Protective effect of histamine microinjected into cerebellar fastigial nucleus on stress gastric mucosal damage in rats.

In the study, we investigated the effect of histamine microinjected into cerebellar fastigial nucleus (FN) on stress gastric mucosal damage (SGMD), and its mechanisms in rats. The model of SGMD was established by restraining and water (21±1°C)-immersion for 3h. The gastric mucosal damage index (GMDI) indicated the severity of gastric mucosal damage. Histamine or receptor antagonist was microinjected into the FN. The decussation of superior cerebellar peduncle (DSCP) and the lateral hypothalamic area (LHA) were destroyed, respectively. The pathological changes of gastric mucosa were evaluated using biological signal acquisition system, Laser-Doppler flowmeter, and western blotting. We found that the microinjection of histamine (0.05, 0.5, and 5µg) into FN significantly attenuated the SGMD, in a dose-dependent manner, whereas, the microinjection of histamine H2 receptor antagonist, ranitidine, and glutamic acid decarboxylase antagonist, 3-mercaptopropionic acid (3-MPA) exacerbated the SGMD. The protective effect of histamine on SGMD was abolished by electrical lesion of DSCP or chemical ablation of LHA. The microinjection of histamine decreased the discharge frequency of the greater splanchnic nerve, and the gastric mucosal blood flow was increased. In addition, the cellular proliferation was enhanced, but the cellular apoptosis was reduced in the gastric mucosa. Also the pro-apoptosis protein, Bax, and caspase-3 were down-regulated, and the anti-apoptosis protein, Bcl-2 was up-regulated following microinjection of histamine. In conclusion, the FN participated in the regulation of SGMD after histamine microinjected into FN, and cerebellar-hypothalamic circuits (include: DSCP, LHA) contribute to the process, which may provide a new therapeutic strategy for SGMD.

Glutamate microinjection into the hypothalamic paraventricular nucleus attenuates ulcerative colitis in rats.

AIM: To investigate the effects of glutamate microinjection into hypothalamic paraventricular nucleus (PVN) on ulcerative colitis (UC) in rats and to explore the relevant mechanisms. METHODS: 2,4,6-Trinitrobenzenesulfonic acid (100 mg/kg in 50% ethanol) was instilled into the colon of adult male SD rats to induce UC. A colonic damage score (CDS) was used to indicate the severity of the colonic mucosal damage. The pathological changes in the colonic mucosa were evaluated using immunohistochemistry, Western blotting, biochemical analyses or ELISA. Ten minutes before UC induction, drugs were microinjected into the relevant nuclei in rat brain to produce chemical stimulation or chemical lesion. RESULTS: Microinjection of glutamate (3, 6 and 12 µg) into the PVN dose-dependently decreased the CDS in UC rats. This protective effect was eliminated after kainic acid (0.3 µg) was microinjected into PVN or into the nucleus tractus solitarius (NTS) that caused chemical lesion of these nuclei. This protective effect was also prevented when the AVP-V1 receptor antagonist DPVDAV (200 ng) was microinjected into the NTS. The discharge frequency of the vagus was markedly decreased following microinjection of glutamate into the PVN. Microinjection of glutamate into the PVN in UC rats significantly increased the cell proliferation and anti-oxidant levels, and decreased the apoptosis and Bax and caspase 3 expression levels and reduced the pro-inflammatory factors in the colonic mucosa. CONCLUSION: The activation of hypothalamic PVN exerts protective effects against UC, which is mediated by the NTS and vagus. The effects may be achieved via anti-oxidative, anti-apoptotic, and anti-inflammatory factors.

Muscimol microinjection into cerebellar fastigial nucleus exacerbates stress-induced gastric mucosal damage in rats.

AIM: To investigate the effects of microinjection of the GABA(A) receptor agonist muscimol into cerebellar fastigial nucleus (FN) on stress-induced gastric mucosal damage and the underlying mechanism in rats. METHODS: Stress-induced gastric mucosal damage was induced in adult male SD rats by restraining and immersing them in cold water for 3 h. GABA(A) receptor agonist or antagonist was microinjected into the lateral FN. The decussation of superior cerebellar peduncle (DSCP) was electrically destroyed and the lateral hypothalamic area (LHA) was chemically ablated by microinjection of kainic acid. The pathological changes in the gastric mucosa were evaluated using TUNEL staining, immunohistochemistry staining and Western blotting. RESULTS: Microinjection of muscimol (1.25, 2.5, and 5.0 µg) into FN significantly exacerbated the stress-induced gastric mucosal damage in a dose-dependent manner, whereas microinjection of GABA(A) receptor antagonist bicuculline attenuated the damage. The intensifying effect of muscimol on gastric mucosal damage was abolished by electrical lesion of DSCP or chemical ablation of LHA performed 3 d before microinjection of muscimol. Microinjection of muscimol markedly increased the discharge frequency of the greater splanchnic nerve, significantly increased the gastric acid volume and acidity, and further reduced the gastric mucosal blood flow. In the gastric mucosa, further reduced proliferation cells, enhanced apoptosis, and decreased anti-oxidant levels were observed following microinjection of muscimol. CONCLUSION: Cerebellar FN participates in the regulation of stress-induced gastric mucosal damage, and cerebello-hypothalamic circuits contribute to the process.

Protection of ghrelin postconditioning on hypoxia/reoxygenation in gastric epithelial cells.

AIM: To investigate the protective effect and mechanisms of ghrelin postconditioning against hypoxia/reoxygenation (H/R)-induced injury in human gastric epithelial cells. METHODS: The model of H/R injury was established in gastric epithelial cell line (GES-1) human gastric epithelial cells. Cells were divided into seven groups: normal control group (N); H/R postconditioning group; DMSO postconditioning group (DM); ghrelin postconditioning group (GH); D-Lys3-GHRP-6 + ghrelin postconditioning group (D + GH); capsazepine + ghrelin postconditioning group (C + GH); and LY294002 + ghrelin postconditioning group (L + GH). 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to detect GES-1 cell viability. Hoechst 33258 fluorochrome staining and flow cytometry were conducted to determine apoptosis of GES-1 cells. Spectrophotometry was performed to determine release of lactate dehydrogenate (LDH). Protein expression of Bcl-2, Bax, Akt, and glycogen synthase kinase (GSK)-3ß was determined by western blotting. Expression of vanilloid receptor subtype 1 (VR1), Akt and GSK-3ß was observed by immunocytochemistry. RESULTS: Compared with the H/R group, cell viability of the GH group was significantly increased in a dose-dependent manner (55.9% ± 10.0% vs 69.6% ± 9.6%, 71.9% ± 17.4%, and 76.3% ± 13.3%). Compared with the H/R group, the percentage of apoptotic cells in the GH group significantly decreased (12.38% ± 1.51% vs 6.88% ± 0.87%). Compared with the GH group, the percentage of apoptotic cells in the D + GH group, C + GH group and L + GH groups significantly increased (11.70% ± 0.88%, 11.93% ± 0.96%, 10.20% ± 1.05% vs 6.88% ± 0.87%). There were no significant differences in the percentage of apoptotic cells between the H/R and DM groups (12.38% ± 1.51% vs13.00% ± 1.13%). There was a significant decrease in LDH release following ghrelin postconditioning compared with the H/R group (561.58 ± 64.01 U/L vs 1062.45 ± 105.29 U/L). There was a significant increase in LDH release in the D + GH, C + GH and L + GH groups compared with the GH group (816.89 ± 94.87 U/L, 870.95 ± 64.06 U/L, 838.62 ± 118.45 U/L vs 561.58 ± 64.01 U/L). There were no significant differences in LDH release between the H/R and DM groups (1062.45 ± 105.29 U/L vs 1017.65 ± 68.90 U/L). Compared with the H/R group, expression of Bcl-2 and Akt increased in the GH group, whereas expression of Bax and GSK-3ß decreased. Compared with the GH group, expression of Bcl-2 decreased and Bax increased in the D + GH, C + GH and L + GH groups, and Akt decreased and GSK-3ß increased in the L + GH group. The H/R group also upregulated expression of VR1 and GSK-3ß and downregulated Akt. The number of VR1-positive and Akt-positive cells in the GH group significantly increased, whereas the number of GSK-3ß-positive cells significantly decreased. These effects of ghrelin were reversed by capsazepine and LY294002. CONCLUSION: Ghrelin postconditioning protected against H/R-induced injury in human gastric epithelial cells, which indicated that this protection might be associated with GHS-R, VR1 and the PI3K/Akt signaling pathway.

Lateral hypothalamic area mediated the protective effects of microinjection of glutamate into interpositus nucleus on gastric ischemia-reperfusion injury in rats.

We investigated the protective effects of chemical stimulation of cerebellar interpositus nucleus (IN) on gastric ischemia-reperfusion injury (GI-RI) and its possible regulatory mechanisms in rats. Gastric mucosal damage index (GMDI) indicated the severity of gastric mucosal injuries. Transferase dUTP nick end labeling (TUNEL) staining and proliferating cell nuclear antigen (PCNA) were performed to assess gastric mucosal cell apoptosis and proliferation. Microinjection of glutamate into IN markedly attenuated GI-RI. Either chemical lesion of IN or electrical ablation of the decussation of superior cerebellar peduncle (DSCP) obviously aggravated GI-RI. The protective effects of IN were reversed with the pretreatments of microinjection of 3-mercaptopropionic acid into IN or Bicuculline into lateral hypothalamic area (LHA), individually. The discharge frequency and intensity of greater splanchnic nerve (GSN) decreased and gastric mucosal blood flow increased after chemical stimulation of IN. The apoptosis of positive cells of gastric mucosa was decreased by chemical stimulation of IN, whereas proliferation increased. The gastric juice volume, acidity, and total acid output were all decreased after the chemical stimulation of IN. These results indicated that IN participates in regulation of GI-RI and is a specific area in central nervous system for exerting protective effects on GI-RI. DSCP, LHA and GSN may involve in this process. Apoptosis and proliferation may mediate this protective process in rats too.

Lateral hypothalamic area mediated the aggravated effect of microinjection of Baclofen into cerebellar fastigial nucleus on stress gastric mucosal damage in rats.

Cerebellum, primarily believed as a subcortical somatic motor center, is increasingly considered to be implicated in visceral activities. However, little is known about its regulation on gastrointestinal organs. In this research, we investigated the aggravated effect of microinjection of gamma-aminobutyric acid receptor subtype B (GABA(B)R) agonist, Baclofen into cerebellar fastigial nucleus (FN) on stress gastric mucosal damage (SGMD) and its possible regulatory mechanism. The gastric mucosal damage index was chosen to indicate the severity of gastric mucosal injure. Immunohistochemistry and transferase-mediated dUTP-biotin nick-endlabeling (TUNEL) methods were used to detect the variations of lateral hypothalamic area (LHA) and gastric mucosa. It had been demonstrated that FN participates in regulation of SGMD via its GABA(B)R and GABA neural pathway, which passes through the decussation of superior cerebellar peduncle and projects to the GABA receptors in LHA. Meanwhile, celiac sympathetic nerve involves in this process via mediating neural discharge, which results in the decrease of gastric mucosal blood flow. Additionally, apoptosis, proliferation and oxidation in gastric mucosa, and gastric acid contribute in the mechanism. It could be expected that these results might suggest insights to the cerebellar and hypothalamic function, and the treatment of gastrointestinal diseases.