虾青素对大鼠烧伤后早期急性肾损伤的保护作用研究

发布时间：2018-02-15 19:56

本文关键词： 烧伤急性肾损伤氧化应激凋亡炎症反应虾青素　出处：《浙江大学》2017年博士论文　论文类型：学位论文

【摘要】：目的重度烧伤患者伤后早期面临较高的继发多器官损伤风险,而肾脏又是极易受损的脏器之一。成人烧伤后早期急性肾损伤的发生率较高,且与预后密切相关。因此烧伤后早期急性肾损伤的及时诊断与干预,对于改善患者的预后具有重要意义。本研究拟利用经典的大鼠重度烧伤模型为研究对象,探讨烧伤后早期急性肾损伤的发生发展以及相应损伤机制的变化规律,寻找潜在的干预时间窗。并评估虾青素(Astaxanthin,ATX)作为潜在治疗手段的应用价值,探索其具体作用机制。方法本研究利用热水浴法构建经典的大鼠重度烧伤模型,模拟烧伤后的机体环境,通过组织学观察、血标本检测、免疫组化、qRT-PCR、Western blot等技术手段观察评估烧伤后急性肾损伤的发生发展,同时观测相关损伤机制,如氧化应激、细胞凋亡、炎症反应的时间变化规律,寻找干预研究的最佳时间窗。此外,根据相关文献选取低、中、高三种ATX剂量,从组织学、分子生物学等角度探索ATX对大鼠烧伤后早期急性肾损伤的潜在保护作用及作用机制。最后,根据前期结果,利用分子生物学手段系统探究与虾青素的肾保护作用潜在相关的分子信号调控基础。结果研究发现,烧伤大鼠伤后早期(72 h内)肾小管损伤评分明显升高,并伴随血肌酐、血NGAL等指标的同步变化,提示急性肾损伤的出现。而氧化应激、细胞凋亡、局部炎症反应三者参与介导了烧伤大鼠早期急性肾损伤的发生发展,并随时间变化,其中伤后48小时时,大鼠肾组织损伤、肾功能减退及相应损伤机制变化相对更为明显,提示其可能是后续研究观察及干预的最佳时间窗。应用虾青素治疗烧伤大鼠,可以明显缓解烧伤引起的肾小管损伤评分及血肌酐、血NGAL水平的升高,并呈剂量依赖性作用。此外,虾青素还可以显著降低烧伤引起的氧化应激指标(氧化还原电位及丙二醛,MDA)的升高,并恢复肾组织内源性过氧化酶(超氧化物歧化酶SOD及过氧化氢酶CAT)的活性。虾青素还可以明显减少肾组织细胞凋亡、下调线粒体凋亡通路相关蛋白(Akt/Bad/Cytochrome C/cleavedcaspase-9/3)的磷酸化或表达。虾青素另可剂量依赖性地减少烧伤诱导的肾组织炎症介质(MPO、IL-1β、IL-6等)表达增加,并可下调烧伤后增加的TLR4的表达及其下游IKK、IκB、NF-κB的磷酸化。结论(1)大鼠重度烧伤后早期肾组织结构呈损伤趋势,并伴肾功能减退,提示急性肾损伤的发生,而这一过程与烧伤引起的氧化应激水平增高、细胞凋亡及局部组织炎症反应增加等损伤机制变化相关。(2)虾青素可以有效缓解重度烧伤后大鼠肾组织损伤及功能减退,其肾保护作用与其对伤后肾组织升高的氧化应激、细胞凋亡及炎症反应水平的缓解相关,并呈一定的剂量相关性。(3)虾青素对烧伤后急性肾损伤的保护作用可能通过调控线粒体相关凋亡信号通路及炎症相关TLR4/NF-KB信号途径来实现。
[Abstract]:Objective: patients with severe burn are at high risk of secondary multiple organ injury in the early stage of burn, and kidney is one of the most vulnerable organs. The incidence of acute renal injury in adult burn patients is higher than that in adults. Therefore, the timely diagnosis and intervention of early acute renal injury after burn is of great significance to improve the prognosis of the patients. To investigate the occurrence and development of early acute renal injury after burn and the changes of the corresponding injury mechanism, to find the potential intervention time window, and to evaluate the application value of astaxanthin ATX as a potential therapeutic method. Methods the classical rat model of severe burn was established by hot water bath method, and the body environment after burn was simulated. The blood samples were detected by histological observation. The occurrence and development of acute renal injury after burn injury were observed by immunohistochemical technique, such as qRT-PCR blot, and the mechanism of injury, such as oxidative stress, cell apoptosis and time change of inflammatory reaction, were observed. To find the best time window for the intervention study. In addition, according to the relevant literature, three doses of ATX were selected from histology. To explore the potential protective effect and mechanism of ATX on early acute renal injury after burn in rats. Molecular biological methods were used to explore the molecular signal regulation underlying the renal protection of astaxanthin. The results showed that the score of renal tubular injury was significantly increased within 72 hours after burn injury, and was accompanied by serum creatinine. The synchronous changes of serum NGAL and other indexes suggested the appearance of acute renal injury, while oxidative stress, apoptosis and local inflammatory reaction were involved in the pathogenesis and development of acute renal injury in burn rats. At 48 hours after injury, the changes of renal tissue injury, renal function decline and the corresponding injury mechanism were more obvious in rats, which suggested that astaxanthin might be the best time window for further observation and intervention, and astaxanthin was used to treat burn rats. It can obviously relieve the increase of renal tubular injury score, creatinine and serum NGAL level induced by burn in a dose-dependent manner. Astaxanthin also significantly decreased the increase of oxidative stress index (redox potential and malondialdehyde (MDA) MDAs) induced by burn. The activity of endogenous peroxidase (superoxide dismutase SOD and catalase CAT) in renal tissue was restored. Astaxanthin could also significantly reduce the apoptosis of renal tissue cells. It down-regulated the phosphorylation or expression of Akt / Bad-Cytochrome C / cleavedcaspase-9 / 3). Astaxanthin also decreased the expression of MPOO- IL-1 尾 IL-6 in a dose-dependent manner. It can also down-regulate the expression of TLR4 and phosphorylation of I 魏 B in the downstream of IKKN I 魏 B in burn rats. Conclusion the early renal tissue structure of rats with severe burn showed a tendency of injury and renal function decreased, suggesting the occurrence of acute renal injury. However, this process is related to the increase of oxidative stress, apoptosis and the increase of inflammatory reaction in local tissues. Astaxanthin can effectively alleviate the renal injury and decrease of function after severe burn in rats. Its renal protective effect was related to the remission of oxidative stress, apoptosis and inflammatory response to the increase of renal tissue after injury. The protective effect of astaxanthin on acute renal injury after burn may be realized by regulating mitochondrial apoptosis signaling pathway and inflammatory related TLR4/NF-KB signaling pathway.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R644

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