糖尿病神经病理性痛大鼠脊髓背角USP49和FKBP51的表达与作用

Expressions of ubiquitin-specific proteinase 49 and FK506 binding protein 51 in spinal dorsal horns and their roles in rats with diabetic neuropathic pain

目的 研究泛素特异性蛋白酶49(USP49)和FK506结合蛋白51(FKBP51)在糖尿病神经病理性痛(DNP)大鼠脊髓背角中的表达及作用。 方法 SD雄性大鼠通过高糖高脂饮食饲养和腹腔注射1%链脲佐菌素建立2型糖尿病大鼠模型并按照随机数字表法分为DNP组、对照短发夹RNA(shRNA)组和USP49沉默组,每组10只。3组大鼠分别鞘内注射生理盐水、Ad-scrambled shRNA(3×10^8 PFU/mL)、Ad-USP49-shRNA(3×10^8 PFU/mL)。另取10只正常大鼠作为对照组,不做任何处理。分别于造模前1 d、造模后1 d、7 d、鞘内注射后1、3、7 d测定大鼠热缩足潜伏期(TWL)和机械缩足反应阈(MWT),并检测背角神经元USP49、FKBP51、糖皮质激素受体α(GRα)mRNA和蛋白的表达水平。 结果 (1)造模前1 d,4组大鼠TWL差异无统计学意义(P>0.05)。鞘内注射后3 d、7 d,USP49沉默组TWL明显长于DNP组,短于对照组,差异有统计学意义(P<0.05)。(2)造模前1 d,4组大鼠MWT差异无统计学意义(P>0.05)。鞘内注射3 d、7 d后,USP49沉默组MWT明显高于DNP组,低于对照组,差异有统计学意义(P<0.05)。(3)与DNP组比较,USP49沉默组USP49 mRNA、FKBP51 mRNA表达显著下降,GRα mRNA表达显著上升,差异有统计学意义(P<0.05)。(4)与DNP组比较,USP49沉默组USP49蛋白、FKBP51蛋白表达明显降低,GRα蛋白明显上升,差异均有统计学意义(P<0.05)。 结论 特异性沉默脊髓背角神经元USP49表达可改善DNP大鼠热痛敏反应和机械痛敏反应,其机制可能与USP49介导的FKBP51蛋白去泛素化有关。

Objective To delineate the expressions of ubiquitin-specific proteinase 49 (USP49) and FK506 binding protein 51 (FKBP51) in the spinal dorsal horns and their roles in rats with diabetic neuropathic pain (DNP). Methods Male SD rats were fed with high glucose and high fat diet with an intraperitoneal injection of 1% streptozocin to establish DNP rat models. DNP rats were allocated into DNP group, control shRNA group (KC group) and knockdown USP49 group (KD group, n=10);saline, Ad-scrambled shRNA (3×10^8 PFU/mL) and Ad-USP49-shRNA (3×10^8 PFU/mL) were intrathecally injected into the rats of the three groups. Another 10 normal rats were chosen as control group (C group). Thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) were measured one d before model establishment, one and 7 d after model establishment, one, three and 7 d after intrathecal injection. The mRNA and protein expressions of USP49, FKBP51 and glucocorticoid receptor α (GRα) in dorsal horn neurons were detected by real time-PCR and Western blotting. Results (1) TWL showed no significant difference among the four groups one d before model establishment (P>0.05);three and 7 d after intrathecal injection, TWL in KD group was significantly prolonged as compared with that in the DNP group, and significantly shortened as compared with that in C group (P<0.05). (2) MWT showed no significant difference among the four groups one d before model establishment (P>0.05);three and 7 d after intrathecal injection, MWT in KD group was significantly higher as compared with that in the DNP group, and significantly lower as compared with that in C group (P<0.05). (3) As compared with DNP group, KD group had significantly decreased mRNA expressions of USP49 and FKBP51, and statistically increased mRNA GRα expression (P<0.05). (4) As compared with DNP group, KD group had significantly decreased protein expressions of USP49 and FKBP51, and statistically increased protein GRα expression (P<0.05). Conclusion Specific knockdown of USP49 expression is associated with improvement of heat hyperalgesia and mechanical hyperalgesia in DNP rats, which may be attributed to the de-ubiquitination of FKBP51 by USP49.