【摘要】：亞甲藍(methyleneblue,MB)是一種吩噻嗪鹽,臨床上用來治療高鐵血紅蛋白血癥及氰化物中毒等。現(xiàn)有研究表明,MB在線粒體中可以充當電子載體增強呼吸鏈功能。本研究利用過氧化氫(hydrogen peroxide,H202)刺激小鼠腹腔巨噬細胞株RAW264.7引發(fā)氧化應(yīng)激,并建立大鼠離體心、肺缺血再灌注(ischemia-reperfusion,IR)損傷模型,研究MB對氧化應(yīng)激損傷和IR損傷的保護作用及機制。在H202刺激RAW264.7誘發(fā)氧化應(yīng)激的實驗中,0.1 μM和1μM MB處理組細胞存活率升高,乳酸脫氫酶(lactate dehydrogenase, LDH)釋放減少,反映MB能有效減輕細胞損傷;MB處理使H2O2刺激的RAW264.7中活性氧(reactive oxygen species, ROS)產(chǎn)生減少,超氧化物歧化酶(superoxydedismutase,SOD)活性增強;同時MB處理使線粒體膜電位(mitochondrial membrane potential, MMP)升高,三磷酸腺苷(adenosine triphosphate, ATP)生成增加,半胱天冬酶-3活化程度降低,細胞凋亡減少,表明MB有效增強線粒體功能、減少細胞凋亡。在大鼠離體心、肺IR實驗中,大鼠手術(shù)前2 h以2 mg/kg劑量腹腔注射MB處理使IR損傷后的離體心、肺功能改善,病理學損傷減輕,LDH釋放減少,提示MB有效減輕了 IR引起的器官損傷;同時與IR組相比,MB處理使組織中ROS及丙二醛(malondialdehyde,MDA)的產(chǎn)生減少,而SOD活性增強,說明MB能改善氧化應(yīng)激;MB增加IR后心、肺組織中MMP和線粒體中ATP生成,減輕線粒體腫脹,反映出MB對線粒體功能發(fā)揮了保護作用。離體肺IR實驗結(jié)果還表明,MB處理抑制IR損傷后細胞色素C由線粒體向細胞質(zhì)中釋放,使細胞凋亡減少。研究結(jié)果表明MB能減輕H202誘發(fā)的RAW264.7氧化應(yīng)激損傷和IR誘發(fā)的大鼠離體心、肺損傷,其保護機制可能與其能維持呼吸鏈功能、減輕線粒體損傷有關(guān)。
[Abstract]:Methylene blue (methyleneblue,MB) is a phenothiazine, which is used to treat methemoglobin and cyanide poisoning. Existing studies have shown that MB can act as an electronic carrier to enhance respiratory chain function in mitochondria. In this study, oxidative stress was induced by hydrogen peroxide (hydrogen peroxide,H202) stimulation of mouse peritoneal macrophage line RAW264.7, and an isolated rat heart and lung ischemia-reperfusion (ischemia-reperfusion,IR) injury model was established. To study the protective effect and mechanism of MB on oxidative stress and IR injury. In the oxidative stress induced by RAW264.7 stimulated by H202, the survival rate increased and the (lactate dehydrogenase, LDH) release of lactate dehydrogenase decreased in 0.1 渭 M and 1 渭 M MB groups, indicating that MB could effectively reduce cell damage. MB treatment decreased the production of reactive oxygen (reactive oxygen species, ROS) and enhanced the activity of superoxide dismutase (superoxydedismutase,SOD) in RAW264.7 stimulated by H2O2. At the same time, MB treatment increased mitochondrial membrane potential (mitochondrial membrane potential, MMP), adenosine triphosphate (adenosine triphosphate, ATP) production, cysteinase-3 (caspase 3) activation and apoptosis, which indicated that MB could effectively enhance mitochondrial function. Reduce apoptosis. In the isolated heart and lung IR test, the isolated heart of rats was treated with 2 mg/kg intraperitoneal injection of MB 2 hours before operation, the lung function was improved, the pathological injury was alleviated, and the release of LDH was decreased. The results suggest that MB can effectively alleviate the organ damage induced by IR. At the same time, compared with IR group, MB treatment decreased the production of ROS and malondialdehyde (malondialdehyde,MDA), but increased the activity of SOD, indicating that MB could improve oxidative stress. MB increased the production of MMP and ATP in the lung and heart after IR, and alleviated the swelling of mitochondria, which indicated that MB played a protective role on mitochondrial function. The results of IR in vitro lung also showed that MB treatment inhibited cytochrome C release from mitochondria to cytoplasm and decreased apoptosis after IR damage. The results showed that MB could attenuate the oxidative stress of RAW264.7 induced by H202 and the injury of isolated rat heart and lung induced by IR. The protective mechanism of MB may be related to its ability to maintain the function of respiratory chain and alleviate the damage of mitochondria.
【學位授予單位】：江南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R96

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