studied,and its regulatory mechanism has not been clarified.The cerebral cortex is the advanced center of pain perception.As an important part of the cortex,the anterior cingulate cortex(ACC)can regulate cognitive decision-making,emotion and pain emotion,and participate in physiological processes such as motor memory.ACC is also the key center for gut related visceral sensory processing and visceral motor control.It can receive projections from the spinal cord and the nucleus tractus solitarius(NTS),participate in the regulation of visceral activity,emotion and motivation,and is related to the production of functional gastrointestinal disorders.Previous laboratory studies found that restraint immersion stress activated neurons and astrocytes in the nucleus tractus solitarius,indicating that the nucleus tractus solitarius was involved in the process of gastric mucosal damage caused by restraint immersion stress,but whether ACC was involved in the process of gastric mucosal damage caused by restraint immersion stress has not been reported in the literature.Based on the above background,the following questions are raised:1.Can restraint immersion stress in rats activate neurons and astrocytes in ACC?2.What is the regulatory function of ACC on stress gastric ulcer?3.What is the effect of glutamate neurons in ACC on stress gastric mucosal injury?4.The effect of ACC on the expression of c-Fos and GFAP in the nucleus tractus solitarius of rats with restraint immersion?To solve the above problems,four parts of experiments are designed:1.The rats were uniformly divided into control group(RWIS 0h)and experimental group(RWIS 1h,RWIS 3h)for experiment.The expression of neuronal marker c-Fos and astrocyte marker GFAP protein in ACC were detected by immunofluorescence technique to explore whether ACC participated in the process of restraint immersion stress.2.The rats were divided into three groups: the first group was no-load virus control group,which was injected with no-load virus(AAV9-h Syn-m Cherry)+ intrapitoneal injection of Clozapine-N-Oxide(CNO);The second group was normal saline control group,which was injected adeno-associated virus(AAV9-h Syn-h M4D(Gi)-m Cherry)+ intrabitoneal injection of normal saline.The third group was the experimental group,which injected virus(AAV9-h Syn-h M4D(Gi)-m Cherry)+ intrabitoneal injection of CNO to activate h Syn-h M4D(Gi)receptor to inhibit ACC neurons.After three weeks of virus transfection,the rats fasted for 24 h and were given RWIS for 3h after 30 minutes of drug injection.The expressions of c-Fos and GFAP in ACC were detected by immunofluorescence technique.The expression of Claudin-1,Occludin and PCNA protein in gastric wall was detected by protein immunoblotting technology,and the gastric mucosal injury was counted to explore the role of ACC in the gastric mucosal injury caused by restraint immersion stress.3.First,the expression of glutamate vesicle transporter 1(VGLUT1)was detected at different stages of rats’ restraint immersion stress.Inhibition of glutamatergic neurons in the ACC: rats were randomly divided into three groups: the first group of no-load virus control group: injection of no-load virus(AAV9-Ca MKIIa-MCS-m Cherry)by intraperitoneal injection of + CNO;The second group was normal saline control group,which was injected adeno-associated virus(AAV9-Camkia-HM4D(Gi)-m Cherry)+ normal saline.The third group was the experimental group,which was injected with adeno-associated virus(AAV9-Camkia-HM4D(Gi)-m Cherry)+CNO.Three weeks after transfection by virus injection,intraperitoneal injection was performed in the same way as experiment 2.Glutaminergic neurons were inhibited in the experimental group.The expression of c-Fos and GFAP in ACC was detected by immunofluorescence at RWIS 3h after 30 minutes of injection.The expression of Claudin-1,Occludin and PCNA in gastric wall were detected by immunoimprinting technique,and the damage of gastric mucosa was analyzed.Activation of glutaminergic neurons in ACC: The experimental virus was replaced with AAV9-Camkia-HM3D(Gq)-m Cherry during activation of glutaminergic neurons,and other operations were the same.To explore the role of glutaminergic neurons in ACC in gastric mucosal injury induced by RWIS.4.Detection of c-Fos and GFAP expression changes in the NTS of restraint immersion stressed rats using immunofluorescence technique,and explore the effect of inhibition/activation of neurons in ACC on the activity of neurons and astrocytes in NTS of rats with restraint immersion stress.The experimental results show that:1.The expression of c-Fos and GFAP in ACC was significantly increased by the stress of restraint immersion in rats,while the gastric mucosal damage gradually increased with the increase of restraint immersion time.The expression of the closing protein Claudin-1 and the occludin Occludin,among the gastric tight junction proteins,decreased with increasing stress time,and the expression of the proliferating cell nuclear antigen PCNA associated with cell cycle regulatory proteins was significantly reduced.The results showed that ACC participated in the process of restraint immersion stress.2.Chemical genetic technology inhibits the activity of neurons in ACC,significantly reduces the expression of c-Fos and GFAP in ACC of rats under restraint immersion stress,reduces the damage of gastric mucosa,and increases the expression of Occludin,Claudin-1,PCNA protein in gastric wall.It shows that neurons in ACC participate in the process of restraint immersion stress,which aggravates the injury of gastric mucosa.3.In rats subjected to restraint immersion stress,the expression of VGLUT1 in ACC was significantly increased,indicating that glutamatergic neurons in ACC were involved in restraint immersion stress.When glutamatergic neurons were inhibited by chemical genetic techniques,the expression of c-Fos and GFAP in ACC decreased,gastric mucosal injury was reduced,and the expression of Occludin,Claudin-1,and PCNA proteins in the gastric wall increased.In contrast,after excitation of glutamatergic neurons,c-Fos and GFAP expression in ACC increased,gastric mucosal injury was intensified,and gastric wall Occludin,Claudin-1,and PCNA protein expression was further decreased.The results suggest that glutamatergic neurons within the ACC are activated during restraint immersion stress and aggravate stressful gastric mucosal injury.4.Inhibition of neurons within the ACC significantly reduced c-Fos and GFAP expression in the NTS of restraint immersion stressed rats.Inhibition of glutamatergic neurons in the ACC similarly reduced c-Fos and GFAP expression in the NTS after restraint immersion stress.Activation of glutamatergic neurons in the ACC resulted in a further increase in c-Fos and GFAP expression in the NTS of restraint immersion stressed rats.This suggests that ACC may regulate the process of gastric mucosal injury caused by restraint immersion stresse through the regulation of NTS.The present study exemplifies the regulation of gastric function by the rat higher central anterior cingulate cortex,which complements the regulation of gastric ulcer by the anterior cingulate cortex and provides new research ideas for the clinical study of stressful gastric mucosal injury. |
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