Epigenetic Mechanism Regulation For Chronic Inflammatory Pain Via TRPM2 Signaling Pathway In Spinal Cord Mice

Objective To explore the epigenetic mechanisms in regulation of the inflammatory pain mediated by transient receptor potential melastatin 2（TRPM2）in mice.Methods First stage: to investigate the threpeutic efficacy of AGK2 to chronic imflammatory pain induced by CFA in mice.According to the different dose of AGK2（0.2 μg/kg、1 μg/kg、5 μg/kg、25 μg/kg）,Specific pathogen free（SPF）wild type（C57）mice and TRPM2^-/-mice were randomly divided into 6 groups: C57 control group（C57 CON）,C57 inflammatory pain group（C57 CFA）,C57 inflammatory pain + AGK2 group（C57 AGK2）,TRPM2^-/-control group(TRPM2^-/-CON),TRPM2^-/-inflammatory pain group(TRPM2^-/-CFA),and TRPM2^-/-inflammatory pain + AGK2 group(TRPM2^-/-AGK2),（n=6）;each group according to different genders,further divided into two subgroups（a.male,b.female）.Inflammatory pain model was induced by intraplantar injection with 40μl of complete Freund’s adjuvant（CFA）into the plantar surface of the left hind paw.basic MWT,CPWL,PWL and paw edema were measured the day before CFA operation.Continueously intrachecal injection of different doses of AGK2 or NS starts from 3th to 9th day postoperation on 8 o,clock morning and evening.Behavior test in each group measured on 1th,3th,5th,7th,9th day.Second stage: to investigate the threpeutic efficacy of SAHA to chronic imflammatory pain induced by CFA in mice.wild type（C57）mice and TRPM2^-/-mice were randomly divided into 6 groups: C57 control group（C57 CON）,C57 inflammatory pain group（C57 CFA）,C57 inflammatory pain + suberoylanilide hydroxamic acid group（C57 SAHA）,TRPM2^-/-control group(TRPM2^-/-CON),TRPM2^-/-inflammatory pain group(TRPM2^-/-CFA),and TRPM2^-/-inflammatory pain + suberoylanilide hydroxamic acid group(TRPM2^-/-SAHA),（n=6）.SAHA group received intrathecal injection of SAHA 50 mg/kg each morning after nociceptive testing.The mice received the same volume saline treatment in the other 4 groups.The 50% paw withdrawal threshold,the paw withdrawal thermal latency and paw edema were measured after injection of CFA,respectively.Third stage: Fluorescence quantitative PCR analysis SIRTs gene expression,the immunofluorescence double marking and laser confocal method to detect SIRT2/TRPM2 protein expression,the CREB protein expression in each group were measured by westein bloting.Results Results of the first stage: Pain behavior test: CFA procedure lead to significantly decrease of MWT,CPWL,PWL and paw edema from 3th after CFA operation compared with Con group（P < 0.001）,treatment with AGK2,the improvement of pain behavior in mice with AGK2 drug concentrations were significantly positive correlation.The TRPM2^-/-mice showed normal sensitivity to mechanical and thermal stimuli compared with C57 mice（P > 0.05）.after CFA operation,the PWL,CPWL,MWT and paw edema were significantly lower than TRPM2^-/-CON group,but it is still significantly higher than that C57 CFA group（P < 0.001）,after treatment with AGK2,the pain behavior of mice has improved significantly.Results of second stage: In C57 mice,the 50% paw withdrawal threshold and paw withdrawal thermal latency in C57 CFA was attenuated compared with that in C57 CON,C57 SAHA group（P < 0.001）;The paw edema of C57 CFA group was significantly higher than that in C57 CON,C57 SAHA group（P < 0.001）.In TRPM2^-/-mice,the 50% paw withdrawal threshold and paw withdrawal thermal latency were significantly higher than C57 CFA group（P < 0.001）,there were no significant difference in the behavioral responses between TRPM2^-/-CFA and TRPM2^-/-SAHA group（P > 0.05）;the paw edema of inflammatory pain group was significantly lower than that in C57 inflammatory pain group（P < 0.001）.Results of third stage: the SIRT1,2,4,6,7 mRNA expression of C57 CFA group were significantly increased,and the mRNA expression of SIRT3 and SIRT5 were decreased;with the immunofluorescence analysis,the was no significant differences of protein expression between C57 CFA group and TRPM2^-/-CFA group,but compared with C57 CFA group,the TRPM2 almost no expression in TRPM2^-/-CFA group.Conclusion The histone deacetylase inhibitors regulate the inflammatory pain through TRPM2 mediated by epigenetic mechanism.