中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2019, Vol. 14 ›› Issue (10): 1814-1822.doi: 10.4103/1673-5374.257536

• 原著:退行性病与再生 • 上一篇    下一篇

人参皂甙Rb1可保护多巴胺能神经元免受单一脂多糖诱导的炎性损伤

  

  • 出版日期:2019-10-15 发布日期:2019-10-15
  • 基金资助:

    山东省医疗卫生技术发展规划项目(2011HD009)、山东省中医药科技发展规划项目(第2017-163号)、山东省自然科学基金项目(ZR2016HP23)；中国泰安市科技发展规划项目(2017NS0151)

Ginsenoside Rb1 protects dopaminergic neurons from inflammatory injury induced by intranigral lipopolysaccharide injection

Da-Wei Li 1, 2, Fa-Zhan Zhou 3, Xian-Chang Sun 4, Shu-Chen Li 2, Jin-Bin Yang 2, Huan-Huan Sun 2, Ai-Hua Wang 1   

  1. 1 Department of Neurology, Qianfoshan Hospital Affiliated to Shandong University, Jinan, Shandong Province, China
    2 Department of Neurology, The People’s Hospital of Xintai, Xintai, Shandong Province, China
    3 Department of Cardiovascularology, Taian Central Hospital, Taian, Shandong Province, China
    4 Department of Physiology, Taishan Medical University, Taian, Shandong Province, China
  • Online:2019-10-15 Published:2019-10-15
  • Contact: Ai-Hua Wang, MD, doctorli1977@126.com
  • Supported by:

    This work was supported by the Medical and Health Technology Development Plan of Shandong Province of China, No. 2011HD009 (to AHW); the Chinese Medicine Science and Technology Development Plan Project of Shandong Province of China, No. 2017-163 (to AHW); the Natural Science Foundation of Shandong Province of China, No. ZR2016HP23 (to AHW); the Science and Technology Development Plan Project of Taian City of China, No. 2017NS0151 (to XCS).

PDF

113

摘要：

大量研究表明，以小胶质细胞过度激活为特征的神经炎症反应在帕金森病的发病机制中起着关键作用。因此，抑制小胶质细胞的过度激活可能延缓帕金森病的发生或减慢病情的发展，为帕金森病提供一种有效的治疗策略。人参中最有效的成分人参皂苷Rb1(GRb1)在体外实验中已显示了，可通过抑制炎症反应发挥的神经保护作用。实验旨在评价在脂多糖诱导的大鼠帕金森病模型中GRb1的神经保护和抗炎作用。实验将大鼠随机分为4组：对照组：假手术后，连续14 d腹腔注射生理盐水；GRb1组：假手术后连续腹腔注射GRb1(20 mg/kg)14 d；脂多糖组：脂多糖单侧注射大鼠黑质部建立帕金森病大鼠模型，连续14 d腹腔注射生理盐水；GRb1+脂多糖组：脂多糖单侧注射大鼠黑质部，然后再用GRb1腹腔注射处理14 d。实验做了如下分析：(1)为了观察GRb1的治疗效果，对注射脂多糖后15 d的大鼠行为学进行测试发现，与脂多糖组比较，GRb1治疗可显著改善阿扑吗啡诱导的大鼠旋转行为；(2)为了观察脂多糖的神经毒性和GRb1的保护作用，用高效液相色谱法测定了大鼠纹状体中多巴胺及其代谢物的含量发现：与脂多糖组比较GRb1可以明显降低脂多糖注射侧黑质中的多巴胺及其代谢物的含量；(3)为了进一步探讨GRb1对脂多糖诱导的神经毒性保护作用，通过免疫组织化学染色和免疫印迹方法检测各组酪氨酸羟化酶的表达以反映巴胺能神经元的变性发现：脂多糖注射后可导致黑质内多巴胺能神经元数量的大量丢失、酪氨酸羟化酶表达量明显下降，而应用GRb1治疗能显著逆转上述变化；(4)为观察GRb1的神经保护作用是否与脂多糖诱导的小胶质细胞活化有关，实验检测了小胶质细胞的标志物Iba-1的表达发现：脂多糖组动物黑质内Iba-1的表达明显升高，而应用GRb1处理后可有效抑制脂多糖诱导的小胶质细胞过度活化；(5)为了进一步阐明GRb1的作用机制，实验分别用酶联免疫吸附试验和Western blot法检测了大鼠黑质中炎症介质(肿瘤坏死因子α、白细胞介素1β、诱导型一氧化氮合成酶、环氧化酶2和核转录因子κB信号通路中相关蛋白(IB，IKK)的表达发现，与对照组比较，脂多糖组动物黑质各内炎症介质表达量明显增加，磷酸化IB、IKK水平明显增加，而应用GRb1处理后能显著逆转上述改变；(6)上述数据证实：GRb1可抑制黑质内注射脂多糖诱导的炎症反应，保护多巴胺能神经元，该作用可能与其抑制核转录因子κB信号通路有关。实验经中国山东大学实验动物伦理委员会(KYLL-2016-0148)批准，批准日期为2016年4月。

orcid: 0000-0001-5771-2699 (Ai-Hua Wang)

关键词: 神经退行性变, 帕金森病, 人参皂苷Rb1, 神经炎症, 脂多糖, 多巴胺能神经元, 小胶质细胞, 核转录因子&kappa, B, 多巴胺, 酪氨酸羟化酶, 黑质, 神经再生

Abstract:

Accumulating studies suggest that neuroinflammation characterized by microglial overactivation plays a pivotal role in the pathogenesis of Parkinson’s disease. As such, inhibition of microglial overactivation might be a promising treatment strategy to delay the onset or slow the progression of Parkinson’s disease. Ginsenoside Rb1, the most active ingredient of ginseng, reportedly exerts neuroprotective effects by suppressing inflammation in vitro. The present study aimed to evaluate the neuroprotective and anti-inflammatory effects of ginsenoside Rb1 in a lipopolysaccharide-induced rat Parkinson’s disease model. Rats were divided into four groups. In the control group, sham-operated rats were intraperitoneally administered normal saline for 14 consecutive days. In the ginsenoside Rb1 group, ginsenoside Rb1 (20 mg/kg) was intraperitoneally injected for 14 consecutive days after sham surgery. In the lipopolysaccharide group, a single dose of lipopolysaccharide was unilaterally microinjected into the rat substantial nigra to establish the Parkinson’s disease model. Lipopolysaccharide-injected rats were treated with normal saline for 14 consecutive days. In the ginsenoside Rb1 + lipopolysaccharide group, lipopolysaccharide was unilaterally microinjected into the rat substantial nigra. Subsequently, ginsenoside Rb1 was intraperitoneally injected for 14 consecutive days. To investigate the therapeutic effects of ginsenoside Rb1, behavioral tests were performed on day 15 after lipopolysaccharide injection. We found that ginsenoside Rb1 treatment remarkably reduced apomorphine-induced rotations in lipopolysaccharide-treated rats compared with the lipopolysaccharide group. To investigate the neurotoxicity of lipopolysaccharide and potential protective effect of ginsenoside Rb1, contents of dopamine and its metabolites in the striatum were measured by high-performance liquid chromatography. Compared with the lipopolysaccharide group, ginsenoside Rb1 obviously attenuated the lipopolysaccharide-induced depletion of dopamine and its metabolites in the striatum. To further explore the neuroprotective effect of ginsenoside Rb1 against lipopolysaccharide-induced neurotoxicity, immunohistochemistry and western blot assay of tyrosine hydroxylase were performed to evaluate dopaminergic neuron degeneration in the substantial nigra par compacta. The results showed that lipopolysaccharide injection caused a large loss of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra and a significant decrease in overall tyrosine hydroxylase expression. However, ginsenoside Rb1 noticeably reversed these changes. To investigate whether the neuroprotective effect of ginsenoside Rb1 was associated with inhibition of lipopolysaccharide-induced microglial activation, we examined expression of the microglia marker Iba-1. Our results confirmed that lipopolysaccharide injection induced a significant increase in Iba-1 expression in the substantia nigra; however, ginsenoside Rb1 effectivelysuppressed lipopolysaccharide-induced microglial overactivation. To elucidate the inhibitory mechanism of ginsenoside Rb1, we examined expression levels of inflammatory mediators (tumor necrosis factor-α, interleukin-1β, inducible nitric oxide synthase, and cyclooxygenase 2) and phosphorylation of nuclear factor kappa B signaling-related proteins (IκB, IKK) in the substantia nigra with enzyme-linked immunosorbent and western blot assays. Our results revealed that compared with the control group, phosphorylation and expression of inflammatory mediators IκB and IKK in the substantia nigra of lipopolysaccharide group rats were significantly increased; whereas, ginsenoside Rb1 obviously reduced lipopolysaccharide-induced changes on the lesioned side of the substantial nigra par compacta. These findings confirm that ginsenoside Rb1 can inhibit inflammation induced by lipopolysaccharide injection into the substantia nigra and protect dopaminergic neurons, which may be related to its inhibition of the nuclear factor kappa B signaling pathway. This study was approved by the Experimental Animal Ethics Committee of Shandong University of China in April, 2016 (approval number: KYLL-2016-0148).

Key words: nerve regeneration, neurodegeneration, Parkinson’s disease, ginsenoside Rb1, neuroinflammation, lipopolysaccharide, dopaminergic neuron, microglia, nuclear factor kappa B, dopamine, tyrosine hydroxylase, substantia nigra, neural regeneration