Role Of High-affinity Choline Transporter CHT1 In Visceral Hyperalgesia Of Irritable Bowel Syndrome

Part one: Upregulation of the high affinity choline transporter CHT1 in colon relieves stress-induced hyperalgesiaBackground:Irritable bowel syndrome(IBS)is a common gastrointestinal functional disease characterized by hyperalgesia,the mechanism of which remains elusive.The cholinergic system is known to be involved in pain inhibitory pathways in multiple diseases,and its involvement in IBS is unknown.Objective: We aimed to explore whether high affinity choline transporter CHT1,a major determinant of the cholinergic signaling capacity,is involved in regulating intestinal sensations associated with stress-induced visceral pain.Materials and Methods: An IBS Sprague-Dawley(SD)rat model was established by chronic water avoidance stress(WAS).Colonic pathological alterations were detected by hematoxylin-eosin staining.Visceral sensations were determined by scoring the abdominal withdrawal reflex(AWR)and visceromotor response(VMR)magnitude of the electromyogram(EMG)in response to colorectal distension(CRD).Abdominal mechanical hyperalgesia was assessed by counting the number of withdrawal events evoked by applying von Frey filaments(VFFs).Quantitative reverse-transcription polymerse chain reaction(q RT-PCR),western blot,and immunostaining were performed to identify the protein and m RNA levels of CHT1 in the colon,as well as its distribution in the colon.Acetylcholine(ACh)secretion in the colon was determined by enzyme-linked immunosorbent assay(ELISA).Effects of MKC-231(a choline uptake enhancer)and PDTC(a selective inhibitor of nuclear factor-?B(NF-?B)),on visceral pain were examined.Results: After 10 days of WAS exposure,AWR score and VMR magnitude in response to CRD was significantly enhanced and the number of withdrawal events in the VFF test was elevated.Protein and m RNA levels of CHT1 in the colon of WAS rats were considerably increased.The density of CHT1-positive intramuscular cells and myenteric plexus neurons in WAS rats was higher than in controls.PDTC could partly reverse CHT1 upregulation in the colon and relieve pain response in WAS rats.Pharmacological enhancement of CHT1 activity by MKC-231 gavage could relieve the visceral pain of WAS rats by upregulating CHT1 protein expression and enhancing ACh production.Conclusion: CHT1 may exert an anti-nociceptive effect in stress-induced visceral pain by modulating ACh synthesis in the colon and NF-?B signaling pathway mediates the regulation of CHT1 under stress.MKC-231 could be used as a potential drug to treat disorders with hyperalgesia.Part two: Role of the high-affinity choline transporter CHT1 in spinal modulation of stress-induced hyperalgesiaBackground:IBS is a common gastrointestinal functional disease with hyperalgesia,whose mechanism remains elusive.The spinal cholinergic system is known to be involved in pain modulation in multiple diseases,but its involvement in IBS is unknown.Objective: We aimed to determine whether high-affinity choline transporter CHT1-mediated choline uptake and transport,the rate-limiting step of cholinergic circulation,is implicated in spinal modulation of stress-induced hyperalgesia.Materials and Methods: A rat IBS model was established by chronic water avoidance stress.Colonic pathological alterations were assessed histologically and visceral sensations were detected by abdominal withdrawal reflex and visceromotor response to colorectal distension.Abdominal mechanical sensitivity was determined by counting the number of withdrawal events in VFF test.QRT-PCR,western blot,and immunostaining were performed to identify spinal CHT1 expression.Acetylcholine secretion was measured by ELISA and the effects of MKC-231(a choline uptake enhancer)and hemicholinium-3(HC-3,a specific inhibitor of CHT1)on visceral pain were examined intrathecally.Immunohistochemical staining was conducted to assess the function of microglia in the spinal dorsal horn and the effects of minocycline,a specific inhibitor of microglial activation,on visceral pain was examined.Results: After 10 days of WAS,AWR score and VMR magnitude in response to CRD along with the number of withdrawal events in VFF test were significantly increased.Protein and m RNA levels of CHT1 in the spinal cord were markedly elevated after WAS.The density of CHT1-positive cells in the spinal dorsal horn in WAS rats was higher than in controls.Intrathecal administration of HC-3 enhanced the pain response in WAS rats.Conversely,pharmacological enhancement of CHT1 activity by MKC-231 injection intrathecally alleviated the pain in WAS rats by increasing spinal ACh production and inhibiting microglial activation.Conclusion: CHT1 may exert an anti-nociceptive effect in spinal modulation of chronic stress-induced hyperalgesia via elevation of spinal ACh synthesis and suppression of microglial activation in the spinal dorsal horn.MKC-231 could be used as a potential drug to treat disorders with hyperalgesia.Part three: Effect of high affinity choline transporter CHT1 on primary cultured hippocampal neurons and microgliaBackground: Neuronal-microglia interaction is implicated in the modulation of hyperalgesia,in which cholinergic system exerts an important effect.The choline uptake and transport-mediated by high affinity choline transporter CHT1 is known as the rate-limiting step of cholinergic circulation.However,the role of CHT1 on the neuronal and microglial function remains elusive.Objective: We aimed to explore whether high affinity choline transporter CHT1 is involved in the regulation of neuronal and microglial function.Materials and Methods: The primary cultured neurons and microglia were derived from the pregnant SD rats aged 16-18 days and newborn SD rats aged 1-3 days,respectively.The growth state of these cells was observed under inverted microscope,and the purity was identified by immunofluorescence staining.Lipopolysaccharide(LPS)treatment was used to induce the damage model of neurons and microglia.The expression of CHT1 in cultures was determined by q RT-PCR,western blot and immunofluorescence staining.ELISA was performed to decipher ACh secretion in culture and the effects of MKC-231,the choline uptake enhancer,on ACh secretion,pain-related molecules(such as nerve growth factor(NGF)and tropomyosin-related kinase A(Trk A))and cytokines(such as IL-6)in neuronal cultures and microglia were evaluated.Results: The primary neurons and microglia were successfully cultured in our method and the purity and quality of the cultures met the requirements of subsequent experiments.Double-immunofluorescence staining demonstrated the robust expression of CHT1 in neurons and microglia.The CHT1 and GAP43 expression in cultured hippocampal neurons was decreased,and the Trk A expression was increased after LPS treatment,while MKC-231 could partly reverse these changes.In addition,administration of MKC-231 could block the LPS-induced decline of ACh secretion in neurons.LPS treatment could upregulate the transcription of IL-1?,TNF-? and NGF in microglia cultures,and downregulate the transcription of IL-4,elevate the phosphorylation level of p38,and attenuate the release of ACh.However,MKC-231 treatment could partly block the above changes.Conclusion: CHT1 may exert an important protective effect in LPS-induced neuronal injury and microglial activation by affecting the release of ACh,NGF/Trk A signaling pathway activation and neuronal growth,which provides a potential therapeutic target for the treatment of neuroinflammatory diseases.