基于miR-146a、miR-155调控NF-κB信号通路探讨芍药甘草汤治疗颈型颈椎病兔颈肌炎症损伤的作用机制

发布时间：2018-01-10 16:33

本文关键词：基于miR-146a、miR-155调控NF-κB信号通路探讨芍药甘草汤治疗颈型颈椎病兔颈肌炎症损伤的作用机制　出处：《福建中医药大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:通过探讨芍药甘草汤对颈型颈椎病模型兔颈部肌肉组织形态学的改变情况、血清炎症因子TNF-α、IL-1β、IL-6表达水平的改变情况,以及颈部肌肉组织中miR-146a/-155-NF-κB信号通路相关因子表达的影响,揭示芍药甘草汤治疗颈型颈椎病的可能作用机制。方法:30只6月龄新西兰大白兔随机分为高剂量芍药甘草汤组、中剂量芍药甘草汤组、低剂量芍药甘草汤组、模型组和正常组,共5组,每组均6只兔子。除正常组外,其余4组均参照张欣等无创兔颈型颈椎病动物模型的建模方法进行改良造模,8周后分别对5组兔进行相应的干预。高、中、低剂量组兔分别给予相应的高、中、低剂量芍药甘草汤进行灌胃治疗,每只兔每次的灌胃剂量是80ml,每日早、晚各灌胃1次;模型组、正常组不予任何干预措施,正常饲养。连续干预4周,结束后第二天,对五组兔均进行耳缘静脉采血,离心后取血液上清,备用于ELISA检测血清炎症因子TNF-α IL-1β、IL-6的表达水平。采血后处死各组兔子,取其颈后肌组织进行各项指标的检测:颈后肌组织HE染色后形态学观察和比较;Western blotting检测标本中信号通路关键节点MyD88、NF-κBP65 的蛋白表达量;RT-qPCR检测标本中 TLR4、TRAF-6、miR-146a、miR-155的表达。结果:1.颈后肌组织大体肉眼观:与正常组比较,模型组和高、中、低剂量组均出现了不同程度的炎症损伤表现,且模型组损伤程度比高、中、低剂量组重。说明芍药甘草汤治疗颈型颈椎病是有效的。2.形态学比较,颈后肌组织退变程度由轻至重依次是正常组、高剂量组、中剂量组、低剂量组、模型组。说明芍药甘草汤干预可以减轻颈后肌组织炎症损伤程度,且呈剂量依赖性。3.血清炎症因子IL-6、IL-1β、TNF-α表达水平比较:干预前,模型组、高、中、低剂量组与正常组相比,各组间对照均具有显著性统计学意义(P0.01),而模型组、高、中、低剂量组之间进行比较均无差异性(P0.05),说明本次实验造模成功;干预后,高、中、低剂量组的表达水平均明显下降,且模型组与正常组相比有显著差异性(P0.01),模型组、高、中、低剂量组之间相比较,各组间对照均具有显著性统计学意义(P0.01),说明芍药甘草汤可以降低血清炎症因子IL-6、IL-1β、TNF-α的表达水平,且呈剂量依赖性。4.颈后肌组织中MyD88、NF-κBP65蛋白表达比较:干预后,低、中、高剂量组的表达均降低,且模型组与正常组相比具有显著性统计学意义(P0.01),模型组、低、中、高剂量组之间相比,各组间对照也均具有统计学意义(P0.05)。说明芍药甘草汤可以降低颈后肌组织中MyD88、NF-κBP65的蛋白表达,且呈剂量依赖性。5.颈后肌标本中miR-146a、miR-155、TLR4、TRAF-6表达水平比较:干预后,低、中、高剂量组miR-146a的表达水平均升高,miR-155、TLR4、TRAF-6的表达水平均降低,且模型组与正常组相比有显著性统计学意义(P0.01),模型组、低、中、高剂量组之间进行比较,各组间对照也均具有统计学意义(P0.05)。说明芍药甘草汤可以降低颈后肌组织中miR-155、TLR4、TRAF-6的表达,升高miR-146a的表达,且均呈剂量依赖性。结论:1.高、中、低这三种不同剂量的芍药甘草汤治疗方法均对缓解兔颈型颈椎病的炎症损伤有效,且其效果呈剂量依赖性:高剂量组中剂量组低剂量组模型组。2.芍药甘草汤治疗兔颈型颈椎病的可能作用机制之一是通过升高颈后肌组织中miR-146a的表达、降低miR-155的表达而抑制NF-κB信号通路释放TNF-α、IL-1β、IL-6等炎症因子,从而改善颈部炎症损伤,治疗颈型颈椎病。
[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.

【学位授予单位】：福建中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5

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