The Role And Mechanism Of Bile Acid Receptor TGR5 In The Development Of Chronic Pain In Anterior Cingulate Cortex

ObjectiveIn this study,we took the G protein-coupled bile acid receptor 1（TGR5）in the brain of mice as the research object,and constructed the chronic pain model of mice.This study focuses on the role of TGR5 in anterior cingulate cortex（ACC）in the occurrence and development of chronic pain and the potential mechanism of action,which provides a new target for the treatment of chronic pain and provides ideas and theoretical basis for the development of new analgesics.MethodsFirst,established the Spared Nerve Injury（SNI）neuropathic pain model to measure the content of brain bile acid in mice,and then LCA(100 mg·kg^-1),CDCA(200 mg_·kg^-1)and UDCA(200 mg_·kg^-1)were given to SNI mice.Mouse behavior changes were detected.The distribution and expression of TGR5 in different brain regions of mice were detected by Western blot,and the specific expression cell types of TGR5 in ACC brain region were detected by immunofluorescence staining.In the function verification section,established the SNI models and Complete Freund’s Adjuvant（CFA）chronic inflammatory pain models,Subsequently,the mouse ACC brain region overexpression and knock down TGR5,detected the mouse behavior changes.The behavioral changes of TGR5-KO mice were detected.Whole cell patch clamp technique was used to observe the role of TGR5 in neuronal abnormal excitation induced by SNI.The cell types dependent on the analgesic and antianxiety effects of TGR5 were determined using the Cre tool mouse combined with DIO virus.In the part of mechanism exploration,we used BV2 microglia cell line for experiments,and Western blot combined with immunofluorescence staining experiment to observe the effects of TGR5 agonist INT-777 on the expression of polarity-related proteins Arg-1 and i NOS in microglia cells.The expression of proinflammatory factors in the supernatant of LPS activated microglia cells and INT-777 intervention was determined by Elisa.The changes of Notch1,HES-1,SIRT1,p-STAT3 and STAT3 were detected by Western blot.Finally,administered TGR5agonist INT-777 was to ACC brain region of SNI model mice to detect the behavioral changes of mice and validate the efficacy in vivo.ResultsDecreased levels of natural TGR5 ligand in the brains of SNI mice.The administration of LCA can relieve SNI-induced pain threshold reduction and anxiety-like emotions,while the administration of CDCA or UDCA can only alleviate SNI-induced pain threshold reduction but cannot relieve anxiety-like emotions.The results of Western blot and immunofluorescence staining showed that there was no significant difference in the expression level of TGR5 in the hippocampus and amygdala of SNI mice,but the expression level of TGR5 in ACC was significantly decreased.Immunofluorescence results showed that TGR5 was mainly expressed on excitatory neurons and microglia cells,and a small amount on astrocytes and inhibitory neurons in mouse ACC.Behavioral test results showed that overexpression of TGR5 in the ACC region of mice alleviated the lower pain threshold and anxiety-like emotions induced by SNI and CFA.Behavioral tests showed that TGR5-KO mice showed reduced pain threshold and anxiety-like behavior compared with WT mice.Patch clamp results showed that overexpression of TGR5 in the ACC region of mice could effectively inhibit the abnormal increase of s EPSC frequency and amplitude caused by SNI.And TGR5 mainly depends on microglia to play a major role.In the part of mechanism exploration,Western blot and immunofluorescence staining results showed that LPS group promoted the transformation of BV2 cells to M1 type compared with normal group.Compared with LPS group,BV2 cells transformed to M2 type after treatment with INT-777.The Elisa results showed that TNF-α,IL-6 and IL-1βin BV2 cell culture supernatant in LPS group were significantly higher than in Vehicel group.Compared with LPS group,TNF-α,IL-6and IL-1βwere decreased after treatment with INT-777.Western blot results showed that compared with Vehicel group,the expressions of Notch1,HES1 and p-STAT3/STAT3 in BV2 cells of LPS group were significantly higher,while the expressions of TGR5 and SIRT1 were significantly lower.Compared with LPS group,the expressions of Notch1,HES1 and p-STAT3/STAT3 in BV2 cells were decreased after administration of INT-777,while the expressions of TGR5 and SIRT1 were increased.Administration of INT-777 in the ACC region of mice alleviated SNI-induced pain threshold reduction and anxiety-like emotions.ConclusionOur study revealed that ACC brain region TGR5 is involved in the regulation of neuropathic pain induced by SNI and chronic inflammatory pain induced by CFA,and the activation of TGR5 receptor may relieve neuropathic pain and chronic inflammatory pain by regulating microglia polarization and inhibiting the release of inflammatory factors.Our results not only elucidate the role and regulatory mechanism of TGR5 in chronic pain,but also provide new strategies and drug development targets for the treatment of chronic pain.