本文關(guān)鍵詞：基于miR-146a、miR-155調(diào)控NF-κB信號通路探討芍藥甘草湯治療頸型頸椎病兔頸肌炎癥損傷的作用機制　出處：《福建中醫(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： 芍藥甘草湯 頸型頸椎病 NF-κB 炎癥因子 miRNA

【摘要】：目的:通過探討芍藥甘草湯對頸型頸椎病模型兔頸部肌肉組織形態(tài)學(xué)的改變情況、血清炎癥因子TNF-α、IL-1β、IL-6表達(dá)水平的改變情況,以及頸部肌肉組織中miR-146a/-155-NF-κB信號通路相關(guān)因子表達(dá)的影響,揭示芍藥甘草湯治療頸型頸椎病的可能作用機制。方法:30只6月齡新西蘭大白兔隨機分為高劑量芍藥甘草湯組、中劑量芍藥甘草湯組、低劑量芍藥甘草湯組、模型組和正常組,共5組,每組均6只兔子。除正常組外,其余4組均參照張欣等無創(chuàng)兔頸型頸椎病動物模型的建模方法進(jìn)行改良造模,8周后分別對5組兔進(jìn)行相應(yīng)的干預(yù)。高、中、低劑量組兔分別給予相應(yīng)的高、中、低劑量芍藥甘草湯進(jìn)行灌胃治療,每只兔每次的灌胃劑量是80ml,每日早、晚各灌胃1次;模型組、正常組不予任何干預(yù)措施,正常飼養(yǎng)。連續(xù)干預(yù)4周,結(jié)束后第二天,對五組兔均進(jìn)行耳緣靜脈采血,離心后取血液上清,備用于ELISA檢測血清炎癥因子TNF-α IL-1β、IL-6的表達(dá)水平。采血后處死各組兔子,取其頸后肌組織進(jìn)行各項指標(biāo)的檢測:頸后肌組織HE染色后形態(tài)學(xué)觀察和比較;Western blotting檢測標(biāo)本中信號通路關(guān)鍵節(jié)點MyD88、NF-κBP65 的蛋白表達(dá)量;RT-qPCR檢測標(biāo)本中 TLR4、TRAF-6、miR-146a、miR-155的表達(dá)。結(jié)果:1.頸后肌組織大體肉眼觀:與正常組比較,模型組和高、中、低劑量組均出現(xiàn)了不同程度的炎癥損傷表現(xiàn),且模型組損傷程度比高、中、低劑量組重。說明芍藥甘草湯治療頸型頸椎病是有效的。2.形態(tài)學(xué)比較,頸后肌組織退變程度由輕至重依次是正常組、高劑量組、中劑量組、低劑量組、模型組。說明芍藥甘草湯干預(yù)可以減輕頸后肌組織炎癥損傷程度,且呈劑量依賴性。3.血清炎癥因子IL-6、IL-1β、TNF-α表達(dá)水平比較:干預(yù)前,模型組、高、中、低劑量組與正常組相比,各組間對照均具有顯著性統(tǒng)計學(xué)意義(P0.01),而模型組、高、中、低劑量組之間進(jìn)行比較均無差異性(P0.05),說明本次實驗造模成功;干預(yù)后,高、中、低劑量組的表達(dá)水平均明顯下降,且模型組與正常組相比有顯著差異性(P0.01),模型組、高、中、低劑量組之間相比較,各組間對照均具有顯著性統(tǒng)計學(xué)意義(P0.01),說明芍藥甘草湯可以降低血清炎癥因子IL-6、IL-1β、TNF-α的表達(dá)水平,且呈劑量依賴性。4.頸后肌組織中MyD88、NF-κBP65蛋白表達(dá)比較:干預(yù)后,低、中、高劑量組的表達(dá)均降低,且模型組與正常組相比具有顯著性統(tǒng)計學(xué)意義(P0.01),模型組、低、中、高劑量組之間相比,各組間對照也均具有統(tǒng)計學(xué)意義(P0.05)。說明芍藥甘草湯可以降低頸后肌組織中MyD88、NF-κBP65的蛋白表達(dá),且呈劑量依賴性。5.頸后肌標(biāo)本中miR-146a、miR-155、TLR4、TRAF-6表達(dá)水平比較:干預(yù)后,低、中、高劑量組miR-146a的表達(dá)水平均升高,miR-155、TLR4、TRAF-6的表達(dá)水平均降低,且模型組與正常組相比有顯著性統(tǒng)計學(xué)意義(P0.01),模型組、低、中、高劑量組之間進(jìn)行比較,各組間對照也均具有統(tǒng)計學(xué)意義(P0.05)。說明芍藥甘草湯可以降低頸后肌組織中miR-155、TLR4、TRAF-6的表達(dá),升高miR-146a的表達(dá),且均呈劑量依賴性。結(jié)論:1.高、中、低這三種不同劑量的芍藥甘草湯治療方法均對緩解兔頸型頸椎病的炎癥損傷有效,且其效果呈劑量依賴性:高劑量組中劑量組低劑量組模型組。2.芍藥甘草湯治療兔頸型頸椎病的可能作用機制之一是通過升高頸后肌組織中miR-146a的表達(dá)、降低miR-155的表達(dá)而抑制NF-κB信號通路釋放TNF-α、IL-1β、IL-6等炎癥因子,從而改善頸部炎癥損傷,治療頸型頸椎病。
[Abstract]:Objective: To investigate the changes of morphology of Shaoyaogancao Decoction cervical spondylosis model of rabbit cervical muscle tissue, serum inflammatory factor TNF- alpha, IL-1 beta, the change of the expression level of IL-6, and the expression of miR-146a/-155-NF- in tissues of cervical muscle B signaling pathway related factors, may reveal the mechanism of Shaoyaogancao Decoction in the treatment of neck type cervical spondylosis. Methods: 30 June old New Zealand rabbits were randomly divided into high dose of Shaoyaogancao Decoction group, middle dose of Shaoyaogancao Decoction group, low dose of Shaoyaogancao Decoction group, model group and normal group, a total of 5 groups, each group had 6 rabbits. Except the normal group, the remaining 4 groups are referenced Zhang Xin had no record of cervical spondylosis animal modeling model of rabbit carotid type modified model, after 8 weeks respectively for the 5 groups of rabbits were corresponding intervention. High, low dose group rabbits were given corresponding high, low dose of Shaoyaogancao Decoction by intragastric administration For each rabbit, every time the gavage dose is 80ml, daily early, late each gavage for 1 times; the model group, the normal group without any intervention, normal feeding. Continuous intervention for 4 weeks, second days after the end of the five group of rabbit ear vein blood were centrifuged, supernatant of blood, preparation for the detection of ELISA serum inflammatory factor TNF- alpha IL-1 beta, the expression level of IL-6. The blood of each group were sacrificed after the rabbit, the neck muscle index detection: after morphological observation and comparison of muscle tissue HE staining after carotid; Western signal pathway of blotting detected in the key nodes of MyD88, expression of NF- kappa BP65 protein TLR4; RT-qPCR, miR-146a, TRAF-6 were detected, the expression of miR-155. Results: 1. neck muscle macroscopic view: compared with the normal group, model group and high, low dose group, there are different degrees of inflammation is the degree of injury ratio in the model group, In the low dose group. Description of Shaoyaogancao Decoction in the treatment of cervical spondylosis is.2. morphology effectively, neck muscle degeneration from light to heavy in the normal group, high dose group, middle dose group, low dose group, model group. The results showed that Shaoyaogancao decoction can interfere with the degree of inflammatory injury of muscle tissue reduce the neck after a dose dependent IL-6, serum inflammatory factor.3. IL-1 beta, TNF- alpha expression level comparison: before the intervention, the model group, high, low dose group compared with normal group, the control group were significant statistical significance (P0.01), and model group, high. Between the low dose group showed no difference (P0.05), means that the model was successful; intervention, high expression level in low dose group were significantly decreased in the model group compared with normal group had significant difference (P0.01), model group, high, low dose compared to the group, among the groups of As it showed statistically significant (P0.01), that Shaoyaogancao decoction can reduce serum inflammatory cytokines IL-6, IL-1 beta, the expression level of TNF- alpha, dose-dependent MyD88.4. neck muscle, comparison of expression of NF- kappa BP65 protein: intervention, low expression in high dose group were to reduce, and the model group compared with the normal group had statistical significance (P0.01), model group, low, compared between the high dose group, control groups were statistically significant (P0.05). The results showed that Shaoyaogancao decoction can reduce MyD88 neck muscle, the expression of NF- kappa BP65 protein, and in a dose dependent manner after carotid.5. miR-146a, miR-155 TLR4, muscle specimens, compare the expression level of TRAF-6: after the intervention, low, high dose group, the expression level of miR-146a was increased, miR-155, TLR4, TRAF-6 expression levels were decreased, and the model group compared with normal group had significant statistical significance Yi (P0.01), model group, low, were compared between high dose group and control among the three groups were statistically significant (P0.05). The results showed that Shaoyaogancao decoction can reduce miR-155, neck muscle tissue TLR4, TRAF-6 expression increased the expression of miR-146a, and were in a dose-dependent manner. Conclusion: 1. high, low, the three different doses of Shaoyaogancao decoction treatment was effective for relieving inflammatory injury of rabbit cervical spondylosis, and the effect is dose dependent: one of the possible mechanism of high dose group, middle dose group and low dose group, model group.2. of Shaoyaogancao Decoction in the treatment of rabbit neck type cervical spondylosis the expression of miR-146a is increased by the neck muscle tissue, decreased the expression of miR-155 and inhibition of NF- B signaling pathway and release of TNF- alpha, IL-1 beta, IL-6 and other inflammatory factors, so as to improve the treatment of cervical inflammation, cervical spondylopathy.

【學(xué)位授予單位】：福建中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R285.5

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