Effect Of Nesfatin-1 In The Dorsal Raphe Nucleus On The Development Of Visceral Hypersensitivity And Possible Mechanism

Irritable bowel syndrome(IBS)is a chronic gastroenterological disease of uncertain etiology marked by abdominal pain or discomfort and disorder of bowel movements.Albeit a common disorder,IBS exerts a negative impact on the quality of life and work productivity of patients.While the pathophysiological mechanism of IBS remains incompletely elucidated,visceral hypersensitivity has been established as a key feature of IBS.5-HT(5-hydroxytryptamine)possesses an important role in visceral hypersensitivity in patients with IBS.Altered 5-HT signaling of the central nervous system contributes to the development of IBS—as indicated by the therapeutic effectiveness of both 5-HT3 receptor antagonists and tricyclic antidepressant.Ren et al.reported that visceral hypersensitivity in rats subjected to maternal separation is associated with elevated 5-HT expression in the dorsal raphe nucleus(DRN)in response to stress.In contrast,downregulation of DRN serotonergic activity through electroacupuncture attenuates visceral hypersensitivity in MS rats.Which indicates that DRN has a critical role in visceral pain modulation.Nesfatin-1,a satiety molecule processed from nucleobindin2(NUCB2),is implicated in visceral hypersensitivity in rats and colocalized with 5-hydroxytryptamine(5-HT)in the DRN.Maternal separation in rats contributes to visceral hypersensitivity via elevated expression of 5-HT in the DRN.Intracerebroventricular injection of nesfatin-1 activates DRN 5-HT neurons.We assumed that nesfatin-1 in the DRN has a regulatory role in visceral sensitivity,potentially through activation of DRN 5-HT neurons.In this study,we sought to determine the effect of nesfatin-1 in the DRN on visceral sensitivity using a maternally separated rat model of IBS.5-HT-and tryptophan hydroxylase(TPH,the rate-limiting enzyme for 5-HT synthesis)-immunopositive cells in the DRN were used as markers of 5-HT neurons.Effects of nesfatin-1 on visceral hypersensitivity and DRN 5-HT neurons were studied.Aim To investigate whether nesfatin-1 in the DRN was associated with the pathogenesis of visceral hypersensitivity and the underlying mechanisms by using a maternally separated rat model of IBS.Methods 1.Male newborn Sprague-Dawley rats were randomly allocated either to the maternally seprated group and normally handled group.Pups in the maternally seprated group were placed into individual plastic cages with sawdust bedding in a separate room to isolate them from their mothers for 3 h daily(8:00 AM–11:00 AM)from postnatal days 2 to 21,whereas pups in the normally handled group were undisturbed.Body weights were evaluated when rats were at postnatal day 3th and 8th week respectively.2.Visceral responses to different grades of colorectal distension(CRD)pressures(20,40,60,80 mm Hg)were quantified by abdominal withdrawal reflex(AWR)scores and the area under the curve(AUC)of electromyographic(EMG)magnitude of the obliquus externus abdominis.3.We observed AWR scores and EMG magnitude in response to graded strengths of CRD pressures in maternally seprated and control rats at 8 weeks of age.The expression of nesfatin-1/NUCB2 and TPH in the DRN were determined by immunohistochemistry.4.Rats in maternally seprated group were microinjected with anti-nesfatin-1/NUCB2 or vehicle into the DRN.then,AWR scores and EMG activity were recorded 2 h later.We observed 5-HT and TPH immunoreactivity in the DRN after application of anti-nesfatin-1/NUCB2 or vehicle in maternally seprated rats.6.The normally handled rats were microinjected with nesfatin-1 or vehicle into the DRN.Nesfatin-1 or vehicle was injected daily for 7 consecutive days,then visceral sensitivity was detected at the 7th day by examining AWR score and AUC of EMG.The expression of 5-HT and TPH in the DRN was determined by immunohistochemistry.Results 1.At postnatal 3th week,Body weights in the two groups were not statistically different(P>0.05),however,body weights in the maternally seprated group were significantly lower than that in normally handled group at postnatal 8th week(P<0.05).2.Maternally seprated rats exhibited higher AWR score and more intensive EMG magnitude at strengths of 40,60,and 80 mm Hg CRD pressure when compared to normally handled rats(P<0.05).3.The expression of nesfatin-1-or TPH-positive cells in the DRN of maternally seprated rats were significantly elevated than normally handled rats(P<0.05).4.Intra-DRN injection of anti-nesfatin-1/NUCB2 in the maternally seprated rats resulted in a significant decline in the AWR scores at 40,60,and 80 mm Hg distension pressures and EMG magnitude at all distension pressures,as compared with vehicle-treated rats.(P<0.05).Furthermore,Intra-DRN injection of anti-nesfatin-1/NUCB2 led to a significant reduction of 5-HT-or TPH-immunopositive cells in the DRN of MS rats(P<0.05).5.Intra-DRN injection of nesfatin-1 in normal rats resulted in significantly elevated AWR scores and EMG magnitude at 40,60,and 80 mm Hg distension pressures,relative to vehicle-treated rats(P<0.05).The number of 5-HT-or TPHimmunopositive cells in the DRN was significantly increased as compared with vehicle-treated rats in all sections of the DRN accordingly.Conclusion Nesfatin-1 has critical effects on visceral hypersensitivity;the underlying mechanisms might be related to the activation of DRN 5-HT neurons.