脊髓背角SPARCL1在瑞芬太尼诱发切口痛小鼠痛觉过敏形成中的作用

Role of SPARCL1 in spinal dorsal horns in development of remifentanil-induced hyperalgesia in mice with incisional pain

目的:评价脊髓背角富含半胱氨酸的酸性分泌蛋白类似蛋白1(SPARCL1)在瑞芬太尼诱发切口痛小鼠痛觉过敏形成中的作用。方法:健康雄性C57BL/6J小鼠48只,8~10周龄,体重18~22 g,采用随机数字表法分为6组(n=8):对照组(C组)、切口痛组(I组)、瑞芬太尼组(R组)、切口痛+瑞芬太尼组(I+R组)、切口痛+瑞芬太尼组+阴性对照siRNA组(I+R+N组)和切口痛+瑞芬太尼+SPARCL1-siRNA组(I+R+S组)。I+R+N组和I+R+S组分别鞘内注射1×108 IFU/ml阴性对照siRNA和SPARCL1-siRNA 10μl,C组、I组、R组、I+R组鞘内注射生理盐水10μl,6组均注射1次/d,连续3 d。待稳定转染后,C组和I组尾静脉注射生理盐水0.1 ml,R组、I+R组、I+R+N组和I+R+S组尾静脉注射瑞芬太尼10μg/kg(用生理盐水稀释至0.1 ml),6组均连续注射4次,间隔15 min。I组、I+R组、I+R+N组和I+R+S组于第1次尾静脉给药后制备切口痛模型。分别于输注生理盐水或瑞芬太尼前24 h(T 0)、输注停止后3、6、24和48 h(T 1-4)时测定机械缩足反应阈(MWT)和热甩尾潜伏期(TWL)。于T 4时测定痛阈后处死小鼠,取L 4-6节段脊髓背角组织,分别采用Western blot法和qRT-PCR法检测SPARCL1及其mRNA的表达水平。结果:与C组比较,I+R组和I+R+N组T 1-4时MWT降低,TWL缩短,I组、R组和I+R+S组T 2-4时MWT降低,TWL缩短,R组、I+R组、I+R+C组脊髓背角SPARCL1及其mRNA表达上调(P<0.05或0.01);与I组或R组比较,I+R组MWT降低,TWL缩短,脊髓背角SPARCL1及其mRNA表达上调(P<0.01);与I+R组比较,I+R+S组T 1-4时MWT增加,TWL延长,脊髓背角SPARCL1及其mRNA表达下调(P<0.05或0.01),I+R+C组上述各指标差异无统计学意义(P>0.05)。结论:SPARCL1活性增强参与了瑞芬太尼诱发切口痛小鼠痛觉过敏的形成。

Objective To evaluate the role of secreted protein acidic and rich in cysteine like protein 1(SPARCL1)in spinal dorsal horns in the development of remifentanil-induced hyperalgesia in mice with incisional pain.Methods Forty-eight healthy male C57BL/6J mice,aged 8-10 weeks,weighing 18-22 g,were divided into 6 groups(n=8 each)using a random number table method:control group(group C),incisional pain group(group I),remifentanil group(group R),incisional pain plus remifentanil group(group I+R),incisional pain plus remifentanil plus negative control group(group I+R+N),and incisional pain plus remifentanil plus SPARCL1-siRNA group(group I+R+S).In I+R+N and I+R+S groups,1×10^8 IFU/ml negative control siRNA and SPARCL1-siRNA 10μl were intrathecally injected,respectively,once a day for 3 consecutive days.Normal saline 10μl was intrathecally injected once a day for 3 consecutive days in C,I,R and I+R groups.After transfection was stable,normal saline 0.1 ml was intravenously injected through the tail vein for 4 consecutive times at 15 min interval in C and I groups,and remifentanil 10μg/kg(diluted to 0.1 ml in normal saline)was intravenously injected via the tail vein for 4 consecutive times at 15 min interval in R,I+R,I+R+N and I+R+S groups.The incisional pain model was established after the first administration via the tail vein in R,I+R,I+R+N and I+R+S groups.The mechanical paw withdrawal threshold(MWT)and thermal paw withdrawal latency(TWL)were measured at 24 h before infusing normal saline or remifentanil(T0)and 3,6,24 and 48 h after stopping infusion(T1-4).Animals were sacrificed after measuring pain threshold at T4,and L4-6 segments of the spinal cord were removed for determination of the expression of SPARCL1 protein and mRNA by Western blot and quantitative real-time polymerase chain reaction,respectively.Results Compared with group C,MWT was significantly decreased and TWL was shortened at T1-4 in I+R and I+R+N groups and at T2-4 in I,R and I+R+S groups,and the expression of SPARCL1 protein and mRNA was significantly up-regulated in R,I+R and I+R+C groups(P<0.05 or 0.01).Compared with group I and group R,MWT was significantly decreased,TWL was shortened,and the expression of SPARCL1 protein and mRNA was up-regulated in group I+R(P<0.01).Compared with group I+R,MWT was significantly increased and TWL was prolonged at T1-4,and the expression of SPARCL1 protein and mRNA was down-regulated in group I+R+S(P<0.05 or 0.01).Conclusion Enhanced activity of SPARCL1 in the spinal dorsal horns is involved in the development of remifentanil-induced hyperalgesia in mice with incisional pain.