| Objective: Orexins(Orexin-A and Orexin-B)are secreted by the hypothalamus and they are mainly involved in feeding and reward regulation.In particular,Orexin-A plays an important role in this regulation process.In the hypothalamus,the neurons expressing Orexin mRNA are mainly distributed in the Lateral Hypothalamus and the Perifornical Area.Studies have found that Orexin-A can promote rodent feeding by microinjection into Ventricle,Paraventricular Nucleus,LHA or PFA.Although intracerebroventricular injection of Orexin-B also produced a similar feeding effect like Orexin-A,but intranuclear injection of Orexin-B produced no effect.Another study found that the promoting effect of Orexin-A or Orexin-B is dependent on the Orexin-A receptor(OX-1R).Therefore,this study was to observe the expression of Orexin-A and Orexin-1 receptor(OX-1R)immunoreactive cells and their mRNA in LHA,and further explore the effect of Orexin-A on LHA glucose-sensitive(GS)neurons,feeding and stomach function and potential mechanisms.To compare and analyze the difference in the regulation effect of Orexin-A in normal,obese and obese resistant rats.The purpose is to supplement and improve the theory of energy balance regulation of Orexin-A,and provide new research ideas for clinical obesity treatment.Methods: Male healthy SD rats(170 ± 10 g)were fed with high-fat diet(80 g of basic diet,10 g of yolk powder and 10 g of lard per 100 g).After 2 weeks,the rats were ranked according to their weight gain.The rats in the middle 1/3 of the order were fed with basic diet as normal rats,the rats in the upper and lower 1/3 of the order were fed with high-fat diet for 6 weeks.6 weeks later,the rats fed with high fat diet were divided into obese rats group(ranked at the top 1/3)and obesity resistant rats group(ranked at the bottom 1/3),and the rats in the middle 1/3 were discarded.The Orexin-A and OX-1R immunopositive cells in LHA of normal,obesity and obesity-resistant rats was analyzed and compared by immunofluorescence histochemical staining.RT-PCR was used to analyze and compare the expression of Orexin-A and OX-1R mRNA in LHA of normal,nutritional obesity and obesity-resistant rats.The effects of Orexin-A and melanocortin 3/4 receptor inhibitor SHU9119 on the discharge activity of glucose-sensitive neurons and the regulation of melanocortin 3/4 receptor signaling pathway in normal,obesity and obesity-resistant rats were observed by single-cell extracellular discharge recording.The effects of Orexin-A and SHU9119 on the intake,gastric motility and gastric emptying of normal,obesity and obesity resistant rats were observed and the regulation of?-MSH signaling pathway was also observed.Normal,obesity and obesity-resistant rats were randomly divided into 6 groups:(1)NS group: LHA injection of NS;(2)Orexin-A group: microinjection of 100 pmol Orexin-A in LHA;(3)SB-334837 group: microinjection of 1 nmol SB-334837 in LHA;(4)SHU9119 group: microinjection of 0.1 ?g SHU9119 in LHA;(5)Orexin-A + SB-334837 group: microinjection 1 nmol SB-334837+ 100 pmol Orexin-A into LHA;(6)Orexin-A + SHU9119 group: LHA was microinjected 0.1 ?g SHU9119 +100 pmol Orexin-A into LHA.The total volume of each group was 0.5 ?L.Results: The results of fluorescence immunohistochemistry showed that there was no significant difference in the expression of Orexin-A in the LHA of obesity,obesity-resistant and normal rats(P > 0.05).Compared with normal rats,the number of OX-1R immunoreactive cells in LHA of obesity and obesity-resistant rats increased significantly(P < 0.05-0.01),and the increase was more significant in obesity-resistant rats than in obesity rats(P < 0.05).The results of PCR showed that there was no significant difference in the expression of Orexin-A mRNA in the LHA of obesity,obesity-resistant and normal rats(P > 0.05).Compared with normal rats,the expression of OX-1R mRNA in LHA of obesity and obesity-resistant rats increased significantly(P < 0.05-0.01),and the increase was more significant in obesity-resistant rats than in obesity rats(P < 0.05).After LHA was microinjected to Orexin-A in normal rats,the discharge frequency of glucose-excitatory neurons decreased from 5.93 ± 1.84 Hz to 3.78 ± 0.73 Hz(P < 0.05);the discharge frequency of glucose-inhibitory neurons increased from 5.47 ± 1.64 Hz to 7.62 ± 1.84 Hz(P < 0.05).The excitation or inhibition effect of Orexin-A on glucose-sensitive neurons could be completely blocked by pre-injection of SB-334867.The excitatory or inhibitory effects of of Orexin-A on glucose-sensitive neurons were enhanced by pre-injection of SHU9119(P < 0.05).After microinjection Orexin-A into LHA of obesity rats,the discharge frequency of glucose-excitatory neurons decreased from 6.12 ± 1.68 Hz to 3.24 ± 0.97 Hz(P < 0.05);the discharge frequency of glucose-inhibitory neurons increased from 5.26 ± 1.82 Hz to 8.19 ± 2.37 Hz(P < 0.05).The excitatory or inhibitory effects of Orexin-A on glucose-sensitive neurons could be completely blocked by pre-injection of SB-334867.The excitatory or inhibitory effects of Orexin-A on glucose-sensitive neurons were further enhanced by pre-injection of SHU9119(P < 0.05).After microinjection Orexin-A into LHA of obesity-resistant rats,the discharge frequency of glucose-excitatory neurons decreased from 5.86 ± 1.94 Hz to 3.07 ± 0.84 Hz(P < 0.05),and the discharge frequency of glucose-inhibitory neurons increased from 5.39 ± 1.48 Hz to 8.19 ± 2.42 Hz(P < 0.05).Pre-injection of SB-334867 could completely blocked excitatory or inhibitory effects of Orexin-A on glucose-sensitive neurons.The excitatory or inhibitory effects of Orexin-A on glucose-sensitive neurons were further enhanced by pre-injection of SHU9119(P < 0.05).Compared with normal rats,the excitation or inhibition effects of Orexin-A on glucose-sensitive neurons in obesity and obesity-resistant rats were significantly enhanced(P < 0.05),and the stimulation effects of SHU91191 on Orexin-A were significantly enhanced(P < 0.05),while the excitation or inhibition effects of Orexin-A on glucose-sensitive neurons in obesity-resistant rats after microinjection of LHA were significantly enhanced than in obesity rats(P < 0.05).Orexin-A was microinjected into LHA,the food intake of normal,obesity and obesity-resistant rats increased significantly in 0-2 h(P < 0.05).The increase of intake in obesity and obesity-resistant rats was higher than that in normal rats(P < 0.05),and the increase of intake in obesity-resistant rats was higher than that in obesity rats(P < 0.05).Pre-injection of SB-334867 completely blocked the promoting effect of Orexin-A on feeding in rats(P > 0.05).Pre-injection of SHU9119 were enhanced the promoting effect of Orexin-A on feeding in rats(P < 0.05).The frequency and amplitude of gastric motility in rats after microinjection of Orexin-A into LHA were significantly increased(P < 0.05),and the effect could be completely blocked by SB-334867.Compared with the Orexin-A group,the frequency and amplitude of gastric motility in rats treated with Orexin-A+SHU9119 significantly promoting(P < 0.05).After microinjection of Orexin-A into LHA,the MI% of rats significantly increased(P < 0.05).The increase of obesity and obesity-resistant rats was higher than that in normal rats(P < 0.05),and the increase of obesity-resistant rats was higher than that in obesity rats(P < 0.05).The promotion of Orexin-A on% MI in rats was completely blocked by SB-334867(P < 0.05)and promoting by SHU9119(P < 0.05).Microinjection of Orexin-A into LHA can promote gastric emptying in rats(P < 0.05).Orexin-A can promote gastric emptying in obesity and obesity-resistant rats more than in normal rats(P < 0.05).Orexin-A can promote gastric emptying in obesity-resistant rats more than in obesity rats(P < 0.05).The effect of Orexin-A on gastric emptying could be completely blocked by SB-334867.Compared with Orexin-A group,the effect of Orexin-A + SHU9119 on gastric emptying in rats increased significantly(P < 0.05).Conclusion: Orexin-A may participate in the regulation of the excitatory activity of LHA glucose-sensitive neurons or feeding and gastric function in the hypothalamus of normal,obese and obesity resistant rats.This effect may be achieved through the OX-1R signaling pathway.The melanocortin 3/4 receptor signaling pathway may also be involved in the regulation of this process;the above-mentioned regulatory effects of Orexin-A are more pronounced in obese and obese resistant rats,it may be associated with increased expression of OX-1R in LHA in obese and obese resistant rats. |
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