Ginsenoside Rg1对脊神经损伤修复的促进作用及机制研究
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摘要
研究背景:
脊神经损伤在脊柱外科非常常见。脊柱退行性疾病、外科手术操作及外伤等都可能对脊神经造成压迫、牵拉、挫伤等，造成不同程度的运动、感觉功能障碍，严重影响患者的生活质量。然而，脊神经损伤修复相关治疗的临床效果尚不理想，脊神经损伤仍然是临床面临的重大难题。
脊神经损伤属于周围神经损伤的范畴。目前，中药及其提取物的相关研究是周围神经损伤修复领域中的热点之一。研究表明，自人参中提取的人参皂苷Rg1（Ginsenoside Rg1, GRg1）对中枢神经系统损伤具有营养、保护及抑制神经元凋亡等作用。据此推测，GRg1的神经保护作用也有可能适用于周围神经系统。但是，目前GRg1的相关研究主要集中于脑保护方面，GRg1在周围神经损伤中的应用及机制研究很少。我们认为，将GRg1应用于周围神经损伤修复，有望提高周围神经损伤再生修复的治疗效果。
研究目的:
在大鼠周围神经损伤模型中研究GRg1对周围神经损伤的治疗效果。同时，在离体条件下针对GRg1对外周神经胶质细胞（雪旺细胞：Schwanncells, SCs）损伤条件下各种生物学行为的调控作用进行深入探索，以进一步揭示GRg1促进周围神经损伤后再生修复的可能机制。
材料方法:
1.制备SD大鼠坐骨神经挫裂伤模型，以不同剂量GRg1进行治疗（高/低剂量），同时设立甲钴胺对照组和生理盐水对照组，通过神经形态计量学指标、荧光金逆行标记、神经电生理检测、动物行为学指标（坐骨神经指数）以及靶器官形态学指标等评价大鼠神经再生修复、功能恢复及靶器官情况等。
2.制备SCs氧化损伤模型，给予GRg1，并设立甲钴胺组、氧化损伤组和正常组作为对照。应用分光光度计测定丙二醛(malondialdehyde, MDA)和超氧化物歧化酶（superoxide dismutase, SOD）等指标以明确SCs脂质氧化损伤的情况，应用DAPI染色和MTT试验明确受损SCs增殖的情况，应用流式细胞仪明确受损SCs凋亡的情况，利用RT-PCR、Western blot及ELISA等技术明确受损SCs合成和分泌NGF、BDNF等神经营养因子的情况。结果：
1.高剂量GRg1组的神经再生修复和功能恢复情况（神经形态计量学指标、荧光金逆行标记、神经电生理检测）明显优于其它组，坐骨神经指数和靶肌肉的形态等恢复良好，最为接近正常。
2. GRg1组SCs的氧化损伤水平、细胞凋亡数量明显低于其他组，而其细胞增殖、细胞活力以及NGF、BDNF等神经营养因子的合成和分泌显著高于其他组。
结论：
GRg1可以有效促进周围神经损伤再生修复并加快神经功能的恢复。同时，GRg1可以显著降低受损SCs的氧化损伤水平，提高其增殖和细胞活力，抑制细胞凋亡，并促进NGF、BDNF等神经营养因子的合成和分泌。上述GRg1对损伤条件下SCs的调控作用可能是GRg1促进外周神经再生和神经功能恢复的机制之一。
Backgrounds:
Spinal nerve injury is very common in the field of spinal surgery. There aremany reasons that might cause the spinal nerve being compressed, stretched orcrushed, resulting in different levels of motor dysfunction and sensorydysfunction. The life quality of patients is severely lowered. However, thetherapeutic effect of spinal nerve injury is not good. Spinal nerve injury is still aclinical challenge for surgeons.
Spinal nerve injury belongs to the category of peripheral nerves injury. Atpresent, the research of Chinese herbal medicine and its extracts is one of hotresearch topics. The present research shows that, Ginsenoside Rg1(one ofginseng extracts) might have trophism and protective effects on the centralnervous system, and can inhibit apoptosis of neurons. Presumably, this kind ofneuro-protective effects of GRg1may also apply to the peripheral nervoussystem. But the current research about GRg1is focused mainly on brainprotection. The research about application of GRg1in peripheral nerve injuryand the mechanism has been rare. We presume that, the application of GRg1inperipheral nerve injury may promote the nerve regeneration.
Objective:
To determine whether GRg1can promote the nerve regeneration inperipheral nerve injury. In addition, to reveal the mechanism underlying thebeneficial effect of GRg1on peripheral nerve repair, further studies wereperformed to identify the effect of GRg1on the biological behaviors ofneuroglia cell of peripheral nerve system (Schwann cells, SCs) after injury.
Materials and Methods:
1. A model of sciatic nerve crush injury in rats was established. Differentdoses of GRg1were administrated (high/low dose). Mecobalamin or saline wasadministrated to the control groups. The nerve regeneration, functional recoveryand condition of the target organ were investigated by nerve morphometricanalysis, retrograde labeling, electrophysiological studies, behavioral tests ofstepping (The sciatic nerve function index, SFI), and histological appearance ofthe target muscles.
2. A SCs oxidative damage model was established. GRg1wereadministrated after the oxidative damage. Mecobalamin or saline wasadministrated to the control groups. The oxidative damage level of the injuredSCs was investigated by examining the malondialdehyde (MDA) andsuperoxide dismutase (SOD) using spectrophotometer. The growth of injuredSCs was characterized using DAPI staining and a MTT assay. The apoptosis ofthe injured SCs was examined by a cell apoptosis assay using flow cytometer.The mRNA and protein levels of NGF and BDNF in injured SCs were assayedby RT-PCR and Western blotting, and the amount of NGF and BDNF secretedwas determined by an ELISA assay.
Results:
1. The nerve regeneration and functional recovery (nerve morphometricanalysis, retrograde labeling, electrophysiological studies) in the high-dose GRg1group were better than other groups. The sciatic nerve function index (SFI)and histological appearance of the target muscles recovered well in thehigh-dose GRg1group, which were the most close to the normal group.
2. The level of oxidative damage and apoptosis in the GRg1group wassignificantly lower than control groups. The proliferation and vitality in theGRg1group were significantly better than control groups. In addition, theexpression and secretion of NGF and BDNF by the injured SCs in GRg1groupis significantly more than control groups and normal group.
Conclusion:
GRg1can effectively promote the nerve regeneration and functionalrecovery in peripheral nerve injury. At the same time, GRg1can reduce theoxidative damage on the injured SCs, promote the proliferation and vitality, andinhibit the apoptosis. The expression and secretion of NGF and BDNF by theinjured SCs were also significantly enhanced. The regulatory effect of GRg1oninjured SCs mentioned above might partially contribute to the beneficial effectof GRg1on nerve regeneration and functional recovery in peripheral nerveinjury.
脊神经损伤在脊柱外科非常常见。脊柱退行性疾病、外科手术操作及外伤等都可能对脊神经造成压迫、牵拉、挫伤等，造成不同程度的运动、感觉功能障碍，严重影响患者的生活质量。然而，脊神经损伤修复相关治疗的临床效果尚不理想，脊神经损伤仍然是临床面临的重大难题。
脊神经损伤属于周围神经损伤的范畴。目前，中药及其提取物的相关研究是周围神经损伤修复领域中的热点之一。研究表明，自人参中提取的人参皂苷Rg1（Ginsenoside Rg1, GRg1）对中枢神经系统损伤具有营养、保护及抑制神经元凋亡等作用。据此推测，GRg1的神经保护作用也有可能适用于周围神经系统。但是，目前GRg1的相关研究主要集中于脑保护方面，GRg1在周围神经损伤中的应用及机制研究很少。我们认为，将GRg1应用于周围神经损伤修复，有望提高周围神经损伤再生修复的治疗效果。
研究目的:
在大鼠周围神经损伤模型中研究GRg1对周围神经损伤的治疗效果。同时，在离体条件下针对GRg1对外周神经胶质细胞（雪旺细胞：Schwanncells, SCs）损伤条件下各种生物学行为的调控作用进行深入探索，以进一步揭示GRg1促进周围神经损伤后再生修复的可能机制。
材料方法:
1.制备SD大鼠坐骨神经挫裂伤模型，以不同剂量GRg1进行治疗（高/低剂量），同时设立甲钴胺对照组和生理盐水对照组，通过神经形态计量学指标、荧光金逆行标记、神经电生理检测、动物行为学指标（坐骨神经指数）以及靶器官形态学指标等评价大鼠神经再生修复、功能恢复及靶器官情况等。
2.制备SCs氧化损伤模型，给予GRg1，并设立甲钴胺组、氧化损伤组和正常组作为对照。应用分光光度计测定丙二醛(malondialdehyde, MDA)和超氧化物歧化酶（superoxide dismutase, SOD）等指标以明确SCs脂质氧化损伤的情况，应用DAPI染色和MTT试验明确受损SCs增殖的情况，应用流式细胞仪明确受损SCs凋亡的情况，利用RT-PCR、Western blot及ELISA等技术明确受损SCs合成和分泌NGF、BDNF等神经营养因子的情况。结果：
1.高剂量GRg1组的神经再生修复和功能恢复情况（神经形态计量学指标、荧光金逆行标记、神经电生理检测）明显优于其它组，坐骨神经指数和靶肌肉的形态等恢复良好，最为接近正常。
2. GRg1组SCs的氧化损伤水平、细胞凋亡数量明显低于其他组，而其细胞增殖、细胞活力以及NGF、BDNF等神经营养因子的合成和分泌显著高于其他组。
结论：
GRg1可以有效促进周围神经损伤再生修复并加快神经功能的恢复。同时，GRg1可以显著降低受损SCs的氧化损伤水平，提高其增殖和细胞活力，抑制细胞凋亡，并促进NGF、BDNF等神经营养因子的合成和分泌。上述GRg1对损伤条件下SCs的调控作用可能是GRg1促进外周神经再生和神经功能恢复的机制之一。
Backgrounds:
Spinal nerve injury is very common in the field of spinal surgery. There aremany reasons that might cause the spinal nerve being compressed, stretched orcrushed, resulting in different levels of motor dysfunction and sensorydysfunction. The life quality of patients is severely lowered. However, thetherapeutic effect of spinal nerve injury is not good. Spinal nerve injury is still aclinical challenge for surgeons.
Spinal nerve injury belongs to the category of peripheral nerves injury. Atpresent, the research of Chinese herbal medicine and its extracts is one of hotresearch topics. The present research shows that, Ginsenoside Rg1(one ofginseng extracts) might have trophism and protective effects on the centralnervous system, and can inhibit apoptosis of neurons. Presumably, this kind ofneuro-protective effects of GRg1may also apply to the peripheral nervoussystem. But the current research about GRg1is focused mainly on brainprotection. The research about application of GRg1in peripheral nerve injuryand the mechanism has been rare. We presume that, the application of GRg1inperipheral nerve injury may promote the nerve regeneration.
Objective:
To determine whether GRg1can promote the nerve regeneration inperipheral nerve injury. In addition, to reveal the mechanism underlying thebeneficial effect of GRg1on peripheral nerve repair, further studies wereperformed to identify the effect of GRg1on the biological behaviors ofneuroglia cell of peripheral nerve system (Schwann cells, SCs) after injury.
Materials and Methods:
1. A model of sciatic nerve crush injury in rats was established. Differentdoses of GRg1were administrated (high/low dose). Mecobalamin or saline wasadministrated to the control groups. The nerve regeneration, functional recoveryand condition of the target organ were investigated by nerve morphometricanalysis, retrograde labeling, electrophysiological studies, behavioral tests ofstepping (The sciatic nerve function index, SFI), and histological appearance ofthe target muscles.
2. A SCs oxidative damage model was established. GRg1wereadministrated after the oxidative damage. Mecobalamin or saline wasadministrated to the control groups. The oxidative damage level of the injuredSCs was investigated by examining the malondialdehyde (MDA) andsuperoxide dismutase (SOD) using spectrophotometer. The growth of injuredSCs was characterized using DAPI staining and a MTT assay. The apoptosis ofthe injured SCs was examined by a cell apoptosis assay using flow cytometer.The mRNA and protein levels of NGF and BDNF in injured SCs were assayedby RT-PCR and Western blotting, and the amount of NGF and BDNF secretedwas determined by an ELISA assay.
Results:
1. The nerve regeneration and functional recovery (nerve morphometricanalysis, retrograde labeling, electrophysiological studies) in the high-dose GRg1group were better than other groups. The sciatic nerve function index (SFI)and histological appearance of the target muscles recovered well in thehigh-dose GRg1group, which were the most close to the normal group.
2. The level of oxidative damage and apoptosis in the GRg1group wassignificantly lower than control groups. The proliferation and vitality in theGRg1group were significantly better than control groups. In addition, theexpression and secretion of NGF and BDNF by the injured SCs in GRg1groupis significantly more than control groups and normal group.
Conclusion:
GRg1can effectively promote the nerve regeneration and functionalrecovery in peripheral nerve injury. At the same time, GRg1can reduce theoxidative damage on the injured SCs, promote the proliferation and vitality, andinhibit the apoptosis. The expression and secretion of NGF and BDNF by theinjured SCs were also significantly enhanced. The regulatory effect of GRg1oninjured SCs mentioned above might partially contribute to the beneficial effectof GRg1on nerve regeneration and functional recovery in peripheral nerveinjury.
引文
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