TRPC6通过CREB在糖尿病痛性神经病变中的作用
发布时间：2019-06-13 17:01
【摘要】：目的:糖尿病痛性神经病变(painful diabetic neuropathy,PDN)是一种发生在糖尿病患者中以机械刺激性疼痛为特点的疾病。长期糖尿病机械性痛(mechanical allodynia,DMA)对患者的生活造成了诸多不便。本实验以链脲佐菌素(Streptozotocin,STZ)诱导大鼠建立糖尿病痛性神经病变模型,研究经典瞬时受体电位通道6(Transient Receptor Potential Canonical,TRPC6)、下游cAMP反应元件结合蛋白(cAMP response element binding protein,CREB)在模型大鼠背根神经节(dorsal root ganglia,DRG)中的表达水平及药物干预后的表达水平变化,及其与大鼠机械性痛觉敏感阈值变化的相互关系。旨在揭示PDN发病的分子生物学原理,为PDN的临床治疗及预防提供新思路和实验依据。方法:(1)将50只清洁级、健康、成年、雄性的斯普拉格-杜勒(Sprague-Dawley,SD)大鼠适应性喂养1周后,随机将大鼠分为CON组6只、DM组44只,给予DM组大鼠STZ腹腔注射建立DM大鼠模型,CON组则仅予以相同剂量的柠檬酸盐缓冲液;(2)分别于造模前及造模后第3、7、14、21d测定大鼠血糖、体重与后爪机械性回缩阈值(paw withdrawal threshold,PWT);(3)第3d时,以血糖值≥16.7mmol/L为标准筛选DM模型大鼠;(4)第14d时,用von Frey丝测定大鼠PWT值的变化,按其比造模前基线值下降50%为标准筛选DMA模型大鼠;(5)第15d时,给予CON组及DMA模型组大鼠行鞘内置管术,术后连续肌内注射青霉素预防性抗感染治疗3天,并将DMA模型大鼠随机分为三组(DMA组、DMA+溶媒组、DMA+SKF96365组),置管后第3天开始鞘内注射SKF96365(65ng/kg),连续干预5天;(6)分别运用实时定量PCR(quantitative real-time PCR,qRT-PCR)技术和Western blot技术检测各DMA模型组及CON组大鼠DRGs中TRPC6、pCREB的mRNA及蛋白表达水平。结果:(1)造模前大鼠的血糖、体重、PWT基线值不存在明显差异(P0.05);(2)STZ注射后第3d,DM组约89%的大鼠血糖显著升高(血糖16.7 mmol/L),与CON组相比,多饮、多食、多尿、消瘦等糖尿病三多一少症状明显;(3)STZ注射后第14d,DM大鼠约52%出现明显的双侧后爪机械刺激性痛(即为DMA模型大鼠),PWT值较CON组显著下降(P0.01),并持续降低至第21d(P0.01);(4)与CON组相比,DMA组大鼠DRGs中TRPC6与pCREB的mRNA和蛋白表达水平明显上调(P0.01),DMA组与DMA+溶媒组大鼠DRGs中的TRPC6、pCREB的mRNA和蛋白表达水平无明显差异(P0.05),DMA+SKF96365组大鼠DRGs中TRPC6、pCREB的mRNA和蛋白表达水平较DMA+溶媒组有所下降(P0.01)。结论:STZ诱导的DMA大鼠DRGs中TRPC6表达上调,与疼痛呈正相关,DMA+SKF96365组TRPC6表达下调,与疼痛的变化相一致,TRPC6可能参与了糖尿病机械痛的发病过程。TRPC6与pCREB表达水平变化相关,提示TRPC6可能是通过调节下游核转录调节蛋白pCREB表达,加重疼痛。
[Abstract]:Objective: diabetic painful neuropathy (painful diabetic neuropathy,PDN) is a disease characterized by mechanical irritating pain in diabetic patients. Long-term diabetic mechanical pain (mechanical allodynia,DMA) causes a lot of inconvenience to the life of patients. In this study, streptozotocin (Streptozotocin,STZ) was used to establish diabetic painful neuropathy model in rats. The expression level of classical transient receptor potential channel 6 (Transient Receptor Potential Canonical,TRPC6) and downstream cAMP response element binding protein (cAMP response element binding protein,CREB) in dorsal root ganglion (dorsal root ganglia,DRG) of model rats and the expression level after drug intervention were studied. And its relationship with the changes of mechanical pain sensitivity threshold in rats. The purpose of this study was to reveal the molecular biological principle of PDN and to provide new ideas and experimental basis for clinical treatment and prevention of PDN. Methods: (1) after adaptive feeding of 50 clean, healthy, adult and male Sprague-Dawley,SD rats for one week, the rats were randomly divided into CON group (n = 6) and DM group (n = 44). The DM rat model was established by intraabdominal injection of STZ in DM group, while the CON group was only given the same dose of citrate buffer. (2) on the 3rd, 7th and 14th day after modeling, respectively, the blood glucose, body weight and mechanical retraction threshold (paw withdrawal threshold,PWT); (3 of the posterior claw were measured, the DM model rats were screened with blood sugar value 鈮，
本文编号：2498671
[Abstract]:Objective: diabetic painful neuropathy (painful diabetic neuropathy,PDN) is a disease characterized by mechanical irritating pain in diabetic patients. Long-term diabetic mechanical pain (mechanical allodynia,DMA) causes a lot of inconvenience to the life of patients. In this study, streptozotocin (Streptozotocin,STZ) was used to establish diabetic painful neuropathy model in rats. The expression level of classical transient receptor potential channel 6 (Transient Receptor Potential Canonical,TRPC6) and downstream cAMP response element binding protein (cAMP response element binding protein,CREB) in dorsal root ganglion (dorsal root ganglia,DRG) of model rats and the expression level after drug intervention were studied. And its relationship with the changes of mechanical pain sensitivity threshold in rats. The purpose of this study was to reveal the molecular biological principle of PDN and to provide new ideas and experimental basis for clinical treatment and prevention of PDN. Methods: (1) after adaptive feeding of 50 clean, healthy, adult and male Sprague-Dawley,SD rats for one week, the rats were randomly divided into CON group (n = 6) and DM group (n = 44). The DM rat model was established by intraabdominal injection of STZ in DM group, while the CON group was only given the same dose of citrate buffer. (2) on the 3rd, 7th and 14th day after modeling, respectively, the blood glucose, body weight and mechanical retraction threshold (paw withdrawal threshold,PWT); (3 of the posterior claw were measured, the DM model rats were screened with blood sugar value 鈮，
本文编号：2498671
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