体外冲击波对糖尿病神经痛大鼠脊髓磷酸化蛋白质组学的影响

Effect of extracorporeal shock wave on phosphoproteomics of spinal cord in rats with diabetic neuralgia

目的评价体外冲击波对糖尿病神经痛大鼠脊髓磷酸化蛋白质组学的影响。方法SPF级健康雄性SD大鼠36只,2月龄,体质量200~250 g。采用随机数字表法分为3组(n=12):对照组(C组)、糖尿病神经痛组(D组)和体外冲击波+糖尿病神经痛组(E组)。C组大鼠持续普通饲料喂养,D组和E组大鼠高糖高脂喂养8周后,腹腔注射链脲佐菌素35 mg/kg,注射后3、7和14 d时尾静脉随机血糖均>16.7 mmol/L,大鼠机械缩足反应阈(MWT)和热缩足潜伏期(TWL)≤85%基线值即视作大鼠糖尿病神经痛模型制备成功。E组于造模后进行体外冲击波,冲击量1000冲击/次,频率10 Hz,能量1.0 bar,1次/周,共4次。于造模前(T_(0))、造模后1、2、3和4周(T_(1-4))时测定MWT和TWL。最后一次冲击后麻醉处死大鼠取腰段脊髓组织行蛋白质组学分析,免疫组化法检测胶质纤维酸性蛋白(GFAP)、IL-1β和TNF-α的表达。结果与C组比较,D组和E组T_(1-4)时MWT和TWL降低(P<0.05);与D组比较,E组T_(1-4)时MWT和TWL升高(P<0.05)。磷酸化蛋白质组学筛选结果,D组与C组共筛选出差异磷酸化蛋白284个,E组与C组共筛选出差异磷酸化蛋白282个,E组与D组共筛选出差异磷酸化蛋白303个,磷酸化mGluR5表达差异有统计学意义(P<0.05)。免疫组化结果显示,与C组比较,D组脊髓GFAP、IL-1β和TNF-α表达上调(P<0.05);与D组比较,E组脊髓GFAP、IL-1β和TNF-α表达下调(P<0.05)。结论体外冲击波减轻大鼠糖尿病神经痛的机制与抑制星形胶质细胞激活及mGluR5过度磷酸化有关。

ObjectiveTo evaluate the effect of extracorporeal shock wave on the phosphoproteomics of the spinal cord in rats with diabetic neuralgia.MethodsThirty-six healthy male SPF-grade Sprague-Dawley rats,aged 2 months,weighing 200-250 g,were divided into 3 groups(n=12 each)using the random number table method:control group(group C),diabetic neuralgia group(group D),and extracorporeal shock wave+diabetic neuralgia group(group E).The rats were continuously fed a common diet in group C,while the rats were fed a high-sugar and high-fat diet for 8 weeks in D and E groups.Streptozotocin 35 mg/kg was intraperitoneally injected,and the successful induction of diabetic neuralgia was defined as the blood glucose>14.6 mmol/L and the mechanical paw withdrawal threshold(MWT)and thermal paw withdrawal latency(TWL)≤85%of baseline values.Group E received extracorporeal shock wave treatment after developing the model,with 1,000 shocks per session at a frequency of 10 Hz and an energy of 1.0 bar,once per week for a total of 4 sessions.The MWT and TWL were measured before developing the model(T_(0))and at 1,2,3 and 4 weeks after developing the model(T_(1)-T_(4)).After the last extracorporeal shock wave treatment,the rats were anesthetized and sacrificed,and lumbar spinal cord tissues were obtained for proteomic analysis and for detection of the expression of glial fibrillary acidic protein(GFAP),interleukin-1beta(IL-1β),and tumor necrosis factor-alpha(TNF-α)(by immunohistochemistry).ResultsCompared with group C,the MWT and TWL were significantly decreased at T_(1)-T_(4) in D and E groups(P<0.05).Compared with group D,the MWT and TWL were significantly increased at T_(1)-T_(4) in group E(P<0.05).The results of phosphoproteomics screening revealed 284 differentially phosphorylated proteins in D and C groups,282 in E and C groups,and 303 in E and D groups(P<0.05).The results of immunohistochemistry showed that the expression of GFAP,IL-1βand TNF-αwas significantly up-regulated in group D compared with group C(P<0.05);the expression of GFAP,IL-1βand TNF-αwas significantly down-regulated in group E compared with group D(P<0.05).ConclusionsThe mechanism by which extracorporeal shock wave alleviates diabetic neuralgia is related to inhibition of astrocyte activation and excessive phosphorylation of mGluR5 in rats.