不同频率间歇低氧下大鼠肝脏损伤机制及抗氧化剂Tempol的干预作用

发布时间：2018-06-01 19:10

本文选题：睡眠呼吸暂停 + 间歇低氧　；参考：《天津医科大学》2012年硕士论文

【摘要】：研究背景与目的阻塞性睡眠呼吸暂停综合征(Obstructive sleep apnea syndrome, OSAS)是以睡眠状态下反复出现的呼吸暂停和血氧饱和度下降为特征,即慢性间歇低氧(Chronic intermittent hypoxia, CIH), CIH是OSAS造成机体损害的主要病理生理学基础。OSAS是一种氧化应激性疾病,同时也是一种炎症性疾病。目前研究认为,OSAS模式的CIH会导致肝脏细胞呈现氧化应激状态进而激活NF-κB及其介导的炎性通路引起一系列炎症介质的变化,最终导致肝脏损害,引起非酒精性脂肪性肝病(Nonalcoholic fatty liver disease, NAFLD),甚至非酒精性脂肪性肝炎(Nonalcoholic steatohepatitis, NASH)。为此,本研究模拟OSAS建立CIH大鼠模型,观察不同频率CIH下大鼠肝脏损伤情况和可能机制及抗氧化剂Tempol的干预作用,以便为临床上预防和治疗OSAS器官合并症提供探索和理论依据。内容 1.不同频率间歇低氧下大鼠肝脏氧化应激和炎症反应的研究 2.抗氧化剂Tempol对CIH下大鼠肝脏损伤的干预作用方法通过向计算机控制的低氧舱中,循环充入不同时间的纯氮气和正常氧浓度的压缩空气,分别造成不同频率的间歇低氧(10次/h、20次/h、30次/h、40次/h)及持续常氧环境。将72只雄性成年Wistar大鼠随机分为间歇低氧组(IH1、IH2、IH3、IH4,频率依次为10、20、30、40次/h)；Tempol干预组：30T1组(30次/h间歇低氧暴露开始第1天即给予Tempol干预,Tempol剂量为：100mg×kg-1×d+生理盐水稀释后腹腔注射,稀释比例为Tempol:生理盐水=1：10,即1ml10%Tempol/Kg体重),30T2组(30次/h间歇低氧暴露第29天开始给予Tempol干预,Tempol剂量同30T1组),生理盐水对照组：30N1组(30次/h间歇低氧暴露开始第1天即给予等体积生理盐水做对照,即1ml0.9%NS/Kg体重),30N2组(30次/h间歇低氧暴露第29天开始给予等体积生理盐水做对照),以及常氧对照组(NC组),共9组,每组8只大鼠,间歇低氧暴露环境最低氧浓度均为5%,常氧对照组始终维持21%的氧浓度。Tempol和生理盐水均于大鼠每天进入低氧舱前半小时腹腔注射,每天1次。暴露6周结束后,各组大鼠均麻醉后股动脉放血处死取出肝脏。肝脏石蜡切片HE染色观察肝细胞形态变化,化学法测定肝脏匀浆中SOD、T-AOC的活性,ELISA法测定MDA、GSH-PX、NF-κB、 ICAM-1的水平。结果第一部分 1.肝脏组织病理：NC组未见明显肝脏损害,不同频率IH组均有部分肝组织细胞胞浆稀疏,核深染,随着低氧频率的升高肝脏出现细胞水肿,甚至少量炎性细胞浸润。表明慢性间歇低氧可以导致大鼠肝脏损害的发生 2.不同频率间歇低氧组和常氧对照组大鼠肝脏MDA、SOD、GSH-PX、T-AOC水平比较：不同频率IH组MDA水平均高于NC组(P均0.05),SOD、 GSH-PX、T-AOC活性均低于NC组(P均0.05)；除IH3组与IH4组比较以上指标均无显著差异外(P分别为0.552,0.700,0.737,0.947),其余IH组两两比较以上指标差异均有统计学意义(P均0.05),且随着低氧频率的升高,MDA水平逐渐升高,SOD、GSH-PX、T-AOC活性逐渐下降。 3.不同频率间歇低氧组和常氧对照组大鼠肝脏NF-κB、ICAM-1水平比较：除IH1组与NC组比较NF-κB、ICAM-1水平差异无统计学意义外(P分别为0.143,0.633),其余IH组NF-κB、ICAM-1水平均高于NC组(P均0.05)；除IH3组与IH4组比较NF-κB水平及IH1组和IH2组、IH3组和IH4组比较ICAM-1水平差异无统计学意义外(P分别为0.103,0.06,0.524),其余IH组两两比较NF-κB ICAM-1水平差异均有统计学意义(P均0.05),且随着低氧频率的升高,NF-κB、ICAM-1水平也逐渐升高。 4.双变量相关分析法显示：MDA与SOD、GSH-PX、T-AOC均呈负相关关系(r分别为-0.721,-0.713,-0.653,P均为0.000),NF-κB与MDA、GSH-PX分别呈正相关(r=0.789,P=0.000)、负相关(r=-0.766,P=0.000)关系,NF-κB与ICAM-1呈正相关关系(r=0.667,P=0.000)。第二部分 1.肝脏组织病理：30T1组、NC组未见明显肝脏损害,30T2组可见轻微肝细胞胞膜破坏,胞浆稀疏,IH3组、30N1组、30N2组可见肝细胞胞膜破坏,细胞水肿,胞浆稀疏,核深染。表明Tempol可以预防慢性间歇低氧下大鼠肝脏损伤的发生,且干预越早,作用越明显。 2. Tempol干预组、生理盐水对照组和常氧对照组大鼠肝脏MDA、SOD、GSH-PX、T-AOC水平比较：30T1组、30T2组较IH3组、30N1组、30N2组MDA水平均下降(P均0.05),SOD、GSH-PX、T-AOC活性均升高(P均0.05)；且30T1组与NC组比较以上指标均无显著差异(P分别为0.209,0.117,0.222,0.413)；但30T1组和30T2组比较以及30T2组和NC组比较以上指标差异均有统计学意义(P均0.05)；IH3组、30N1组、30N2组两两比较以上指标差异均无统计学意义(P均0.05)。 3. Tempol干预组、生理盐水对照组和常氧对照组大鼠肝脏NF-κB、ICAM-1水平比较：30T1组、30T2组较IH3组、30N1组、30N2组NF-κB、ICAM-1水平均下降(P均0.05)；30T1组、30T2组、NC组两两比较NF-κB、ICAM-1水平差异均有统计学意义(P均0.05),IH3组、30N1组、30N2组两两比较上述指标差异均无统计学意义(P均0.05)。结论 1.不同频率CIH通过氧化应激和炎症反应共同造成大鼠肝脏损害,且随着间歇低氧频率的增加肝脏损伤也逐渐加重,但并不是频率越高,肝脏损伤越重,即肝脏损伤程度与CIH频率并不完全呈线性关系。 2.CIH过程中产生的ROS的蓄积进而引起的氧化应激反应可能作为始动因素进而激活氧化还原敏感转录因子NF-κB及其介导的炎症通路,影响炎症介质的释放,二者共同造成CIH下大鼠肝脏损伤的发生 3.抗氧化剂Tempol可以干预CIH所导致的氧化应激反应的发生,进而抑制NF-κB的激活,预防和缓解CIH肝损害,但尚不能完全逆转既存的肝损害。
[Abstract]:Research background and purpose
Obstructive sleep apnea syndrome (OSAS) is characterized by recurrent apnea and decreased oxygen saturation in the state of sleep, namely, chronic intermittent hypoxia (Chronic intermittent hypoxia, CIH), CIH is the main pathophysiological basis for OSAS to cause damage to the body, and.OSAS is a kind of oxidative stress. OSAS model CIH can cause oxidative stress in liver cells and activate NF- kappa B and its mediated inflammatory pathway to cause a series of inflammatory mediators, resulting in liver damage and non alcoholic fatty liver disease (Nonalcoholic fatty liver diseas). E, NAFLD), even nonalcoholic steatohepatitis (Nonalcoholic steatohepatitis, NASH). To this end, this study simulated OSAS to establish a CIH rat model to observe the liver damage and possible mechanism under the different frequencies of CIH and the possible mechanism of the antioxidant Tempol, so as to provide exploration and treatment for the clinical prevention and treatment of OSAS organ complication. On the basis.
content
1. oxidative stress and inflammatory response in rats liver under intermittent hypoxia with different frequencies
2. intervention effect of antioxidant Tempol on liver injury induced by CIH in rats
Method
In a computer controlled hypoxic chamber, the cycle was filled with compressed air of pure nitrogen and normal oxygen concentration at different times, resulting in intermittent hypoxia at different frequencies (10 /h, 20 times /h, 30 /h, 40 /h) and continuous constant oxygen environment. 72 male adult Wistar rats were divided into intermittent hypoxia group (IH1, IH2, IH3, IH4, and the frequency was 10,2). 0,30,40 /h); Tempol intervention group: 30T1 group (30 times /h intermittent hypoxia exposure began to give Tempol intervention, Tempol dose was: 100mg * kg-1 x d+ saline diluted peritoneal injection, dilution ratio was Tempol: physiological saline =1:10, namely weight). Pre, Tempol dose and 30T1 group), saline control group: group 30N1 (first days of 30 /h intermittent hypoxia exposure, i. e. 1ml0.9%NS/Kg weight), 30N2 group (30 times of /h intermittent hypoxia exposure to equal volume of normal saline as control), and normal oxygen control group (NC group), a total of 9 groups, 8 rats in each group. The lowest oxygen concentration in the intermittent hypoxic environment was 5%, and the oxygen concentration of 21% in the normal oxygen control group was always maintained at.Tempol and the normal saline was intraperitoneally injected into the abdominal cavity for 1 times a day before the hypoxic capsule was entered. After 6 weeks of exposure, the rats in each group were killed and removed from the liver after the anaesthesia. The liver paraffin section HE staining was used to observe the liver fine. Cell morphology changes, the activity of SOD and T-AOC in liver homogenate were measured by chemical method, and the levels of MDA, GSH-PX, NF- B and ICAM-1 were measured by ELISA.
Result
Part one
1. liver histopathology: no obvious liver damage was found in group NC. There were some liver tissue cells with sparse cytoplasm and deep staining in IH groups at different frequencies. The liver edema and even a small amount of inflammatory cell infiltration were found in the liver with the increase of hypoxia frequency.
2. the levels of MDA, SOD, GSH-PX and T-AOC in the liver of rats with different frequencies of intermittent hypoxia group and normal oxygen control group were compared: MDA level in IH group at different frequencies was higher than that in group NC (P 0.05), SOD, GSH-PX, T-AOC activity were lower than that of NC group (0.05). 22 compared with the above indicators, the differences were statistically significant (P = 0.05), and with the increase of hypoxia frequency, MDA levels gradually increased, while SOD, GSH-PX and T-AOC activities gradually decreased.
3. the levels of NF- kappa B and ICAM-1 in the liver of rats with different frequencies of intermittent hypoxia group and normoxic control group were compared: there was no significant difference in ICAM-1 level between IH1 and NC groups (P respectively 0.143,0.633), and the level of NF- kappa of the rest IH group was higher than that of the group (all 0.05). Compared with group IH4, the difference of ICAM-1 level was not statistically significant (P was 0.103,0.06,0.524, respectively), and the difference of NF- kappa B ICAM-1 level in the other IH group 22 was statistically significant (P 0.05), and as the frequency of hypoxia increased, NF- kappa B, ICAM-1 level also increased gradually.
4. the bivariate correlation analysis showed that MDA had a negative correlation with SOD, GSH-PX and T-AOC (r was -0.721, -0.713, -0.653, P 0 respectively), and NF- kappa B was positively correlated with MDA.
The second part
1. liver histopathology: group 30T1, group NC had no obvious liver damage, group 30T2 showed slight hepatocyte membrane damage, cytoplasm sparsely, IH3, 30N1, 30N2 group, cell membrane destruction, cell edema, cytoplasm sparsity, deep nucleus staining, indicating that Tempol can prevent the occurrence of liver injury in rats with chronic intermittent hypoxia, and the earlier the intervention, the more effective the intervention, the more effective the action, the more effective. Obviously.
In 2. Tempol intervention group, the liver MDA, SOD, GSH-PX, T-AOC levels were compared in the normal saline control group and the normal oxygen control group: 30T1 group, 30T2 group was lower than IH3 group, 30N1 group, 30N2 group MDA level (P are 0.05). 2,0.413), but compared with group 30T1 and group 30T2, and compared with group 30T2 and NC group, there were significant differences (P 0.05), and there was no significant difference in the above indexes in IH3 group, 30N1 group and 30N2 group 22 (P 0.05).
3. Tempol intervention group, normal saline control group and normal oxygen control group rats liver NF- kappa B, ICAM-1 level compared: 30T1 group, 30T2 group IH3 group, 30N1 group, 30N2 group NF- kappa B, ICAM-1 levels were all decreased (0.05), 22 groups, 22 groups, differences were statistically significant (0.05), group, group, 22 group There was no significant difference between the above indexes (P 0.05).
conclusion
1. the liver damage in rats was caused by oxidative stress and inflammatory response at different frequencies of CIH, and the liver damage increased with the increase of intermittent hypoxia frequency, but the higher the frequency, the heavier the liver injury, that is, the degree of liver injury was not completely linear with the frequency of CIH.
The oxidative stress induced by the accumulation of ROS in the 2.CIH process may act as a starting factor to activate the redox sensitive transcription factor NF- kappa B and its mediated inflammatory pathway, which affects the release of the inflammatory mediators, and the two causes the liver injury in the rat under CIH.
3. antioxidant Tempol can interfere with the occurrence of oxidative stress caused by CIH, inhibit the activation of NF- kappa B and prevent and alleviate the liver damage of CIH, but it can not completely reverse the existing liver damage.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R766

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