泮托拉唑对急性肺损伤模型大鼠和人肺微血管内皮细胞损伤的作用及作用机制

目的探讨泮托拉唑对急性肺损伤(ALI)模型大鼠和人肺微血管内皮细胞(HPMECs)损伤的作用及作用机制。方法将48只SD大鼠随机分为正常对照组,脂多糖组,阳性对照组,泮托拉唑低、高剂量组和泮托拉唑高剂量+氯喹组,各8只。除正常对照组外,其余各组大鼠均腹腔注射5 mg/kg脂多糖复制ALI模型;阳性对照组大鼠腹腔注射2 mg/kg地塞米松,泮托拉唑低、高剂量组分别腹腔注射泮托拉唑26,104 mg/kg,泮托拉唑高剂量+氯喹组注射104 mg/kg泮托拉唑和10 mg/kg氯喹,正常对照组和脂多糖组大鼠均腹腔注射等体积生理盐水。取对数生长期的HPMECs,随机分为正常对照组,脂多糖组,阳性对照组,泮托拉唑低、高剂量组和泮托拉唑高剂量+氯喹组。除正常对照组(给予等体积生理盐水)外,其余各组细胞添加100μg/mL脂多糖,以构建HPMECs损伤模型,阳性对照组添加100 nmol/L地塞米松,泮托拉唑低、高剂量组细胞分别添加泮托拉唑40,80μg/mL,泮托拉唑高剂量+氯喹组添加80μg/mL泮托拉唑和5μmol/L氯喹。采用苏木精-伊红染色法检测大鼠肺组织的病理学变化,测定肺组织湿/干重比值(W/D);采用TUNEL染色法检测大鼠肺组织细胞凋亡情况;采用酶联免疫吸附法检测肺组织和HPMECs细胞中肿瘤坏死因子-α(TNF-α)、白细胞介素6(IL-6)、白细胞介素1β(IL-1β)水平;采用CCK-8法检测细胞活性;采用蛋白免疫印迹法检测肺组织和HPMECs细胞中LC3、Beclin1、雷帕霉素靶蛋白(mTOR)、磷酸化mTOR(p-mTOR)的表达水平。结果与脂多糖组比较,阳性对照组,泮托拉唑低、高剂量组大鼠肺组织病理学评分、W/D值均显著降低(P <0.05),大鼠血清和HPMECs细胞中TNF-α,IL-6,IL-1β水平均显著降低(P <0.05),TUNEL阳性细胞数均显著减少(P <0.05),细胞凋亡率和p-mTOR/mTOR表达水平均显著降低(P <0.05),LC3Ⅱ/LC3Ⅰ和Beclin1表达水平均显著升高(P <0.05)。结论泮托拉唑可通过mTOR信号通路调控自噬,在ALI模型大鼠和HPMECs损伤中发挥保护作用。

Effect of Pantoprazole on Acute Lung Injury Rats and Injury of Human Pulmonary Microvascular Endothelial Cells Injury and Its Mechanism

Objective To investigate the effect of pantoprazole on acute lung injury(ALI) rats and human pulmonary microvascular endothelial cell(HPMECs) injury and its mechanism.Methods A total of 48 SD rats were randomly divided into the normal control group,lipopolysaccharide(LPS) group,positive control group,pantoprazole low-and high-dose groups and pantoprazole highdose+chloroquine group,with eight rats in each group.ALI model was established by intraperitoneal injection of 5 mg/kg of LPS except the normal control group(equal volume of normal saline).The rats in the positive control group were intraperitoneally injected with 2 mg/kg of dexamethasone,the rats in the pantoprazole low-and high-dose groups were intraperitoneally injected with 26,104 mg/kg of pantoprazole,the rats in the pantoprazole high-dose+chloroquine group was injected with 104 mg/kg of pantoprazole and 10 mg/kg of chloroquine,and the rats in the normal control group and LPS group were intraperitoneally injected with equal volume of normal saline.HPMECs during the logarithmic growth period were randomly divided into the normal control group,LPS group,positive control group,pantoprazole low-and high-dose groups and pantoprazole high-dose+chloroquine group.HPMECs injury model was established by adding 100 μg/mL of LPS to cells in other groups except the normal control group(equal volume of normal saline).100 nmol/L of dexamethasone was added to cells in the positive control group,and 40 μg/mL and 80 μg/mL of pantoprazole were added to cells in pantoprazole low-and high-dose groups respectively.80 μg/mL of pantoprazole and 5 μmol/L of chloroquine were added to cells in pantoprazole high-dose+chloroquine group.The pathological changes of lung tissue of rats were detected by the hematoxylin-eosin staining,and the wet/dry weight ratio(W/D) of lung tissue was measured.The apoptosis of rat lung tissue was detected by the TUNEL staining.The levels of tumor necrosis factory(TNF-α),interleukin-6(IL-6) and interleukin 1β(IL-1β) in lung tissue and HPMECs were detected by the enzymelinked immunosorbent assay(ELISA).The cell activity was detected by the CCK-8 method.The expression levels of LC3,Beclinl,mammalian target of rapamycin(mTOR) and phosphorylated mTOR(p-mTOR) in lung tissue and HPMECs cells were detected by the Western blot.Results Compared with those in the LPS group,the lung histopathological score and W/D ratio of rats were significantly lower(P <0.05),the levels of TNF-α,IL-6 and IL-1β in serum of rats and HPMECs were significantly lower(P <0.05),the number of TUN EL positive cells was significantly fewer(P <0.05),the apoptosis rate and p-mTOR/mTOR expression level were significantly lower(P <0.05),while the expression levels of LC3 Ⅱ/LC3 Ⅰ and Beclinl were significantly higher in the positive control group and pantoprazole low-and high-dose groups(P <0.05).Conclusion Pantoprazole can regulate autophagy through the mTOR signaling pathway and play a protective role in A LI model rats and HPMECs injury.