加味桂枝茯苓汤通过PINK1/Parkin信号通路调控线粒体自噬途径减轻PDPN大鼠坐骨神经损伤的作用机制

Mechanism of Jiawei Guizhi Fuling Decoction in Alleviating Sciatic Nerve Injury in PDPN Rats by Regulating Mitophagy Through PlNK1/Parkin Signaling Pathway

摘要:

目的 观察加味桂枝茯苓汤（JGFD）通过PTEN诱导假定激酶1（PINK1）/帕金森病蛋白（Parkin）信号通路调控线粒体自噬途径减轻痛性糖尿病周围神经病变（PDPN）大鼠坐骨神经损伤的作用机制。方法 通过腹腔注射链脲佐菌素（STZ）制备PDPN模型。成模后随机分为JGFD高、中、低剂量组（JGFD-H、JGFD-M、JGFD-L，39.6、19.8、9.9 g·kg^-1·d^-1）、阳性药组（硫辛酸胶囊，LA，50 mg·kg^-1·d^-1）、模型组（PDPN），并设立空白组（CON）。成模后持续给药干预8周。分别检测干预第0、2、4、8周大鼠体质量和空腹血糖，第0、8周大鼠机械痛阈值和热痛阈值，第8周大鼠运动神经传导速度；苏木素-伊红（HE）染色观察坐骨神经组织形态；透射电镜观察线粒体和自噬小体超微结构；蛋白免疫印迹法（Western blot）检测坐骨神经组织PINK1、Parkin、p62、酵母Atg6同系物（Beclin-1）、微管相关蛋白1轻链3（LC3）蛋白表达；实时荧光定量聚合酶链式反应（Real-time PCR）检测坐骨神经组织PINK1、Parkin、p62、Beclin-1、LC3 mRNA水平。结果 与CON组比较，PDPN组大鼠各时间点体质量显著降低，空腹血糖显著升高，机械痛阈值和热痛阈值显著缩短，运动神经传导速度显著减慢，坐骨神经组织PINK1、Parkin、Beclin-1、LC3蛋白和mRNA表达显著降低、p62蛋白和mRNA表达显著升高（P<0.01）；坐骨神经神经纤维水肿，节段性脱髓鞘；髓鞘板层结构排列松散紊乱，线粒体明显肿胀，电子密度减低，嵴断裂消失，未见典型自噬溶酶体和自噬小体结构。与PDPN组比较，JGFD各剂量组体质量明显升高，空腹血糖明显降低（P<0.05，P<0.01）；JGFD各剂量组、LA组机械痛阈值和热痛阈值明显延长，运动神经传导速度明显增快，坐骨神经组织PINK1、Parkin、Beclin-1、LC3蛋白和mRNA表达明显升高、p62蛋白和mRNA表达明显降低（P<0.05，P<0.01）；坐骨神经病理损伤均有不同程度改善；髓鞘形态结构较为完整，线粒体外膜完整或者轻微水肿，JGFD-M组、JGFD-H组可见自噬溶酶体结构。与LA组比较，JGFD-H组体质量明显升高，空腹血糖明显降低，运动神经传导速度明显增快，坐骨神经组织PINK1蛋白和PINK1、Parkin、Beclin-1 mRNA表达明显升高，p62 mRNA表达明显降低（P<0.05，P<0.01）。结论 加味桂枝茯苓汤可能通过调控PINK1/Parkin信号通路，激活线粒体自噬，减轻PDPD大鼠坐骨神经损伤。

Abstract:

Objective To observe the mechanism of Jiawei Guizhi Fuling decoction （JGFD） in alleviating sciatic nerve injury in painful diabetic peripheral neuropathy （PDPN） rats by regulating mitophagy through the PTEN-induced putative kinase 1 （PINK1）/Parkin signaling pathway.Method The PDPN model was established by intraperitoneal injection of streptozotocin （STZ）. After modeling， the rats were randomly divided into JGFD high， medium， and low dose groups （JGFD-H， JGFD-M， JGFD-L； 39.6， 19.8， 9.9 g·kg^-1·d^-1， respectively）， a positive drug group （lipoic acid capsules， LA； 50 mg·kg^-1·d^-1）， and a model group （PDPN）. A blank control group （CON） was established. Drug intervention was administered continuously for 8 weeks after modeling. Measurements included body weight and fasting blood glucose of PDPN rats at weeks 0， 2， 4， and 8， mechanical pain threshold and thermal pain threshold at weeks 0 and 8， and motor nerve conduction velocity at week 8. Hematoxylin-eosin （HE） staining was used to observe the morphology of sciatic nerve tissue. The ultrastructure of mitochondria and autophagosomes was observed by transmission electron microscopy. Western blot was performed to detect the protein expression levels of PINK1， Parkin， p62， Beclin-1， and LC3 in sciatic nerve tissue. Additionally， real-time quantitative PCR （Real-time PCR） was performed to detect the mRNA expression levels of PINK1， Parkin， p62， Beclin-1， and LC3 in sciatic nerve tissue.Result Compared with the CON group， the PDPN group showed a significant decrease in body weight at all time points， a significant increase in fasting blood glucose， significantly shortened mechanical pain and thermal pain thresholds， and significantly reduced motor nerve conduction velocity. The protein and mRNA expression of PINK1， Parkin， Beclin-1， and microtubule-associated protein light chain 3（LC3） in sciatic nerve tissue was significantly reduced， while p62 protein and mRNA expression was significantly increased （P<0.01）. Pathological changes included edema of sciatic nerve fibers， segmental demyelination， loose and disordered arrangement of the myelin sheath layers， significant swelling of mitochondria， reduced electron density， disappearance of cristae， and absence of typical autophagosome and autolysosome structures. Compared with the PDPN group， each JGFD dose group showed a significant increase in body weight and a significant reduction in fasting blood glucose （P<0.05， P<0.01）. The mechanical pain threshold and thermal pain threshold were significantly prolonged， and motor nerve conduction velocity was significantly increased across all JGFD and LA groups. The expression levels of PINK1， Parkin， Beclin-1， and LC3 proteins and mRNA in sciatic nerve tissue were significantly increased， while p62 protein and mRNA expression levels were significantly decreased （P<0.05， P<0.01）. Pathological damage to the sciatic nerve was alleviated to varying degrees， with a relatively intact myelin sheath morphology and intact or slightly edematous outer mitochondrial membrane. Autophagolysosome structures were observed in the JGFD-M and JGFD-H groups. Compared with the LA group， the JGFD-H group showed a significant increase in body weight， a significant reduction in fasting blood glucose， a significant increase in motor nerve conduction velocity， a significant increase in PINK1 protein expression and PINK1， Parkin， and Beclin-1 mRNA expression in sciatic nerve tissue， and a significant decrease in p62 mRNA expression （P<0.05， P<0.01）.Conclusion JGFD may alleviate sciatic nerve injury in PDPN rats by activating mitophagy through the regulation of the PINK1/Parkin signaling pathway.