Mechanistic Studies on the Improvement of Diabetic Peripheral Neuropathy by Plantamajoside via Promoting the PINK1/Parkin Mitochondrial Autophagy Pathway
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摘要:
目的 研究大车前苷（plantamajoside，PMS）对糖尿病周围神经病变（diabetic peripheral neuropathy，DPN）的疗效并从线粒体自噬探讨其作用机制。 方法 C57BL/6J小鼠随机分为6组（n = 10）：正常组（Control）、模型组（Model）、阳性药物组（LA）、大车前苷低（L-PMS）、中（M-PMS）、高（H-PMS）剂量组，高糖高脂饮食配合腹腔注射链脲佐菌素构建DPN模型。模型构建成功后，灌胃给药干预4周，取坐骨神经，HE染色和Nissl染色观察其病理学变化；通过试剂盒检测坐骨神经组织中氧化应激指标SOD、MDA、GSH-Px和炎症因子IL-1β、IL-6、TNF-α； Western blotting法检测小鼠坐骨神经VDAC1、TOM20、COX IV、PINK1、Parkin、自噬蛋白Beclin1、LC3、P62表达。 结果 PMS可剂量依赖性地改善DPN小鼠的行为学指标，减轻坐骨神经的病理损伤，使坐骨神经纤维排列紧密，髓鞘结构清晰可见，着色均匀，施旺细胞以及尼氏体数量增加。与模型组比较，M-PMS组、H-PMS组均可提高SOD、GSH-Px的表达水平（P < 0.05），降低MDA表达（P < 0.05）。与模型组比较，M-PMS组、H-PMS组可降低IL-1β、IL-6、TNF-α表达（P < 0.05）。与模型组比较，L-PMS组、M-PMS组、H-PMS组均可不同程度地降低VDAC1、TOM20与COX IV蛋白表达水平（P < 0.05）。与模型组比较，L-PMS组、M-PMS组、H-PMS组均可不同程度地使PINK1、Beclin1、Parkin与LC3蛋白表达上升（P < 0.05），P62蛋白表达下降（P < 0.05）。 结论 PMS可能通过促进PINK1/Parkin通路介导的线粒体自噬，缓解氧化应激相关的炎症损伤，在改善 DPN 小鼠神经损伤方面发挥作用。 -
关键词:
- 糖尿病周围神经病变 /
- 大车前苷 /
- 线粒体自噬 /
- PINK1/Parkin /
- 坐骨神经
Abstract:Objective To investigate the efficacy of plantamajoside （PMS） on diabetic peripheral neuropathy （DPN） and to explore its mechanism of action from mitochondrial autophagy. Methods Mice （C57BL/6J） were randomly divided into 6 groups（n = 10）: normal group （Control）, model group （Model）, positive drug group (LA), and low （L-PMS）, medium （M-PMS）, and high （H-PMS） dosage groups. High-sugar and high-fat diet with intraperitoneal injection of streptozotocin was used to duplicate the DPN model. After successful model duplication, the intervention was administered by gavage for 4 weeks. Sciatic nerve was taken, and pathological changes were observed by HE and Nissl staining; oxidative stress indexes SOD, MDA, GSH-Px and inflammatory factors such as IL-1β, IL-6, TNF-αin sciatic nerve tissues were detected by kits, and the expression of VDAC1 , TOM20, COX IV, PINK1, Parkin, and autophagy proteins of Beclin1, LC3, P62 in mouse sciatic nerves was detected by Western blotting . Results PMS dose-dependently improved the behavioral indexes of DPN mice, reduced the pathological damage of sciatic nerve, and resulted in tightly arranged sciatic nerve fibers, clearly visible myelin structure, uniform coloration, and increased number of Schwann cells as well as Nissl bodies. Compared with the model group, both the M-PMS group and the H-PMS group increased the expression levels of SOD and GSH-Px （P < 0.05）, while decreased the expression of MDA （P < 0.05）; the M-PMS group and the H- PMS groups reduced the expression of IL-1β, IL-6, and TNF-α （P < 0.05）; the L-PMS group, M-PMS group, and H-PMS group reduced the expression levels of VDAC1, TOM20, and COX IV proteins （P < 0.05）; the L-PMS group, M-PMS group, and H-PMS group could differentially increase the expression of PINK1, Beclin1, Parkin, and LC3 proteins （P < 0.05）, and decrease the expression of P62 proteins （P < 0.05）. Conclusion PMS can play a role in ameliorating neurological injury in DPN mice by promoting PINK1/Parkin pathway-mediated mitochondrial autophagy and alleviating oxidative stress-related inflammatory injury. -
Key words:
- Diabetic peripheral neuropathy /
- Plantamajoside /
- Mitochondrial autophagy /
- PINK1/Parkin /
- Sciatic nerve
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图 1 各组小鼠机械痛阈、热痛反应时间、运动神经传导速度、感觉神经传导速度比较[（$\bar x \pm s $），n = 10]
A：各组小鼠机械痛阈统计学分析；B：各组小鼠热痛反应时间统计学分析；C：各组小鼠运动神经传导速度统计学分析；D：各组小鼠感觉神经传导速度统计学分析。与Control组比较，## P < 0.01；与Model组比较，* P < 0.05，** P < 0.01。
Figure 1. Comparison of mechanical pain threshold，thermal pain reaction time，motor nerve conduction velocity，and sensory nerve conduction velocity in the mice of each group[（$\bar x \pm s $），n = 10]
图 2 各组小鼠坐骨神经病理损伤的显微镜观察图
A：各组小鼠坐骨神经HE染色图；B：各组小鼠坐骨神经Nissl染色图。蓝色箭头所指为神经纤维，黑色箭头所指为神经纤维中的施旺细胞（500×）。
Figure 2. Microscopic view of pathological damage to the sciatic nerve in various groups of the mice
图 3 各组小鼠MDA、SOD、GSH-Px、IL-1β、IL-6、TNF-α比较[（$ \bar x \pm s$），n = 10]
A：小鼠血清SOD表达统计学分析；B：小鼠血清MDA表达统计学分析；C：小鼠血清GSH-Px表达统计学分析；D：小鼠血清IL-1β表达统计学分析；E：小鼠血清IL-6表达统计学分析；F：小鼠血清TNF-α表达统计学分析。注：与Control组比较，## P < 0.01；与Model组比较，* P < 0.05，** P < 0.01。
Figure 3. Comparison of IL-1β，IL-6，TNF-α，MDA，SOD，GSH-Px in the mice of each group[（$\bar x \pm s $），n = 10]
图 4 各组小鼠线粒体形态结构蛋白表达水平的变化[（$\bar x \pm s $），n = 3]
A：各组小鼠线粒体形态结构蛋白表达；B：各组小鼠VDAC1蛋白表达统计学分析；C：各组小鼠TOM20蛋白表达统计学分析；D：各组小鼠COX IV蛋白表达统计学分析。与Control组比较，## P < 0.01；与Model组比较，* P < 0.05，** P < 0.01。
Figure 4. Changes in the expression levels of mitochondrial morphological structure proteins in various groups of the mice[（$\bar x \pm s $），n = 3]
图 5 各组小鼠PINK1/Parkin 介导线粒体自噬蛋白水平的变化[（$\bar x \pm s $），n = 3]
A：各组小鼠线粒体自噬蛋白表达；B：各组小鼠PINK1蛋白表达统计学分析。C：各组小鼠Parkin蛋白表达统计学分析；D：各组小鼠Beclin1蛋白表达统计学分析；E：各组小鼠LC3蛋白表达统计学分析；F：各组小鼠P62蛋白表达统计学分析。注：与Control组比较，## P < 0.01；与Model组比较，* P < 0.05，** P < 0.01。
Figure 5. Changes in PINK1/Parkin-mediated mitochondrial autophagy protein levels in various groups of the mice[（$\bar x \pm s $），n = 3]
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