Protective effects of methane saturated saline on endotoxin-induced acute lung injury in rats

doi:10.3969/j.issn.2095-4344.0599

Abstract

Abstract:

BACKGROUND: Methane has been shown to have significant antioxidant and anti-apoptotic effects in many diseases, such as intestine, liver, myocardium and other ischemia/reperfusion injury. The protective effect of methane on acute lung injury (ALI) has not been studied.
OBJECTIVE: To investigate the protective effect of methane saturated saline on ALI induced by endotoxin in rats and its possible mechanism.
METHODS: ALI in rats was induced by endotoxin (20 mg/kg). The experiment was divided into blank, control (methane saturated saline), ALI, ALI and methane (ALI treated by methane saturated saline) groups. After 24 hours, the wet/dry weight ratio of lung tissue was detected. Hematoxylin-eosin staining and electron microscopy were conducted. The contents of tumor necrosis factor α, interleukin 6, and interleukin 1β in alveolar lavage, and activity of malondialdehyde and superoxide dismutase in lung tissue were determined by ELISA. Cell apoptosis was measured by TUNEL. The content of caspase-3 in lung tissue was detected by western blot assay.
RESULTS AND CONCLUSION: There was no significant difference in each index between blank and control groups. Compared with the blank group, the wet/dry weight ratio, inflammatory reaction in lung tissue, oxidative stress and apoptotic rate were significantly increased in the ALI and ALI and methane groups (P < 0.05). Compared with the ALI group, the wet/dry weight ratio, inflammatory cytokines, apoptotic rate, caspase-3 expression and malondialdehyde content were significantly decreased, and superoxide dismutase activity was significantly increased in the ALI and methane group (P < 0.05). In summary, methane saturated saline can treat ACI caused by endotoxin by reducing the level of oxidative stress in lung tissue, inhibiting the expression of inflammatory factors and reducing apoptosis.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Methane, Endotoxins, Acute Lung Injury, Tumor Necrosis Factor-alpha, Interleukin-6, Interleukin-1beta, Malondialdehyde, Superoxide Dismutase, Tissue Engineering

CLC Number:

R446
R318

Meng Qingxi, Wang Weiheng, Sun Aijun, Xi Yanhai, Ye Xiaojian. Protective effects of methane saturated saline on endotoxin-induced acute lung injury in rats[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(3): 396-403.

Figures/Tables 3

2.3 肺组织的苏木精-伊红染色和电子显微镜检测内毒素处理1 h后大鼠开始出现活动度减少，精神差，痢疾等反应。急性肺损伤+CH4组与急性肺损伤组相比，上述症状较为轻微，大鼠活动度及精神状态尚可。组织学检查大体观察可见空白组和对照组大鼠肺组织表面光滑，呈粉红色，无液体渗出及出血点。光学显微镜下可见肺组织结构清晰，肺泡壁薄，肺泡内未见液体渗出，见图2A，B。电镜下观察肺泡的超微结构可见肺泡上皮细胞具有典型的线粒体及内质网结构，见图3A，B。内毒素处理组大鼠肺组织大体观察可见整个肺部充血水肿，表面粗糙，间有点状斑片状坏死。光学显微镜下见肺间质水肿，弥漫性出血，广泛炎性细胞浸润，肺泡壁增厚，内有大量液体渗出，见图2C。电镜下可见1型肺泡细胞出现典型的空泡形成，去颗粒，线粒体肿胀，核周池肿胀内质网变粗糙，2型肺泡上皮细胞可见典型的板层小体空泡和管状髓鞘脱落，见图3C。急性肺损伤+CH4组大鼠肺组织大体可见中度肺水肿，表面点状坏死灶，光学显微镜下可见肺泡出血及炎细胞浸润，肺水肿、肺泡出血及炎细胞浸润但较急性肺损伤组为轻，见图2D。电镜下肺泡上皮细胞超微结构明显好于急性肺损伤组，见图3D。 2.4 甲烷饱和生理盐水对急性肺损伤大鼠支气管肺泡灌洗液细胞的影响见图4A所示，生理状况下大鼠支气管肺泡灌洗液中主要的细胞为中性粒细胞、巨噬细胞及淋巴细胞。甲烷饱和生理盐水处理对正常支气管肺泡灌洗液中细胞数量和种类影响不大(P > 0.05)，见图4B。内毒素处理后，支气管肺泡灌洗液中细胞总量明显增加(P < 0.05)，见图4C，其中以中心性粒细胞和巨噬细胞增多为主，而甲烷饱和生理盐水处理可以明显降低内毒素导致的支气管肺泡灌洗液中细胞总量及中性粒细胞和巨噬细胞的数量(P < 0.05)，见图4D，E。 2.5 甲烷饱和生理盐水对急性肺损伤大鼠支气管肺泡灌洗液中炎症因子的影响见图5所示，对照组和空白组相比炎症因子质量浓度差异无显著性意义(P > 0.1)，表明富甲烷生理盐水对正常大鼠肺部的炎症因子无显著影响；急性肺损伤组和急性肺损伤+CH4组的支气管肺泡灌洗液中肿瘤坏死因子α、白细胞介素6和白细胞介素1β的质量浓度显著上升(P < 0.05)，而经甲烷饱和生理盐水治疗后的急性肺损伤+CH4组与急性肺损伤组相比各炎症因子的质量浓度有显著降低(P < 0.05)，但仍明显高于对照组和空白组(P < 0.05)。说明饱和甲烷生理盐水可以通过抑制肺部炎症因子的产生和分泌，降低由于内毒素导致的急性肺损伤。 2.6 甲烷饱和生理盐水对急性肺损伤大鼠肺组织氧化应激的影响各组处理24 h后，肺组织中丙二醛和超氧化物歧化酶的含量测定结果见图6所示，空白组与对照组相比，丙二醛和超氧化物歧化酶的含量差异无显著性意义(P > 0.1)。急性肺损伤和急性肺损伤+CH4组与空白组相比肺组织中丙二醛含量显著升高，超氧化物歧化酶的含量显著降低(P < 0.05)；急性肺损伤+CH4组与急性肺损伤组相比，丙二醛的含量显著降低，而超氧化物歧化酶含量显著升高(P < 0.05)，但仍然未达到空白组与对照组水平(P < 0.05)。表明甲烷对正常大鼠肺组织中的丙二醛和超氧化物歧化酶的含量无显著影响，而急性肺损伤造模后，肺组织中丙二醛的含量显著增高，而超氧化物歧化酶含量显著降低。甲烷治疗急性肺损伤可以显著的降低肺组织中丙二醛含量，提高超氧化物歧化酶含量。 2.7 甲烷饱和生理盐水对急性肺损伤大鼠肺组织凋亡的影响如图7所示，TUNEL染色时，正常细胞染色后细胞核蓝绿色，而凋亡细胞的细胞核被染成黄褐色。肺组织TUNEL染色结果显示：各组均可见棕黄色、棕褐色的凋亡细胞核，但Blank与对照组棕黄色细胞核出现率很低，细胞凋亡较少，且两组差异无显著性意义(P > 0.05)。而急性肺损伤组和急性肺损伤+CH4组中棕黄色凋亡细胞核出现率明显高于空白组(P < 0.05)，而急性肺损伤+CH4组与急性肺损伤组相比，凋亡细胞的比例明显降低(P < 0.05)。肺组织Western blot检测caspase-3蛋白在空白组与对照组中的表达无明显差异(P > 0.05)，见图8，这与TUNEL结果相符。而在急性肺损伤组和急性肺损伤+CH4组中其表达明显增加(P < 0.05)。急性肺损伤+CH4组与急性肺损伤组相比，caspase-3的表达量显著降低(P < 0.05)。由以上结果可知，甲烷对正常大鼠肺组织的细胞凋亡没有显著影响，急性肺损伤造模可以显著的增加肺组织细胞的凋亡，饱和甲烷生理盐水处理可以显著的降低肺组织细胞的凋亡。"

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中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程