自噬对炎性痛大鼠痛敏和星形胶质细胞活化的影响

The effect of autophagy on hyperalgesia and astrocytic activation in rats with inflammatory pain

目的 探讨自噬对炎性痛大鼠疼痛行为学和星形胶质细胞活化的影响.方法 清洁级雄性SD成年大鼠78只,按随机数字表法分为对照组（n=12）、模型组（n=42）、自噬诱导剂雷帕霉素（Rap）预处理组（n=12）和自噬抑制剂3-甲基腺嘌呤（3-MA）预处理组（n=12）.经足底皮下注射完全弗式佐剂（CFA）100μL复制大鼠炎性痛模型;对照组注射100μL生理盐水.Rap预处理组和3-MA预处理组于制模前1 h经腹腔注射Rap 10 mg/kg或3-MA 15 mg/kg;对照组和模型组注射等量生理盐水.于制模前和制模后6、12、24 h及3 d取模型组大鼠6只,取脊髓L4~6组织,采用蛋白质免疫印迹试验（Western Blot）检测自噬蛋白膜型微管轻链相关蛋白3-Ⅱ（LC3-Ⅱ）、自噬相关基因Beclin-1表达;各组分别取6只大鼠,于制模后6、12、24 h及3 d、7 d观察疼痛行为学指标机械缩足反应阈（MWT）和热缩足潜伏期（TWL）的变化;各组另取6只大鼠,于制模后24 h取脊髓组织,采用免疫荧光法,于共聚焦显微镜下观察星形胶质细胞的变化及胶质纤维酸性蛋白（GFAP）阳性表达,并采用Western Blot检测GFAP表达量.结果 ① 炎性痛可诱发大鼠脊髓自噬蛋白LC3-Ⅱ和Beclin-1的表达增加,24 h达峰值（A值分别为0.59±0.07、0.51±0.06）,随后逐渐下降.② 模型组大鼠MWT、TWL随时间延长呈先降低后升高趋势,24 h达谷值〔MWT（g）：17.8±1.9,TWL（s）：6.8±0.4〕,且12 h起即明显低于对照组〔12 h MWT（g）：21.5±2.4比43.4±5.1,TWL（s）：12.0±1.1比17.6±1.2,均P〈0.05〕,制模后3 d起有所升高.与模型组比较,给予自噬诱导剂Rap 12 h起,MWT即明显增加（g：36.8±4.9比21.5±2.4）,TWL即明显延长（s：14.3±1.1比12.0±1.1,均P〈0.05）;而给予自噬抑制剂3-MA 12 h起,MWT即进一步减少（g：18.6±1.9比21.5±2.4）,TWL即进一步缩短（s：8.4±0.6比12.0±1.1,均P〈0.05）.③ 共聚焦显微镜下显示,对照组星形胶质细胞无明显变化,仅有少量GFAP表达.炎性痛可诱导星形胶质细胞活化,表现为胶质细胞肥大、增生、胶质网络变形,且GFAP表达较对照组明显增加（A值：0.54±0.09比0.16±0.02,P〈0.05）.自噬诱导剂Rap可减轻胶质细胞活化,抑制GFAP表达,与模型组比较差异有统计学意义（A值：0.33±0.06比0.54±0.09,P〈0.05）;而自噬抑制剂3-MA可进一步加重胶质细胞的活化,GFAP表达较模型组进一步上调（A值：0.73±0.08比0.54±0.09,P〈0.05）.结论 自噬参与了炎性痛大鼠疼痛行为学和胶质细胞的激活过程.

Objective To evaluate the influence of autophagy on pain behavioristics and astrocytic activation in rats with inflammatory pain.Methods Seventy-eight clean male Sprague-Dawley （SD） adult rats were randomly divided into four groups： control group （n = 12）, model group （n = 42）, autophagy inducer rapamycin （Rap） pretreatment group （n = 12） and autophagy inhibitor 3 methyladenine （3-MA） pretreatment group （n = 12）. The inflammatory pain rat model was reproduced by subcutaneous injection of freund＇s complete adjuvant （CFA） 100μL at foot sole, whilein control group, the same volume 0.9% normal saline 100μL was injected at the same site. One hour before modeling, Rap 10 mg/kg and 3-MA 15 mg/kg were intraperitoneally injected in rats in Rap and 3-MA pretreatment groups respectively, and the same volume of 0.9% normal saline was injected intraperitoneally in rats of control and model groups. Before modeling and 6, 12, 24 hours and 3 days after modeling, the L4-L6 spinal cord tissue was harvested from 6 rats in model group, and autophagy protein membrane microtubule-associated protein 1 light chain 3 Ⅱ （LC3 Ⅱ） and autophagy related gene Beclin-1 expressions were detected by Western Blot in the tissue; the changes of pain behavioral indexes mechanical withdraw threshold （MWT） and thermal withdrawal latency （TWL,n = 6）, were observed at6, 12, 24 hours and 3 days, 7 days after modeling in the 6 rats taken from each group; in another 6 rats in each group, 24 hours after modeling, L4-L6 spinal cord tissue was collected, immunofluorescence staining was used to observe the changes of astrocytes and the positive expression of glial fibrillary acidic protein （GFAP） under a confocal microscopy, and the protein expression quantity of GFAP was detected by Western Blot in the tissue.Results ① The inflammatory pain could induce the increase of rat autophagy protein LC3Ⅱ and Beclin-1 expressions in spinal cord tissue, reaching their peaks at 24 hours （A value： 0.59±0.07, 0.51±0.06, respectively）, and then they were gradually decreased. ② With the prolongation of time, in the model group MWT was gradually decreased, TWL was gradually shortened, they reached their valley values at 24 hours after modeling [MWT （g）： 17.8±1.9, TWL （s）： 6.8±0.4], and from 12 hours they were significantly decreased compared with those in control group [12hours MWT （g）： 21.5±2.4 vs. 43.4±5.1, TWL （s）： 12.0±1.1 vs. 17.6±1.2, bothP 〈 0.05], after modeling for 3 days they were increased; Compared with model group, 12 hours after autophagy inducer Rap was given, MWT was significantly increased （g： 36.8±4.9 vs. 21.5±2.4,P 〈 0.05）, TWL was significantly prolonged （s： 14.3±1.1 vs. 12.0±1.1,P 〈 0.05）; from 12 hours after autophagy inhibitor 3-MA was given, MWT was further reduced （g： 18.6±1.9 vs. 21.5±2.4, P〈0.05）, TWL was further shortened （s： 8.4±0.6 vs. 12.0±1.1,P 〈 0.05）. ③ Confocal microscopic findings showed, there was no significant acstrocytic change, and only litter GFAP expression was seen in control group. In model group, the inflammatory pain induced astrocyte activation, manifesting glial cell hypertrophy, hyperplasia, gelatinousnetwork deformation, and GFAP expression was obviously increased compared with that in the control group （A value： 0.54±0.09 vs. 0.16±0.02,P 〈 0.05）. Since autophagy inducer Rap can decrease astrocyte activation and inhibit GFAP expression, there was statistical significant difference between Rap pretreatment and model groups （A value： 0.33±0.06 vs.0.54±0.09,P 〈 0.05）; autophagy inhibitor 3-MA can further aggravate astrocytes activation and up-regulate GFAP expression in 3-MA pretreatment group （A value： 0.73±0.08 vs. 0.54±0.09,P 〈 0.05）.Conclusion Autophagy participates in the process of astrocytic activation and pain behavioristics in rats with inflammatory pain.