褪黑素通过缓解氧化应激促进糖尿病周围神经病变神经修复的作用机制

The mechanism of melatonin promotes nerve repair in diabetic peripheral neuropathy by alleviating oxidative stress

摘要

目的 探讨褪黑素在糖尿病周围神经病变中对神经修复的作用机制。方法 将SD大鼠分为对照组、模型组和褪黑素治疗组, 腹腔注射链脲佐菌素 (60 mg/kg) 建立糖尿病周围神经病变模型, 褪黑素治疗组腹腔注射褪黑素 (10 mL/kg) 治疗。收集背根神经节组织, 通过苏木精-伊红染色观察神经细胞形态变化, 实时荧光定量聚合酶链式反应和Western blot检测线粒体融合蛋白2 (Mfn2)、动力相关蛋白1 (DRP1)、葡萄糖调节蛋白78 (Grp78)、PTEN诱导假定激酶1 (Pink1)、蛋白激酶R样内质网激酶 (PERK)、p47磷酸化蛋白 (p47)、神经营养因子 (NT) 以及神经生长因子 (NGF) 的mRNA和蛋白表达水平。在体外实验中, 模型组对背根神经细胞进行高糖处理以建立损伤模型, 褪黑素处理组给予褪黑素 (0.5 mmol/L) 处理。利用试剂盒检测背根神经细胞中活性氧 (ROS) 水平, JC-1染色检测线粒体膜电位, 荧光探针检测Ca ²⁺, 检测Mfn2、DRP1、Pink1、Grp78、PERK、p47、NT和NGF的表达水平。结果 通过血糖>16.7 mmol/L且尾神经感觉传导速度下降验证糖尿病周围神经病变模型制造成功。与对照组比较, 模型组背根神经节组织中部分神经细胞的核质界限不清晰、胞质染色较淡, 而褪黑素治疗组神经细胞形态相对模型组有所恢复。与对照组比较, 模型组DRP1、Grp78、PERK、p47、NT和NGF的mRNA和蛋白表达水平升高, Mfn2和Pink1的mRNA和蛋白表达水平降低 (P<0.05), 褪黑素治疗组中上述mRNA和蛋白表达水平得到改善 (P<0.05)。在体外实验中, 与对照组背根神经细胞比较, 高糖组中ROS水平增加, 线粒体膜电位和Ca ²⁺水平降低, DRP1、Grp78、PERK、p47、NT和NGF的mRNA和蛋白表达水平升高, Mfn2和Pink1的mRNA和蛋白表达水平降低 (P<0.05)。与高糖组比较, 褪黑素处理组中ROS水平减少, 线粒体膜电位和Ca ²⁺水平升高, Mfn2和Pink1的mRNA和蛋白表达水平升高, DRP1、Grp78、PERK、p47、NT和NGF的mRNA和蛋白表达水平降低 (P<0.05)。结论 褪黑素通过调节内质网应激和氧化应激相关蛋白的表达, 缓解了糖尿病周围神经病变中的线粒体功能并促进神经修复, 提示其在糖尿病周围神经病变管理中的潜在治疗作用。

Abstract

Objective To investigate the mechanisms by which melatonin promotes nerve repair in diabetic peripheral neuropathy. Methods Sprague-Dawley rats were divided into a control group, a model group, and a melatonin treatment group. Diabetic peripheral neuropathy was induced by intraperitoneal injection of streptozotocin (60 mg/kg). Rats in the melatonin treatment group received melatonin (10 mL/kg, intraperitoneally). Dorsal root ganglia tissues were collected, and hematoxylin-eosin staining was used to observe morphological changes in neurons. The mRNA and protein expression levels of mitochondrial fusion protein 2 (Mfn2), dynamin-related protein 1 (DRP1), glucose-regulated protein 78 (Grp78), PTEN-induced kinase 1 (Pink1), protein kinase R-like endoplasmic reticulum kinase (PERK), phosphorylated p47 phox (p47), neurotrophin (NT), and nerve growth factor (NGF) were detected by Real-time quantitative polymerase chain reaction and Western blot. In vitro, DRG neurons were treated with high glucose to establish a cellular injury model, while the melatonin treatment group received melatonin (0.5 mmol/L). Reactive oxygen species (ROS) levels were measured using assay kits, mitochondrial membrane potential (MMP) was assessed by JC-1 staining, and Ca ²⁺ levels were detected with fluorescent probes. The expression of Mfn2, DRP1, Pink1, Grp78, PERK, p47, NT, and NGF were examine. Results The successful establishment of diabetic peripheral neuropathy model was verified by blood glucose levels > 16.7 mmol/L and decreased sensory conduction velocity of caudal nerve. Compared with the control group, dorsal root ganglia neurons in the model group showed indistinct nuclear-cytoplasmic boundaries, and pale cytoplasm, while neuronal morphology was relatively restored in the melatonin group. In the model group, mRNA and protein levels of DRP1, Grp78, PERK, p47, NT, and NGF were significantly increased, whereas those of Mfn2 and Pink1 were decreased (P<0.05), the mRNA and protein expression levels were improved in the melatonin treatment group (P<0.05). In vitro, compared with the control group, high-glucose-treated dorsal root ganglia neurons showed increased ROS levels, decreased MMP and Ca ²⁺ levels, upregulation of DRP1, Grp78, PERK, p47, NT, and NGF, and downregulation of Mfn2 and Pink1 (P<0.05). In contrast, melatonin treatment reduced ROS levels, increased MMP and Ca ²⁺ levels, upregulated Mfn2 and Pink1, and downregulated DRP1, Grp78, PERK, p47, NT, and NGF (P<0.05). Conclusion Melatonin alleviates mitochondrial function and promotes nerve repair in diabetic peripheral neuropathy by modulating the expression of proteins involved in endoplasmic reticulum stress and oxidative stress. These findings suggest that melatonin has potential therapeutic value in the management of diabetic peripheral neuropathy.

关键词

糖尿病周围神经病变 / 褪黑素 / 线粒体功能 / 氧化应激 / 神经修复

Key words

Diabetic peripheral neuropathy / Melatonin / Mitochondrial function / Oxidative stress / Nerve repair

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基金资助

^*新疆维吾尔自治区自然科学基金(2022D01C317)