| Objective: The prevalence of diabetes is increasing year by year,which has become a global health problem.It has been reported that the endocannabinoid system(ECS)plays an important role in regulating appetite,sugar and lipid metabolism.For example,ECS activation can effectively enhance appetite,reduce glucose uptake of skeletal muscle,promote fat storage in adipose tissue,liver tissue and skeletal muscle tissue.It is an abnormal accumulation of energy,which eventually leads to obesity,which is in the maintenance machine.The homeostasis of body energy metabolism,improvement of obesity,insulin resistance and other chronic diseases play an important role.Cannabinoid(CB)is mainly involved in the regulation of central feeding behavior by activating cannabinoid receptor(CBR),especially type 1 cannabinoid receptor(CB1R)is considered to be the target molecule of many cannabinoids.Recent studies have shown that the hippocampus,which mainly regulates learning and memory,has an important influence on the occurrence and development of diabetes and its complications.It has been found that pyramidal cell morphology is abnormal in the hippocampus of diabetic db/db mice.In the hippocampus,CB1 R and glucagon receptor(GLP-1R)are co located,so it is of great significance to explore whether GLP-1R signaling pathway is involved in the regulation of feeding and gastric function in diabetic rats.The aim of this study is to observe the effect of cannabinoid in CA1 area of hippocampus on the intake and gastric motility of diabetic rats and its potential mechanism by means of single extracellular discharge recording,microinjection of nucleus and in vivo gastric motility,so as to provide reliable experimental basis for the prevention and treatment of metabolic disorders such as diabetes and obesity.Methods: 1.To prepare the model of type I diabetic rats: SD rats(220-250 g)were randomly selected and fasted for 12 hours before the model was made.A single intraperitoneal injection of 65 mg / kg of streptozotocin(STZ,Sigama)was made.72 hours later,the tail vein blood was taken to measure fasting blood glucose,the blood glucose was higher than 7.0 mmol/L and the postprandial blood glucose was higher than 11.1 mmol/L.2.The effects of microinjection of cannabinoid,cannabinoid receptor antagonist rimonabant or GLP-1 receptor agonist exendin-4 into the hippocampal CA1 area on the discharge of gastric stretch sensitive neurons in diabetic rats were observed by single extracellular recording.3.Microinjection of cannabinoid,rimonabant or exendin-4 into hippocampal CA1 area to observe the effect of 0-4h and 4-8h food intake in normal and diabetic rats.In this part,normal rats(n = 36)and diabetic rats(n = 36)were randomly divided into 6 groups(n = 6):(1)NS group(0.5 μl NS in hippocampal CA1 area),(2)CB group(0.5 μl 0.5 μL 4 μg CB in hippocampal CA1 area),(3)rimonabant group(0.5 μL 3 μg rimonabant in hippocampal CA1 area),(4)exendin-4 group(0.5 μL 2.5 μg Exendin-4 in hippocampal CA1 area)(5)CB + rimonabant group(0.5 μ L 3 μ g rimonabant + 0.5 μL 4 μ CB mixture was injected into hippocampal CA1 area),(6)CB + exendin-4 group(0.5 μl 2.5 μg exendin-4 + 0.5 μL 4 μg CB mixture was injected into hippocampal CA1 area).The food intake of rats at 0-4 h and 4-8 h after administration was measured by feeding measuring cage.4.The changes of gastric motility in normal and diabetic rats were observed by microinjection of cannabinoid,rimonabant or exendin-4 into CA1 area of hippocampus.In this part,normal rats(n = 36)and diabetic rats(n = 36)were randomly divided into 6 groups(n = 6):(1)NS group(0.5 μl NS in hippocampal CA1 area),(2)CB group(0.5 μl 0.5 μL 4 μg CB in hippocampal CA1 area),(3)rimonabant group(0.5 μL 3 μg rimonabant in hippocampal CA1 area),(4)exendin-4 group(0.5 μL 2.5 μg Exendin-4 in hippocampal CA1 area)(5)CB + rimonabant group(0.5 μ L 3 μg rimonabant + 0.5 μL 4 μg CB mixture was injected into hippocampal CA1 area),(6)CB + exendin-4 group(0.5 μl 2.5 μg exendin-4 + 0.5 μL 4 μg CB mixture was injected into hippocampal CA1 area).The drugs were injected into the CA1 area of hippocampus of rats through catheterization,and the effects on the amplitude and frequency of gastric contraction were observed.The gastric contractile signal is transmitted from the stress receptor to the gastrointestinal motility transducer,where it is converted into an electrical signal input computer,and the gastrointestinal motility data is analyzed by the powerlab multi-channel biological signal acquisition and processing system.5.To observe and compare the expression of cannabinoid receptor in CA1 area of hippocampus between normal and diabetic rats by fluorescence immunohistochemistry.Results: 1.The effect of microinjection of cannabinoid into CA1 area of hippocampus on the firing activity of GD neurons in normal and diabetic rats:(1)A total of 109 firing neurons were recorded in the CA1 region of the hippocampus of 35 normal rats,69 of which(69 / 109,63.30%)responded to gastric stretch and were identified as GD responsive neurons.Among the 69 GD responsive neurons,37(37 / 69,53.62%)were identified as gd-e neurons,while the other 32(32 / 69,46.38%)were identified as gd-i neurons.Among the 37 gd-e neurons,CB was microinjected into the hippocampal CA1 region,while 26(29 / 48,60.42%)were significantly decreased(P < 0.05,7.07 ± 1.15 Hz).Among the 32 gd-i neurons,CB was microinjected into CA1 area of hippocampus,and the discharge frequency of 19 neurons increased significantly(P < 0.05,5.92 ± 1.27 Hz vs.2.53 ± 0.68 Hz),with an average increase of 57.26 ± 3.23%(P < 0.05).If the CB1 R antagonist rimonabant is injected into the CA1 area of hippocampus in advance,the inhibition / excitation induced by CB can be completely blocked,indicating that CB may regulate the electrophysiological characteristics of GD neurons by acting on its receptor CB1R;in addition,if the GLP-1 receptor agonist exendin-4 is injected into the CA1 area of hippocampus in advance,the inhibition or excitation of gd-e neurons by CB and CB can be partially blocked.The results showed that GLP-1 signaling pathway might be involved in the regulation of CB on the excitability of gd-e or gd-i response neurons in CA1 of normal rats.(2)A total of 127 firing neurons were recorded in CA1 area of hippocampus of 35 diabetic rats,86 of which(86 / 127,67.72 %)responded to gastric stretch and were identified as GD responsive neurons.Of the 86 GD responsive neurons,48(48 / 86,55.81 %)were identified as gd-e neurons,while the other 38(38 / 86,44.19 %)were identified as gd-i neurons.Among the 48 gd-e neurons,CB was microinjected into hippocampal CA1 area,and the firing frequency of 29 neurons was significantly reduced(P < 0.01,8.76 ± 1.21 Hz vs.1.41 ± 0.6 Among the 38 gd-i neurons,CB was microinjected into CA1,and the discharge frequency of 17 neurons increased significantly(P < 0.01,2.96 ± 0.58 Hz vs.5.57 ± 1.09 Hz),with an average increase of 88.18 ± 2.7 %.The excitatory effect of CB on GD neurons in diabetic rats could be completely blocked by the injection of rimonabant into CA1 area of hippocampus in advance(P < 0.05),suggesting that the excitatory / inhibitory effect of CB on GD neurons might be related to the CB1 R signal pathway in CA1 area of hippocampus in diabetic rats;in addition,if the injection of exendin-4,a GLP-1 receptor agonist,into CA1 area of hippocampus in advance,CB could inhibit gd-e neurons.The excitatory effect on gd-i neurons could be partially blocked(P < 0.05),but there was no significant change in the discharge activity of GD responsive neurons when rimonabant or exendin-4 were injected into the hippocampal CA1 area of diabetic rats(P > 0.05).It is suggested that GLP-1 signaling pathway may also be involved in the regulation of excitability of gd-e or gd-i response neurons in CA1 region of hippocampus of diabetic rats by CB.(3)The results showed that the inhibitory effect of CB on GD neurons in diabetic rats was significantly higher than that in normal rats(68.03 ± 6.15 % vs.83.90 ± 4.2 %,P < 0.05),and the excitatory effect of CB on GD neurons in diabetic rats was significantly stronger than that in normal rats(57.26 ± 3.23 % vs.88.18 ± 2.7 %,P < 0.05)2.The effect of microinjection of cannabinoid into CA1 area of hippocampus on the food intake of normal and diabetic rats:(1)In normal rats,compared with NS group,CB injection in hippocampal CA1 area can significantly increase the 0-4h food intake of rats,but has no significant effect on the 4-8h food intake;if the mixture of rimonabant and CB is injected in hippocampal CA1 area,the CB promoting food intake disappears;if the mixture of CB and exendin-4 is injected in hippocampal CA1 area,the 0-4h food intake of rats is significantly reduced compared with CB alone.However,there was no significant effect on food intake of rats by injecting alone rimonabant or exendin-4(P > 0.05).(2)In diabetic rats,compared with NS group,CB injection in hippocampal CA1 area significantly increased the food intake at 0-4h,but there was no significant change in the food intake at 4-8h(P > 0.05);if the mixture of rimonabant and CB was injected into hippocampal CA1 area,the feeding promoting effect of CB could be blocked;if the mixture of CB and exendin-4 was injected into hippocampal CA1 area,the food intake at 0-4h was significantly reduced compared with CB injection alone.Similarly, there was no significant effect on the feeding of rats by injecting alone rimonabant or exendin-4(P > 0.05).(3)The results showed that compared with the normal rats,the intake of CB diabetic rats injected into CA1 area of hippocampus increased significantly in 0-4h(P < 0.05).3.The effect of microinjection of cannabinoid into CA1 area of hippocampus on gastric motility in normal and diabetic rats:(1)In normal rats,the amplitude and frequency of gastric contraction increased significantly(P < 0.05)when CB was microinjected into hippocampal CA1 area.If the mixture of rimonabant and CB was injected into hippocampal CA1 area,the effect of CB on gastric motility(amplitude and frequency of contraction)could be completely blocked(P < 0.05);if the mixture of CB and exendin-4 was injected into hippocampal CA1 area,the effect of CB on gastric motility decreased(P < 0.05).However,there was no significant effect on gastric motility in rats by single injection of rimonabant or exendin-4(P > 0.05).(2)In diabetic rats,the amplitude and frequency of gastric contraction were significantly increased by microinjection of CB into hippocampal CA1(P < 0.05);if CB + rimonabant mixture was injected into hippocampal CA1,the effect of CB on gastric motility(amplitude and frequency of contraction)could be completely blocked(P < 0.05);in addition,if CB + exendin-4 mixture was injected into hippocampal CA1,the effect of CB on gastric motility could be partially blocked.It was broken(P < 0.05).Single injection of rimonabant or exendin-4 had significant effect on gastric motility(P > 0.05).(3)The results showed that there was no significant difference in gastric motility index between normal and diabetic rats(P > 0.05)when ns was injected into hippocampal CA1 area,and CB was injected into hippocampal CA1 area,the gastric motility index of diabetic rats increased more significantly(P < 0.05).4.The results of fluorescence immunohistochemistry showed that there were red fluorescent CB1 R immunoreactive neurons in CA1 area of hippocampus in normal and diabetic rats,but the expression of CB1 R immunoreactive cells in CA1 area of hippocampus in diabetic rats(24.66 ± 4.78 / 0.1mm2,P < 0.05)was significantly higher than that in normal rats(17.35 ± 2.19 / 0.1mm2,P < 0.05).It is further confirmed that there is a cannabinoid target in the CA1 region of hippocampus,and the effect of cannabinoid on feeding and gastric motility regulation in diabetic rats is stronger than that in normal rats,which may be related to the increased expression of CB1 R.Conclusions: CB is involved in the activity of discharge,feeding and regulation of gastric motility of GD reactive neurons in CA1 area of hippocampus in normal and diabetic rats,which may be mainly realized by activating CB1 R pathway.The effect of cannabinoid on excitability,feeding or gastric motility of GD neurons in diabetic rats is stronger than that in normal rats,which may be related to the increase of CB1 R expression in CA1 area of hippocampus,which is also cannabinoid.The morphological basis was provided by the effects of feeding promotion and gastric motility. |
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