| Objective: Nesfatin-1 is from nuclear histones2(Nucleobindin?,NUCB2)cleavage product,widely distributes in the central and peripheral organs.Studies showed that Nesfatin-1 plays a role in the regulation of food intake,cardiovascular,reproductive function,gastrointestinal function and insulin secretion.In view of the fact that Nesfatin-1 has an important influence on the feeding which is closely related to growth,therefore,the purpose of this research is to study the expression and role of Nesfatin-1 protein and mRNA in the growth axis of rat,which providing scientific theoretical basis for further researching for Nesfatin-1/NUCB2 in regulation of the animal growth.Methods:(1)Five female adult SD rats(220-250g).Hypothalamus,pituitary,pancreas,liver,muscle,stomach and duodenum were rapidly collected after decapitation.The tissues from the rats which were fixed in 4% paraformaldehyde,and processed for conventional paraffin,and cut into slices with five-?m-thick slice by a microtome,and then stained with immunohistochemistry PV-9000 two-step method.The slices were observed under an optical microscope and photographed,and then analyzed using the software JEDA801 D.(2)Five SD rats at 10 d,25d,42 d and 80 d were used,respectively.The blood samples were taken from jugular venous,and hypothalamus was collected after decapitation.The level of NUCB2 mRNA in hypothalamus and the concentration of Nesfatin-1 were detected by the real-time fluorescent quantitative RT-PCR(qRT-PCR)and enzyme-linked immunosorbent assay,respectively.(3)Fifteen female rats at 25 d were randomly divided into three groups,the test group(n=5)and the control group(n=10).There are 5 rats without any treatment were fed to the vaginal opening,recording time(42.67±1.83).The rats were treated with intraperitoneal injection of 1% 0.5ml pentobarbital sodiumanesthesia,and then fixed in the rat brain stereotaxic apparatus,the test group received intracerebroventricular injection of 10?l Nesfatin-1(containing 50pmol),the control animals were injected with 10?l physiological saline.Fed them into the test group rats vaginal opening time(31.83±1.27d),all rats were sacrificed,the hypothalamus,pituitary,liver and pancreas were taken,the NUCB2 mRNA and growth hormone-releasing hormone(GHRH)and somatostatin(SS)mRNAs in the hypothalamus,growth hormone(GH)mRNA in the pituitary,growth hormone receptor(GHR)and insulin-like growth factors(IGF-1)mRNAs in the liver,insulin(Ins)mRNA in the pancreas were detected by qRT-PCR.Results:(1)Nesfatin-1 was mainly distributed in the hypothalamic paraventricular nucleus,dorsomedial nucleus,periventricular nucleus,lateral area,arcuate nucleus,supraoptic nucleus,anterior pituitary gland,pancreatic islet and acinar,liver,gastric mucosal glands,duodenal villus and intestinal glands.The analysis showed that the average optical density of Nesfatin-1 in periventricular nucleus and islet was significantly more than in any other tissues(P<0.05),while the average optical density of Nesfatin-1 in pancreas,liver and stomach was significantly lower than in other organs(P<0.05),the average optical density was higher in dorsomedial hypothalamic nucleus,lateral paraventricular and supraoptic nucleus,arcuate nucleus than in the pituitary gland(P<0.05).(2)NUCB2 mRNA level in the hypothalamus of rats was variable in different development stages,that is,the lowest expression was found at 10 d and the highest at 25 d.In addition,NUCB2 mRNA in hypothalamus of rats at 25 d was significantly higher compared to 10 d,42d and 80d(P<0.05),the concentration of Nesfatin-1 in serum from 10 d to 80 d gradually increased,but there was no significant difference between 25 d and 42 d rats(P>0.05).(3)Intracerebroventricular injection of Nesfatin-1 to female rats accelerated puberty onset,but didn't affect the body weight of rat,while inhibited NUCB2,GHRH,GH,GHR and IGF-1 mRNA levels in hypothalamus,pituitary,liver and pancreas,respectively(P<0.05)and promoted the expression of Ins mRNA in the pancreas(P>0.05).Conclusion:(1)Nesfatin-1 is widely distributed in hypothalamus,adenohypophysis liver,pancreas,stomach and duodenum.The different expression of Nesfatin-1 was found in different development stages,different tissues and area in the same tissue.(2)The concentration of Nesfatin-1 increase with the increase of age in rat,but the peak expression of NUCB2 mRNA in the hypothalamus of rat at 25 d is found.(3)Nesfatin-1 reduce the levels NUCB2 mRNA in growth axis,GHRH and SS mRNA in the hypothalamus,GHand GHR mRNA in the pituitary and the IGF-1 mRNA in liver,and increase the expression of Ins mRNA,which indicates that tNesfatin-1 regulates the function of the growth axis. |
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