The Role Of Detgth Receptor 5 In Homeostasis Of Colonic Myenteric Neurons In Mice And Its Mechanism

Background and aim:Intestinal nervous system(ENS)plays an important role in the regulation of intestinal movement,sensation and other physiological functions.The disorder of ENS is involved in various intestinal diseases.Due to the influence of intestinal contents,intestinal microbes,and intestinal motility,neurons in the ENS continue to undergo apoptosis,while the intestinal nerve precursor cells(ENPCs)canproliferate and differentiate to form new neurons tocompensate the loss of neurons and maintain the stability of the structure and function of the ENS.The mechanism of ENPCs proliferate and differentiate is complex and still unclear,it is important to explore its mechanism.Death receptor 5(DR5)is a member of the tumor necrosis factor receptor(TNFR)superfamily,whichcaan be activated by tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)to trigger apoptosis of tumor cells,but has no toxicity to normal cells.Recently,it has been found that the activation of DR5 in some cells did not mediate death signals,but promoted cell proliferation and differentiation.DR5 might participate in the proliferation of central neurons during central nervous system development process,but its specific role remained to be clarified.DR5 was expressed in the myenteric plexus,but there was no report about its role in myenteric plexus.Did DR5 participate in the maintenance of myenteric neurons?To clarify this problem,we designed to use DR5 gene knockout mice(DR5/-mice)and to explore the roleof DR5 on the colonic myenteric neurons and its mechanism through in vivo experiments and in vitro ENPCs culture,and further study whether DR5 was involved in the injury of colonicmyenteric plexus in colitis.Results:1.DR5 was expressed in the colonic myenteric plexus.DR5 gene knockout promted the causesed abnormal expression of various neurons and neurotransmitters in the myenteric plexus.The number of nitric oxide synthase(nNOS)positive neurons and its neurotransmitter nitric oxide(NO)in the myenteric plexus increased significantly after the DR5 gene was knocked out;Choline acetyltransferase(ChAT)positive cholinergic neurons and their neurotransmitter acetylcholine also increased significantly after DR5 gene knockout;The inhibitory neurotransmitter y-aminobutyric acid,the expression of its synthesis rate-limiting enzyme GAD65/67 increased.The gene expression of another inhibitory neurotransmitter vasoactive intestinal peptide(VIP)also increased.2.The myenteric plexus participated in the regulation of intestinal motility.Consistent with the change of the neurons in the myenteric plexus,DR5 gene knockout causesed the colonic motility abnormality mediated by the myenteric plexus.Compared with WT mice,DR5/-mice had decreased contractility of longitudinal colon muscle strips,displayed by decreased peak systolic tension and reduced area under the curve,but DR5 gene knockout did not affect the frequency of colon contraction.There was no difference between DR5-/-mice and WT mice in the myogenic contraction of colon muscle strips induced by carbachol gradient.Electrical field stimulation(EFS)could affect the movement of intestine via inducing the release of neurotransmitters in the colonic myenteric plexus.The colonic relaxation induced by EFS in DR5-/-mice was significantly stronger than that in WT mice.The nNOS inhibitor NPLA completely blocked the colonic relaxation response induced by EFS in WT mice,inhibibted but did not completely block the colonic relaxation induced by EFS in DR5-/-mice.The results suggested that the abnormal colon movement after DR5 gene knockout was related to the changes of neurons and neurotransmitters in the myenteric plexus.3.DR5 gene knockout did not affect the expression of pro-apoptotic protein Cleavedcapase3 in myenteric plexus,but the number of Nestin-positive(Nestin+)ENPCs and βTubulin-positive(β-Tubulin+)neurons increased,the protein expression of Nestin and βTubulin was also increased.The cell number of ENPCs from DR5-/-mice increased faster than that from WT mice during ENPCs culturing in vitro.In the cultured ENPCs from DR5-/-mice,the number of Nestin+ cells and the proportion of EdU(5-Ethynyl-2’-deoxyridine)positive ENPCs were significantly higher than that from WT mice.The number of nestin+ ENPCs and the proportion of EdU+ENPCs were significantly decreased following the small molecule selectvie DR5 agonist Bioymifi administration in vitro cultured ENPCs for 5 days.7 days colonic infusion of Bioymifi caused the decreasing of Nestin+ENPCs and β-Tubulin+neurons in WT mice.The results suggested that DR5 gene knockout might lead to the decreasing of colonic myenteric neurons via enhancing the proliferation of ENPCs.Accordingly,activation of DR5 inhibited the proliferation of ENPCs in the colonic myenteric plexus.4.DR5 participated in the regulation of the proliferation of colonic ENPCs in myenteric plexus through acting on BMP2-p-Smad1/5 pathway.4.1 The protein expression of Atoh1,c-Myc,Hes1,and p-Smad1/5 related to neuronal proliferation in the longitudinal muscle myenteric plexus of DR5-/-mice was increased.7 days colonic infusion of Bioymifi in WT mice prompted the the expression of p-Smad1/5 and Hesl in colonic longitudinal muscle myenteric plexus,while the expression of Atoh1 and c-Myc did not change.On the 5th day of ENPCs culture,the expression of p-Smad1/5 in DR5 gene knockout ENPCs was significantly higher than that in ENPCs from WT mice,while there was no different of Atoh1 and c-Myc between ENPCs from DR5-/-mice and WT mice.On the 5th day of ENPCs culture,there was nearly no expression of Hes1 in ENPCs,and there was expression of Hes1 and β-Tubulin+ neuron on the 10th day of cultured ENPCs.4.2 BMP pathway inhibitor LDN193189 could inhibit p-Smad activation induced by BMPs.LDN193189 treatment eliminated the difference in p-Smad1/5 expression between ENPCs from DR5-/-mice and from WT mouse ENPCs in vitro.LDN193189 treatment also decreased the number of the Nestin+ENPCs induced by DR5 knockout and there was no difference in the number of ENPCs between ENPCs from DR5-/-mice and from WT mice.Intraperitoneal injection of LDN193189 could also abolished the difference of Nestin+ENPCs,β-Tubulin+neurons,ChAT+and nNOS+neurons between DR5-/-mice and WT mice.The results suggested that DR5 participated in the proliferation regulation of ENPCs in the colonic myenteric plexus depending on the expression of p-Smad1/5.4.3 Compared with cultured ENPCs from WT mice,the expression of BMP ligand BMP2 gene and protein in DR5 gene knockout ENPCs were up-regulated.Immunohistochemistry showed that the expression of BMP2 in the myenteric plexus of DR5-/-mice was significantly higher than that of WT mice.In vivo Bioymifi administration declined the expression of BMP2 protein in colonic myenteric plexus.5.The up-regulation of DR5 was involved in the injury of colitis myenteric plexus during acute colitis.5.1 2.5%dextran sodium sulfate(DSS)induced acute colitis in mice,resulted in decreased body weight,increased disease activity index and mucosal damage scores,as well as shortened colonic length.and increased Compared to the control group,the number of β-Tubulin+ neurons and Nestin+ENPCs in the colonic myenteric plexus in colitis mice decreased significantly.5.2 The expression of DR5 in myenteric neurons of acute colitis model was up-regulated comparted with the control group.5.3 Under physiological conditions,iNOS labeled M1 macrophages were rarely seen in the intramuscular plexus,while in the model of acute colitisthe expression of iNOS labeled M1 macrophages in the myenteric plexus increased.LPS induced M1 polarization of macrophages.The supernatant of M1 macrophages up-regulated the expression of DR5 in cultured ENPCs and inhibited the expression of Nestin gene.However,the supernatant of M1 macrophages did not affect the expression of Nestin in ENPCs from DR5-/-mice.Conclusions:1.DR5 gene knockout promoted the proliferation of ENPCs in colonic intermuscular plexus,which in turn resulted the up-regulation of the myenteric neurons numbers and abnormalities in colonic motor function.2.DR5 was expressed in the ENPCs of colonic intermuscular plexus.ENPCs DR5 gene knockout promoted ENPCs proliferation via up-regulating the expression of BMP2 and pSmad1/5 in ENPCs.Accordingly,selective activation of DR5 in ENPCs inhibited the proliferation of ENPCs proliferation by downregulating the BMP2 expression,which then inhibiting the activation of p-Smad1/5.(3)M1 macrophages increased in the myenteric plexus in the colitis model.The supernatant of M1 macrophages could up-regulated the expression of DR5 in ENPCs and downregulated the expression of Nestin.The increased expression of DR5 caused inhibition of ENPCs proliferation,which might was one of the reasons for the injury of the myenteric plexus in colitis.