局麻药激活TRPV1通道介导糖尿病小鼠神经毒性损伤的机制研究

Local anesthetics induce calcium overloading in neurons and result in neurotoxicity in diabetes. However, it remains unclear how the local anesthetics elicit calcium overloading under diabetic conditions. TRPV1, a cation channel sensitive to capsaicin and heat, has been newly found to be directly activated by lidocaine in sensory neurons in vitro. Importantly, the expression of TRPV1 is up-regulated under diabetic conditions, and enhanced spontaneous calcium influx was observed in capsaicin-sensitive neurons in diabetic rat model. We hence hypothesize that the local anesthetics-induced neurotoxicity in diabetes is due to the up-regulation and hyperactivation of TRPV1 channels. Indeed, our preliminary data have shown that more sensory neurons were activated by lidocaine in hyperglycemia culture condition than in normal culture condition, whereas TRPV1-deficient neurons did not respond to lidocaine. To further determine the role of TRPV1 in local anesthetics-induced neurotoxicity in vivo, we will establish diabetic models in wild-type and TRPV1-deficient mice, and their response to lidocaine using behavior, electrophysiology, and calcium imaging techniques. Furthermore, we will determine whether pharmacological blockade of TRPV1 alleviates anesthetics neurotoxicity in diabetes using the TRPV1 specific antagonist, AMG-9810. The dose-effect curve for AMG-9810 treatment will be measured in cultured neurons in vitro and in diabetic mice in vivo. These studies will provide novel insight on the neural mechanism underlying anesthetics-induced neurotoxicity in diabetes, and suggest a novel therapeutic strategy for preventing and treating peripheral neurotoxicity in diabetes.

神经元钙离子内流、钙超载与局麻药增强糖尿病患者的神经毒性损伤密切相关，但局麻药通过何种机制增加细胞钙内流，目前尚无定论。已知糖尿病大鼠中，对辣椒素敏感的背根神经节细胞自发性钙内流增加。辣椒素的受体TRPV1为感觉神经纤维上非选择性阳离子通道，主要传导热与痛觉，可被局麻药利多卡因直接激活，其表达可因病理状态而上调。钙成像预实验发现，高糖环境下，利多卡因致钙浓度增加的背根神经节细胞数目增多，且为TRPV1阳性；TRPV1基因敲除的小鼠无此现象。本项目拟建立野生型与TRPV1基因敲除的糖尿病小鼠动物模型，采用动物行为学、免疫组化、定量RT-PCR、钙成像及电生理方法，探讨局麻药是否通过糖尿病状态下“上调”或“高敏”的TRPV1受体，导致钙超载而增强其神经毒性损伤；并测定TRPV1特异性拮抗剂AMG-9810的体内外量效曲线，从而为防治局麻药加重糖尿病神经毒性损伤提供理论依据及药物作用靶点。

局麻药广泛用于神经阻滞、椎管内麻醉及术后镇痛，但可引发短暂性神经综合征、痛觉过敏等神经并发症，严重时可导致细胞通透性改变、细胞水肿、线粒体功能障碍、诱导细胞凋亡程序等，即局麻药神经毒性损伤。糖尿病患者局麻药所致神经毒性损伤增强，表现为相关并发症增加，但其细胞学机制尚未明确。神经元内钙离子浓度增加可导致细胞内钙超载，对局麻药神经毒性敏感性增强，诱发细胞损伤与细胞凋亡及相应信号通路开放，被认为是局麻药神经毒性损伤的重要原因。本课题采用野生型小鼠（WT）与TRPV1基因敲除型小鼠（Trpv1-/-）的原代培养背根神经节细胞，应用钙成像技术验证布比卡因是否通过TRPV1介导细胞内钙超载；并通过建立体外高糖损伤模型，比较WT与Trpv1-/-小鼠的DRG神经元对布比卡因所导致钙超载的差异，同时检测高糖环境下TRPV1通道的活性变化。为进一步研究糖尿病情况下使用布比卡因致神经毒性损伤增强的细胞学机制，建立WT与Trpv1-/-的2型糖尿病小鼠动物模型，采用动物行为学、免疫荧光染色、定量RT-PCR、蛋白质免疫印迹与钙成像方法，进一步了解布比卡因通过TRPV1介导神经毒性损伤尤其是糖尿病神经毒性损伤增强的特点。本课题得出以下重要结果：1.布比卡因可致小鼠DRG神经元细胞内钙离子超载，与TRPV1相关，且为细胞外钙离子依赖。2. 高糖环境下，布比卡因致DRG细胞钙超载增加与TRPV1活性增加相关。3. 非糖尿病状态下，布比卡因所致钙离子超载、细胞凋亡及机械痛觉过敏与TRPV1相关；糖尿病状态下，布比卡因所致钙离子超载、细胞凋亡及机械痛觉过敏增强，该现象与糖尿病状态TRPV1活性增加相关。实验结果从整体动物与细胞分子水平阐述了局麻药通过糖尿病状态下”高敏“的TRPV1受体，导致钙超载而增强其神经毒性损伤，为防治局麻药加重糖尿病神经毒性损伤提供理论依据及药物作用靶点。