The Study Of The Role And Mechanism Of Liver X Receptor ? In CFA-Induced Chronic Inflammatory Pain

Background and AimsChronic pain is defined as a neuroepigenetic disorder caused by persistent tissue inflammation or nerve injury under various pathological conditions,which is accompanied by lasting,multifaceted maladaptations ranging from gene modulation to synaptic dysfunction and emotional disorders.Inflammation,tissue injury and/or nerve injury-induced changes of gene expression in sensory neurons from the dorsal root ganglion,dorsal horn of spinal cord and pain-associated brain regions are thought to participate in chronic pain genesis.However,how these changes occur?Lots of efforts have been made to look for genetic mechanisms regulating gene expression.It is evidenced that multiple mechanisms are involved in regulation of gene expression,among them,epigenetic regulation,including DNA methylation,histone modification and small molecular RNA,has been widely proved to be effective in controlling gene expression.Epigenetic modifications may last for years or even generations and participate in the regulation of pain hypersensitivity under chronic pain conditions.Central sensitization plays a key role in the process of chronic pain,which is associated with the activation of a distributed group of structures,including the somatosensory cortex,insular cortex,and the anterior cingulate cortex?ACC?.ACC attracts more and more attention in pain research which plays key roles in pain modulation,learning,memory,and attention.Multiple mechanisms are involved in the process of the central sensitization of chronic pain.Neuroinflammation is a critical factor responsible for the development of chronic pain,causing the changes of neurotransmitter metabolism,neuroendocrine function and abnormal neuroplasticity in central nervous system?CNS?,leading to hyperalgesia and pain chronicity in the brain.Our previous work revealed a significant enhancement in neurotransmitter release probability of synapses in ACC from mice with chronic pain.More and more evidence linked central sensitization to abnormal gene expression in cells processing nociceptive signaling in CNS.Studies demonstrated that many genes undergo expression changes at mRNA and protein levels in tissues or cells of pain circuitry during the development or maintenance of persistent pain.Liver X receptors?LXRs?,including LXR?/NR1h3 and LXR?/NR1h2,belong to the nuclear receptor superfamily of transcription factors.LXRs have emerged as important regulators controlling cellular and whole-body cholesterol homeostasis,as well as important regulators of inflammation gene expression and innate immunity.LXR?is expressed predominantly in organs of lipid metabolism,whereas LXR?is expressed ubiquitously in most of the physical system and plays a crucial role in the immune system and CNS.LXR?knockout did not cause fundamental developmental defects in mice,whereas LXR?deletion led to impaired cerebral cortex lamination,loss of DA neurons in substantia nigra,anxiety,and impaired behavior responses.Furthermore,LXR?was also expressed in spinal cord,and male LXR?^-/-mice suffered from adult-onset motor neuron degeneration,whereas LXR?activation by T0901317 protected the spinal cord from injury.GW3965,another synthetic full agonist for both LXRaand LXRbisoforms,can readily cross the blood-brain barrier to exert its specific actions in brain.LXRs activation by T0901317 and GW3965 alleviated mechanical allodynia in neuropathic pain and delayed obesity-induced allodynia in mice.Notably,the role of LXRs in chronic pain remains largely unknown.Our study intends to establish chronic inflammatory pain?CIP?mice model induced by Complete Freund's adjuvant?CFA?and combines methods including animal behavior tests,electrophysiology,molecular biology,and epigenetics to?1?investigate the roles of LXRs in the pathogenesis of CIP;?2?explore the possible mechanisms involved in GW3965-mediated analgesic effects in CIP;?3?determine the subtype of LXRs involved in GW3965-mediated analgesic effects;?4?illuminate the epigenetic mechanisms involved in regulating the expression of LXR?in ACC in CIP mice.Based on above researches,we aim to evaluate the possibility of LXR?as a new drug target for the treatment of chronic pain,and provide theoretical basis for the clinical research and drug development of chronic pain.Methods1.The role of LXRs activation in pain behavior of CIP model mice1.1 The distribution and cell expression pattern of LXRs in ACCImmunofluorescent staining was used to detect the expression pattern of LXRs?LXRaand LXR??in ACC region as well as that of cell specificity.LXR?was co-labeled with neuronal marker?-tubulin III,astrocyte marker GFAP,and microglia marker Iba-1,respectively.LXR?was co-labeled with excitatory neuron marker CAMK IIa,and inhibitory neuron marker GAD67 respectively by immunofluorescence staining to determine LXR?distribution of neuronal type in ACC.1.2 The changes of LXRs expressions in ACC of CIP miceA CIP model was established by CFA?CFA:0.9%normal saline=1:1,10ml?hindpaw injection in mice.The mechanical pain and thermal pain behavior of mice were measured at day 1,3,5,7 and 14 after CFA injection by Von Frey and a plantar analgesia device.The frozen brain slices containing ACC and ACC brain tissues were collected and subjected to Western blot and immunohistochemical staining to check the expression changes of LXRaand LXR?in ACC of CIP model.1.3 The effects of LXRs agonist GW3965 on pain behaviors in mice with CIPThe effects of GW3965 administration as pre-treatment or post-treatment on CFA-induced mechanical allodynia and thermal hyperalgesia of CIP mice were evaluated by Von Frey and a plantar analgesia device.1.4 The effects of GW3965 on the expression and function of LXRs in ACC of CIP miceThe effects of GW3965 pre-treatment on the expression of LXRs in ACC of CIP mice were evaluated by Western blot.The expression of LXRs target genes,ABCA1 and ApoE,in ACC and serum of CIP mice after GW3965 treatment were tested by ELISA.1.5 The effects of knockdown of LXRs expression via lentivirus infection in ACC on pain behaviors and locomotor activities of miceLentiviral expression vectors?short hairpin RNA for LXRa,shLXRa;short hairpin RNA for LXR?,shLXR??for LXRaand LXR?and their negative control?shNC?were designed and produced.The lentiviral shLXRaand shLXR?were microinjected into the bilateral ACC stereotacticly.Two weeks later,the knockdown effects on the expression of LXRaand LXR?proteins in ACC were measured using Western blot.The changes of pain behaviors in mice were detected after knockdown of LXRaand LXR?in ACC via shLXRaand shLXR?infection by Von Frey and a plantar analgesia device.Meanwhile,the locomotor activities of mice were detected by open field test and rotarod test to ensure the accuracy of pain behavior detection1.6 To determine the subtype of LXRs for GW3965 to exert analgesic effects in CIP miceCIP model was established after knockdown of LXRaand LXR?in mice ACC and GW3965 pre-treatment.The effects of shLXRaand shLXR?on GW3965-mediated analgesic effects were checked by Von Frey and a plantar analgesia device.2.The mechanism of LXRs activation-mediated analgesic effects in CIP mice2.1 Changes of glial cells activation in ACC region of CIP miceWestern blot was used to detect the changes of astrocyte marker GFAP,and microglia marker Iba-1 in ACC of mice with CIP.2.2 The effects of GW3965 on mitogen-activated protein kinase?MAPKs?signaling pathway in ACC of CIP miceWestern blot was used to detect the effects of GW3965 on the changes of p-ERK,p-JNK and p-p38 in ACC from CIP model mice.2.3 To determine the different LXRs subtypes in GW3965-mediated inhibition of MAPKs activationThe CIP model was established after knockdown of LXRaand LXR?in mice ACC via lentivirus shRNA infection and GW3965 pre-treatment.The effects of shLXRaand shLXR?on GW3965-mediated regulation of MAPKs in CIP mice were checked by Western blot.2.4 The effects of GW3965 on nuclear factor-?B?NF-?B?signaling pathway in ACC of CIP miceCo-Immunocoprecipitation?Co-IP?was used to detect the physical interaction between LXR?and NF-?B p65 in ACC.The effects of GW3965 treatment on nuclear translocation of p65 and p50,and p-I?B?expression in ACC induced by CIP were detected by immunofluorescence and Western blot.2.5 The effects of GW3965 on pro-inflammatory cytokine release in ACC of CIP miceThe release of pro-inflammatory mediator TNF-ain ACC and serum from CIP mice before and after GW3965 treatment were detected by ELISA.2.6 The effects of GW3965 on the enhanced synaptic transmission in ACC of CIP miceWhole cell patch clamp was used to detect the changes of neuronal excitatory synaptic transmission,including the frequency and amplitude of AMPAR-mediated miniature excitatory postsynaptic currents?mEPSCs?and excitatory postsynaptic currents?EPSCs?induced by different voltages,that was Input-Output?I-O?curve in ACC brain slices of CIP mice with or without GW3965 perfusion.3.The mechanism of histone modification in regulating LXR?expression in ACC of CIP mice3.1 The expression of HDACs in ACC of mice with CIPThe expression changes of HDACs,including HDAC2 and HDAC5 in ACC of CIP mice were detected by Western blot.3.2 The effects of HDAC inhibitor SAHA on the expression levels of global acetylated histones and Lxr?mRNA in cortical neuronsPrimary cultured cortical neurons were treated with SAHA or DMSO,the expression changes of acetylated histone H3?AcH3?and acetylated histone H4?AcH4?were detected using Western blot and the Lxr?mRNA expression changes were detected by real-time PCR?RT-PCR?.3.3 To determine the binding site of acetylated histones on Lxr?promoterThe total length of 2,000 bp upstream of the Lxr?transcription start site was analyzed,and four pairs of highly specific primers for Lxr?were designed.Chromatin immunoprecipitation?ChIP?analysis was carried out to test the enrichment of acetylated histones at Lxr?promoter region in cortical neurons,validating by comparison to IgG control ChIP and promoter amplicon sequencing.3.4 The effects of SAHA on the expression levels of acetylated histones on Lxr?promoterCultured cortical neurons were treated with SAHA or DMSO,and the enrichment of AcH3 and AcH4 at Lxr?promoters were analyzed and quantified by ChIP and RT-PCR to determine the regulatory effects of HDAC inhibitor on Lxr?transcription and expression.Results1.The role of LXRs activation in pain behavior of CIP model mice1.1 The distribution and cell expression pattern of LXRs in ACCImmunofluorescent staining results showed that LXR?was widely expressed in ACC,whereas the expression of LXRawas very limited in ACC.LXR?colocalized mainly in glutamatergic neurons,moderately in GABAergic neurons,less in microglia and astrocyte in ACC region.1.2 The changes of LXRs expression in ACC of CIP miceCompared with Sham group,the expression level of LXR?was significantly reduced in ACC of CIP mice,while the expression level of LXRawas very low and did not change significantly in CIP mice1.3 The effects of LXRs agonist GW3965 on pain behaviors in mice with CIPPain behavior tests showed that GW3965 administration as pre-treatment or post-treatment,alleviated the mechanical allodynia and thermal hyperalgesia of CIP mice significantly.1.4 The effects of GW3965 on the expression and function of LXRs in ACC of CIP miceWestern blot analysis showed that GW3965 did not change the expression of LXRs in ACC of CIP mice.ELISA analysis showed that GW3965 increased expression of ABCA1and ApoE,which are target genes for LXRs,suggesting that LXRs were activated by GW3965.1.5 The effects of knockdown of LXRs expression via lentivirus infection in ACC on pain behaviors and locomotor activities of miceWestern blot analysis showed that the expression levels of LXR?and LXR?were knocked down via the transfection of shLXR?and shLXR?in the bilateral ACC respectively.Pain behavior tests that both shLXRaand shNC groups presented no differences in response threshold to mechanical and thermal stimuli.However,shLXR?group exhibited hyperalgesia but not allodynia.At the same time,no significant changes of locomotor activities were observed in each group after shRNA infection in ACC.1.6 To determine the subtype of LXRs for GW3965 to exert analgesic effects in CIP micePain behavior tests showed that knockdown of LXR?expression in bilateral ACC completely eliminated GW3965-mediated analgesic effects in CIP mice;whereas,lentiviral shLXR?did not affect GW3965-mediated analgesic effects.Moreover,lentiviral shLXR?enhanced CFA-induced hyperalgesia.The collected data suggested that GW3965exerted analgesic effects by activating LXR?subtype.2 The mechanism of LXRs activation-mediated analgesic effects in CIP mice2.1 Changes of glial cells activation in ACC region of CIP miceWestern blot analysis showed that the expression of astrocyte marker,GFAP,was increased at day 1,3 and 5 after CFA injection in ACC,while,the expression of microglia marker,Iba-1,did not change significantly in ACC of CIP mice.2.2 The effects of GW3965 on MAPKs signaling pathway in ACC of CIP miceWestern blot analysis showed that CFA injection could induce a higher amount of phosphorylation of ERK and JNK,but not p38 phosphorylation in ACC compared with the Sham group.GW3965 administration inhibited the enhanced p-ERK and p-JNK in ACC of CIP mice.2.3 Determination of different LXRs subtypes in GW3965-mediated inhibition of MAPKs activationWestern blot analysis showed that knockdown of LXR?in ACC partially abolished the inhibitory effects of GW3965 on MAPKs phosphorylation;Whereas,knockdown of LXRadid not influence the effects of GW3965 on MAPKs phosphorylation.2.4 The effects of GW3965 on NF-?B signaling pathway in ACC of CIP miceCo-immunoprecipitation?Co-IP?assay validated that LXR?physically interacted with NF-?B p65 in ACC.Western blot analysis showed that CFA injection induced an increased level of p-I?B?,nuclear p65 and p50,and a decreased expression of cytoplasmic p65 and p50 correspondingly in ACC.GW3965 treatment obviously reversed the enhanced nuclear translocation of p65 and p50,corrected the elevated p-I?B?upon CFA injury.2.5 The effects of GW3965 on pro-inflammatory cytokine release in ACC of CIP miceELISA analysis showed that CFA injection led to the increase of TNF-?release in mice ACC.GW3965 treatment obviously reversed the enhanced release of TNF-?and increased expression of ABCA1 and ApoE,which are target genes for LXRs.2.6 The effects of GW3965 on the enhanced synaptic transmission in ACC of CIP miceThe whole cell patch clamp recording showed that the slope of the I-O curve and the frequency,but not amplitude,of AMPAR-mediated mEPSCs were significantly potentiated in slices from CFA-injected mice compared with the Sham group.GW3965perfusion reversed the enhanced slope of I-O curve and strengthened mEPSC frequency.3 The mechanism of histone modification in regulating LXR?expression in ACC of CIP mice3.1 The expression of HDACs in ACC of mice with CIPWestern blot analysis showed elevated HDAC5 expression,but not HDAC2expression,upon CFA injury in ACC compared with Sham group,and HDAC5 expression were negatively correlated with that of LXR?.3.2 The effects of HDAC inhibitor SAHA on the expression levels of global acetylated histones and Lxr?mRNA in cortical neuronsWestern blot analysis showed that SAHA treatment induced global histone acetylation,including AcH3 and AcH4 levels in primary cultured cortical neurons.RT-PCR results showed that incubation of cultured neurons with SAHA led to an induction of Lxr?mRNA expression compared with the Sham group.3.3 Determination of the binding site of acetylated histones on Lxr?promoterChIP analysis revealed that AcH3 and AcH4 bound to the regions of about 1,900 bp,1,600 bp and 350 bp,but not 1,100 bp upstream of the transcription start site of Lxr?in cultured cortical neurons.3.4 The effects of SAHA on the expression levels of acetylated histones on Lxr?promoterChIP and RT-PCR analysis showed that levels of AcH3 but not AcH4 at Lxr?promoter I,II and IV were robustly induced by SAHA treatment for 1 h,followed by a gradual decrease in 24 h after SAHA treatment in cortical neurons.The results suggested that SAHA induced the transcriptional activity of Lxr?by increasing the level of AcH3 in the promoter region of Lxr?.ConclusionIn conclusion,our study revealed that reduction of LXRbexpression or LXRbdysfunction was involved in neuroinflammatory response and central sensitization in CIP.The analgesia effect of GW3965 was mediated by the activation of LXRb,and epigenetic regulation of LXRbwas involved in the pathogenesis of CIP.Our research provides,for the first time,a novel epigenetic mechanism that the reduced LXR?level by histone modification accounts for the etiology of chronic pain.In brief,collected data showed as followings:?1?LXR?deficit in ACC was involved in CIP.?2?LXR?knockdown by shRNA in ACC led to hyperalgesia.?3?LXR?activation by agonist GW3965 exerted analgesic effects by inhibiting inflammation responses and enhanced excitatory neurotransmission in ACC.?4?Elevated HDAC5 expression was negatively correlated with that of LXR?in ACC in CIP.?5?HDAC5 bound to Lxr?promoter and triggered histone deacetylation,resulting in the reduction of Lxr?transcription.Therefore,our results might provide not only a better understanding of the role and regulatory mechanism of LXR?in CIP,but also new strategies and a novel therapeutic target for chronic pain.