麻醉药物预处理、同时处理、后处理对急性肺损伤大鼠的免疫调节机制的研究

Organ protection is an important issue in perioperative medicine. Preconditioning, postconditioning and conditioning are well known perioperative approaches, rendering organs less vulnerable to injury through the application of tissue-protective agents. Anesthetics are known as immunomodulatory substances in inflammatory processes induced by ischemia-reperfusion injury or hypoxia-reoxygenation but also in situations of acute pulmonary inflammation. Acute lung injury (ALI) and acute respiratory distress syndrome(ARDS) are a major cause of acute respiratory failure in critically ill patients. While drug treatment is investigated intensively, no pharmocological approach has yet been established. Patients suffering from ALI/ARDS most often need to be ventilated mechanically, and therefore remain sedated in intensive care unit. The overall effect of sedatives and anesthetics on this disease is unclear. The lung represents a site for the invasion of various bacteria or bacterial products. Along with alveolar macrophages, pulmonary epithelial cells are the first cells to be challenged by pathogenic microorganisms. Lipopolysaccharide (LPS) triggers a physical association between the cluster of differentiation 14 (CD14) and Toll-like receptor 4 (TLR4). Inflammatory response to endotoxin is largely mediated through CD14 and TLR4. The activation of CD14 and TLR4 leads to a proinflammatory cascade including the expression of proinflammatory mediators, such as tumor necrosis factor-a (TNF-a). CD14 and TLR4 have been found on alveolar type II cells (AT II ) and could, thus, play an important role in innate immune response in the alveolar surface area. Therefore, we designed a study to explore whether ALI is improved by nowadays commonly used anesthetics(midazolam, remifentanil, dexetomidate and sevoflurane) pre-administration, co-administration and post-administration and to elucidate its possible mechanism of action in LPS-induced ALI in anesthetized, ventilated rats as well as in AT II cells. In vitro assay: different kinds of anesthetics are pre-administered, co-administered, and post- administered in LPS-induced ALI rats. Lung pathology, CD14 and TLR4 gene and protein expression as well as cytokine in BALF are detected. In vivo assay: different kinds of anesthetics are pre-administered, co-administered, and post- administered in LPS-induced AT II cells. CD14 and TLR4 gene and protein expression as well as cytokines in supernatant are detected.

器官保护是围术期的重要待研究问题。麻醉药物的使用对免疫系统功能直接或间接地产生不同影响。免疫功能失常会引起术后和重症监护病人长时间的感染和败血症。各种麻醉药物越来越多地应用于临床麻醉和重症监护病房，而关于它们对机体免疫机制的研究尚不足。脂多糖作为诱导肺损伤常用的介质，其作用机理在逐渐阐明，但其具体免疫学机制仍在探讨和研究中。本课题拟通过大鼠的在体实验和离体肺泡II型上皮细胞的实验，研究目前常用麻醉药物包括咪唑安定、雷米芬太尼、右美托咪定、七氟烷预处理、同时处理和后处理对脂多糖诱导的肺损伤大鼠肺组织及肺泡II型上皮细胞免疫调节机制的影响，尤其是核因子-κB上游的CD14和TLR4基因及蛋白表达的影响，已期进一步明确其作用机理,及麻醉药物的预处理、同时处理和后处理对急性肺损伤的免疫调节的不同。

背景：免疫功能的异常可能导致患者术后出现长时间的感染，并引发败血症，严重者会导致感染性休克以及多种器官的功能衰竭直至死亡。丙泊酚是临床最常用的静脉全身麻醉药物之一，因此丙泊酚对机体免疫功能的影响受到越来越多医务工作者的关注。.研究内容：我们选择HUVEC（人脐静脉血管内皮细胞）对其进行不同处理。根据实验对照原则，共分为6组。第一组：生理盐水30min；第二组：生理盐水30min后加入LPS（1μg/μl）；第三组：异丙酚10μM处理30min后加入LPS（1μg/μl）；第四组：异丙酚25μM处理30min后加入LPS（1μg/μl）；第五组：异丙酚50μM处理30min后加入LPS（1μg/μl）；第六组：异丙酚100μM处理30min后加入LPS（1μg/μl）。LPS处理3小时后收取细胞进行western blot和real-time PCR实验检测。结果发现，与生理盐水对照组相比，LPS在蛋白和RNA水平上促进TLR4的表达；而伴随着异丙酚浓度的增加，LPS促进TLR4表达的作用逐渐减弱即异丙酚抑制TLR4的表达呈浓度梯度趋势。.结果：通过TARGETSACN靶基因预测软件，我们筛选出调控TLR4转录的miRNA可能是miR-21；芯片筛查结果也提示miR-21的可能靶点为TLR4。我们在HUVEC中转染miR-21 mimics和miR-21 mimics Ncontrol，48小时后应用real-time PCR 检测转染效率同时检测TLR4的转录水平，miR-21mimics细胞组中的TLR4转录水平明显下调。我们在HUVEC中转染miR-21 inhibitor和miR-21 inhibitor Ncontrol，48小时后应用real-time PCR 检测转染效率同时检测TLR4的转录水平， miR-21 inhibitor促进TLR4的转录。我们进行上述6组实验分组之后，收取细胞应用real-time PCR方法检测miR-21的表达情况。结果显示：与生理盐水对照组相比，LPS下调miR-21的RNA表达，这与之前的研究报道一致；同时，我们发现伴随着异丙酚浓度的增加，LPS下调miR-21表达的作用逐渐减弱即异丙酚上调miR-21的作用呈浓度梯度趋势。上述结果提示我们：LPS下调miR-21 而异丙酚可以上调miR-21，其中miR-21的靶基