本文關(guān)鍵詞：犀角地黃湯對缺血性卒中體外炎癥反應(yīng)信號通路的干預(yù)研究　出處：《南京中醫(yī)藥大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 缺血性卒中 犀角地黃湯 氧糖剝奪/復(fù)氧 PC12細(xì)胞 炎癥 TLR信號通路

【摘要】：研究目的從體外實(shí)驗(yàn)的角度,研究犀角地黃湯對腦缺血再灌注損傷的神經(jīng)保護(hù)作用,以及與炎癥反應(yīng)相關(guān)的TLR4/MyD88信號通路在犀角地黃湯神經(jīng)保護(hù)效應(yīng)中的作用。通過本課題的研究,以期深化中醫(yī)瘀熱理論在缺血性卒中治療中的指導(dǎo)意義,并為缺血性卒中的治療提供新的策略。研究方法首先,本課題采用高效液相色譜法(HPLC)同時測定犀角地黃湯中主要成分芍藥苷、芍藥內(nèi)酯苷、氧化芍藥苷的含量,建立犀角地黃湯的質(zhì)量控制分析方法。其次,本研究以PC12細(xì)胞為研究載體,在體外通過氧糖剝奪和復(fù)氧方法模擬腦缺血再灌注損傷。實(shí)驗(yàn)中將PC12細(xì)胞分為空白對照組(control)、模型對照組(OGD/R)、犀角地黃湯組(XJDH)和芍藥苷組(Paeoniflorin)�？瞻讓φ战M細(xì)胞正常培養(yǎng),不經(jīng)過OGD/R處理;模型對照組細(xì)胞經(jīng)OGD/R處理,但不加任何藥物干預(yù);犀角地黃湯組是加入犀角地黃湯干預(yù),在CGD/R條件下培養(yǎng)細(xì)胞;芍藥苷組是加入犀角地黃湯的活性成分芍藥苷,在OGD/R條件下培養(yǎng)細(xì)胞。藥物與細(xì)胞孵育一定時間后,通過MTT實(shí)驗(yàn)及LDH釋放實(shí)驗(yàn),考察犀角地黃湯或芍藥苷對細(xì)胞活力的影響;ELISA法測定各組細(xì)胞炎癥因子IL-1β、IL-6和TNF-α的釋放水平,并檢測細(xì)胞內(nèi)caspase-3的濃度,考察犀角地黃湯或芍藥苷神經(jīng)保護(hù)作用與炎癥、凋亡的關(guān)系。通過Western Blot實(shí)驗(yàn),研究犀角地黃湯或芍藥苷對TLR4、MyD88、NF-κB、TRAF6、p-ERK、p-JNK、Akt和cleaved caspase-3等蛋白表達(dá)水平的影響,初步探討犀角地黃湯神經(jīng)保護(hù)作用的可能機(jī)制。研究結(jié)果HPLC實(shí)驗(yàn)結(jié)果顯示,犀角地黃湯中芍藥苷、芍藥內(nèi)酯苷、氧化芍藥苻三種物質(zhì)的含量分別為0.232±0.031%、0.276±0.018%、0.019±0.002%。所建的分析方法操作簡便快速準(zhǔn)確,可為犀角地黃湯的質(zhì)量控制提供依據(jù)。氧葡萄糖剝奪復(fù)氧后,與正常培養(yǎng)PC12細(xì)胞相比,OGD后PC12細(xì)胞活性下降43.75±2.4%。提前給予0.4,0.2,0.1 mg/mL犀角地黃湯處理,細(xì)胞活力分別為91.9 ± 2.6%,97.3 ± 3.5%,66.9 ± 1.8%,與模型對照組比較均具有顯著性差異。LDH檢測實(shí)驗(yàn)結(jié)果表明,犀角地黃湯可以減少細(xì)胞內(nèi)LDH的釋放,改善OGD/R導(dǎo)致的PC12細(xì)胞損傷。ELISA實(shí)驗(yàn)數(shù)據(jù)表明,OGD/R能夠促進(jìn)炎癥介質(zhì)1L-1β、1L-6和TNF-α的產(chǎn)生,而犀角地黃湯和芍藥苷均可顯著抑制相關(guān)炎癥介質(zhì)的釋放。進(jìn)一步機(jī)制研究表明,犀角地黃湯和芍藥苷上調(diào)PC12細(xì)胞中TLR4,MyD88,TRAF6和NF-κB表達(dá),降低細(xì)胞caspase-3含量,上調(diào)cleaved caspase-3水平,對MAPK和Akt相關(guān)通路蛋白p-ERK1/2、p-JNK沒有顯著性影響。研究結(jié)論(1)犀角地黃湯能夠減輕OGD/R對PC12細(xì)胞的損傷,起到神經(jīng)保護(hù)作用;(2)犀角地黃湯通過調(diào)控TLR4/MyD88通路,降低炎癥因子釋放,起到神經(jīng)保護(hù)作用;(3)中醫(yī)瘀熱理論在缺血性卒中的研究中具有重要價值,而犀角地黃湯有望為缺血性卒中提供新的治療方法。
[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- 偽, 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 鹵0.031 0.276 鹵0.018 and 0.019 鹵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 鹵2.4 after OGD. The cell viability was 91.9 鹵2.6 and 97.3 鹵3.5, respectively. The cell viability was 66.9 鹵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- 偽. 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.
【學(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R285

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