第一临床医学院知识库

目的  通过慢性间歇性低氧（CIH）合并高脂饮食的大鼠模型模拟阻塞性睡眠呼吸暂停低通气综合症（OSAHS）合并肥胖患者，探讨CIH和高脂饮食对大鼠心脏组织损伤以及胞外-5'-核苷酸酶（CD73）表达的影响，并以复氧模型模拟临床持续正压通气（CPAP）治疗研究复氧的干预作用，为OSAHS的临床治疗提供新的思路。

方法  建立慢性间歇性低氧合并高脂饮食的大鼠模型。将48只SPF级健康雄性Wistar大鼠，根据随机数字表法随机分为4组，每组12只。 A组：空白对照组（常氧及普通饮食）；B组：高脂组（常氧及高脂饮食）；C组：间歇性低氧组（间歇性低氧及普通饮食）；D组：间歇性低氧合并高脂饮食组（间歇性低氧及高脂饮食）。A组和C组投放普通饲料，B组和D组投放高脂饲料喂养。C组和D组置于8h/d的间歇性低氧环境中，同时给予A组和B组间歇性压缩空气。在实验6周后,每组随机选取6只大鼠，取血清检测血清甘油三酯（TG）、低密度脂蛋白胆固醇（LDL-C）水平、取大鼠心脏组织经苏木精-伊红（HE）染色后在显微镜下观察心脏病理结构变化，免疫组化和蛋白印迹法检测蛋白CD73在各组大鼠心肌细胞中的表达水平。大鼠原饮食条件不变，复氧2周，再次重复上述检测，并进行分析。

结果  CIH 6周后结果：1.与A组比较，B、C、D组大鼠血清TG和LDL-C水平均升高，其中D组显著升高，差异均有统计学意义（均P<0.05）。2.HE结果显示：A组心肌细胞未见明显异常；B组和C组中可见部分心肌纤维排列紊乱，局灶性变性坏死；D组心肌细胞损伤最为明显，可见肌纤维排列紊乱，纵横纹不清，部分肌纤维明显溶解。3.免疫组化显示：与A组比较，B、C、D组心肌CD73蛋白表达水平均明显升高（均P<0.05），其中D组CD73蛋白表达水平升高显著(P<0.01)。4.蛋白印记也显示出一致的结果：与A组比较，B、C组 CD73蛋白表达水平均明显升高（均P<0.05），其中D组CD73蛋白表达水平升高更为显著（P<0.01）。复氧2周后结果：1.与复氧前比较，C组和D组大鼠血清TG和LDL-C水平均有升高，且差异均有统计学意义（P<0.05）。2.HE结果显示：A组:心脏组织未见明显异常。B组可见部分肌纤维排列紊乱，纵横纹不清，局灶性变性坏死。C组仍可见部分肌纤维排列紊乱，纵横纹不清，但局灶性坏死较复氧前减少；D组大鼠心肌细胞仍可见心肌纤维排列紊乱，纵横纹不清，局部肌纤维溶解变性坏死，但坏死病灶范围较复氧前减少。3.免疫组化显示：与复氧前比较，C组和D组大鼠心肌CD73的蛋白表达均显著下降 (P<0.01)，且差异具均具有统计学意义。4.蛋白印迹也显示出一致的结果：与复氧前比较，C组和D组大鼠CD73的蛋白表达水平均下降 (P<0.05 )，且差异均具有统计学意义。

结论  1.慢性间歇性低氧和高脂饮食均可以引起大鼠脂代谢紊乱和心肌的病理损伤，在慢性间歇性低氧合并高脂饮食情况下，这种代谢紊乱与心肌的病理损伤更为严重。

2.慢性间歇性低氧和高脂饮食均可以引起大鼠心肌组织CD73表达增加，在慢性间歇性低氧合并高脂饮食情况下，大鼠心肌组织CD73表达增加更为显著。

3.2周的复氧并不能改善大鼠脂代谢紊乱，但可以改善心肌组织的病理损伤并且使大鼠心肌组织CD73表达水平显著下降。

Objective  Obstructive sleep apnea-hypopnea syndrome (OSAHS) combined with obese patients was studied in a rat model of chronic intermittent hypoxia (CIH) combined with a high-fat diet to investigate the effects of CIH and high-fat diet on rat heart tissue injury and extracellular- 5'-nucleotidase (CD73) expression. The effect of reoxygenation in the treatment of clinical continuous positive airway pressure (CPAP) therapy with a reoxygenation model provide new ideas for the clinical treatment of OSAHS.

Methods  A rat model of chronic intermittent hypoxia combined with a high-fat diet was established. 48 SPF grade male Wistar rats were randomly divided into 4 groups according to the random number table method, 12 in each group. Group A: Control group (normoxic and common diet); Group B: High-fat group (normoxic and high fat diet); Group C: Intermittent hypoxia (intermittent hypoxia and common diet); Group D: Intermittent hypoxia combined with high fat diet (intermittent hypoxia and high fat diet). Group A and group C were given normal feed; group B and group D were given high-fat diet feed. Groups C and D were placed in an intermittent hypoxic environment of 8 h/d, while groups A and B were given intermittently compressed air. Six weeks after the experiment, 6 rats were randomly selected from each group. Serum levels of triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) were measured. Heart tissue of rats was treated with hematoxylin-eosin. After HE staining, the pathological changes of the heart were observed under a microscope. Immunohistochemistry and Western blot were used to detect the expression level of protein CD73 in rat cardiomyocytes. The original dietary conditions of the rats were unchanged and reoxygenated for 2 weeks. The above tests were repeated and analyzed.

Results  The results after CIH for 6 weeks: 1. Compared with group A, the levels of serum TG and LDL-C in group B, C, and D increased, and group D significantly increased, and the differences were statistically significant (all P<0.05). 2. The results of HE showed that there was no obvious abnormality in the cardiomyocytes of group A. Part of the myocardium fibers were disordered and focal degeneration and necrosis were seen in group B and C. The damage of myocardium was most obvious in group D. The damage of myocardial cells in group D was the most obvious. The disorder of muscle fibers was observed, the cross-grain pattern was unclear, and some muscle fibers dissolved.3. Immunohistochemistry showed that compared with group A, the expression of CD73 protein in group B, C, and D was obviously increased (P<0.05), and the expression of CD73 protein in group D was significantly increased (P<0.01). 4. Western blotting also showed consistent results: Compared with group A, the expression levels of CD73 protein in group B and C were obviously increased (all P<0.05), and the expression level of CD73 protein in group D was significantly increased (P< 0.01). After reoxygenation for 2 weeks, the results were as follows: 1. Compared with pre-reoxygenation, serum TG and LDL-C levels in group C and group D increased, and the differences were statistically significant (all P<0.05). 2. The HE results showed: In group A: no obvious abnormalities was seen in heart tissue. In group B, some muscle fibers were seen to be disordered, with unclear cross-sectional pattern and focal degeneration and necrosis. In group C, some muscle fibers were still seen to be disordered with unclear cross-sectional pattern, but focal necrosis was decreased before reoxygenation. In group D, myocardium still showed disordered myocardial fibers, unclear cross-sectional pattern, and local muscle fiber degeneration and necrosis. The extent of necrotic lesions decreased before reoxygenation. 3. Immunohistochemistry showed that compared with before reoxygenation, the protein expression of CD73 in myocardium of group C and group D were significantly decreased (P<0.01), and the difference was statistically significant. 4. Western blot also showed consistent results: compared with before reoxygenation, the protein expression of CD73 in group C and group D decreased (P<0.05), and the differences were statistically significant.

Conclusion  1. Chronic intermittent hypoxia and high-fat diet can cause lipid metabolism disorder and myocardial pathological damage in rats. Under chronic intermittent hypoxia combined with high-fat diet, this metabolic disorder and myocardial pathological damage were more serious. 2. Both of chronic intermittent hypoxia and high-fat diet can induce the increase of CD73 expression in rat cardiac muscle tissue. Under chronic intermittent hypoxia combined with high-fat diet, the expression of CD73 in myocardium was increased significantly. 3. Reoxygenation for 2 weeks did not improve the disorder of lipid metabolism in rats, but it could improve the pathological damage of myocardial tissue and significantly reduce the expression of CD73 in myocardial tissue of rats.