| Objective:Obesity has become a major public health problem.Overweight and obesity are closely to many social problems and psychological problems,which affect people’s quality of life and reduce their life expectancy.Endogenous opioid peptides are synthesized in vivo and widely distributed in the brain,nerves and peripheral tissues of animals,mainly including enkephalin,dynorphin,endorphin,endorphin and orphanin.Opioid peptides have many receptor subtypes,including μ-opioid receptor receptor(MOR)is an important component involved in physiological regulation of energy metabolism,gastrointestinal activities and immune function Endomorphin(EM)has the strongest affinity for opioid peptide receptors.There are two subtypes of endomorphin,endomorphin-1(EM-1)and endomorphin-2(EM-2).Microinjection of EM-1 into the lateral ventricle could promote the feeding of mice in a dosedependent manner.The amygdala of the limbic system is mainly involved in the regulation of emotion,reward behavior,learning and memory.In recent years,more and more studies have shown that the amygdala,especially the central amygdaloid(CeA),also plays an important role in the regulation of energy metabolism.Studies have shown that most neurons in the central amygdala have μ-opioid receptor expression.Is EM-1 involved in the regulation of different feed intake in rats? Is it involved in the regulation of gastric sensory afferent and gastric motor efferent activities? Is this regulation related to obesity? Its potential mechanism? There is no report.The purpose of this study is to explore the effects of EM-1 microinjection in CeA on the discharge activities of neurons receiving gastric afferent information in the amygdala,as well as the regulation of gastric motility and food intake in rats and its potential mechanism,and to analyze and compare the differences of the above effects between normal and obese rats.The purpose of this study is to supplement and enrich the central mechanism of obesity,and to find out a new direction for the prevention and treatment of energy metabolism disorders such as obesity.Methods:(1)5-week-old healthy male Wistar rats were fed with high-fat diet(45% fat)for 6 w to establish diet induced obesity rats.The body weight of rats was regularly recorded,and the rats of body weight more than 20% of the the normal diet group were considerd as obese rats.(2)By single cell discharge recording,the effects of microinjection of EM-1 or μ-opioid receptor antagonist(D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2,CTOP)into amygdala on the activity of Gastric Distension(GD)sensitive neurones in the normal and obese rats were observed.(3)To observe and compare the effects of microinjection of EM-1 into CeA on gastric motility and food intake(standard,high-sugar,high-fat and high-protein diet)between the normal and obese rats.(4)Western Blot and immunofluorescence histochemistry were used to compare the expression of EM-1 and μ-opioid receptor protein in the CeA of the normal and obese rats.(5)The binding capacity of μ-opioid peptide receptor to EM-1 ligand in CeA of normal and obese rats was detected by radioligand-receptor binding assay.Results:(1)The effects of EM-1 on the firing activity of GD neurons in CeA.The results showed that there were GD sensitive neurons in CeA,including gastric distension inhibitory(GD-I)neurons and gastric distension excitatory(GD-E)neurons.Microinjection of EM-1 into CeA could stimulate 70.37% GD-I neurons in normal rats and 73.02% in obese rats,but inhibit 64.05% GD-E neurons in normal rats and 70.83% in obese rats.Pre-injection of μ-opioid receptor antagonist CTOP could weaken the excitatory or inhibitory effect of EM-1 on GD neurons in CeA.The excitatory or inhibitory effect of EM-1 on GD neurons in obese rats was significantly stronger than that in normal rats(P < 0.05).(2)Effects of microinjection of EM-1 into CeA on gastric motility in ratsThe gastric motility amplitude(P < 0.05)and gastric motility frequency(P < 0.05)of normal and obese rats were significantly increased by CeA microinjection of EM-1;Microinjection of EM-1 + CTOP mixture with CeA could block the gastric motility effect of EM-1 in rats(P < 0.05).Injection of CTOP alone with CeA had no significant change in gastric motility in normal or obese rats(P > 0.05).Gastric motility index [MI% =(area under the curve after administration)/(area under the curve before administration)× 100%] analysis showed that there was no significant change in MI% in obese NS group compared with normal NS group (P > 0.05);However,the MI% of normal(P < 0.05)or obese rats(P < 0.05)increased significantly after CeA microinjection of EM-1;Further adopt MI% change rate [MI% change rate(%)=(MI% EM-1-MI% NS)/ MI% NS × 100%] analysis showed that compared with normal rats,EM-1 had a more significant effect on increasing MI% in obese rats(P < 0.05).(3)Effect of CeA microinjection of EM-1 on the gastric emptying in ratsMicroinjection of EM-1 into the CeA significantly increased the gastric emptying in normal(P < 0.05)or obese rats(P < 0.05);μ-opioid receptor antagonist CTOP could block the promoting effects of EM-1 on the gastric emptying in normal(P < 0.05)or obese rats(P < 0.05);Compared with normal rats,EM-1 had a more significant effect on gastric emptying in obese rats(P < 0.05).(4)Effect of microinjection of EM-1 into CeA on feeding in ratsThe intake of standard diet(P < 0.05),high-fat diet(P < 0.05)or high protein diet(P < 0.05)in normal or obese rats increased significantly after 0-2 h and 2-4 h of CeA microinjection of EM-1,but there was no significant change in food intake at 4-6 h(P > 0.05);However,there was no significant change in the high sugar diet intake for 0-2 h,2-4 h or 4-6 h in normal or obese rats after CeA microinjection of EM-1(P > 0.05);Microinjection of EM-1 + CTOP into CeA could block the feeding promoting effect of EM-1 in CeA rats(P > 0.05).Statistical analysis showed that the intake of high fat diet(P < 0.05)or high protein diet(P < 0.05)in normal or obese rats was significantly more than that in normal rats;The intake of high sugar diet in the obese rats was also significantly higher than that of normal diet(P < 0.05).The increase and change rate of high-fat(P < 0.05)or high protein(P < 0.05)feed intake was significantly higher than that of standard feed intake in normal or obese rats.Further statistical analysis showed that the intake of high-fat diet(P < 0.05),high sugar diet(P < 0.05)or high protein diet(P < 0.05)increased significantly in obese rats than in normal rats;When obese rats were injected with CeA,the increase and change rate of feed intake of promoting standard(P < 0.05),promoting high fat(P < 0.05)or promoting high protein(P < 0.05)for 0-2 h or 2-4 h was also significantly higher than that of normal rats.(5)Expression of CeA EM-1 and μ-opioid receptor proteinFluorescence immunohistochemical study showed that there were EM-1 and μ-opioid receptor immunoreactive cells expression in the CeA of normal and obese rats;The expression of EM-1 immunoreactive cells in the CeA of obese rats did not change significantly compared with normal rats(P > 0.05),but the expression of μ-opioid receptor immunoreactive cells increased significantly(P < 0.05).The results of Western blot showed that there were EM-1 and μ-opioid receptor in CeA of normal and obese rats.The expression of μ-opioid receptor protein was significantly higher than that of the normal rats(P < 0.05),but the expression of EM-1 protein did not change significantly(P > 0.05).(6)Binding of CeA EM-1 ligand to μ-opioid receptorThe study of radioligand receptor binding showed that the specific binding activity of μ-opioid receptor to ligand EM-1 in the CeA of obese rats was significantly higher than that of the normal rats(P < 0.05).Conclusion:There are neurons that can receive sensory afferent information from stomach and respond to EM-1 in the CeA,and EM-1 in CeA can participate in the modulation of excitability of GD neurons,gastric motility and selective palatable food intake through μ-opioid receptor signal pathway,and this regulatory effect is more significant in obese rats.The increase of gastric motility and palatable food intake in obese rats may be related to the increased expression of μ-opioid peptide receptor in CeA and the increased binding capacity of EM-1 ligand to μ-opioid peptide receptor in CeA. |
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