慢性间歇性缺氧对大鼠颏舌肌线粒体的影响以及脂联素的干预机制

发布时间：2019-06-25 08:13

【摘要】：目的: 关于慢性间歇性缺氧（CIH）对颏舌肌功能的影响以及脂联素的干预机制研究至今尚未完全阐明。本课题通过建立慢性间歇性缺氧大鼠模型，研究（1）CIH对大鼠颏舌肌线粒体结构和功能的影响；（2）脂联素对CIH诱导的大鼠颏舌肌线粒体损伤的干预调节作用以及可能的机制研究，从而为防治阻塞性睡眠呼吸暂停低通气综合征提供新的途径。方法: 本研究首先建立大鼠间歇性缺氧模型，30只健康雄性Sprague Dawley(SD)大鼠，8周龄，体重180～200g，随机分组，分为正常对照组（Control组）、慢性间歇性缺氧组（CIH组）和慢性间歇性缺氧组+脂联素组（CIH+AD组），每组10只。造模成功后，首先检测不同处理组大鼠血清脂联素水平的变化。接着通过透射电子显微镜、定量real-time PCR、琥珀酸脱氢酶（SDH）染色法、细胞色素氧化酶（COX）染色法和Western Blot观察不同处理组之间大鼠颏舌肌线粒体合成、超微结构和氧化能力的改变。在明确CIH对大鼠颏舌肌线粒体的损伤后，我们探讨了脂联素的干预作用以及可能的机制研究。机制研究主要检测脂联素介导的信号通路对大鼠颏舌肌线粒体的影响以及不同处理组间颏舌肌肌纤维改变，检测主要依赖于Western blot和real-time PCR技术。机制研究重点观察了脂联素是对LKB1-AMPK-PGC1-α通路的激活情况。结果: CIH造模成功后，我们检测了大鼠血清脂联素水平。CIH组大鼠血清脂联素浓度为(1051.7±201.8)ng/ml，与对照组（浓度（2356±276.7）ng/ml）相比明显降低；CIH+AD组脂联素浓度为（1934.7±253.3）ng/ml，明显高于CIH组，差异具有统计学意义。结果表明：慢性间歇性缺氧可以引发大鼠血清脂联素水平降低，而当给予外源性脂联素后，CIH+AD组血清脂联素水平显著升高，但仍略低于对照组。为了阐明CIH对颏舌肌功能的影响，我们着重研究的了CIH对线粒体合成、超微结构和功能的影响。实验结果显示：（1）间歇性缺氧35天后，大鼠颏舌肌线粒体含量降低，参与线粒体合成重要调控基因（如PGC1-α、Esrrα、Nrf1、Cycs）的mRNA表达显著下降。以上的结果均提示间歇性缺氧35后大鼠颏舌肌线粒体合成发生障碍。（2）慢性间歇性缺氧条件下，大鼠颏舌肌肌膜下和肌纤维间线粒体的面积变小，线粒体的内外膜结构变得紊乱，基质电子密度显著降低，线粒体嵴排列不规则，嵴数量减少，嵴变短，这些说明CIH诱导了颏舌肌线粒体超微结构的损伤。（3）间歇性缺氧条件下，与线粒体功能成正相关基因的转录水平显著降低，SDH染色和COX染色提示CIH组较对照组，SDH和COX阳性细胞明显减少。以上结果均说明CIH环境下，大鼠颏舌肌线粒体功能显著受损，肌纤维的氧化能力明显减弱。接着，我们研究了脂联素对CIH诱导的颏舌肌线粒体损伤的影响。实验结果显示：（1）当给予外源性脂联素后，CIH+AD组大鼠颏舌肌线粒体的含量显著高于CIH组；基因水平研究发现，CIH+AD组与线粒体合成密切相关基因mRNA表达水平显著高于CIH组。以上结果均提示当给予外源性脂联素后，可以减轻因慢性间歇性缺氧所致的大鼠颏舌肌线粒体合成障碍。（2）当给予外源性脂联素后，CIH+AD组大鼠颏舌肌线粒体超微结构损伤较CIIH组相比，显著改善，主要表现为肌膜下和肌纤维间线粒体的面积较CIH组明显增加，线粒体内外膜结构变得完整，结构清晰，基质形态和结构也较完整，密度适中，线粒体嵴较CIH组排列规整。（3）定量real time-PCR结果提示：CIH+AD组与CIH组比，与线粒体功能呈正相关基因的转录水平显著升高，差异具有统计学意义。SDH染色和COX染色均提示：CIH+AD组与CIH组相比，SDH和COX阳性细胞明显增加。以上结果均表明：当给予外源性脂联素后，明显改善慢性间歇性缺氧所致大鼠颏舌肌线粒体功能损伤，显著提升肌纤维的氧化能力。随后我们检测不同处理组颏舌肌肌纤维的变化。Western Blot检测结果提示慢性间歇性缺氧环境下，Myosin chain1和Troponin I的蛋白表达水平明显低于正常对照组，MHC1基因的mRNA表达水平亦显著低于正常对照组；当给予外源性脂联素干预后，Myosin chain1、Troponin I的蛋白表达水平以及MHC1基因的mRNA表达水平均高于CIH组。以上结果表明慢性间歇性缺氧环境下，颏舌肌I型即氧化型纤维的含量较正常对照组显著降低；当给予外源性脂联素干预后，颏舌肌I型纤维的含量明显增高，即脂联素可以减轻慢性间歇性缺氧所致的氧化型纤维的损伤，从而提升颏舌肌的收缩功能。最后，，我们检测了脂联素介导的信号通路的激活情况。Western Blot检测结果提示慢性间歇性缺氧环境下，LKB1-AMPK-PGC1-α通路的活化被部分抑制；当我们给予外源性脂联素干预后，该通路又被重新激活。因此我们推论：脂联素可能通过激活LKB1-AMPK-PGC1-α通路从而改善慢性间歇性缺氧所致的大鼠颏舌肌结构和功能的损伤。结论: 我们的研究表明，脂联素可能通过显著激活LKB1-AMPK-PGC1a通路改善CIH诱导的颏舌肌线粒体结构和功能的损伤。
[Abstract]:Purpose: The effect of chronic intermittent hypoxia (CIH) on the function of the tongue and the mechanism of the intervention of adiponectin have not been completed yet. The effects of (1) CIH on the structure and function of the mitochondrial structure and function of the rat's tongue muscle were studied by establishing a model of chronic intermittent hypoxia in rats. (2) The effect of adiponectin on the mitochondrial damage of the rat tongue muscle induced by CIH and the possible mechanism In order to provide a new method for the prevention and treatment of obstructive sleep apnea-hypopnea syndrome The way. Methods: In this study, an intermittent hypoxia model was established in rats,30 healthy male Sprague Dawley (SD) rats,8 weeks of age,180 to 200 g of body weight, and randomly divided into normal control group (control group), chronic intermittent hypoxia group (CIH group) and chronic intermittent hypoxia group + adiponectin group (control group). CIH + AD Group)10 rats in each group. After the model was successful, the serum of rats in different treatment groups was first tested. The changes of the level of adiponectin were observed by transmission electron microscopy, quantitative real-time PCR, succinate dehydrogenase (SDH) staining, cytochrome oxidase (COX) staining and Western Blot. And the effect of the intervention of adiponectin was discussed. The mechanism study mainly detected the effect of adiponectin-mediated signaling pathway on the mitochondria of the rat's tongue and the muscle fiber of the tongue in different treatment groups, and the detection mainly depended on Western blot and real-t. The mechanism study focused on the effect of adiponectin on LKB1-AMPK-PGC1- path of passage Results: The CIH model was successful. The level of serum adiponectin in the rat was detected. The serum adiponectin concentration in the CIH group was (1051.7-201.8) ng/ ml, and the concentration of adiponectin in the CIH + AD group was significantly lower than that of the control group (2356-26.7) ng/ ml. The concentration of adiponectin in the CIH + AD group was (1934.7-253.3) ng/ ml, which was significantly higher than that of the control group. The results showed that the level of adiponectin in the serum of the rats can be reduced by chronic intermittent hypoxia, and the serum adiponectin in the CIH + AD group after administration of the exogenous adiponectin In order to clarify the effect of the CIH on the function of the tongue, we focus on the CIH. The effects of mitochondrial synthesis, ultrastructure and function were studied. The results showed that: (1) After 35 days of intermittent hypoxia, the content of the mitochondria in the rat's tongue was decreased, and the important regulatory genes involved in the synthesis of mitochondria (such as PGC1-1, Esrr, Nrf1) were involved. And Cycs) showed a significant decrease in the expression of mRNA. (2) Under the condition of chronic intermittent hypoxia, the area of the mitochondria between the muscle and the muscle fibers of the rat tongue is smaller, the inner and outer membrane structures of the mitochondria become disordered, the electron density of the matrix is obviously reduced, and the line particles the volume of the body is irregular, the number of the elements is reduced, the volume of the body is shortened, H. The ultrastructure of the mitochondria in the tongue of the tongue was induced. (3) Under the condition of intermittent hypoxia, the level of transcription of the gene related to the function of the mitochondria was significantly lower, and the staining of SDH and the staining of the COX-2 in the CIH group were significantly lower than that in the control group. In group, SDH and COX-positive cells were significantly reduced. The results indicated that the function of the mitochondrial function of the rat's tongue in the presence of CIH Significantly, the oxidation ability of the muscle fibers was significantly reduced. Then, we studied the fat The effects of adiponectin on the mitochondrial injury induced by CIH were studied. The results showed that: (1) When the exogenous adiponectin was given, the content of the mitochondria in the papillary muscle of the CIH + AD group was significantly higher than that of the CIH group, and the level of the gene found that the CIH + AD group and the mitochondria were closely combined. The expression level of the related gene was significantly higher than that of the CIH group. (2) When the exogenous adiponectin was given, the ultrastructure of the mitochondria in the papillary muscle of the CIH + AD group was significantly improved compared with that of the CIIH group, and the main performance was that the area of the mitochondria in the muscle membrane and the muscle fiber was higher than that of the CIH group. in addition, that internal and external membrane structure of the mitochondria become complete, the structure is clear, the shape and the structure of the matrix are also complete, The results of quantitative real time-PCR showed that the CIH + AD group and the CIH group were in positive correlation with the function of the mitochondria. There was a significant increase in the level of transcription, the difference was of statistical significance, and both the SDH staining and the COX staining showed: CIH + AD group and CI Compared with group H, the positive cells of SDH and COX-positive cells increased significantly. The mitochondrial function is damaged, and the oxidation capacity of the muscle fiber is obviously improved. The results of the Western Blot test indicated that the level of protein expression of Myosin chain1 and Troponin I was significantly lower than that in the control group. The expression of MHC1 gene was also significantly lower than that in the control group. After the intervention of exogenous adiponectin, the level of protein expression of Myosin chain1, Troponin I was The results showed that the content of the oxidized fibers was significantly lower in the control group than in the normal control group under the condition of chronic intermittent hypoxia, and the expression of the MHC1 gene was significantly lower in the control group than in the normal control group. After the intervention of the source of adiponectin, the content of the I-shaped fibers in the tongue of the tongue of the tongue is obviously increased, that is, the adiponectin can reduce the chronic intermittent hypoxia. The resulting oxidized fiber is damaged, thus promoting the contraction function of the papillary muscle. Finally, we examined the activation of the signal pathway mediated by adiponectin. The results of the Western Blot test suggest that the activation of LKB1-AMPK-PGC1-1 pathway is partially inhibited in the chronic intermittent hypoxia environment. System; this pathway was re-activated when we were given an exogenous adiponectin intervention. So we concluded that adiponectin may be derived from the activation of LKB1-AMPK-PGC1-1 pathway. and improve Chronic intermittent hypoxia-induced damage to the structure and function of the tongue of the rat's tongue. Conclusion: We have shown that adiponectin may activate LKB1-AM significantly by significantly activating LKB1-AM.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R766

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