| Background Neuropathic pain is a worldwide clinical problem and difficult to treat.Traditional therapies only alleviate the syndroms partly and may cause severe adverse effects. Epigenetic regulation including histone modification has been revealed to play a vital role in the process of neuropathic pain. Recent research found that G9a?a histone methytransferase, was upregulated after nerve injury and contributed to both the development and maintenance of neuropathic pain through the upregulation of MOR and Kv1.2 in the dorsal root ganglia(DRG). But how is G9 a increased after nerve injury is not known. CCAAT/enhancer-binding protein-?(C/EBP?), as an important transcription factor, was proved to transactivate G9 a during 3T3-L1 preadipocyte differentiation. However, whether C/EBP? contributes to the increase of G9 a in the DRG and participates in neuropathic pain is still unkown.Objective To investigate the effect of C/EBP? in the process of neuropathic pain and its exact mechanism.Methods1. To investigate the expression of C/EBP? during neuropathic pain, mice were conducted with chronic construction injury(CCI) and tissues of DRG and spinal cord were collected at 0, 3, 7 and 14 day post surgery. Then western blot was used to check the protein level of C/EBP? in these tissues.2. To investigate whether C/EBP? participated in the development and maintenance of neuropathic pain, mice were injected with C/EBP? si RNA in the DRG 4 days before the conduction of CCI surgery or 5 days post CCI. Locomotor function and pain related behaviors including paw withdrawal thresholds in response to mechanical stimuli and paw withdrawal latencis in response to heat and cold stimuli were then observed.3. To mimic the high expression of C/EBP? induced by CCI, mice were injected with r AAV5- C/EBP? on the leftside of L3 and L4 DRGs. Then locomotor function, spontaneous pain and pain-related behaviors including paw withdrawal thresholds in response to mechanical stimuli and paw withdrawal latencis in response to heat and cold stimuli were observed till 8 weeks post injection.4. To investigate how C/EBP? participated in neuropathic pain, the m RNA and protein levels of C/EBP?, G9 a, MOR and Kv1.2 were checked with RT-PCR and Western Blot. Chromatin immunoprecipitation analysis was performed to see the direct binding of C/EBP? and G9 a gene in the DRG. Immunohistochemistry and single cell RT-PCR were implemented to check the co-localization of C/EBP?with G9 a, MOR and Kv1.2.5. To investigate the role of G9 a in the regulation of MOR and Kv1.2 by C/EBP?,primary cell culture was practiced with DRGs from G9 a knockout and wild type mice. r AAV5- C/EBP? was used to increase the expression of C/EBP? in cultured cells and protein levels of C/EBP?, G9 a, MOR and Kv1.2 were checked after cell culture and virus infection.Results:1. The protein level of C/EBP? was increased in the injured DRGs but not the contralateral side of DRGs or ipsilateral spinal cord after CCI.2. Previous injection of C/EBP? si RNA in the DRG inhibited CCI induced mechanical allodynia, thermal hyperalgesia and cold allodynia without affecting the locomotor function.3. DRG microinjection of C/EBP? si RNA effectively alleviated CCI induced mechanical allodynia, thermal hyperalgesia and cold allodynia without affecting the locomotor function.4. Mimicking the high expression of C/EBP? in the DRG induced mice with mechanical allodynia, thermal hyperalgesia, cold allodynia and spontaneous pain.5. CCI increased the m RNA and protein levels of C/EBP? and G9 a, and decreased the expression of MOR and Kv1.2 in the injured DRGs. Inhibiting C/EBP?expression in the DRG could reverse expression changes of G9 a, MOR and Kv1.2induced by CCI. Overexpression of C/EBP? in the DRG could upregulate the m RNA and protein levels of G9 a and downregulate the expression of MOR and Kv1.2.6. C/EBP? could bind directly to the promotor of G9 a in the DRG and nerve injury enhanced this binding.7. C/EBP? could co-localize with G9 a, MOR or Kv1.2 in the DRG cells.8. Knocking out G9 a in the DRG inhibited the regulation of MOR and Kv1.2 by C/EBP?.Conclusion:C/EBP? played an important role in the initiation and maintenance of neuropathic pain through the regulation of G9 a, MOR and Kv1.2. |
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