Effect Of Nesfatin-1 In The Amygdala On The Development Of Visceral Hypersensitivity And Possible Mechanism

Irritable bowel syndrome(IBS)is a common functional gastrointestinal disease in outpatients of gastroenterology,which is characterized by bloating,abdominal pain or discomfort with the change of bowel habits but without a detectable structural or biochemical abnormalities.To date,the pathophysiology of IBS is multifactorial and only partly understood.Visceral hypersensitivity,which has been thought as a biomarker of IBS,has been widely researched in recent years.Both central and peripheral mechanisms have been suggested to be involved in the development of visceral hypersensitivity.Stress and the dysfunction of hypothalamic-pituitary-adrenal(HPA)axis have been both demonstrated to be involved in the pathogenesis and development of IBS.Studies in rodents have demonstrated that the amygdala,the core site of anxiety regulation,also involved in the development via glucocorticoid receptor(GR)and mineralocorticoid receptor(MR)pathways.Nesfatin-1,a newly identified satiety molecule was derived from cleavage of the calcium-binding protein nucleobindin2(NUCB2).This peptide contains 1-82 amino acids,which was widely distributed in stress associated brain nucleus,such as the hypothalamic supraoptic nucleus(SON),paraventricular nucleus(PVN),nucleus of the solitary tract(NTS),locus coeruleus(LC).In addition,nesfatin-1 was also detected in the limbic system including amygdaloid nuclei.Nesfatin-1 has been confirmed to be participated in the activation of HPA axis.Central nesfatin-1 was also involved in the mediation of anxiety-and/or fear-related responses.But to date,little is known about the effect of nesfatin-1 on visceral sensitivity.In another study,microinjection of corticosterone into the amygdala increase the expression of corticotropin-releasing factor(CRF),our previous study has demonstrated that central nesfatin-1 may be involved in the development of visceral hypersensitivity through CRF-CRFR1 pathway in rats.Whether nesfatin-1 in the amygdala has impact on the pathogenesis of visceral hypersensitivity and what is the possible mechanism? The purpose of the present study was to investigate the action of nesfatin-1 in the amygdala on visceral sensitivity and possible mechanism of this effect.The animal model of visceral hypersensitivity was established by maternal separation.Aim To investigate whether nesfatin-1 in the amygdala was involved in the development of visceral hypersensitivity and the possible mechanism.Methods 1.Neonatal male Sprague-Dawley rats were randomly divided into maternal separation group and normal control group.Rats in maternal separation group were separated from their dams as a whole litter during 3 hours every day over a period of 14 days between postnatal days 2 and 16.Separations were conducted between 8 AM and 11 AM in plastic cages and placed in a separated room.In control group,rats left untouched with their mothers.Body weight of rats in the two groups was monitored at postnatal day 20 and 8th week.2.We measured the abdominal withdrawal reflex(AWR)and the area under the curve(AUC)of electromyographic(EMG)activity of the external oblique under different grade of colorectal distention(CRD)to evaluate visceral sensitivity of rats of the two groups.3.Blood was collected at 8-9 AM.The concentration of serum nesfatin-1 and glucocorticoid(GC)were determined using enzyme-linked immunosorbent assay(ELISA).4.The expression of protein and m RNA level of nesfatin-1/NUCB2,GR and MR in the amygdala of different groups were examined by western-blotting and RT-PCR.5.Rats in maternal separation group were randomly divided into two groups and microinjected with anti-nesfatin-1/NUCB2 or vehicle into the amygdala and examine the AWR score and AUC of EMG again.6.Normal adult rats were randomly divided into 4 groups,which were administered by nesfatin-1,mifepristone(GR antagonist)and nesfatin-1,spironolactone(MR antagonist)and nesfatin-1,and vehicle into the amygdala.Visceral sensitivity was detected 1 week late after injection by examining AWR score and AUC of EMG.The serum level of GC was determined using ELISA.Results 1.Body weight of rats in the maternal separation group and control group was determined at postnatal day 20 and 8th week.At postnatal day 20,the body weight of the two groups was not statistically different(P>0.05),however,the body weight of maternal separation rats was significantly lower than that in control group at postnatal 8th week(P<0.05).2.The mean AWR score and AUC of EMG in maternal separation rats was significantly higher than that in non-handed rats at the distension pressure level of 40,60 and 80 mm Hg(P<0.05).3.The concentration of serum nesfatin-1 in maternal separation rats was significantly higher than normal control rats(P<0.05),the level of serum GC was also significantly higher in maternal separation group than control group(P<0.01).4.In maternal separation rats,the level of serum nesfatin-1 was positive correlated with serum GC level.What's more,the serum nesfatin-1 concentration was also positive correlated with AWR score and AUC of EMG under different CRD pressure.5.The expression of protein and m RNA level of nesfatin-1/NUCB2 in the amygdala of maternal separation rats were significantly elevated than control rats(P<0.05).6.In the present study,we found no statistically difference of the expression of protein and m RNA of GR and MR in the amygdala between maternal separation and control rats.7.In maternal separation rats,after microinjection of anti-nesfatin-1/NUCB2,the mean AWR score and AUC of EMG were significantly lower than that rats received vehicle(P<0.05).8.Normal rats that received nesfatin-1 showed higher level of mean AWR score and AUC of EMG than vehicle at the distension pressure level of 20,40,60 and 80 mm Hg(P<0.05).However,when compare to nesfatin-1-treated rats,administered mifepristone and nesfatin-1,spironolactone and nesfatin-1 into the amygdala showed decreased mean AWR score and AUC of EMG at all CRD pressure,which were similarly with that received vehicle.9.The serum level of nesfatin-1-treated rats was significantly higher than that rats in vehicle treated group(P<0.05).Conclusion Nesfatin-1 in the amygdala may be involved in the development of visceral hypersensitivity,which may be increase serum GC level and posible be involved in the GR and(or)MR pathways.