脊髓神经元SAM68-TRPV1信号通路在小鼠糖尿病神经病理性痛中的作用

Role of spinal cord neuron SAM68-TRPV1 signaling pathway in diabetic neuropathic pain in mice

目的评价脊髓神经元有丝分裂相关蛋白-68 kDa(SAM68)-瞬时受体电位香草素亚族1(TRPV1)信号通路在小鼠糖尿病神经病理性痛(DNP)中的作用。方法SPF级雄性C57BL/6小鼠40只,8周龄,体质量18~22 g,采用腹腔注射链脲佐菌素0.12 mg/g制备糖尿病模型,以后足机械痛阈(MWT)较造模前下降≥50%为DNP造模成功。糖尿病造模后4周时取DNP小鼠24只,采用单纯随机抽样方法分为3组(n=8):DNP组、SAM68抑制组(KD组)和病毒空载对照组(VC组);随机取MWT下降<50%的糖尿病小鼠8只作为非DNP组(ND组),随机取正常小鼠8只作为对照组(NC组)。糖尿病造模后4周时,KD组和VC组分别于脊髓腰膨大处注射SAM68基因沉默病毒和空载病毒。于糖尿病造模前和造模后4、6周测定MWT。在造模后6周MWT测定结束后处死小鼠,取脊髓组织,采用Western blot法检测SAM68和TRPV1的表达,免疫荧光观察其在脊髓的表达定位。结果与NC组和ND组相比,DNP组造模后4和6周时MWT下降,脊髓组织SAM68和TRPV1表达上调(P<0.05);与DNP组相比,KD组造模后6周时MWT升高,脊髓组织SAM68和TRPV1表达下调(P<0.05),VC组上述指标差异无统计学意义(P>0.05)。SAM68与TRPV1表达于脊髓同一区域神经元。结论脊髓神经元SAM68-TRPV1信号通路的激活参与小鼠DNP的病理生理机制。

Objective To evaluate the role of spinal cord neuron Src-associated-in-mitosis-68-kDa(SAM68)-transient receptor potential vanilloid-1 channel(TRPV1)signaling pathway in diabetic neuropathic pain(DNP)in mice.Methods Forty SPF male C57BL/6 mice,aged 8 weeks,weighing 18-22 g,were used in this study.Diabetes mellitus was induced by intraperitoneal streptozotocin 0.12 mg/g,and successful DNP model was defined as decrease in the mechanical paw withdrawal threshold(MWT)of the hind limb≥50% of the baseline value.Twenty-four mice with DNP at 4 weeks after developing the model were divided into 3 groups(n=8 each)using a simple random sampling:DNP group,SAM68 knocked down group(KD group)and virus control group(VC group).Eight diabetic mice with decrease in MWT<50%were randomly selected as non-DNP group(ND group),and 8 normal mice were randomly selected as control group(NC group).At 4 weeks after developing the diabetes mellitus model,SAM68 gene silencing virus and empty virus were injected into the lumbar enlargement of the spinal cord in KD group and VC group,respectively.MWT was measured before developing the diabetes mellitus model and at 4 and 6 weeks after developing the diabetes mellitus model.The mice were sacrificed after the end of MWT measurement at week 6 after developing the model,spinal cord tissues were collected and the expression of SAM68 and TRPV1 was detected by Western blot,and their localization in the spinal cord was observed by immunofluorescence.Results Compared with NC and ND groups,the MWT was significantly decreased at 4 and 6 weeks after developing the model,and the expression of SAM68 and TRPV1 in spinal cord tissues was up-regulated in DNP group(P<0.05).Compared with DNP group,the MWT was significantly increased at 6 weeks after developing the model,the expression of SAM68 and TRPV1 in spinal cord tissues was down-regulated,and no significant change was found in the parameters mentioned above in VC group(P>0.05).SAM68 and TRPV1 were expressed in neurons in the same region of the spinal cord.Conclusions Activation of SAM68-TRPV1 signaling pathway in spinal cord neurons is involved in the pathophysiological mechanism of DNP in mice.