热休克预处理对严重烫伤大鼠胃黏膜细胞线粒体结构和功能的影响

目的 探讨热休克预处理(HS)对严重烫伤大鼠胃黏膜细胞线粒体的保护作用及机制.方法 观察热休克预处理对严重烫伤大鼠胃黏膜细胞超微结构及线粒体超氧化物歧化酶(mSOD)和线粒体细胞色素氧化酶(mCCO)活力的影响.将Wistar大鼠随机分为烫伤组(40只):烫伤后即制作急性胃黏膜损伤模型,另取8只大鼠不致伤作为空白对照(伤前);HS组(40只):于烧伤前20 h行热休克预处理,另取8只行热休克预处理不烫伤,作为实验对照(伤前).各组于伤后3、6、12、24、48 h处死大鼠(每组每时相点8只大鼠)后留取标本检测大鼠胃黏膜损伤指数(UI)、胃黏膜细胞超微结构改变,热休克蛋白(HSP)60、HSP70、mSOD、mCCO变化,并进行相关分析.结果 大鼠严重烫伤后胃黏膜损害明显(伤后24 h UI值12.7±1.9),胃黏膜细胞超微结构改变显著,mSOD和mCCO活力随烧伤时间延长而降低,伤后24 h分别降至0.64±0.05、0.28±0.04.热休克预处理能诱导烫伤大鼠胃黏膜HSP60和HSP70表达显著增加.伤后24 h分别为0.53±0.08和0.64±0.09,明显高于烫伤组0.31±0.04(P＜0.05)和0.27±0.04(P＜0.01),并明显减轻大鼠严重烫伤后急性胃黏膜损害(伤后24 h UI值6.3±0.7 vs 12.7±1.9,P＜0.01);热休克预处理能显著降低胃黏膜mSOD的消耗(伤后24 h值293±32 vs 108±11,P＜0.01),提高mCCO活性(伤后24 h值0.64±0.05vs 0.28±0.04,P＜0.01);电镜观察显示热休克预处理大鼠胃黏膜细胞超微结构受损程度减轻,线粒体改变不明显.相关分析结果显示,mSOD与HSP70呈显著正相关(r=0.436,P＜0.05);mCCO与HSP60呈显著正相关(r=0.679,P＜0.05).结论 热休克预处理对严重烫伤大鼠胃黏膜细胞线粒体具有保护作用,可显著减轻大鼠烫伤后急性胃黏膜损害,保护线粒体机制与HSP60、HSP70保护抗氧化酶mSOD及呼吸酶mCCO活性密切相关.

Influence of heat shock preconditioning on structure and function of mitochondria in gastric mucosa of severely burned animals: experiment with rats

OBJECTIVE: To investigate the influence of heat shock (HS) preconditioning on the ultra-structure of gastric mucosal cells and the activity of cytochrome oxidase (CCO) and superoxide dismutase (SOD) in mitochondria isolated from gastric mucosa of burned rats in order to investigate its protective mechanism on burn-induced acute gastric mucosal lesion. METHODS: Ninety-six Wistar rats were divided randomly into two groups:(1) burn model group (n =40) undergoing burning and made into model of acute gastric mucosal lesion; and then subdivided into 5 equal subgroups: 3, 6, 12, 24, and 48 h after burning; (2) normal control group (n = 8); (3) HS preconditioning + burn group (n = 40), undergoing burning 24 h after HS preconditioning; and (4) HS preconditioning group (n = 8), without burn as experimental control A number of rats were sacrificed and laparotomized before and 3, 6, 12, 24, and 48 h after the burnings. Specimens were obtained from the gastric antrum, bodies if stomach and gastric lesion to undergo microscopy and determination of the ulcer index ( UI ). Streptavidin-peroxidase immunohistochemistry was used to detect the protein expression of heat shock protein (HSP)-60 and HSP-70. Mitochondria were isolated and the activities of CCO and SOD in mitochondria were measured. The dynamic postburn changes in micro and ultra structure of gastric mucosal cells of scalded rats were observed. RESULTS: The rats of the burn group showed conspicuous gastric mucosal lesions. The UI levels at every time point of the HS group were all the lowest in comparison with other groups. The expression levels of HSP70 and HSP60 of the HS group, especially those at the time points 3, 6, 12, and 24 h were significantly higher than those of the burn group (P <0.05 or P <0.01). The activities of mCCO and mSOD of the HS group did not decrease remarkably at any time point in comparison with the control groups, and the activities of mCCO and mSOD at the time points 6, 12, and 24 h of the HS group were all significantly higher than those of the burn group (P <0.05 of P <0.01). Electron microscopy showed that the ultrastructural lesion was mild and alteration of gastric mucosal mitochondria was not significant in the HS group compared with those of the burn group. A positive correlation was shown between SOD and HSP70 (r = 0.436, P <0.05) and also between CCO and HSP60 (r =0.679, P <0.05). CONCLUSION: HS preconditioning ameliorates the burn-induced acute gastric mucosal lesion and has a protective role to gastric mucosal mitochondria. The role of HS preconditioning on mitochondria is associated with the protection of the activity of antioxidase and CCO by HSP60 and 70.