Study Of The Mechanism Of ChREBP Protein On Neuropathic Pain Alleviation By Inhibiting Neuroinflammation

Background:The spinal cord is the primary center for pain message transmission and integration.The microglia in the spinal dorsal horn are rapidly activated upon detection of peripheral nerve injury,and release a variety of substances such as cytokines,which play a crucial role in the occurrence and development of neuropathic pain.Recent studies have reported that transcriptome sequencing of the spinal cord in SNI(Spared Nerve Injury,SNI)rats revealed an increased expression of ChREBP.However,it has not been reported whether the increased expression of ChREBP is related to NPP.Another study reported that ChREBP protein in macrophages has a significant inhibitory effect on inflammation.So,whether the ChREBP protein in macrophage-like microglia also has a significant anti-inflammatory effect,and whether ChREBP in the spinal dorsal horn mediates the occurrence and development of NPP by regulating the inflammatory response is still unknown.In this study,the microglial-mediated inflammatory response in the spinal dorsal horn was used as the entry point.Through genetic engineering and bioinformatics methods,the role of ChREBP in the occurrence and development of NPP and its molecular mechanism were explored to provide new targets for the treatment of pain.MethodsChapter 1:Construct SNI-induced mechanical allodynia rats and detect the 50%Paw withdraw threshold(50%PWT)before and after SNI 3,7,14,and 21 days.Real-time quantitative polymerase chain reaction(qPCR),western blot,and immunofluorescence were used to detect the changes in the expression of ChREBP in the L4-L6 spinal dorsal horn before and after SNI.Immunofluorescence was used to detect the cellular localization of ChREBP in the spinal dorsal horn.Chapter 2:The spinal stereotactic microinjection method(hereinafter referred to as microinjection)is used to deliver interfering ChREBP adeno-associated virus or control vector,and SNI modeling is performed on the 28th day after microinjection.On the 7th day after SNI,the expression of ChREBP in the spinal dorsal horn was detected by qPCR,immunoblotting and immunofluorescence.The expression of inflammatory factors TNF-?,IL-1?,IL-6 was detected by qPCR.The change of 50%PWT was detected before and after microinjection.Chapter 3:Microinjection was performed to deliver coding ChREBP adeno-associated virus or control vector,and SNI modeling was performed on the 28th day after injection.On the 7th day after SNI,the expression of ChREBP in spinal dorsal horn was detected by qPCR,western blot and immunofluorescence.The expression of inflammatory factors was detected by qPCR.The change of 50%PWT was detected before and after microinjection.Chapter 4:Bioinformatics methods were applied to predict the upstream transcription factors and potential binding sites of ChREBP.The reporter plasmid of full-length of ChREBP promoter and the reporter plasmid containing the predicted binding site fragments were co-transfected with the plasmid encoding the predicted target gene(cJun)and microglia,respectively,and subjected to double luciferase assay.The plasmid encoding cJun or control plasmid was transfected with microglia,and then the abundance of ChREBP promoter fragments enriched by immunoprecipitation of cJun antibody was detected by chromatin immunoprecipitation-qPCR(ChIP-qPCR)and agarose gel electrophoresis.Chapter 5:After SNI surgery,qPCR,immunoblotting,and immunofluorescence were applied to detect changes in cJun expression.Changes in cJun,ChREBP,inflammatory factors,and 50%PWT were assessed after intrathecal injection of cJun small interfering RNA,with or without overexpression of ChREBP.In addition,changes in cJun,ChREBP,inflammatory factors,and 50%PWT were detected after intrathecal injection of lentivirus encoding cJun,with or without downexpression of ChREBP.Chapter 6:In the LPS-induced microglia,ChREBP was knocked down or overexpressed to identify the effect of ChREBP on inflammation.The inflammation cytokines were detected by enzyme-linked immunosorbent assay.Moreover,cJun was down-or up-regulated to verify the activation of ChREBP and to elucidate the effect on inflammation.Results:The expression of ChREBP protein in the ipsilateral L4-L6 spinal dorsal horn of SNI surgery was increased,and it was co-stained with the microglia marker IBA-1 and the neuron marker NeuN.Microinjection of interfering ChREBP adeno-associated virus knocked down the ChREBP expression and promoted the inflammatory response in the spinal dorsal horn and mechanical allodynia.In contrast,micro injection of encoding ChREBP adeno-associated virus up-regulated the ChREBP expression and inhibited the inflammatory responses in the spinal dorsal horn and mechanical allodynia.cJun up-regulates the fluorescence activity of luciferase reporter of the ChREBP promoter fragment 3 which containing binding site 3.Immunoprecipitation of the cJun antibody enriched to more fragments 3 than the other fragments of the ChREBP promoter.Knockdown of cJun in the SDH of SNI rats inhibited ChREBP and inflammatory respons.Moreover,knockdown of cJun while overexpressing Mlxipl improved inflammation and mechanical allodynia to a greater extent compared with knockdown of cJun alone.Upregulation of cJun in the SDH promoted ChREBP and inflammatory respons.Additionally,overexpression of cJun while konckdown of Mlxipl promoted inflammation and mechanical allodynia to a greater extent compared with upregulation of cJun alone.Besides,cJun was co-stained with ChREBP by the detection of immunofluorescence double staining.Down-regulation of ChREBP stimulated inflammatory response in LPS-induced microglia,while up-regulation of ChREBP hampered inflammatory response.Additionally,up-regulation of cJun promoted ChREBP and inflammation response.In contrast,down-regulation of cJun inhibited ChREBP and inflammation.Conclusion:CJun in the SDH was activated after peripheral nerve injury.On the one hand,cJun directly aggravated neuropathic pain by promotion of microglia-derived inflammation.On the other hand,cJun increased the transcription of ChREBP in the spinal dorsal horn.ChREBP in turn alleviated mechanical allodynia by inhibition of inflammation response.ChREBP provided a protective mechanism for the development and progression of NP by inhibiting neuroinflammation in the SDH.Targeting ChREBP in the spinal dorsal horn might be a new target and effective strategy for the treatment of NP.