高压氧通过调节氧化防御对高原脱习服小鼠心脏的保护作用

    Protective effect of hyperbaric oxygen on the heart in high-altitude de-acclimatization mice by regulating oxidative defense

      摘要
    • 摘要:
      目的 探讨高压氧（hyperbaric oxygen，HBO）治疗对高原脱习服（high-altitude de-acclimatization，HADA）模型小鼠心脏组织的氧化防御作用。
      方法 雄性昆明小鼠36只，采用随机数字法分为3组，正常对照（normal control， NC）组、高原脱习服（high-altitude de-acclimatization，HADA）组、高压氧治疗（hyperbaric oxygen treatment， HBOT）组，每组12只。动物模型制备使用小动物低舱模拟海拔5 000 m高原环境，舱内饲养14 d出舱后连续给予3 d高压氧治疗；于高压氧治疗后1 d和3 d分别取心脏组织备用。组织匀浆与活性氧(reactive oxygen species，ROS)荧光染色进行氧化损伤及抗氧化能力评价；组织HE染色观察心脏组织病理学改变；Masson染色评价心脏组织胶原沉积和纤维化；TUNEL染色评价心肌细胞凋亡程度，血红素加氧酶-1（heme oxygenase-1，HO-1）与核因子E2相关因子2（nuclear factor erythroid 2-related factor 2，Nrf2）免疫荧光染色观察HO-1与Nrf2阳性细胞比率。
      结果 HE染色可见HADA组小鼠心肌细胞变性坏死明显，成纤维细胞浸润，心脏组织间隙明显增宽；病理评分结果表明，HADA组较对照组评分增高，HBOT组较HADA组病理评分明显降低（P<0.05）；Masson染色可见HADA组小鼠心肌组织间隙纤维化染色面积较NC组明显增加，HBOT组较HADA组纤维化面积明显减少（P<0.05）；组织匀浆氧化损伤及抗氧化能力检测结果显示，HADA组丙二醛（malondialdehyde，MDA）， 一氧化氮（nitric oxide，NO）， 一氧化氮合酶（nitric oxide synthase，NOS）， 诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）含量较NC组明显升高，HBOT组较HADA组明显降低（P<0.05）；HADA组超氧化物歧化酶(superoxide dismutase，SOD)， 谷胱甘肽过氧化物酶(glutathione peroxidase，GPX)， 过氧化氢酶(catalase，CAT)， 总抗氧化能力（total antioxidant capacity，T-AOC）含量较NC组明显降低，HBOT组较HADA组显著升高（P<0.05）；ROS免疫荧光染色结果，HADA组荧光强度较正常NC组显著升高，HBOT组较HADA组荧光强度显著降低（P<0.05）。TUNEL染色结果显示，HADA组较NC组阳性细胞数显著增加，而HBOT组比HADA组阳性细胞数明显减少，同时随着时间延长阳性细胞数增加（P<0.05）；HO-1与Nrf2免疫荧光染色结果显示，HADA后HO-1与Nrf2阳性细胞较NC组显著减少，而HBOT后HO-1与Nrf2阳性细胞表达增加（P<0.05）。
      结论 HBOT通过减轻心脏组织病理损伤及心肌细胞的凋亡对HADA所致的心脏组织起到保护作用，其主要的保护机制与减轻氧化损伤有关。

       

      Abstract:
      AIM  To investigate the oxidative defense effect of hyperbaric oxygen therapy on cardiac tissue of high-altitude de-acclimatization model mice.
      METHODS  According to the random number table, 36 male Kunming mice were evenly divided into 3 groups, respectively normal control (NC) group, high-altitude de-acclimatization (HADA) group, and hyperbaric oxygen treatment (HBOT) group. After 14 days of feeding in a hypobaric chamber stimulating the altitude of 5 000 m, these animal models received HBOT for 3 days. Their heart tissue was taken at the 1 d and 3 d. The oxidative damage and antioxidant capacity were assessed by tissue homogenate and ROS fluorescence staining. Cardiac histopathological changes were observed through HE staining. The collagen deposition and fibrosis in heart tissue were assessed by Masson staining. The apoptosis of cardiomyocytes was assessed by TUNEL staining, and the ratio of HO-1 and Nrf2 positive cells was observed through HO-1 and Nrf2 immunofluorescence staining.
      RESULTS  HE staining showed obvious degeneration and necrosis of cardiomyocytes, infiltration of fibroblasts and widening of heart tissue gap in the HADA group. Pathological assessment demonstrated that the scores of the HADA group were higher than those of the NC group, while the scores of the HBOT group were significantly lower than those of the HADA group (P<0.05). Masson staining revealed that the fibrosis area in the myocardial space of mice in the HADA group significantly increased compared with the NC group, whereas the fibrosis area in the HBOT group significantly reduced compared with the HADA group (P<0.05). The results of oxidative damage and antioxidant capacity of tissue homogenate showed that the content of MDA, NO, NOS and iNOS in the HADA group was significantly higher than that in the NC group, and the content of HBOT group was significantly lower than that in the HADA group (P<0.05); The content of SOD, GPX, CAT and T-AOC in the HADA group were significantly lower than those in the NC group, and the content of HBOT group were significantly higher than those in HADA group (P<0.05); ROS immunofluorescence staining showed that the fluorescence intensity of the HADA group was significantly higher than that of the NC group, and the fluorescence intensity of the HBOT group was significantly lower than that of the HADA group (P<0.05). TUNEL staining results showed that the number of positive cells in the HADA group was significantly higher than that in the NC group, while the number of positive cells in the HBOT group was significantly lower than that in the HADA group. Meanwhile, it increased with the extension of time (P<0.05); Immunofluorescence staining showed that HO-1 and Nrf2 positive cells decreased significantly following HADA, while the expression of such cells increased after HBOT (P<0.05).
      CONCLUSION  HBOT has a protective effect on cardiac tissue by mitigating pathological injury and apoptosis of cardiomyocytes. The main protective mechanism is related to mitigating of oxidative damage.

       

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