| Objective: The cause of obesity is closely related to environmental and genetic factors.Ghrelin,also known as auxin,is the only brain-gut peptide secreted by the periphery and acting on the feeding center(then the hypothalamus),which plays an important role in the regulation of food intake,energy expenditure and metabolism in rats.Studies have reported that intracerebroventricular(icv.)injection of ghrelin can significantly increase food intake in rats,weight loss by gastrectomy,serum ghrelin levels decreased.Therefore,ghrelin is closely related to obesity and appetite.In this study,fluorescence immunohistochemistry,feeding selection,gastrointestinal transit and motor behavior were used to analyze and compare the expression and similarity of LHA ghrelin receptor in normal rats,obese(DIO)rats and obesity-resistant(DR)rats.The effects of LHA ghrelin on feeding,feeding choices,spontaneous activity,and gastrointestinal transit,and the underlying mechanisms of ghrelin in these three groups of rats,The purpose is to supplement and improve ghrelin's theory of energy balance regulation,and provide new research ideas for clinical obesity treatment.Methods: 1.Construction of a nutritional obesity and obesity resistant rat model: Healthy male Sprague-Dawley rats(180-220 g),high-fat diet(15 g per 100 g of fat,including 2.5 g of vegetable oil and 12.5 g of lard)were housed for 2 weeks,and normal rats were screened according to body weight.In the middle 1/3),it was changed to normal feed(5 g of fat per 100 g,including 2.5 g of vegetable oil and lard),and the remaining rats continued to feed high-fat diet for 6 weeks and weighed.The first 1/3 of the high-weight rats were defined as nutritional obese rats.The lower 1/3 of the rats with low body weight were defined as obese resistant rats,while the middle 1/3 of the rats were discarded;2.The effect of LHA microinjection of ghrelin on the selective food intake of normal rats,DIO rats and DR rats was observed by LHA tube administration: Twenty normal,DIO or DR rats were randomly divided into 4 groups(n=5).(1)NS group: LHA microinjection of 0.5 ?l NS;(2)ghrelin group: LHA microinjection 0.5 ?l 15 nmol / L ghrelin;(3)[D-Lys3]-GHRP-6 group,LHA microinjection 0.5 ?l 28 nmol / L ghrelin receptor antagonist [D-Lys3]-GHRP-6;(4)[D-Lys3]-GHRP-6 + ghrelin group: LHA microinjection 0.25 ?l 28 nmol/L [D-Lys3]-GHRP-6 + 0.25 ?l 15 nmol/L ghrelin.After LHA injection,compare and analyze the similarities and differences between normal,DIO and DR rats on normal diet,high-fat diet and high-sugar diet;3.The effects of microinjection of ghrelin on the spontaneous activity(SPA)of normal rats,DIO rats and DR rats were observed by LHA tube administration: Twenty normal,DIO or DR rats were randomly divided into 4 groups(n=5).(1)NS group;(2)ghrelin group;(3)[D-Lys3]-GHRP-6 group;(4)[D-Lys3]-GHRP-6 + ghrelin group.(Specific dosage see above 2).After injecting different drugs into rat LHA,the horizontal direction(X-axis,Y-axis),vertical direction(Z-axis)and total activity and effects of the rats were observed through a rat SPA box;4.To observe the effect of microinjection of ghrelin into the LHA on the gastrointestinal transit rate of normal,DIO and DR rats: Twenty normal,DIO or DR rats were randomly divided into 4 groups(n=5).(1)NS group;(2)ghrelin group;(3)[D-Lys3]-GHRP-6 group;(4)[D-Lys3]-GHRP-6 + ghrelin group.(Specific dosage see above 2).Microinjection of ghrelin into the LHA,after 2 h,0.3 ml of 6% carmine red methylcellulose solution was orally administered,and the time until the first red feces was discharged was recorded;5.The expression and similarity of ghrelin receptors in LHA of normal rats,DIO rats and DR rats were analyzed and compared by immunohistochemical staining.Results: 1.Effect of LHA microinjection of ghrelin on different food intake in rats: The results showed that in normal rats,compared with NS group,LHA microinjection of ghrelin,rats' normal diet,high-fat diet and high-sugar diet were significantly increased(P<0.05),but compared with normal diet,high fat Dietary and high-sugar diets increased significantly more(P<0.05).However,there was no significant difference between high-fat diet and high-sugar diet(P>0.05).LHA was microinjected with ghrelin receptor antagonist [D-Lys3]-GHRP-6.Rats' normal diet,high-fat diet and high-carbohydrate diet were significantly decreased(P<0.05).LHA pre-injection of [D-Lys3]-GHRP-6 completely blocked the feeding effect of ghrelin on rats(P<0.05).Comparison between groups in the normal rat group,the DIO rat group,or the DR rat group showed that Compared with normal rats,DIO(P<0.05)and DR(P<0.05)rats preferred high-fat or high-sugar foods;The normal diet(P<0.05),high-fat diet(P<0.05)and high-glucose diet(P<0.05)of LHA microinjection ghrelin,DIO rats and DR rats were significantly higher than normal rats;However,there was no significant difference between the normal diet(P>0.05),high-fat diet(P>0.05)and high-glucose diet(P>0.05)between DIO rats and DR rats.It is suggested that the feeding effect of ghrelin in three groups of rats is not exactly the same,that is,the ghrelin-promoting effect is more significant in DIO and DR rats,and the specific mechanism needs further investigation.2.The effect of LHA microinjection of ghrelin on spontaneous activity in rats: Comparison results in the normal rat group,DIO rat group or DR rat group showed that,compared with the NS group,LHA microinjection of ghrelin,normal rats,DIO rats or DR rats significantly increased in the direction of X-axis(horizontal axis)and Y-axis(longitudinal axis)for 24 h(P<0.05).The total activity also increased significantly(P<0.05),but there was no significant change in the Z-axis(vertical axis)direction(P>0.05).Comparison between groups in the normal rat group,the DIO rat group,or the DR rat group showed that,compared with normal or DIO rat NS group,LHA injection of NS,DR rats showed a significant increase in X-axis(P < 0.05),Y-axis(P < 0.05)and total activity(P < 0.05)at 24 h;Compared with normal or DIO rat ghrelin group,LHA injected ghrelin,DR rats had the most significant increase in activity in the X-axis(P<0.05)or Y-axis(P<0.05)direction.The increase in total activity at 24 h was also significantly higher than that in normal or DIO rats(P<0.05);It is suggested that one of the causes of obesity resistance in DR rats may be related to the increase in spontaneous activity;LHA ghrelin promoted rat feeding and increased spontaneous activity in rats,further confirming that ghrelin is involved in energy balance regulation.However,the reason why this effect is more prominent in DIO rats is still unclear and needs further investigation.3.Effect of microinjection of ghrelin on gastrointestinal transit rate in rats: The results showed that the gastrointestinal transit rate of DIO rats(P<0.05)was significantly slower than that of normal rats,but the gastrointestinal transit rate of DR rats(P<0.05)was significantly accelerated.Comparison results in the normal rat group,DIO rat group or DR rat group showed that Compared with the NS group,LHA is injected with ghrelin,The gastrointestinal transit rates of normal rats(P<0.05),DIO rats(P<0.05)or DR rats(P<0.05)were significantly accelerated;Compared with the NS group,LHA was injected with [D-Lys3]-GHRP-6,and the gastrointestinal transport rate of these three groups was significantly slowed down(P<0.05).If LHA was microinjected with [D-Lys3]-GHRP-6+ghrelin mixture,ghrelin promoted the gastrointestinal transit rate of these three groups was completely blocked compared with the ghrelin group(P<0.05).Comparison between groups in the normal rat group,the DIO rat group,or the DR rat group showed that,compared with the normal NS group,the gastrointestinal transit rate of DIO rats was significantly slower(P<0.05),but the gastrointestinal transit rate of DR rats was significantly faster(P<0.05).Compared with the normal ghrelin group,the gastrointestinal transit rate of the DR group was the most significant(P<0.05),and the average transport rate increased by 38.03%.The gastrointestinal transit rate of the normal group increased by 15.71%,and the gastrointestinal transit rate of the DIO group increased by 19.24%.The results suggest that DR rats can resist obesity caused by high-fat diet,which may be related to faster gastrointestinal transit rate;Both endogenous and exogenous ghrelin can participate in the inhibition of obesity formation by increasing gastrointestinal transit rate 4.Fluorescence immunohistochemistry showed that compared with normal rats,the expression of ghrelin receptor immunoreactive cells in DIO rats(P<0.05)and DR rats(P<0.05)was significantly increased.However,there was no significant difference in the expression of DIO rats compared with DR rats(P>0.05).It is suggested that ghrelin promotes food intake,spontaneous activity or gastrointestinal transit in DIO or DR rats,which may be related to the increased expression of ghrelin receptor in LHA.Conclusions: Ghrelin is involved in the selective feeding,spontaneous activity and regulation of gastrointestinal transport function in normal,obese and obese resistant rats,which may be achieved by mainly activating the ghrelin receptor signaling pathway;Ghrelin is strongly involved in feeding,spontaneous activity or gastrointestinal transit in DIO or DR rats,which may be related to the increased expression of ghrelin receptor in LHA. |
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