睡眠呼吸暂停模式间歇低氧大鼠炎症损伤机制、心肌细胞caspase-3表达水平及抗氧化干预的研究

发布时间：2018-05-11 06:32

本文选题：阻塞性睡眠呼吸暂停综合征 + 间歇低氧　；参考：《天津医科大学》2012年博士论文

【摘要】：阻塞性睡眠呼吸暂停综合征(OSAS)是一种伴随心血管疾病严重后果的常见疾病。OSAS心血管并发症的发病机制尚不明确,可能是涉及各种不同机制的多因素过程。OSAS的病理生理特点为间歇低氧,这种低氧再氧合类似于缺血再灌注,会产生炎症反应和组织细胞损伤。因此导致血管内皮损伤和功能紊乱的炎症过程在OSAS心血管并发症的发病机制中可能发挥了关键作用。另外,各种心血管疾病的最终结局为心室重构和心力衰竭,心肌细胞凋亡是心室重构和心力衰竭发生的重要细胞分子学基础,而细胞凋亡的过程实际上是caspases不可逆有限水解底物的级联放大反应过程,caspase-3是caspases级联“瀑布”下游最关键的凋亡蛋白酶。因此,本研究建立不同频率间歇低氧动物模型,观察血清循环炎性标志物水平及心肌组织不同转录因子活性,探讨间歇低氧及低氧频率与炎症反应之间的关系,明确IH诱发炎症损伤的机制；观察IH条件下心肌细胞caspase-3蛋白表达水平,探讨IH诱发心脏损伤的细胞学机制；并应用抗氧化剂干预抑制炎症反应,为OSAS心血管并发症的预防和治疗提供新的方向和实验依据。内容 1.不同频率间歇低氧大鼠炎症损伤机制的研究 2.抗氧化剂Tempol对间歇低氧大鼠炎症损伤的干预效应 3.间歇低氧大鼠心肌细胞caspase-3蛋白表达水平及抗氧化剂Tempol的干预效应方法第一部分：将成年雄性Wistar大鼠分别暴露于不同频率IH(IH1组：10次/h、IH2组：20次/h、IH3组：30次/h、IH4组：40次/h)和持续常氧(SC组)环境下,并采用标准饲养(NC)组进行对照,共暴露6周。应用ELISA法检测血清炎性标志物TNF-α、IL-6、IL-8、CRP、ICAM-1、VEGF和抗炎细胞因子IL-10浓度；应用Western Blot法检测心肌细胞核内转录因子NF-κBP65、心肌组织HIF-1α的蛋白水平,Real-time PCR法检测大鼠心肌组织c-fos mRNA表达水平。第二部分：将IH(频率为30次/h)暴露的成年雄性Wistar大鼠分成两组,应用Tempol分别从暴露开始时及暴露4周后进行早期(]H3T0组)和晚期(IH3T组)干预,共暴露6周。观察上述炎症指标及不同转录因子水平的变化情况,并与IH3组和SC组进行比较。第三部分：对IH3T0组大鼠,应用免疫组化法测定心肌细胞caspase-3蛋白水平,并与IH3组和SC组进行比较。结果第一部分： 1、与SC组、NC组比较,各频率IH组血清炎性标志物TNF-αIL.8、IL-6、CRP、ICAM-1和VEGF水平明显升高,抗炎因子IL.10明显降低,F值分别为9.679、24.461、19.359、30.278、27.318、22.564和20.594,P值均为0.000。 2、与SC组、NC组比较,各频率IH组心肌细胞核内NF-κBP65、心肌组织HIF.1α、c-fos mRNA水平明显升高,F值分别为35.089、24.934和22.950,P值均为0.000。 3、不同频率IH组间比较,随频率增高,TNF-α、IL-8、IL-6、CRP和ICAM-1水平以及NF-κBP65、c-fos mRNA水平逐渐升高(11-13组与IH1组比较均P0.01),IL.10逐渐降低(IH3组与IH1组比较P0.01)。但IH3组与IH4组间上述指标没有差异(P0.05),峰值(或谷值)在频率为30次/h时出现。NF-κBP65水平与TNF-α、IL-8、IL-6、CRP和ICAM-1水平呈正相关(r值分别为0.519、0.574、0.608、0.659和0.576,P值分别为0.002、0.001、0.000、0.000和0.003)；与IL-10水平呈负相关(r=-0.618,P=0.000)。 4、不同频率IH组间比较,随频率增高,VEGF水平及HIF.1α水平持续升高。HIF.1α水平与VEGF水平呈正相关(r=0.661,P=0.000)。第二部分： 1、IH3T0组和IH3T组上述炎症标志物及转录因子水平低于IH3组,抗炎因子水平高于IH3组。 2、IH3T0组和IH3T组上述炎症标志物、抗炎因子及转录因子水平与SC组比较差异有统计学意义(P0.05)。第三部分： 1、与SC组相比,IH3组心肌细胞caspase-3蛋白水平显著增高(P0.01)。 2、IH3T0组心肌细胞caspase-3蛋白水平均明显低于IH3组(P0.01)。 3、IH3组心肌细胞caspase-3表达水平与NF-κd3P65蛋白水平呈正相关(r=0.864,P值分别为0.006)。结论 1.IH可以导致炎症和抗炎系统失衡,从而引起系统性和心脏组织局部炎症损伤。 2.IH引起的炎症损伤有一定的间歇低氧频率依赖性。但炎症反应与间歇低氧频率之间并不呈线性关系,炎症反应随频率增高逐渐增强,到一定频率后不再增高,说明机体代偿机制和适应性反应的存在。 3.IH可引起不同转录因子的激活,主要包括NF-κB, HIF-1α和AP-1,其中中低度IH频率优势激活NF-κB、AP-1介导的炎症通路,启动并放大炎症反应；而过高IH频率则优势激活HIF-1α介导的适应性通路,这有可能是机体的保护性机制。 4.1H可能通过活化NF-κB,转录调节促凋亡基因的表达,介导细胞凋亡通路中间蛋白caspase-3的活化。 5.抗氧化剂通过调节氧化/抗氧化平衡来调节炎症/抗炎系统平衡,可以减轻IH诱导的炎症损伤,抑制心肌细胞凋亡,对IH损伤有保护效应。
[Abstract]:Obstructive sleep apnea syndrome (OSAS) is a common disease associated with serious cardiovascular disease, the pathogenesis of.OSAS cardiovascular complications is not clear. It may be the pathophysiological characteristics of.OSAS, which involves various mechanisms of various mechanisms, which is characterized by intermittent hypoxia. This hypoxic reoxygenation is similar to that of ischemia reperfusion. Inflammatory reactions and tissue cell damage. Therefore, the inflammatory processes that lead to vascular endothelial damage and dysfunction may play a key role in the pathogenesis of OSAS cardiovascular complications. In addition, the final outcome of various cardiovascular diseases is ventricular remodeling and heart failure, and myocardial apoptosis is the reconstitution of ventricular remodeling and heart failure. The process of cell apoptosis is in fact the cascade amplification reaction process of caspases irreversible limited hydrolytic substrates, and caspase-3 is the most critical apoptotic protease in the cascade cascade of cascades in the caspases cascade. Therefore, this study establishes a different frequency intermittent hypoxia animal model to observe the level of serum circulating inflammatory markers and to observe the level of the serum circulating inflammatory markers and to observe the level of the serum circulating inflammatory markers and to observe the level of the serum circulating inflammatory markers. The activity of different transcription factors in myocardium was used to explore the relationship between intermittent hypoxia and hypoxia frequency and inflammatory response, and to clarify the mechanism of IH induced inflammatory damage. The expression of caspase-3 protein in cardiac myocytes under IH conditions was observed and the cytological mechanism of IH induced heart damage should be explored, and the anti inflammatory intervention should be used to inhibit the inflammatory reaction, which was the OSAS heart. It provides a new direction and experimental basis for prevention and treatment of vascular complications.
content
1. the mechanism of inflammatory injury induced by intermittent hypoxia in rats at different frequencies.
2. intervention effect of antioxidant Tempol on inflammatory injury induced by intermittent hypoxia in rats
3. the expression level of caspase-3 protein in myocardial cells of intermittent hypoxia rats and the intervention effect of antioxidant Tempol
Method
Part 1: adult male Wistar rats were exposed to different frequencies of IH (group IH1: 10 times /h, IH2 group: 20 /h, IH3 group, 30 /h, IH4 group: 40 /h) and constant oxygen (SC group) environment, and the standard feeding (NC) group was exposed for 6 weeks. The concentration of F and anti inflammatory cytokine IL-10, the Western Blot method was used to detect the transcription factor NF- kappa BP65, the protein level of HIF-1 a in myocardium, and Real-time PCR method was used to detect c-fos mRNA expression level in rat myocardium.
The second part: the adult male Wistar rats exposed to IH (frequency 30 times /h) were divided into two groups. Tempol was exposed to early (]H3T0) and late (IH3T) for 6 weeks from exposure to exposure and 4 weeks after exposure. The changes of the above inflammatory indices and different transcriptional levels were observed and compared with those in the IH3 group and the SC group.
The third part: in group IH3T0, the level of caspase-3 protein in myocardial cells was detected by immunohistochemistry, and compared with group IH3 and SC.
Result
Part one:
1, compared with group SC and group NC, the levels of serum inflammatory markers, TNF- a IL.8, IL-6, CRP, ICAM-1 and VEGF were significantly increased in each frequency IH group, and the anti inflammatory factor IL.10 decreased obviously, and F was 9.679,24.461,19.359,30.278,27.318,22.564 and 20.594.
2, compared with group SC and group NC, NF- kappa BP65, HIF.1 A and c-fos mRNA in myocardial nuclei of group IH were significantly increased, F values were 35.089,24.934 and 22.950 respectively, P values were 0.000..
3, compared with the frequency of IH, the level of TNF- alpha, IL-8, IL-6, CRP and ICAM-1, NF- kappa BP65, c-fos mRNA increased gradually with the frequency of different frequencies, and the level of c-fos mRNA increased gradually (compared with the IH1 group). There is a positive correlation between the level of.NF- kappa BP65 and the levels of TNF- alpha, IL-8, IL-6, CRP and ICAM-1 (R values are 0.519,0.574,0.608,0.659 and 0.576 respectively, P values are 0.002,0.001,0.000,0.000 and 0.003 respectively), and are negatively correlated with IL-10 levels.
4, compared with the IH group, with the increase of frequency, the level of VEGF and HIF.1 alpha continued to rise. The level of.HIF.1 alpha was positively correlated with the level of VEGF (r=0.661, P=0.000).
The second part:
1, the levels of inflammatory markers and transcription factors in group IH3T0 and group IH3T were lower than those in group IH3, and the levels of anti-inflammatory factors were higher than those in group IH3.
2, the levels of inflammatory markers, anti-inflammatory factors and transcription factors in group IH3T0 and group IH3T were significantly different from those in group SC (P0.05).
The third part:
1, compared with group SC, the level of caspase-3 protein in myocardial cells of IH3 group was significantly higher (P0.01).
2, the level of caspase-3 protein in myocardial cells of group IH3T0 was significantly lower than that of group IH3 (P0.01).
3, the expression level of Caspase-3 in myocardial cells of IH3 group was positively correlated with the level of NF- kappa d3P65 protein (r=0.864, P value was 0.006).
conclusion
1.IH can lead to imbalance between inflammatory and anti-inflammatory systems, resulting in systemic and cardiac tissue inflammatory injury.
The inflammatory injury caused by 2.IH has a certain frequency dependence of intermittent hypoxia. However, there is no linear relationship between the inflammatory response and the frequency of intermittent hypoxia. The inflammatory response increases gradually with the increase of frequency, and no longer increases after a certain frequency, indicating the survival of the body's compensatory mechanism and the adaptive response.
3.IH can cause activation of different transcription factors, mainly including NF- kappa B, HIF-1 alpha and AP-1, in which the frequency of middle and low IH activates NF- kappa B, AP-1 mediated inflammatory pathways, initiates and amplifies the inflammatory response, while the frequency of high IH activates the adaptive pathway mediated by HIF-1 alpha, which may be the protective mechanism of the organism.
4.1H may regulate the expression of Pro apoptotic genes through activation of NF- kappa B, and mediate the activation of intermediate protein caspase-3 in apoptotic pathway.
5. antioxidants regulate inflammatory / anti-inflammatory balance by regulating oxidation / antioxidant balance, which can reduce the inflammatory damage induced by IH, inhibit the apoptosis of myocardial cells and protect the IH damage.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R766

【引证文献】