| Objective: To investigate the effect and underlying mechanism of ghrelin on hypothalamic arcuate nucleus gastric distention sensitive neurons and gastric motility in diabetic rats.Methods: The rats were randomly assigned to the control group and the diabetes group(DM).The rats in the DM group were fed with the high-fat diet for 20 weeks and free access to water.After 8 weeks of diet intervention,the rats received one intraperitoneal injection of low-dose streptozocin(STZ)(30 mg/kg,Sigma),while the control group were injected with citrate buffer solution(pH 4.4,1 ml/kg).Four weeks after the STZ injection,the rats were deprived of food for 12 h.Diabetes was successfully induced in rats with fasting blood glucose levels ≥ 7.8 mmol/L and postprandial blood glucose ≥ 11.1 mmol/L.Electrophysiological experiments were conducted to observe the discharge activity of the gastric distention sensitive neurons of the arcuate nucleus in normal rats and diabetic rats by single cell external discharge recording: the normal rats and diabetic rats were anesthetized then placed in the stereotaxic apparatus,and glass microelectrodes were placed in the arcuate nucleus according the brain stereopsis,The glass microelectrode were filled with 15 nmol/L ghrelin or 15 nmol/L des-acyl ghrelin,28 nmol/L ghrelin receptor antagonist [dLys-3]-GHRP-6 or 45 nmol/ L GHSR-1a specific antagonist BIM28163 and injections of equal volume and saline were used as controls,respectively.The changes of discharge activity of gastric distention sensitive neurons in the arcuate nucleus after injection of drugs were observed.In vivo gastric motility test in conscious rats: the rats were anesthetized and placed in stereotaxic apparatus,and the brain atlas was referenced.The stainless steel sleeves were placed in the arcuate nucleus of normal rats or diabetic rats,and the 0.5 μL ghrelin(0.025 pmol,0.25 pmol or 2.5 pmol),0.5 μL [d-Lys-3]-GHRP-6(2.5 pmol),0.5 μL 1.25 pmol BIM28163(n = 8)or 0.5 μL mixed solution 2.5 pmol [d-Lys-3]-GHRP-6 or 1.25 pmol BIM28163 + 0.25 pmol ghrelin was respectively slowly injected via the cannula.The amplitude and frequency of gastric motility were observed in rats.The expression of GHSR-1 in the arcuate nucleus of normal rats and diabetic rats was observed and compared by fluorescence immunohistochemistry.The rats were anesthetized and perfused with hearts,then the 15 μm brain slices were made.Fluorescence was performed by immunofluorescence staining,and the expression of GHSR-1 in the arcuate nucleus was observed under fluorescent microscope.Results: Electrophysiological studies showed that microinjection of ghrelin into the arcuate nucleus could significantly affect the discharge activity of gastric distention sensitive neurons.After microinjection of ghrelin into the arcuate nucleus of the normal rats,the discharge frequency of 47 neurons in 68 gastric distention sensitive excitatory(GD-E)neurons increased from 1.83 Hz±0.38 Hz to 3.26 Hz±0.75 Hz(P<0.05),with an average increase of 40.21% ±5.21%;In the 55 gastric distention sensitive inhibitory(GD-I)neurons,33 neurons discharge was inhibited,the discharge frequency decreased from 1.57 Hz±0.39 Hz to 0.97 Hz±0.34 Hz(P<0.05),with an average decrease of 57.72%±5.71%.Microinjection of ghrelin in the arcuate nucleus of diabetic rats,discharge frequency of 13 neurons in 37 GD-E neurons in the arcuate nucleus(13/37,35.1%)increased from 1.98 Hz±0.35 Hz to 2.45 Hz± 0.61 Hz(P<0.01),compared with saline control group,the difference was statistically significant(P<0.01).Compared with normal rats,the rate of discharge of GD-E neurons in diabetic rats decreased significantly(21.73%±4.31% vs.40.21%±5.21%,P<0.01).The discharge frequency of 18 GD-I neurons in the arcuate nucleus in diabetic rats(18/29,62.06%)decreased from 1.26 Hz ±0.42 Hz to 0.62 Hz± 0.21 Hz(P<0.01),the average decline of 53.7%±8.71%,significantly higher than those in the saline group(4.69%±1.12%,P<0.01).Compared with normal rats,the discharge frequencies of GD-I neurons in diabetic rats(53.72%±8.71% vs.57.72%±5.71%,P < 0.05)were significantly different.Pre-microinjection of [d-lys-3]-GHRP-6 or BIM28163 into arcuate nucleus the,the effect of ghrelin on the discharge frequency of gastric distention sensitive neurons was completely blocked.The [d-lys-3]-GHRP-6 or BIM28163 alone had no siginificantly effect on the firing frequency of gastric distention sensitive neurons(P>0.05).In vivo gastric motility studies showed that microinjection of ghrelin into the arcuate nucleus significantly enhanced gastric motility in normal rats and diabetic rats,and was in dose dependent(P<0.05-0.01).The effect of ghrelin on gastric motility in diabetic rats was significantly weaker than that in normal rats(P>0.05).The ghrelin induced gastric motility effect can be completely blocked by [d-lys-3]-GHRP-6 or BIM28163.Fluorescence immunohistochemistry study showed that GHSR-1a positive cells were found in the arcuate nucleus of normal rats and diabetic rats(P<0.05).Compared with normal rats,the expression of GHSR-1a in the arcuate nucleus was decreased of diabetic rats.Conclusion: The ghrelin can be involved in the regulation of excitability of the gastric distention sensitive neurons in the arcuate nucleus of normal rats and diabetic rats,and promote the movement of the stomach in normal rats and diabetic rats.This effect may be achieved via the ghrelin receptor signaling pathway.But in normal rats,the regulation effect of ghrelin was significantly higher than that of diabetic rats.It is suggested that ghrelin can participate in the regulation of gastric afferent information and participate in the regulation of gastric motility information in normal rats and diabetic rats.The excitability of gastric distention sensitive neurons and the decrease of gastric motility response to ghrelin in diabetic rats may be related to the decrease of ghrelin receptor expression in arcuate nucleus of diabetic rats.This study provides a basis for the targeted treatment of diabetes in the future. |
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