辛伐他汀抑制烟雾吸入引起的大鼠急性肺损伤及其机制研究

发布时间：2018-06-26 04:25

本文选题：烟雾吸入损伤 + TNF-α　；参考：《郑州大学》2017年硕士论文

【摘要】：背景和目的烧伤伴有吸入性肺损伤能引起多种器官障碍进而出现较高的发病率及死亡率。成立照料烧伤患者所需成本对医疗制度是一种巨大的负担。在过去十年中,减低烧伤死亡率已经取得重要进展,但治疗仍远非理想。烟雾吸入性损伤所导致的急性肺损伤(ALI)是以肺水肿、肺组织缺氧及中性粒细胞浸润为特征,其主要发病率及致死率多与休克、败血症、缺血再灌注等有关,是常见的临床问题。这些急性炎症反应也可以概括为炎症细胞聚集,蛋白质泄漏至肺泡中,致间质水肿,破坏上皮的完整性。而NF-kB需要许多细胞因子转录,包括TNF-α、IL-1β。在急性肺损伤早期阶段,这些细胞因子被细菌内毒素通过NF-KB信号并释放。并且,凋亡通路所出现的异常调控在急性肺损伤中肺组织失去正常功能细胞中具有重要作用。辛伐他汀为羟甲基戊二酰辅酶A(HMG-COA)还原酶抑制剂,近年来大量实验发现,辛伐他汀不但有降脂作用,在急性肺损伤中还表现出多效性,包含促进炎症细胞凋亡、抗炎、改善血管内皮功能等。有文献报道他汀类中辛伐他汀可阻断许多炎性因子和细胞因子的释放和功能表达,如TNF-a、IL-6、NF-kB等,通过降低这些炎症反应介质在血清中的表达,进而抑制局部和全身的炎症反应。因此本实验通过研究辛伐他汀对烟雾所致吸入性肺损伤大鼠炎症及抗炎介质的影响,观察其对烟雾所致吸入性肺损伤的抑制作用。实验一烟雾吸入性肺损伤大鼠模型的制备方法将48只清洁级大鼠按随机化原则分为四组,分别为对照组、6min组、7min组、8min组,每组12只。采纳自制烟雾致伤设备分别给予6min组、7min组、8min组大鼠行对应时间的烟雾吸入性损伤,烟雾致伤后分别于6h、12h、24h这三个时相进行观察各组大鼠临床表现、存活率及烟雾致伤后24h存活大鼠的肺组织病理情况。结果1.临床表现:正常组大鼠静息状态下无明显异常,致伤后的所有大鼠均出现呼吸急促,频率达120-140次/分,口唇紫绀,鼻腔及口唇可见黑色烟灰沉着,被毛湿润,结膜充血,6min组伤后活动饮食逐渐正常;7min组大鼠活动减少,精神萎靡不振,摄食减少,伴有喘鸣,部分大鼠逐渐恢复正常;8min组大鼠于致伤后活动明显减少,精神明显萎靡不振,摄食明显减少,伴有喘鸣。2.存活率:烟雾致伤后四组大鼠存活率对照,7min组与6min组、正常组对照三个时相的大鼠存活率无明显差异(P0.05);8min组较6min组、正常组三个时相的大鼠存活率低(P0.05),于24h较7min组的大鼠存活率低(P0.05)。3.烟雾致伤24h后各组存活大鼠的肺组织病理学评分对比,三组与正常组大鼠肺组织病理学评分均有统计学意义龴P0.05㖞,7min组大鼠肺组织病理学评分高于6min组龴P0.05㖞,8min组大鼠肺组织病理学评分高于7min组龴P0.05㖞。结论参考其他烟雾所致吸入性损伤模型的制备基础上,本实验采用以木屑为燃烧原料,自制致伤室模拟火灾现场,以大鼠为对象制作烟雾所致吸入性损伤模型,致伤7min组大鼠能至中重度烟雾吸入性肺损伤,存活率较高,且该模型操作简单,重复性高,成本低廉,能满足本课题研究需要。实验二辛伐他汀抑制烟雾吸入引起的大鼠急性肺损伤及其机制研究方法健康成年SD大鼠54只,随机化原则分为正常组、盐水组、辛伐他汀组,辛伐他汀组大鼠烟雾致伤后,用灌胃器灌入实验药物—辛伐他汀50mg/kg,每日一次,盐水组大鼠烟雾致伤后灌入与辛伐他汀组相同量的生理盐水,正常组作为基础值,不做任何处理。三组大鼠分别于灌液后6h、24h、48h这三个时相采取大鼠动脉血离心后血清、左肺肺泡灌洗液上清应用ELISA法测定TNF-a、IL-6的含量,并取右肺上叶组织提取蛋白后应用Western Blot法测定TNF-a、IL-6、NF-kB的蛋白表达情况。于灌液48h后取右肺下叶行HE病理染色。各组各时相大鼠6只。结果1.烟雾吸入性肺损伤后,盐水组、辛伐他汀组血清及肺泡灌洗液中TNF-α、IL-6的含量均明显升高,在不同观察时相均明显高于正常组(P0.05),辛伐他汀治疗组血清及肺泡灌洗液中TNF-α、IL-6含量与盐水组相比,除6h时相外,其它三个时相均有显著降低(P0.05)。2.盐水组、辛伐他汀组TNF-α、IL-6及NF-kB蛋白表达较正常组明显升高,有统计学差异(P0.05);而辛伐他汀组与盐水组相比,除6h时相外,TNF-α、IL-6及NF-kB蛋白的表达均明显减少,有统计学差异(P0.05)。3.灌液48h后显微镜下正常组大鼠肺泡腔清晰、完整、洁净,肺泡间隔匀称无肿胀,间质无炎性细胞聚集;盐水组大鼠肺组织有明显充血、出血,肺泡结构破坏,肺泡间隔增厚,间质有大量的炎症细胞浸润等;辛伐他汀组病理改变均较盐水组明显减轻。结论辛伐他汀能降低烟雾吸入性损伤大鼠肺组织及血清中TNF-a、IL-6和NF-kB的水平,对大鼠早期烟雾肺损伤有保护作用。
[Abstract]:Background and objective burns accompanied by inhaled lung injury can cause a variety of organ disorders and higher morbidity and mortality. The cost of caring for burn patients is a huge burden on the medical system. In the past ten years, the reduction of burn mortality has been an important development, but the treatment is still far from ideal. Smoke inhalation loss Acute lung injury (ALI) caused by injury is characterized by pulmonary edema, hypoxia and neutrophil infiltration in the lung. The main morbidity and mortality are associated with shock, septicemia, and ischemia reperfusion, which are common clinical problems. These acute inflammatory reactions can also be included in the accumulation of inflammatory cells, protein leaks into the alveoli, and caused to the alveoli. NF-kB requires a lot of cytokine transcription, including TNF- alpha, IL-1 beta. In the early stage of acute lung injury, these cytokines are transmitted by the bacterial endotoxin through the NF-KB signal. And the abnormal regulation of the apoptotic pathway in the lung tissue loses the normal function of the lung tissue in acute lung injury. Action. Simvastatin is a hydroxymethyl amyl two acyl coenzyme A (HMG-COA) reductase inhibitor. In recent years, a large number of experiments have found that simvastatin not only has the effect of lowering lipid, but also shows a pleiotropic effect in acute lung injury, including promoting inflammatory cell apoptosis, anti-inflammatory, and improving vascular endothelial function. The release and functional expression of many inflammatory factors and cytokines, such as TNF-a, IL-6, NF-kB, and so on, inhibit the expression of these inflammatory mediators in the serum and then inhibit the local and systemic inflammatory response. The preparation method of the model of smoke inhalation lung injury was divided into four groups according to the randomization principle of 48 rats. The control group, 6min group, 7min group, group 8min, group 8min, each group of 12 rats were given the 6min group, 7min group and 8min group, respectively. The smoke inhalation injury at the time of smoke inhalation and the three phases of 6h, 12h and 24h were observed respectively after the smoke injury. The survival rate and the lung histopathology of the 24h surviving rats after the smog injury were observed. Results the 1. clinical manifestations were as follows: the normal group rats had no obvious abnormality in the resting state, and all the rats after the injury occurred respiratory shortness. The frequency of 120-140 times / minutes, cyanosis of lips, nasal and lip black soot, wet hair, conjunctiva hyperemia, 6min group after injury, active diet gradually normal, 7min group rats activity decreased, mental malaise, less food, accompanied by wheezing, some rats gradually recovered normal; group 8min rats after injury significantly decreased activity, mental brightness after injury obviously, spirit Ming rats after injury obviously decreased, mental brightness after injury, mental Ming rats after injury obviously decreased, mental brightness after injury, mental Ming rats after injury obviously reduced activities after injury, mental Ming after injury, mental Ming rats after injury obviously decreased, mental brightness after injury, mental Ming after injury, mental Ming obviously decreased after injury, mental Ming rats after injury obviously decreased after injury, mental Ming rats after injury obviously decreased, mental brightness after injury, mental Ming rats after injury obviously reduced activities after injury, mental Ming after injury, mental bright rats after injury obviously decreased, mental Ming after injury, mental Ming after injury, mental Ming obviously decreased after injury, mental Ming rats after injury significantly decreased, mental after injury, mental Ming rats after injury obviously decreased, mental brightness after injury, mental Ming after injury, mental bright rats after injury obviously decreased, mental Ming after injury, mental Ming after injury significantly reduced The survival rate of the four rats in the four groups after the smoke injury was compared, the survival rate of the rats in the 7min group and the 6min group and the control group of the normal group were not significantly different (P0.05). The survival rate of the rats in the group 8min was lower than that of the normal group (P0.05), and the survival rate of the 24h compared to the 7min group was low (P0.0, P0.0, P0.0, P0.0, P0.0, and P0.0), and the survival rate was lower in the 24h than the 7min group (P0.0). 5) compared with the lung histopathology score of the survival rats in each group after.3. smoke injury 24h, the pathological score of lung tissue in the three group and the normal group was statistically significant? P0.05? The lung histopathological score of group 7min rats was higher than that of the 6min group? P0.05? The lung histopathological score of group 8min rats was higher than that of the 7min group? P0.05? Conclusions reference to the other smog. On the basis of the preparation of inhalation damage model, this experiment uses wood chips as combustion materials, self-made injury chamber to simulate fire scene. The model of inhalation injury caused by smoke is made in rats, and the rats in group 7min can be injured to moderate to severe smoke inhalation lung injury, the survival rate is high, and the model is simple to operate, high repeatability and low cost. Experimental two simvastatin inhibits the acute lung injury of rats induced by smoke inhalation and its mechanism research methods: 54 healthy adult SD rats. The principle of randomization is divided into normal group, saline group, simvastatin group, simvastatin group and simvastatin 50mg/kg after smog injury in simvastatin group. Once a day, the rats in the saline group were injected with the same amount of normal saline with the simvastatin group after the smoke injury. The normal group was used as the base value and did not do any treatment. The three groups of rats were divided into the three phases of 6h, 24h, and 48h after the irrigation. The left lung alveolar lavage supernatant was used to determine the content of TNF-a, IL-6, and the left lung alveolar lavage fluid supernatant. The protein expression of TNF-a, IL-6, NF-kB was measured by Western Blot method after extracting the right upper lobe tissue. After 48h, HE pathological staining was performed in the right lower lobe of the lung. 6 rats in each group were observed. The results showed that the levels of TNF- A and IL-6 in the serum and alveolar lavage fluid in the 1. smoke inhalation group, in the simvastatin group, in the simvastatin group and in the serum and alveolar lavage fluid were significantly increased. Compared with the normal group (P0.05), the content of TNF- alpha and IL-6 in the serum and alveolar lavage fluid of simvastatin group was significantly lower than that of the saline group. The other three phases were significantly decreased (P0.05).2. saline group, and the expression of TNF- alpha, IL-6 and NF-kB protein in simvastatin group was significantly higher than that in the normal group (P, P). There was a statistically significant difference (P). 0.05) 0.05) but compared with the saline group, the expression of TNF- alpha, IL-6 and NF-kB in the simvastatin group was significantly reduced, and there was a statistically significant difference (P0.05).3. perfusion 48h after 48h, the alveolar cavity in the normal group was clear, complete, clean, and the alveolar septum was unswollen and interstitial without inflammatory cell aggregation; the lung tissue of the saline group was obviously filled. Blood, bleeding, alveolar structure damage, alveolar septum thickening, mass of inflammatory cell infiltration, and the pathological changes of simvastatin group were significantly lower than that of saline group. Conclusion simvastatin can reduce the level of TNF-a, IL-6 and NF-kB in lung tissue and serum of smoke inhalation injury rats, and have protective effect on early smoke and lung injury in rats.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R563.8

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