异常黑胆质成熟剂对烧伤创面早期进行性加深的作用研究

发布时间：2018-06-06 14:48

本文选题：异常黑胆质成熟剂 + 烧伤　；参考：《新疆医科大学》2016年博士论文

【摘要】：目的：通过构建大鼠梳状烫伤模型模拟二度以上烧伤创面早期进行性加深的病理生理过程,系统观察不同剂量异常黑胆质成熟剂(Abnormal Savda Munziq,ASMq)对烧伤创面进行性加深过程的作用,探讨潜在的剂量效应及相关作用机制。方法：首先构建大鼠烧伤创面进展模型,即以200～220g Sprague-Dawley (SD)大鼠为研究对象,戊巴比妥钠麻醉后,将订制的长方体铜块(切面为20mm×10mm)浸于100℃水中5分钟,再置于脱毛后的大鼠背部皮肤持续20秒,间隔5mm建立四个创面,取创面间隙及部分创面组织(20mm×9mm)为淤滞区代表进行研究。在通过不同时间段大体及HE染色观察确认烧伤创面早期进展性变化。再利用RT-PCR、ELISA、TUNEL染色、免疫组化/荧光染色等方式,从分子水平探讨烧伤创面早期进展性加深的机制,并找出可能的干预时间点。其次,利用不同剂量ASMq灌胃给药,从组织学、分子水平观察其对早期烧伤创面的作用,及其对相关损伤机制的影响。最后利用特异性分子信号抑制剂来明确可能介导ASMq保护作用的信号通路。结果：第一部分实验中,根据大体观及HE染色显微镜下观察,我们发现深二度烧伤后,大鼠淤滞区充血并呈坏死倾向,可见两直接烧伤损伤创面呈融合趋势。HE染色后镜下观察显示,烧伤后72小时内,随着时间变化,大鼠烧伤创面淤滞区皮肤组织内氧化应激水平逐步升高,内源性抗氧化酶系(GPx,SOD)水平因消耗增加而较正常对照组明显降低(P0.05)。烧伤后48小时,氧化应激水平达到高峰。与此同时,自由基产生的主要两条途径的关键酶--黄嘌呤氧化酶(Xanthine Oxidase, XO)及属于还原型烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate, NADPH)氧化酶的非吞噬细胞氧化酶4(non-phagocytic cell oxidase 4, Nox4)在烧伤后均呈现明显的表达增加(P0.05)。烧伤后大鼠淤滞区组织中炎症介质(MPO, IL-1β, IL-6)的分布与释放随时间明显增加(P0.05),48小时时最为明显。而淤滞区组织中活化的NF-κB表达也随时间表达增加(P0.05),各时间组与正常对照组均有显著差异。此外,烧伤后淤滞区皮肤组织出面明显的细胞凋亡增加,伴随着活化的凋亡相关蛋白-裂解的含半胱氨酸的天冬氨酸蛋白水解酶3/9(cleaved cysteinyl aspartate specific proteinase, Cleaved Caspase 3/9,CC3/9)表达上调。第二部分实验中,我们观察到ASMq治疗可以有效减轻淤滞区皮肤组织损伤。此外,ASMq剂量相关的降低组织内MDA水平,并显著提高烧伤后降低的内源性抗氧化酶系(GPx, SOD)水平(P0.05),同时剂量相关的降低XO及Nox4表达(P0.05)。ASMq还可缓解淤滞区组织中炎症介质释放(P0.05),并上调NF-κB活化水平(P0.05),高剂量的ASMq治疗作用更加明显,而LPS干预可以逆转高剂量ASMq的治疗作用。另外,ASMq治疗后淤滞区组织细胞凋亡明显减少,CC3/9的分布与表达也明显降低(P0.05),ASMq作用呈剂量依赖效应；Erk激活阻滞剂PD98059有效逆转高剂量ASMq治疗对淤滞区组织细胞凋亡的缓解作用及CC3/9表达下调(P0.05)。第三部分,我们调查了对可能参与ASMq作用的信号通路,实验结果结果显示：1)ASMq治疗可以减少NF-κB活化,中、高剂量ASMq作用更加显著,高剂量最为明显(P0.01)；2)ASMq治疗可以影响线粒体相关凋亡信号通路中Bad磷酸化及Bcl-xL.细胞色素C(Cytochomre C, Cyto C)的表达。高、中、低剂量ASMq治疗均可以明显增加Bad磷酸化(P0.01),呈剂量相关作用(P0.01)。而对于Bcl-xL,中、高剂量的ASMq的上调作用更加明显。中、高剂量ASMq治疗可以显著下调烧伤引起的Cyto C表达增加,高剂量的作用更加明显。高剂量ASMq对Bad磷酸化及Bcl-xL、细胞色素C(Cyto C)的表达的影响,均可依被PD98059逆转,提示Erk激活在ASMq作用中起重要调节作用；3)根据已有的文献,ASMq可能对Ras/Erk/p90RSK信号级联存在调节作用,而这一级联也是线粒体凋亡信号通路上游重要的调节信号级联。免疫荧光染色显示,随着剂量增加,ASMq治疗可以显著提高磷酸化的Erk (p-Erk)分布,而Western blotting结果也显示,低、中、高剂量ASMq均可进一步显著增加Erk磷酸化激活,并呈剂量依赖增加(P0.01),PD98059可以显著抑制ASMq对Erk的激活作用(P0.01)。此外,中、高剂量ASMq均可显著上调Ras表达水平,且高剂量组作用更加明显(P0.01),而PD98059对Ras表达没有影响(P0.05)。高、中、低剂量的ASMq对p90RSK的表达均有显著的上调作用,与Erk磷酸化激活相对应,并呈剂量相关作用,而Erk抑制剂PD98059可显著下调高剂量ASMq增加的p90RSK表达水平。结论：(1)早期的二度以上烧伤创面存在一个动态的进行性加深过程,初始创面附近皮肤组织有损伤进展扩大倾向,这一过程与氧化应激水平及细胞凋亡增加及炎症反应加重等病理生理变化相关；2)ASMq可以有效缓解烧伤创面早期进行性加深过程中的组织结构损伤和氧化应激水平、炎症反应及细胞凋亡的增加,其作用与剂量相关；3)ASMq对烧伤创面早期进行性加深的治疗作用,可能主要通过影响氧化应激、Ras/Erk/p90RSK介导的线粒体凋亡途径及NF-kB介导的炎症反应来实现。
[Abstract]:Objective: to simulate the pathophysiological process of the early progressive burn wound of two degree burn wound by building a rat model of comb like scald, and to observe the effect of different doses of Abnormal Savda Munziq (ASMq) on the progressive deepening process of burn wound, and explore the potential dose effect and related mechanism. First, the rat model of burn wound was constructed, which was 200 ~ 220g Sprague-Dawley (SD) rats. After pentobarbital sodium anaesthesia, the cupric cupric cuboid (20mm x 10mm) was immersed in 100 centigrade water for 5 minutes. Then the skin of the rat's back after hair removal was kept for 20 seconds, and four wounds were established by interval 5mm, and the gap between the wound and the wound was taken. Part of the wound tissue (20mm x 9mm) was the representative of the stagnation area. The early progressive changes in the burn wound were confirmed by gross and HE staining in different time periods. Then RT-PCR, ELISA, TUNEL staining, immunofluorescence staining and other methods were used to investigate the mechanism of the early progressive deepening of the burn wound from the molecular level, and to find out possible The intervention time points. Secondly, using different doses of ASMq to give the medicine, from the histology and the molecular level, to observe the effect on the early burn wound and its effect on the related damage mechanism. Finally, we use the specific molecular signal inhibitor to identify the signal pathway that may mediate the protective effect of ASMq. Observation under the HE staining microscope, we found that after deep two degree burn, the stasis area of rats was congested and necrotic. It was seen that the two direct burn wounds were observed under the fusion trend and observed under the microscope. Within 72 hours after the burn, the level of oxidative stress in the skin tissue of the burn wound area increased gradually as time changed, and the level of oxidative stress in the skin tissue of the burn wound was gradually increased. The level of GPx (SOD) was significantly lower than that in the normal control group (P0.05). 48 hours after the burn, the level of oxidative stress reached its peak. At the same time, the key enzymes of the two main routes of free radicals - xanthine oxidase (Xanthine Oxidase, XO) and the archetypal nicotinamide adenine dinucleotide phosphate ( Nicotinamide adenine dinucleotide phosphate, NADPH) oxidase 4 (non-phagocytic cell Oxidase 4, Nox4) increased significantly after burn (P0.05). The distribution and release of inflammatory mediators (MPO, IL-1 beta,) in the tissues of the rats after burn increased with time, 48 hours. The expression of activated NF- kappa B increased with time (P0.05), and there was a significant difference between each time group and the normal control group. In addition, the apoptotic cell apoptosis increased obviously in the skin tissue after the burn, accompanied by the activated apoptosis related protein - cysteine - containing aspartate protein hydrolase 3 The expression of /9 (cleaved cysteinyl aspartate specific proteinase, Cleaved Caspase 3/9, CC3/9) was up-regulated. In part second, we observed that ASMq therapy could effectively reduce skin tissue damage in the stagnation area. OD) level (P0.05), and dose-dependent reduction of XO and Nox4 expression (P0.05).ASMq can also relieve the release of inflammatory mediators in the tissues of the stagnant region (P0.05), and increase the activation level of NF- kappa B (P0.05). The effect of high dose ASMq therapy is more obvious, and LPS intervention can reverse the therapeutic effect of high dose. The distribution and expression of CC3/9 also decreased significantly (P0.05), and the effect of ASMq was dose-dependent; Erk activating blocker PD98059 could effectively reverse the effect of high dose ASMq therapy on the apoptosis of tissue cells and down regulation of CC3/9 expression (P0.05). The third part, we investigated the signaling pathways involved in the possible participation of ASMq. The experimental results showed that: 1) ASMq therapy could reduce the activation of NF- kappa B, and the effect of high dose ASMq was more significant, and the highest dose was most obvious (P0.01); 2) ASMq therapy could affect the expression of Bad phosphorylation and Bcl-xL. cytochrome C (Cytochomre C, Cyto). The increase of Bad phosphorylation (P0.01) showed a dose-dependent effect (P0.01). For Bcl-xL, the up regulation of high dose ASMq was more obvious. High dose ASMq therapy could significantly decrease the expression of Cyto C caused by burn, and the high dose effect was more obvious. High dose ASMq on Bad phosphorylation and Bcl-xL, cytochrome C (cytochrome C) expression The effect can be reversed by PD98059, suggesting that Erk activation plays an important role in the role of ASMq; 3) according to the existing literature, ASMq may regulate the cascade of Ras/Erk/p90RSK signals, and this cascade is also an important cascade of modulation signals upstream of the mitochondrial apoptosis signaling pathway. Immunofluorescence staining shows that with the increase of dose, ASM Q treatment could significantly increase the Erk (p-Erk) distribution of phosphorylation, and Western blotting results also showed that low, medium and high dose ASMq could further increase the activation of Erk phosphorylation, with a dose-dependent increase (P0.01), PD98059 could significantly inhibit the activation of ASMq to Erk (P0.01). The effect of the high dose group was more obvious (P0.01), while PD98059 had no effect on the expression of Ras (P0.05). High, middle and low doses of ASMq had a significant up-regulated effect on the expression of p90RSK, corresponding to the activation of Erk phosphorylation, and a dose-dependent effect, while Erk inhibitor PD98059 significantly lowered the p90RSK expression level of the high dose ASMq increase. (1) there is a dynamic progressive deepening process of burn wounds above two degrees in the early stage, and the skin tissue in the vicinity of the initial wound surface has a tendency to expand damage. This process is related to the oxidative stress level, the increase of apoptosis and the aggravation of inflammatory reaction, and 2) ASMq can effectively relieve the early progressive burn wound. The damage of tissue structure and oxidative stress, inflammation and apoptosis are increased in the process, and the effect is related to the dose. 3) the effect of ASMq on the early progressive deepening of burn wound may be realized mainly through the effect of oxidative stress, Ras/Erk/p90RSK mediated apoptosis pathway and NF-kB mediated inflammatory reaction.
【学位授予单位】：新疆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R644

【参考文献】