L-甲硫氨酸通过调节脊髓DNA甲基化水平减轻甲醛溶液所致急性炎性痛机制研究

背景　甲硫氨酸能够促进DNA甲基化的发生，DNA甲基化参与疼痛的发生发展和维持。急性炎性痛是临床常见症状，控制不及时会转化成慢性炎性痛，外源性补充甲硫氨酸可能通过调节DNA甲基化参与调节急性炎性痛，改善患者疼痛症状。目的　本研究采用甲醛溶液诱导急性炎性痛模型大鼠，观察注射L-甲硫氨酸（L-MET）是否会减轻大鼠足底急性炎性痛并探讨其机制，以期为寻找新的疼痛生物标志物和开发理想的镇痛新药提供理论依据。方法　2017年7月-2018年12月，将24只健康成年清洁级雄性Sprague-Dawley（SD）大鼠按照随机数字表法分为A组（0.9%氯化钠溶液+0.9%氯化钠溶液组）、B组（L-MET+0.9%氯化钠溶液组）、C组（0.9%氯化钠溶液+2 g/L甲醛溶液组）、D组（L-MET+2 g/L甲醛溶液组），每组6只。B组、D组腹腔注射L-MET，2次/d，总量不超过0.18 mg/kg，连续注射3 d；A组、C组注射等量0.9%氯化钠溶液。C组、D组左后足足跖部皮下注射2 g/L甲醛溶液20 μl，制作甲醛溶液所致急性炎性痛模型，大鼠足部肿胀并会出现相应的抬足舔足行为视为模型制作成功；A组、B组注射等量0.9%氯化钠溶液。全程记录给药后60 min大鼠行为学，并记录疼痛次数，每隔3 min为1个观察时段，共分20个观察时段。行为学检测结束后，将大鼠处死，取脊髓L4~L6之间脊髓组织，检测大鼠脊髓全基因组DNA甲基化水平及大鼠脊髓DNA甲基化转移酶（DNMT）1、DNMT2、DNMT3a、DNMT3b RNA水平。结果　A组、B组大鼠无明显不适异常反应；C组、D组大鼠出现躁动不安、注射足抬起不着地、舔咬或抖动注射足等反应，其疼痛行为反应呈典型的双相变化，从注射后即刻开始，持续3~5 min的急性疼痛时相（第一时相），5~10 min的静息期，随后出现可持续0~45 min的继发性疼痛时相（第二时相）。C组、D组大鼠各时间点疼痛次数均多于A组、B组（P<0.05）；D组大鼠6~39 min疼痛次数少于C组（P<0.05）。B组、D组大鼠脊髓全基因组DNA甲基化水平高于A组（P<0.05）；C组、D组大鼠脊髓全基因组DNA甲基化水平低于B组（P<0.05）；D组大鼠脊髓全基因组DNA甲基化水平高于C组（P<0.05）。C组、D组大鼠脊髓DNMT3a、DNMT3b RNA水平高于A组、B组（P<0.05）；D组大鼠脊髓DNMT3a RNA水平低于C组，DNMT3b RNA水平高于C组（P<0.05）。结论　L-MET对于甲醛溶液所致急性炎性痛模型大鼠具有明显镇痛作用，其机制与脊髓全基因组DNA甲基化水平以及DNMT水平的变化有关。

L-Methionine Attenuates Acute Inflammatory Pain Caused by Formaldehyde Solution by Regulating the Level of DNA Methylation in Spinal Cord

Background　Methionine can promote the occurrence of DNA methylation，which is involved in the development and maintenance of pain.Acute inflammatory pain is a common clinical symptom，which will turn into chronic inflammatory pain if it is not controlled in time.Exogenous supplementation of methionine may participate in the regulation of acute inflammatory pain by regulating DNA methylation，so as to improve the pain symptoms of patients.Objective　The aim of this study was to investigate the preemptive analgesic effect and mechanism of L-Methionine（L-MET） on acute inflammatory plantar pain induced by formaldehyde solution in rats.Methods　In this study carried out between July 2017 and December 2018，24 healthy adult cleaning grade male SD rats were randomly and evenly divided into groups A（0.9% sodium chloride solution+0.9% sodium chloride solution group），B（L-MET+0.9% sodium chloride solution group），C（0.9% sodium chloride solution+2 g/L formaldehyde solution group） and D（L-MET+2 g/L formaldehyde solution group）.In the first consecutive three days of intervention，via intraperitoneal injection，L-MET was administered to groups B and D（twice daily，total dosage ≤0.18 mg/kg），and 0.9% sodium chloride solution was administered to groups A and C（twice daily，with the amount equal to that of L-MET），respectively.Then，via subcutaneous injection in the plantar of the rear left foot，20 μl formaldehyde solution（with a concentration of 2 g/L） was administered to groups C and D to induce acute inflammatory pain（a successful model was defined as the foot became swelling with foot lifting and licking behaviors），and 0.9% sodium chloride solution was administered to groups A and B（with the amount equal to that of L-MET），respectively.The number of foot lifting and licking was measured within 60 minutes after the second round of injection，with an overall behavioral monitoring period of 60 minutes（consisting of 20 3-minute observation periods）.Rats were sacrificed after the behavioral examination.The spinal cord lumbar（L4-L6） was taken and the tissue DNA and RNA were extracted.The whole genome DNA methylation，DNMT1，DNMT2，DNMT3a，and DNMT3b were in the tissue were measured.Results　Rats in groups A and B showed no obvious abnormal discomfort reactions.Those in groups C and D were restlessness，and they licked and bit or shook the intervened rear left foot，which was lifted but could not touch the ground，demonstrating typical biphasic pain responses：immediately after the injection，the acute pain appeared and lasted for 3-5 minutes（the first phase），then disappeared for 5-10 minutes，and then the secondary pain appeared and lasted for 0-45 minutes（second phase）.The mean frequency of pain in group C was higher than that of groups A and B at each observation period（P<0.05），and so did group D（P<0.05）.The mean frequency of pain within 6-39 minutes of observation in group D was less than that of group C（P<0.05）.Group B showed higher mean whole genome DNA methylation level than groups A，C and D（P<0.05）.Group D showed higher mean whole genome DNA methylation level than groups A and C（P<0.05）.Group C showed higher mean DNMT3a and DNMT3b RNA levels than groups A and B（P<0.05），and so did group D（P<0.05）.Compared with group C，group D had lower mean DNMT3a RNA level but higher mean DNMT3b RNA level（P<0.05）.Conclusion　L-MET has obvious analgesic effect in a rat model of acute inflammatory pain caused by formaldehyde solution，and the mechanism may be related to the whole genome DNA methylation and the level of DNMT in the spinal cord.