The Protective Effect And Underlying Mechanisms Of Oxytocin On Enteric Nervous System Injury

Research BackgroundThe enteric nervous system(ENS)is composed of the ganglion formed by the aggregation of neurons and nerve fibers.Neurons connect with each other forming nervous system which integrates and processes information independently,known as the "micro-brain" of the gastrointestinal tract,regulating important gastrointestinal functions.Recent studies have found that mature enteric neurons still have continuous apoptosis and proliferation.The proliferation is mainly realized by enteric neural precursor cells(ENPCs).The dynamic balance between the mature enteric neurons and ENPCs is the basis of ENS physiological function maintenance.Many factors could lead to structural damage and functional disorders of ENS,such as neurotoxic drugs,neurodegeneration,and so on.Vincristine(VCR)is widely used in treatment of various malignant tumors in clinic.The peripheral neurotoxicity of VCR is an important reason for many patients to stop treatment.After VCR treatment,enteric motor function decreased significantly,which may be related to enteric neurons injury.VCR also triggers enteric immune responses,in which enteric macrophages are important regulators and can be polarized into anti-inflammatory and proinflammatory phenotypes under different conditions.The interaction between enteric macrophages and neurons is involved in the maintenance of ENS function.However,the cause of enteric neurons injury after VCR treatment remains unclear,and whether enteric macrophages play an important role in it is still unknown.Enteric senescence is associated with enteric neurodegeneration.The gastrointestinal transmission time of the elderly was prolonged,and the enteric neurons were injured,which was manifested as reduced number,abnormal morphology and fibrous swelling.At present,most studies on the mechanism of enteric neurodegeneration focus on oxidative stress and inflammatory response,and few studies fucus on the regulation of enteric neuron proliferation.Glial cell derived neurotrophic factor(GDNF)is reduced in neurodegenerative patients.In addition,the proliferation and differentiation of ENPCs,as well as the survival of mature neurons,are affected by GDNF.Therefore,GDNF is currently considered to be an important potential target for neurodegenerative therapy.Oxytocin(OT)is the first structurally identified and chemically synthesized bioactive peptide hormone.In addition to the hypothalamus,OT is also secreted by other organs,including the digestive tract.OT functions biologically by binding to oxytocin receptor(OTR).The expression of OTR is widespread.The classic roles of OT are to stimulate uterine contractions and lactation reflex.In recent years,the effects of OT have been greatly expanded,such as anti-inflammatory,antioxidant,analgesic,sedative,blood pressure regulation,learning and memory,etc.OT is also involved in regulating the proliferation and differentiation of a variety of cells,including glial cells.However,the role of OT in enteric neurons injury and its specific mechanism remain unclear.By establishing two kinds of ENS injury models induced by neurotoxic drugs(VCR)and neurodegenerative changes(senescence),this study revealed the roles and mechanisms of OT in ENS injury induced by VCR and senescence for the first time,further expanded the functions of OT,and provided new ideas for the clinical treatment of ENS injury.Part I The Protective Effect and Underlying Mechanisms of Oxytocin on Enteric Neurons Injury Induced by VincristineObjectivePrevious studies reported that VCR led to neurons injury accompanied by an immune response.OT has neuroprotective function,but its effect on enteric neurons injury induced by VCR remains unclear.This part explored the mechanisms of enteric neurons injury induced by VCR and the role of enteric macrophages in it.At the same time,exogenous OT was administered to explore the role and mechanisms of OT in enteric neurons injury induced by VCR.Methods1.Preparation of animal model(1)Preparation of enteric neurons injury model induced by VCR:The mice were intraperitoneally injected with NS or VCR(0.1 mg/kg)for 10 days.(2)Experiment of enteric macrophage depletion:The mice were intraperitoneally injected with 200 μl control or clodronate liposomes every 3 days,and NS or VCR(0.1 mg/kg)was injected for 10 consecutive days the next day after the first administration of liposomes.(3)Enteric neuroprotective experiment of OT:The mice were intraperitoneally injected with NS or OT(1 mg/kg)for 1 h,and then injected with NS or VCR(0.1 mg/kg)for 10 consecutive days.2.Colonic myenteric neurons isolation and cultureThe longitudinal muscle myenteric plexus(LMMP)was prepared by removing the colonic mucosa,submucosa,circular muscle,and serous membrane successively with anatomical instruments.The LMMP was digested with papain(10 mg/ml)for 55 min and collagenase Ⅱ(1 mg/ml)for 55 min.The myenteric neurons were isolated and cultured in vitro.3.Culture of colonic myenteric neurons with peritoneal macrophages supernatantThe peritoneal macrophages supernatants stimulated by different drugs were collected as conditioned medium and added into colonic myenteric neurons cultured in vitro to observe the enteric neurons injury caused by inflammatory factors.4.The proportions of CD86+pro-inflammatory macrophages and CD206+antiinflammatory macrophages in colonic mononuclear cells were detected by flow cytometry.5.The number of βⅢ-Tubulin+enteric neurons and the length of axons were detected by immunofluorescence.6.The mRNA expression of inflammatory factors in colonic LMMP and RAW264.7 cells was detected by qRT-PCR.7.The differential expression of colonic LMMP mRNA was detected by RNA-Seq.8.The concentration of inflammatory factors in the supernatant of cell culture medium and tissue was detected by ELISA.9.The expression of P38 and P44/42 in RAW264.7 cells and the expression of SGK1,FOX03 and Cleaved caspase-3 in colonic LMMP and conditioned cultured neurons were detected by Western blot.10.The number of apoptotic neurons in colonic tissue was detected by TUNEL staining.11.The motor function of gastrointestinal tract was measured by GITT.Results1.VCR resulted in colonic myenteric neurons injury and intestinal motility reductionAfter 10 days of intraperitoneal injection of VCR(0.1 mg/kg)or NS,the colonic myenteric neurons were isolated and cultured in vitro for 6 days.The results showed that the number of neurons and the length of axons decreased,and intestinal motility reduced after VCR injection.2.VCR regulated the polarization of colonic macrophagesCompared with the control group,the proportion of CD86+pro-inflammatory macrophages increased significantly and that of CD206+anti-inflammatory macrophages decreased slightly after VCR injection.The expression of mRNA and protein of proinflammatory factors IL-1β,IL-6 and TNF-α in colonic LMMP significantly increased after VCR injection,and the mRNA levels of anti-inflammatory factors Arg1 and Il-10 had downward trend.These results indicated that VCR promoted the polarization of colonic macrophages towards pro-inflammatory type.3.The role of macrophages in colonic myenteric neurons injury induced by VCRThe continuous depletion of macrophages during VCR injection was maintained by the use of clodronate liposomes.The results showed that depletion of macrophages during VCR injection significantly improved the number of enteric neurons and axon length,reduced the mRNA and protein levels of IL-1β,IL-6 and TNF-α,and accelerated the intestinal motility.4.The effect and mechanism of VCR on M1-type polarization in RAW264.7 cellsAfter stimulating RAW264.7 cells with VCR concentrations of 10-10 M,10-9 M and 10-8 M for 24 h,the mRNA and protein levels of pro-inflammatory factors were detected.The results showed that all three concentrations of VCR stimulated RAW264.7 cells to M1 type polarization,but 10-9 M VCR had the strongest effect.Moreover,VCR(10-9 M)cooperated with LPS to induce cells to produce more pro-inflammatory factors.VCR stimulated M1 macrophage polarization through increasing phosphorylation of ERK1/2 and P38-MAPK.5.VCR led to the colonic myenteric neurons injury by promoting the secretion of macrophage inflammatory factorsThe supernatant produced by mouse peritoneal macrophages was used as conditioned medium to incubate colonic myenteric neurons.The results showed that compared with the control group,the number of colonic myenteric neurons and the length of axon were significantly decreased when the macrophages supernatant was stimulated by VCR.6.VCR-induced pro-inflammatory macrophages caused colonic myenteric neurons injury via the SGK1-FOX03 pathwayThe LMMPs with or without macrophage depletion during VCR injection were detected by RNA-seq,whose differential genes were analyzed and verified by qRT-PCR.Detection of SGK1 and FOXO3 proteins levels in LMMP lysates showed that VCR-induced proinflammatory macrophages led colonic myenteric neurons injury by reducing the expression of SGK1,p-SGK1 and p-FOXO3/FOXO3.7.VCR-induced pro-inflammatory macrophages accelerated the apoptosis of colonic myenteric neuronsCompared with the control group,in the VCR group,the number of TUNEL+neurons in ganglion region significantly increased,and the Cleaved caspase-3 expression increased in the LMMP lysate.The apoptosis of neurons induced by VCR decreased after macrophage depletion.8.The role of macrophage MAPK pathway in VCR-induced colonic myenteric neurons injury(1)The inhibitors of MAPK pathway reduced the secretion of inflammatory factors in macrophagesThe RAW264.7 cells were preincubated with PD98059(20 μM)and SB203580(10μM)for 1 h to inhibits the activation of ERK1/2 and P38-MAPK pathways,and then stimulated with VCR(10-9 M)alone or in combination with LPS(100 ng/ml)for 24 h.Compared with absence of inhibitors groups,the use of inhibitors significantly decreased the expression of Il6 mRNA in each group.(2)Inhibiting the MAPK pathway of macrophages alleviated VCR-induced colonic myenteric neurons injuryThe peritoneal macrophages were pre-incubated with these two inhibitors and then stimulated with VCR(10-9 M).The supernatant was used as conditioned medium to incubate colonic myenteric neurons.Compared with the VCR group,the number and axon length of neurons incubated with inhibitor pretreated supernatant were significantly increased.Inhibition of ERK1/2 and P38-MAPK pathways in macrophages weakened the inhibition of SGK1-FOXO3 signaling pathway and alleviated the apoptosis of colonic myenteric neurons.9.OT inhibited pro-inflammatory macrophage polarization induced by VCR(1)In vitro,OT inhibited pro-inflammatory polarization of RAW264.7 cells induced by VCRThe RAW264.7 cells were incubated with five concentrations of OT(10-10 M,10-9 M,108 M,10-7 M and 10-6 M)1 h in advance,and then stimulated with VCR(10-9 M)for 24 h to detect the mRNA expression of Il-1β and Il-6,The results showed that 10-8 M,10-7 M and 10-6 M OT inhibited the pro-inflammatory macrophage polarization induced by VCR.(2)In vivo,OT inhibited pro-inflammatory polarization of colonic macrophages induced by VCRThe mice were intraperitoneally injected with OT(1 mg/kg)or NS for 1 h,and then injected with VCR(0.1 mg/kg)or NS for 10 days to detect the expression of pro-inflammatory factors in LMMP.Compared with the VCR group,the pretreatment of OT significantly decreased the mRNA expression of Il-1β and Il-6.10.OT alleviated the injury of colonic myenteric neurons induced by VCRThe mice were intraperitoneally injected with OT(1 mg/kg)or NS for 1 h,and then injected with VCR(0.1 mg/kg)or NS for 10 days to detect the number of neurons in ganglion region.Compared with the VCR group,OT significantly increased the number of neurons in ganglion region,demonstrated that OT alleviated the injury of colonic myenteric neurons induced by VCR.11.The mechanisms of OT alleviating colonic myenteric neurons injury induced by VCRThe mice were intraperitoneally injected with OT(1 mg/kg)or NS for 1 h,and then injected with VCR(0.1 mg/kg)or NS for 10 days.Compared with the VCR group,in the OT group,the phosphorylation levels of P44/42 and P38 were significantly decreased,the expression of SGK1 and p-SGK was increased,the level of p-FOXO3/FOXO3 was increased and the expression of Cleaved caspase-3 was decreased.ConclusionVCR induced pro-inflammatory macrophage polarization through ERK1/2 and P38MAPK pathways,and the pro-inflammatory macrophages caused colonic myenteric neurons injury via the SGK1-FOXO3 pathway.OT inhibited the pro-inflammatory macrophage polarization through the ERK1/2 and P38-MAPK pathways,and promoted the activation of SGK1-FOXO3 pathway to protect the colonic myenteric neurons injury induced by VCR.Part II The Protective Effect and Underlying Mechanisms of Oxytocin on Enteric Neurons and Enteric Neural Precursor Cells Injury Induced by SenescenceObjectiveIt has been reported that senescence led to the decrease of plasma OT and the injury of enteric neurons.However,the effect and mechanisms of OT on enteric neurons and enteric neural precursor cells(ENPCs)injury induced by senescence have not been reported.In this part,by using the mouse model of natural senescence,the mechanisms of enteric neurons and ENPCs injury induced by senescence and the role of OT in it were explored.Methods1.Preparation of animal modelThe young and old mice were intraperitoneally injected with NS or OT(1 mg/kg)for 14 days.In the experiment to inhibit the function of enteric glial cells(EGCs),the old mice were injected intraperitoneally with OT and Fluorocitrate(FC,10 μM/kg)daily for 14 days.2.The ENPCs isolation and cultureThe LMMP was prepared by removing the colonic mucosa,submucosa,circular muscle and serous membrane successively with anatomical instruments.The LMMP was digested with papain(10 mg/ml)for 45 min and collagenase Ⅱ(1 mg/ml)for 40 min.The ENPCs were isolated and cultured in vitro.3.Culture of ENPCs with colonic LMMP supernatantThe colonic LMMP supernatants stimulated by different drugs were collected as conditioned medium and added into ENPCs cultured for 6 days in vitro.4.The EGC CRL2690 cells senescence model was constructed with H2O2,and cell activity was detected by MTT and cell senescence ratio was detected by β-galactosidase.5.The number of βⅢ-Tubulin+enteric neurons and Nestin+ENPC and the expression of p-ERK of EGCs were detected by immunofluorescence.6.The proliferation of ENPCs was detected by Nestin-Cre:tdTomato mice and EdU stainning.7.The mRNA expression of age-related markers,OT-OTR signaling pathway and gliarelated molecules in colonic LMMP and EGC CRL2690 cells were detected by qRT-PCR.8.The concentration of OT and GDNF in colonic tissue was detected by ELISA.9.The expression of P16,Nestin,RET,PI3K and AKT in colonic LMMP was detected by Western blot.10.The motor function of colon was measured by bead arrangement experiment.Results1.Exogenous OT alleviated colonic senescenceThe expression of colonic senescence related markers was detected.The results showed that,compared with the young mice,the expression of P16 and senescence associated secretory phenotype(SASP)was increased in the old mice,while exogenous OT reduced the expression in the old mice.2.The role of OT-OTR signaling system in aging colonThe expression of colonic Otr and Ot mRNA was detected.The results showed that,compared with the young mice,the expression of Otr and Ot in the old mice colon decreased,while exogenous OT increased their expression in the old mice colon.3.OT alleviated the structural and functional injury of ENS induced by senescenceThe number of neurons and Nestin+ENPCs in ganglion region and the colon motility were detected.The results showed that,the number of neurons and ENPCs in the ganglion region of the old mice was reduced,and the colonic motility was impaired.Exogenous OT significantly increased the number of colon neurons and ENPCs,and improved the colonic motility in old mice.4.OT alleviated the injury of colonic myenteric neurons and ENPCs induced by senescence through EGCs(1)The EGCs expressed OTRThe GFAP and OTR in the colonic myenteric plexus were co-located,indicating that EGCs expressed OTR.(2)The effects of OT on aging EGCs and GDNFThe expression of GFAP and GDNF in colon was detected.The results showed that compared with the young mice,the expression of GFAP and GDNF in old mice colon was decreased,while exogenous OT significantly increased the expression in old mice colon.(3)OT was involved in regulating aging EGCs through ERK pathwayThe expression of p-ERK in colonic myenteric EGCs was detected.The results showed that the expression of p-ERK in GFAP+EGCs in colonic myenteric tissues of old mice was decreased compared with that of young mice,and exogenous OT increased the p-ERK expression of GFAP+EGCs in old mice.It was indicated that OT was involved in regulating EGCs GDNF expression by activating ERK signaling pathway.(4)FC inhibited the protective effect of OT on the injury of colonic myenteric neurons and ENPCs induced by senescenceThe old mice were intraperitoneally inj ected with OT and FC(10 μM/kg)daily for 14 days.Compared with the old mice injected with OT,the expression of GFAP in the colon of the old mice injected with FC+OT had no significant change,but the expression of GDNF was decreased,indicating that the use of FC inhibited the function of EGCs and reduced the production of GDNF.The number of neurons and ENPCs in ganglion region was detected.Compared with the old mice injected with OT,the number of neurons and ENPCs in the ganglion region of the old mice injected with FC+OT was decreased.5.The role of OT in H2O2-induced senescence model of EGC CRL2690 cells(1)The role of OT on EGC CRL2690 cells under normal culture conditionThe EGC CRL2690 cells were cultured under normal conditions and incubated with OT at different concentrations(10-10 M,10-9 M,10-8 M,10-7 M,10-6 M)for 2 h to detect the mRNA expression of Otr and Gdnf.The results showed that 10-10 M,10-9 M and 10-8 M OT incubation reduced the expression of Otr,but didn’t affect the expression of Gdnf.(2)The role of OT on EGC CRL2690 cells under H2O2-induced senescence conditionThe EGC CRL2690 cells were stimulated with H2O2 at different concentrations(0 μM,1μM,5 μM,10 μM,25 μM,50 μM,75 μM,100 μM)for 8 h to induce cell senescence.The MTT was used to detect the cell activity.The results showed that 25 μM H2O2 had the biggest effect,and this concentration also induced cells senescence.The cells were incubated with different concentrations of OT(10-9 M,10-8 M,10-7 M,10-6 M)for 2 h in advance,and then stimulated with 25μM H2O2 for 8 h to detect the mRNA expression of Gdnf and SASP.The results showed that 10-8 M OT significantly increased the expression of Gdnf and decreased the expression of SASP.6.OT alleviated the injury of colonic myenteric neurons and ENPCs induced by senescence through GDNF-RET-PI3K-AKT pathway(1)The Nestin+ENPCs proliferated rapidly in vitroThe Nestin+ENPCs were isolated from the colon of Nestin-Cre:tdTomato mice and cultured in vitro for 6 days to observe the changes of ENPCs with time.The results showed that ENPCs proliferated rapidly with time.In addition,the ENPCs isolated from wild mice were stained by EdU and Nestin.The results showed that up to 93.11±1.52%of Nestin+ENPCs cells showed EdU+,indicating that the Nestin+ENPCs proliferated rapidly in vitro.(2)The role of GDNF-RET in OT alleviating ENPCs injury induced by senescenceThe supernatant of LMMP with or without OT(10-8 M)stimulated was used as conditioned medium to culture colonic ENPCs of the old mice colon for 6 days,and the RET inhibitor BLU667(10 nM)was added to observe the proliferation of ENPCs in vitro.The results showed that the addition of RET inhibitors inhibited the effect of OT on ENPCs proliferation.(3)OT-induced GDNF alleviated ENPCs injury induced by senescence through RETPI3K-AKT pathwayThe RET,PI3K and AKT protein expression in LMMP tissue lysate was detected.The results showed that exogenous OT significantly increased the expression of RET and the phosphorylation levels of PI3K and AKT in old mice.ConclusionSenescence resulted in colonic myenteric neurons and ENPCs injury,and colonic motility reduction.Exogenous OT increased the expression of GDNF in aging EGCs through the ERK signaling pathway,and the GDNF alleviated colonic ENPCs injury induced by senescence through RET-PI3K-AKT signaling pathway.