Regulation Of Endogenous Orexin-A On Feeding Behavior In Rats

Objective: To investigate the effects of central injection of orexin-1 receptor antagonist SB-334867 and orexin-A antibody on food intake in rats;To explore whether endogenous orexin-A is involved in the regulation of feeding behavior.Methods: The specificity of orexin antibodies was detected by Western blot analysis,and the distribution of orexin-A immunoreactive neurons and immunoreactive nerve fibers in the brain was analyzed through immunohistochemistry.The feeding rats were fed individually,and the daily food intake wa s calculated.Fasting for 4 hours before the experiment,food and water were available ad libitum.In order to explore the effects of orexin-1 receptor antagonist SB-334867 and orexin-A antibody on food intake in rats,56 rats were randomly divided into 7 groups(n=8):(1)Normal sheep serum group;(2)1.0μl orexin-A antibody group;(3)4.0μl orexin-A antibody group;(4)10.0μl orexin-A antibody group;(5)40.0μl orexin-A antibody group;(6)NS group;(7)5.0μ g /0.5μl SB-334867 group.In order to study the effects of cannabinoid receptor antagonist AM251 on the feeding of rats,36 rats were randomly divided into 4 groups(n=9):(1)physiological saline control group;(2)AM251 group;(3)orexin-A group;(4)AM251+orexin-A group.Each group of rats were injected with the corresponding medicine and placed in t heir cages,and the rats were given pre-weighed food.The remaining food was measured hourly in 8 hours,and the cumulative food intake per hour was calculated.Rescults: Western blot analysis showed that the orexin antibodies of this study may selectively bind to the orexin-A antigen but can not bind to the orexin-B antigen,suggesting that the antibodies used in this study are orexin-A specific antibodies.Immunohistochemical analysis showed that orexin-A immunoreactive neurons were mainly expressed in the lateral hypothalamic area(LHA)and the perofornical area(Pe F)in the central nervous system.In the mesencephalon,diencephalon and afterbrain regions were not detected orexin-A immunoreactive neurons expression.In the parietal cortex,the central and medial amygdaloid nuclei and the hippocampus were detected orexin-A immunoreactive nerve fibers.Especially in the hypothalamic arcuate nucleus,the periventricular hypothalamic nucleus and the paraventricular nucleus of the hypothalamus,orexin-A immunoreactive nerve fibers were expressed more.In the thalamus,orexin-Aimmunoreactive nerve fibers mainly project to the central part of the brain,including the rhomboid nucleus,the thalamus,the paraventricular nucleus,the medial habenular nucleus,and the stria thalamic.In addition,a high expression of nerve fibers was found in the central gray region,locus coeruleus and raphe nuclei of the midbrain.orexin-A immunoreactive nerve fibers are also expressed in the reticular structures of the pons and medulla,the nucleus of the doubt,and the vagus nerve complex.Food intake studies showed that,compared with normal rabbit serum control group,the lateral ventricle injection of 4.0μL,10μL,40μL orexin-A antibody,the cumulative food intake during per hour within 8 hours were significantly decreased and showed a significant dose dependent relationship(P<0.05~0.001).Compared with the NS control group,the intracerebroventricular injection of 5.0μg /0.5μL SB-334867 or cannabinoid receptor antagonists AM251 could significantly reduce the cumulative food intake in rats during per hour within 8 hours(P<0.05~0.001).After the lateral ventricle injection of orexin-A,the cumulative food intake of rats in 0-1h,0-2h,0-4h,0-8h was significantly higher than that in normal saline(NS)control group,the difference was statistically significant(P<0.05).After the lateral ventricle administration of a mixture of cannabinoid receptor antagonists AM251 and orexin-A,the results showed that cannabinoid receptor antagonist AM251 partially blocked the effect of orexin-A induced feeding.The cumulative food intake of AM251+orexin-A group in 1 hours,2 hours,4 hours,and 8 hours decreased significantly(P<0.05).Conclusions: Central endogenous orexin-A can promote feeding;cannabinoid receptor signaling pathway is involved in orexin-A feeding regulation.