人参皂甙Rd抑制脑缺血炎症损伤的作用及机制研究

发布时间：2018-05-20 09:19

本文选题：人参皂甙Rd + 小胶质细胞　；参考：《第四军医大学》2014年博士论文

【摘要】：脑卒中是一种致病机制复杂，严重影响人类生存和生活质量的灾难性疾病。尽管目前在治疗方面取得部分相关进展，但是研究者对于如何保护卒中相关的脑损伤知之甚少。这就导致具有明确神经保护作用，能够安全运用于临床的药物少之又少。因此，开发具有明确保护作用的药物对脑卒中治疗具有重要的意义。人参和三七是亚洲地区应用广泛的传统中草物，悠久的用药史证实用药安全有效。近来研究逐步揭示人参皂甙(ginsenosides, GS)及其有效单体成分GSRb、GSRg、GSRd等具有不同程度的脑损伤保护作用。其中，我们课题组前期的实验工作证实，人参皂甙Rd(ginsenosides Rd, GSRd)能够减轻谷氨酸兴奋性毒性损伤，抑制神经元胞内钙超载，减少脑缺血后的氧化应激损伤，改善线粒体功能方面，具有肯定的神经保护作用。二期和三期的临床试验同样表明GSRd治疗急性缺血性脑卒中患者安全有效。更为重要的是，我们发现在脑缺血急性期后应用GSRd，仍然能发挥有效的脑损伤保护作用；考虑到炎症反应是脑缺血后期重要的病理过程，我们推测GSRd抑制炎症损伤可能是其发挥效应的重要机制之一。因此，本课题旨在研究GSRd对实验性脑缺血后的炎性反应的影响及其可能的作用机制；同时结合GSRd和皮质类固醇的化学结构十分相似的特点，将GSRd和激素类药物的作用及不良反应进行比较，为GSRd临床应用于脑卒中治疗提供理论依据。实验一人参皂甙Rd抑制实验性脑缺血炎性反应的研究目的：探讨GSRd对实验性脑缺血后的炎性反应的影响。方法：分别采用大鼠大脑中动脉阻塞再灌注模型、LPS炎性损伤模型和氧糖剥夺模型模拟脑缺血再灌注炎性损伤，以米诺环素、地塞米松（dexamethasone，DEX）、米非司酮（RU486）作为对照。使用TTC染色测定脑梗死体积，MTT检测GSRd对炎性损伤神经元存活率的影响。使用实时定量PCR技术和液相芯片技术检测GSRd（10mg/Kg）分别对缺血后炎性细胞因子mRNA和蛋白表达的影响。结果：（1）10mg/Kg GSRd预先给药能够有效减少大鼠缺血后的脑梗死体积，提高受损神经元的存活数量。DEX和RU486对缺血后脑梗死体积不产生影响。（2）GSRd、DEX和米诺环素预处理能够减少多种炎性细胞因子mRNA和蛋白的表达，从而减轻缺血后的炎性损伤，但三者的抗炎作用没有显著性差异。（3）RU486可以阻断或部分阻断GSRd和DEX对细胞因子的抑制作用。结论：GSRd预处理能够保护脑缺血模型下的神经元，这种作用可能与其减轻损伤后的炎性反应、抑制细胞因子表达密切相关。实验二人参皂甙Rd通过作用糖皮质激素受体发挥抗炎效应的研究目的：探讨GSRd作用糖皮质激素受体发挥抗炎效应的作用机制。方法： OGD损伤后，使用GSRd（10μM）进行干预后，使用qRT-PCR检测不同干预处理组NFκB p65mRNA的表达。使用Western Blot检测糖皮质激素受体（glucocorticoid receptor，GR）通路关键蛋白（GR、p-IκBα/IκBα、p-p38/p38、NFκB p65）的表达水平。结果：（1）NFκB是一种调控炎性细胞因子表达的重要转录因子。GSRd预处理能够抑制IκBα磷酸化和NFκB P65入核，从而抑制NFκB活性。（2）p38是维持炎性细胞因子稳定表达的重要因子。GSRd预处理能够抑制p38MAPK磷酸化，减低p-p38/p38比值，降低p38活性。（3）GR激活可同时调控其下游的NFκB和p38分子。GSRd预处理能够促进GR转位入核，GR抑制剂RU486可以部分阻断GSRd对缺血后GR通路蛋白表达的调节作用。结论：GSRd可抑制炎症调控因子NFκB和p38的活性，，这种作用可能是与其激活GR密切相关。实验三人参皂甙Rd与糖皮质激素抗炎效应及不良反应的对比研究目的：糖皮质激素的特点是抗炎作用强大和用药不良反应广泛。研究对比GSRd与DEX使用后的抗炎效应及不良反应。方法：分别探讨GSRd和DEX对代谢、免疫和骨形成方面的影响。健康C57/B6小鼠分别注射4周10mg/Kg GSRd和2.5mg/Kg DEX，对比不同干预对小鼠血糖水平，胸腺、脾脏和体重质量的改变；采用胶原诱导关节炎模型对比GSRd和DEX对关节炎症的作用；观察不同浓度GSRd（10nM-10M）和DEX（10nM-10M）对体外培养成骨细胞系MC3T3-E1的增殖和分化影响。结果：（1）和正常组相比，GSRd组小鼠的血糖、胸腺、脾脏、体重无明显变化；相反，DEX组小鼠的血糖升高、胸腺、脾脏和体重质量明显下降（p0.05）。（2）胶原诱导关节炎模型小鼠实验中，GSRd和DEX均可减轻关节炎性细胞浸润，两者之间无显著差异。（3）在成骨细胞培养实验中，GSRd和DEX均有促成骨细胞增殖和成分化的作用，但GSRd的有效剂量范围较广（10nM-1M）。结论：与DEX相比，GSRd的抗炎作用同样强大，不会影响血糖、体重和免疫器官，不会造成骨质疏松，用药安全，是一种极具潜力的神经保护药物。
[Abstract]:The study shows that ginsenoside Rd ( GS ) and its effective monomer components GSRb , GSRg and GSRd have different degrees of brain damage protection .
Considering that inflammatory reaction is an important pathological process in the late stage of cerebral ischemia , we hypothesized that GSRd can inhibit inflammatory injury as one of the important mechanisms for its effect .

Therefore , the aim of this study was to study the effect of GSRd on inflammatory response after experimental cerebral ischemia and its possible mechanism .
Combined with the characteristics of the chemical structure of GSRd and corticosteroid , the effects of GSRd and hormone drugs and adverse reactions were compared , which provided the theoretical basis for the clinical application of GSRd in the treatment of stroke .

Experimental study on the inhibition of ginsenoside Rd on the inflammatory response of experimental cerebral ischemia

Objective : To investigate the effect of GSRd on inflammatory response after experimental cerebral ischemia .

Methods : Rat brain middle cerebral artery occlusion reperfusion model , LPS inflammatory injury model and oxygen sugar deprivation model were used to simulate cerebral ischemia / reperfusion inflammatory injury . The effects of GSRd ( 10 mg / Kg ) on the survival rate of inflammatory cytokines mRNA and protein were measured by TTC staining .

Results : ( 1 ) The pre - administration of 10 mg / kg GSRd can reduce the volume of cerebral infarction after ischemia and improve the survival of injured neurons . ( 2 ) The pretreatment of DEX and RU486 can reduce the expression of various inflammatory cytokines mRNA and protein .

Conclusion : GSRd pretreatment can protect neurons in cerebral ischemia model , which may reduce inflammatory response after injury and inhibit the expression of cytokines .

Experimental study on the anti - inflammatory effect of two - two - two - ginseng saponin Rd by action of glucocorticoid receptor

Objective : To investigate the effect of GSRd on glucocorticoid receptor on anti - inflammatory effect .

Methods : After intervention with GSRd ( 10 渭M ) , the expression of NF - 魏B p65mRNA was detected by qRT - PCR . Western Blot was used to detect the expression of NF - 魏B p65 mRNA in glucocorticoid receptor ( GR ) pathway .

Results : ( 1 ) NF - 魏B is an important transcription factor regulating the expression of inflammatory cytokines . GSRd pretreatment can inhibit the phosphorylation of NF - 魏B and NF - 魏B P65 , thereby inhibiting NF - 魏B activity . ( 2 ) p38 is an important factor to maintain the stable expression of inflammatory cytokines .

Conclusion : GSRd can inhibit the activity of NF - 魏B and p38 , which may be related to the activation of GR .

Comparative study on anti - inflammatory effects and adverse effects of ginsenoside Rd and glucocorticoid

Objective : To study the anti - inflammatory effects and adverse effects of GSRd and DEX .

Methods : The effects of GSRd and DEX on metabolism , immunity and bone formation were investigated .
Collagen - induced arthritis model was used to compare the effects of GSRd and DEX on joint inflammation .
The effects of different concentrations of GSRd ( 10 nM - 10M ) and DEX ( 10 nM - 10M ) on proliferation and differentiation of MC3T3 - E1 were investigated .

Results : ( 1 ) Compared with the normal group , the blood sugar , thymus , spleen and body weight of GSRd group mice were not significantly changed .
( 3 ) GSRd and DEX all had the effects of promoting osteoblast proliferation and differentiation during osteoblast culture experiments , but the effective dose range of GSRd was wider ( 10 nM - 1M ) .

Conclusion : Compared with DEX , GSRd has the same anti - inflammatory effect , which does not affect blood sugar , body weight and immune organs . It does not cause osteoporosis and drug safety .
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R743.3

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