犀角地黄汤对缺血性卒中体外炎症反应信号通路的干预研究

发布时间：2018-01-12 17:26

本文关键词：犀角地黄汤对缺血性卒中体外炎症反应信号通路的干预研究　出处：《南京中医药大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究目的从体外实验的角度,研究犀角地黄汤对脑缺血再灌注损伤的神经保护作用,以及与炎症反应相关的TLR4/MyD88信号通路在犀角地黄汤神经保护效应中的作用。通过本课题的研究,以期深化中医瘀热理论在缺血性卒中治疗中的指导意义,并为缺血性卒中的治疗提供新的策略。研究方法首先,本课题采用高效液相色谱法(HPLC)同时测定犀角地黄汤中主要成分芍药苷、芍药内酯苷、氧化芍药苷的含量,建立犀角地黄汤的质量控制分析方法。其次,本研究以PC12细胞为研究载体,在体外通过氧糖剥夺和复氧方法模拟脑缺血再灌注损伤。实验中将PC12细胞分为空白对照组(control)、模型对照组(OGD/R)、犀角地黄汤组(XJDH)和芍药苷组(Paeoniflorin)。空白对照组细胞正常培养,不经过OGD/R处理;模型对照组细胞经OGD/R处理,但不加任何药物干预;犀角地黄汤组是加入犀角地黄汤干预,在CGD/R条件下培养细胞;芍药苷组是加入犀角地黄汤的活性成分芍药苷,在OGD/R条件下培养细胞。药物与细胞孵育一定时间后,通过MTT实验及LDH释放实验,考察犀角地黄汤或芍药苷对细胞活力的影响;ELISA法测定各组细胞炎症因子IL-1β、IL-6和TNF-α的释放水平,并检测细胞内caspase-3的浓度,考察犀角地黄汤或芍药苷神经保护作用与炎症、凋亡的关系。通过Western Blot实验,研究犀角地黄汤或芍药苷对TLR4、MyD88、NF-κB、TRAF6、p-ERK、p-JNK、Akt和cleaved caspase-3等蛋白表达水平的影响,初步探讨犀角地黄汤神经保护作用的可能机制。研究结果HPLC实验结果显示,犀角地黄汤中芍药苷、芍药内酯苷、氧化芍药苻三种物质的含量分别为0.232±0.031%、0.276±0.018%、0.019±0.002%。所建的分析方法操作简便快速准确,可为犀角地黄汤的质量控制提供依据。氧葡萄糖剥夺复氧后,与正常培养PC12细胞相比,OGD后PC12细胞活性下降43.75±2.4%。提前给予0.4,0.2,0.1 mg/mL犀角地黄汤处理,细胞活力分别为91.9 ± 2.6%,97.3 ± 3.5%,66.9 ± 1.8%,与模型对照组比较均具有显著性差异。LDH检测实验结果表明,犀角地黄汤可以减少细胞内LDH的释放,改善OGD/R导致的PC12细胞损伤。ELISA实验数据表明,OGD/R能够促进炎症介质1L-1β、1L-6和TNF-α的产生,而犀角地黄汤和芍药苷均可显著抑制相关炎症介质的释放。进一步机制研究表明,犀角地黄汤和芍药苷上调PC12细胞中TLR4,MyD88,TRAF6和NF-κB表达,降低细胞caspase-3含量,上调cleaved caspase-3水平,对MAPK和Akt相关通路蛋白p-ERK1/2、p-JNK没有显著性影响。研究结论(1)犀角地黄汤能够减轻OGD/R对PC12细胞的损伤,起到神经保护作用;(2)犀角地黄汤通过调控TLR4/MyD88通路,降低炎症因子释放,起到神经保护作用;(3)中医瘀热理论在缺血性卒中的研究中具有重要价值,而犀角地黄汤有望为缺血性卒中提供新的治疗方法。
[Abstract]:Objective to study the neuroprotective effect of Rhino Corner Rehmannia decoction on cerebral ischemia-reperfusion injury in vitro. And the role of TLR4/MyD88 signaling pathway related to inflammation in the neuroprotective effect of Rhino Corner Rehmannia decoction. In order to deepen the theory of traditional Chinese medicine stasis heat in the treatment of ischemic stroke guidance significance, and provide a new strategy for the treatment of ischemic stroke. First of all, research methods. The content of paeoniflorin, paeoniflorin and oxidized paeoniflorin in Rhino Corner Rehmannia decoction was determined by high performance liquid chromatography (HPLC). To establish a quality control analysis method of Rhino Corner Rehmannia decoction. Secondly, PC12 cells were used as the research carrier in this study. PC12 cells were divided into blank control group and model control group by oxygen deprivation and reoxygenation in vitro. XJDH) and Paeoniflorin (Paeoniflorin). The cells of blank control group were cultured normally without OGD/R treatment. The cells of model control group were treated with OGD/R without any drug intervention. Rhino horn Dihuang decoction group was treated with rhino horn Dihuang decoction and cultured in CGD/R condition. Paeoniflorin group added rhino horseradish Dihuang decoction of the active ingredient paeoniflorin in OGD/R culture conditions. After a certain time of incubation with the drug and cells through MTT and LDH release experiment. To investigate the effect of rhino horn and Rehmannia decoction or paeoniflorin on cell viability; The levels of IL-6 and TNF- 伪, and the concentration of caspase-3 in the cells were measured by ELISA method. To investigate the relationship between the neuroprotective effect of rhino horn and dihuang decoction or paeoniflorin on inflammation and apoptosis. By Western Blot experiment, the effects of rhino horn and dihuang decoction or paeoniflorin on TLR4 and MyD88 were studied. The expression levels of NF- 魏 B TRAF6, p-ERKG, p-JNKK, Akt and cleaved caspase-3. HPLC results showed that paeoniflorin and paeoniflorin were found in the decoction. The content of three substances of Fu Fu were 0.232 卤0.031 0.276 卤0.018 and 0.019 卤0.002, respectively. The analytical method was simple, rapid and accurate. It can provide the basis for the quality control of Rhino Corner Rehmannia decoction. After oxygen and glucose deprivation of reoxygenation, compared with the normal culture of PC12 cells. The activity of PC12 cells decreased by 43.75 卤2.4 after OGD. The cell viability was 91.9 卤2.6 and 97.3 卤3.5, respectively. The cell viability was 66.9 卤1.8%. Compared with the model control group, there were significant differences. The results showed that rhino horn and Dihuang decoction could reduce the release of intracellular LDH. To improve PC12 cell damage induced by OGD/R. Elisa data showed that OGD / R could promote the production of 1 L-1 尾 -1 尾 -1L-6 and TNF- 伪. Both rhino horn dihuang decoction and paeoniflorin could significantly inhibit the release of related inflammatory mediators. Further studies showed that rhino horn dihuang decoction and paeoniflorin upregulated TLR4 and MyD88 in PC12 cells. The expression of TRAF6 and NF- 魏 B decreased the content of caspase-3 and up-regulated the level of cleaved caspase-3. MAPK and Akt related pathway protein p-ERK1 / 2 + -JNK has no significant effect. Conclusion: Rhinoceros Cortex Dihuang decoction can reduce the damage of PC12 cells induced by OGD/R. Play a neuroprotective role; (2) Rhinoceros Corner and Rehmannia decoction can reduce the release of inflammatory factors and play a neuroprotective role by regulating the TLR4/MyD88 pathway; 3) the theory of blood stasis and heat in TCM has important value in the study of ischemic stroke, and the decoction of Rhino Corner Dihuang is expected to provide a new treatment method for ischemic stroke.
【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285

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