清肺通络方干预肺炎支原体致肺氧化应激损伤的Nrf2/ARE调控机制研究

Oxidative stress in cells and organisms has become a new research direction and hot spot in the biological function on lung injury caused by MP infection. The poor effect of anti-infection treatment in children with MPP may be related to the oxidative stress inducing pulmonary apoptosis and exacerbating inflammation. The previous experiment results showed that clearing lung and dredging collaterals method can restrain apoptosis, regulate the level of inflammatory factors and reduce lung injury. However, it is not clear which signal transduction pathway is used for this method. Nrf2/ARE signal pathway plays an important role in anti-inflammation and injury, inhibiting oxidative stress and regulating apoptosis. On the basis of previous studies, we speculate that the key mechanism of clearing lung and dredging collaterals method is to inhibit oxidative stress response by activating Nrf2/ARE signal pathway. In order to verify the above hypothesis, this study intends to induce oxidative stress through MP treatment of alveolar epithelial cells and to explore whether it can resist oxidative stress injury by using clearing lung and dredging collaterals method. The molecular mechanism of antioxidant effect of clearing lung and dredging collaterals method was explored by combining molecular biological methods. Finally, the antioxidant protective effect and mechanism of clearing lung and dredging collaterals method on lung tissue of MPP mice were verified in vivo, so as to probe into the relationship between the theory of “pulmonary collateral stagnation syndrome” and oxidative stress and lung function in TCM, and enrich the modern scientific connotation of TCM in preventing and treating MPP.

细胞和机体的氧化应激在MP感染所致肺损伤的生物学作用现已成为一个新的研究方向和热点。MPP患儿抗感染治疗效果不佳可能与氧化应激触发肺细胞凋亡、加重炎症反应有关。前期实验结果显示清肺通络方能够抑制肺细胞凋亡，调控炎性因子水平，降低肺损伤。然而该方通过哪种信号转导途径发挥作用目前尚不清楚。Nrf2/ARE信号通路在抗炎症及损伤、抑制氧化应激与调节细胞凋亡中发挥重要作用。结合前期研究基础，我们推测清肺通络方通过激活Nrf2/ARE信号通络，抑制氧化应激反应是其治疗MPP的关键机制。为证实以上假说，本课题拟通过MP处理肺泡上皮细胞诱发氧化应激，应用清肺通络方处理，探索其能否抵御氧化应激损伤，结合分子生物学手段探索清肺通络方抗氧化作用的分子机理，最后体内验证清肺通络方对MPP小鼠肺组织的抗氧化保护作用及机理，探讨中医“肺络痹阻”理论与氧化应激和肺功能之间的关系，丰富中医药防治MPP的现代科学内涵。

抗感染治疗一直是MPP治疗方案中的核心部分，大环内酯类抗生素是MPP的首选抗菌药物，然而随着大环内酯类抗生素的广泛使用，MP对其的耐药菌株逐渐增多，重症MPP的发生率和耐药菌株的增加使临床治疗难度加大，寻找新的治疗药物迫在眉睫。前期实验结果显示清肺通络方可减轻肺部炎症，改善肺微循环和通气功能，减轻肺损伤。然而，该方通过哪种信号转导途径发挥作用尚未明确。. MPP患儿抗感染治疗效果不佳可能与氧化应激触发宿主细胞凋亡、加重炎症反应有关。Nrf2/ARE信号通路在抗炎症及损伤、抑制氧化应激与调节细胞凋亡中发挥重要作用。本实验首先从细胞水平验证，说明MP感染可以诱导A549细胞发生氧化还原失衡，而Nrf2/ARE信号通路介导氧化应激的发生，氧化应激又进一步促进宿主细胞凋亡。清肺通络方可以通过调控Nrf2/ARE信号通路抑制MP感染后宿主细胞的氧化应激损伤与凋亡。第二方面，从整体动物水平验证，说明MP感染诱发小鼠肺组织氧化应激损伤，并进一步说明其机制可能与Nrf2/ARE信号通路有关，清肺通络方可以通过调节Nrf2/ARE信号通路来减轻MPP小鼠肺组织氧化应激损伤。我们运用Western Blot、PCR、Annexin V/PI、DCFH-DA荧光探针法、CCK-8等方法，从体内与体外两方面验证了Nrf2/ARE信号通路介导的MPP氧化应激与清肺通络方的干预机制。

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