Pin1介导血管内皮细胞氧化应激反应参与重症中暑急性肺损伤的机制研究

目的:观察热打击后小鼠肺微血管内皮细胞（pulmonary microvascular endothelial cells，PMVECs）和肺组织中肽基-脯氨酰顺/反异构酶1（Pin1）通过调控氧化应激和凋亡形成，参与重症中暑急性肺损伤（acute lung injury，ALI）形成的分子机制。方法:体外实验，建立PMVECs热打击模型，对照组将PMVECs置于标准37 ℃、5% CO 2细胞培养箱，热打击组将细胞置于43 ℃细胞培养箱中进行热打击2 h，热打击后继续在细胞培养箱进行复温（1、3、6、12 h），并使用Pin1抑制剂Juglone（1 μmol/L）预处理细胞1 h。体内实验，通过热打击构建重症中暑小鼠模型，热打击组动物置于仿真气候舱内，舱内温度（35.5±0.5） ℃，湿度（60±5）%，肛温表监测小鼠直肠温度，小鼠体温达到42 ℃即停止热打击，热打击后1、3、6以及12 h处死动物，对照组小鼠始终置于室温（25±0.5） ℃下，并使用Pin1抑制剂Juglone （1 mg/kg）于热打击前连续3 d腹腔注射对小鼠进行预处理。Western blot观察Pin1、cleaved caspase-9和cleaved caspase-3的表达情况；DHE染色，荧光显微镜下观察细胞中O 2-˙水平；ELISA检测肺组织中丙二醛（malondialdehyde，MDA）和超氧化物歧化酶（superoxide dismutase，SOD）水平；HE染色检测各组小鼠的病理改变情况；免疫组化染色检测各组小鼠肺组织中Pin1表达情况；TUNEL染色检测各组小鼠肺组织凋亡情况。 结果:热打击后复温1 h即可诱导PMVECs和肺组织中Pin1的表达，并呈现复温时间依赖性增加（ F=771.6， P<0.05； F=1 035， P<0.05）；热打击后复温3 h开始PMVECs和肺组织中cleaved caspase-9的表达，并呈现复温时间依赖性增加（ F=729.8， P<0.05； F=1 773， P<0.05）；PMVECs和肺组织中cleaved caspase-3在热打击后复温3 h开始表达，随着复温时间的延长其表达不断增多，趋势与cleaved caspase-9一致（ F=1 084， P<0.05； F=1 252， P<0.05）；同时，热打击后导致PMVECs中O 2-˙释放。热打击后小鼠肺组织中氧化-抗氧化系统失衡，表现为热打击后小鼠肺组织中MDA的持续释放（ F=114.2， P<0.05），而SOD水平持续抑制（ F=99.15， P<0.05）。使用Pin1抑制剂Juglone对PMVECs和小鼠进行预处理，发现与热打击组相比，热打击+Juglone组PMVECs和肺组织中Pin1、cleaved caspase-9、cleaved caspase-3均被抑制（均 P<0.05）；使用Pin1抑制剂后明显减轻热打击后PMVECs中O 2-˙的释放，促进重症中暑小鼠肺组织中氧化-抗氧化系统平衡恢复，与热打击组相比，热打击+Juglone组肺组织中MDA被抑制（11.53±0.84）nmol/mL vs （9.65±0.69） nmol/mL， t=12.52， P<0.05]，而SOD明显恢复（41.18±3.45） U/mL vs （57.52±4.83） U/mL， t=5.57， P<0.05]。同时，抑制Pin1表达后可以明显减轻热打击后肺组织的病理损伤，以及抑制凋亡的发生。 结论:初步确认Pin1主要通过介导肺组织和PMVECs的氧化应激反应和凋亡发生，进而参与热打击后肺损伤的形成。

The mechanism of Pin1 mediated oxidative stress of vascular endothelial cells involved in severe heat stroke induced acute lung injury

Objective:To explore the molecules mechanism of Pin1 in severe heat stroke induced acute lung injury by observing Pin1 regulate oxidative stress and apoptosis formation in pulmonary microvascular endothelial cells (PMVECs) and lung tissue in heat stressed mice.Methods:In vitro, a PMVECs heat stress (HS) model was established. In the control group, PMVECs were placed in a standard 37 °C, 5% CO 2 cell incubator; in the HS group, PMVECs were placed in a 43 °C cell incubator for 2 h, then the cells were further incubated at 37 °C for 1, 3, 6 or 12 h. PMVECs were pretreated with Pin1 inhibitor Juglone (1 μmol/L) 1 h before 43 °C of HS. In vivo, a severe heat stroke mouse model was established. In the HS group, the mice were kept at the simulation of climate chamber with temperature (35.5±0.5) °C, humidity (60±5)%, the rectum temperature in mice was measured by the anal rectal temperature table, when the temperature reached 42 °C, the heat exposure was stopped, and the mice were sacrificed at 1, 3, 6 or 12 h after HS. In the control group, the mice were kept at room temperature (25±0.5) °C. Mice received daily intraperitoneal administration of Pin1 inhibitor Juglone (1 mg/kg) for 3 d before HS. The protein level of Pin1, cleaved caspase-9 and cleaved caspase-3 were analyzed by Western blot, the level of O 2-˙ in cells was observed by DHE staining and fluorescence microscopy, the levels of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in lung tissue were measured by ELISA, the pathological changes of mice in different group were detected by HE staining, and the expression of Pin1 in the lung tissue of different groups was detected by immunohistochemical staining, the apoptosis rate of the lung tissue in different groups was tested by TUNEL staining. Results:At 1 h after HS, the protein expression of Pin1 in PMVECs and lung tissue began to increase in a rewarming time-dependent manner ( F=771.6, P<0.05; F=1 035, P<0.05). Cleaved caspase-9 protein in PMVECs and lung tissue started to increase at 3 h post-HS, then increased with a rewarming time-dependent manner ( F=729.8, P<0.05; F=1 773, P<0.05). The protein expression of cleaved caspase-3 in PMVECs and lung tissue also started to increase at 3 h after HS and the expression continued to be increased with prolonged rewarming time, and the trend was consistent with cleaved caspase-9 ( F=1 084, P<0.05; F=1 252, P<0.05). In addition, HS induced the increased release of O 2-˙ from PMVECs, HS induced the imbalance of oxidation-antioxidant system in lung tissue of mice after HS which verified by the continuous release of MDA ( F=114.2, P<0.05) and the continuous inhibition of SOD activity ( F=99.15, P<0.05). Compared with the HS group, pretreatment with Pin1 inhibitor Juglone in PMVECs and mice before HS significantly inhibited the protein expression of Pin1, cleaved caspase-9 and cleaved caspase-3 (all P<0.05), pretreatment with Pin1 inhibitor greatly reduced the release of O 2-˙ in PMVECs after HS, and promoted the restore of the oxidation-antioxidant system balance of lung tissue in mice with severe heat stroke. In addition, compared with the HS group, inhibiting the expression of Pin1 significantly decreased HS induced MDA release (11.53±0.84) nmol/mL vs (9.65±0.69) nmol/mL, t=12.52, P<0.05], promoted the restore of SOD activity (41.18±3.45) U/mL vs (57.52±4.83) U/mL, t=5.57, P<0.05] and improved the pathological damage of lung tissue as well as decreased the occurrence of apoptosis in post-HS mice. Conclusion:It was confirmed that Pin1 is involved in heat stress induced acute lung injury mainly through mediating oxidative stress response and apoptosis.