内质网应激和Ca^(2+)超载在热打击诱导的肺微血管内皮细胞损伤中的作用与机制

Role and mechanism of endoplasmic reticulum stress and Ca^(2+) overload in pulmonary endothelial cell damage induced by heat stress

目的研究内质网应激和Ca^(2+)超载在热打击诱导的肺微血管内皮细胞(PMVECs)损伤中的作用及其可能的机制。方法建立PMVECs温度梯度热打击模型。对照组细胞始终置于标准37℃、5%CO2细胞培养箱孵育,热打击组细胞分别置于39、41、43℃细胞培养箱中热打击2h,热打击后继续在37℃、5%CO2细胞培养箱孵育6h;另取细胞在43℃热打击前分别以20μmol/L钙离子抑制剂BAPTA-AM和50μmol/L环孢霉素A(Cs A)预处理,后续处理同43℃热打击组细胞。Western blotting检测磷酸化的蛋白激酶样内质网激酶(p-PERK)、磷酸化的真核生物翻译起始因子2α(p-e IF2α)、活性转录因子4(ATF4)以及葡萄糖调节蛋白78(GRP78)蛋白表达;流式检测细胞内Ca^(2+)、线粒体膜电位以及线粒体通透性转换孔的变化;Caspase活性试剂盒检测caspase-3变化以反映细胞凋亡情况;Millicell-ERS细胞电阻仪测定细胞的跨上皮细胞电阻(TER)以了解细胞通透性的改变。结果随着热打击温度的增高(41~43℃),PMVECs内p-PERK、p-e IF2α以及ATF4、GRP78蛋白表达增强,细胞内Ca^(2+)水平增高,线粒体通透性转换孔开放,线粒体膜电位下降,细胞通透性增加,凋亡增多,且在43℃热打击组最为明显,与对照组比较差异有统计学意义(P<0.05)。使用Ca^(2+)抑制剂预处理细胞可以促使PMVECs线粒体通透性转换孔、膜电位以及细胞通透性恢复,减少凋亡的发生;使用线粒体保护剂预处理细胞并不能减轻PMVECs的Ca^(2+)释放,但可促进细胞通透性恢复以及减少凋亡发生。结论热打击可激活PMVECs的内质网应激反应,并诱导细胞内Ca^(2+)超载介导的细胞和线粒体损伤,这可能是中暑导致内皮细胞损伤的重要机制之一。

Objective To observe the effect of different temperatures on endoplasmic reticulum stress, calcium overload, mitochondria and cell damage in pulmonary microvascular endothelial cells （PMVEC） induced by heat stress, and clarify the mechanism of endothelial cell injury in the process of heat stress to provide experimental basis for clinical prevention and treatment of heat stree. Methods Heat stress model of PMVEC cell was set up. Control group cells were incubated at 37℃, 5%CO2, while heat stress group cells were incubated at 39℃, 41℃, 43℃ for 2h, respectively, then further incubated at 37℃, 5%CO2 for 6h. Pretreatment of cells with 20μmol/L BAPTA-AM or 50μmol/L CsA before heat stress at 43℃. The protein levels of p-PERK, PERK p-eIF2a, eIF2a, ATF4 and GRP78 were analyzed by Western blotting. Intracellular Ca^2＋, mitochondrial membrane potential and the changes in mitochondrial permeability transition pore were investigated by flow cytometry. The change of caspase-3 was detected by Caspase Assay Kit. Millicell-ERS Volt-Ohm Meter and Accessories was used for determining the changes of transepithelium electrical resistance （TER）. Results Compared with the control group, with the increase of heat stress temperature （41-43℃）, the phosphorylation of p-PERK and p-eIF2a protein and the expressions of ATF4 and GRP78 proteins were gradually activated, intracellular Ca^2＋ increased, MPTP pore was opened, mitochondrial membrane potential decreased, cell permeability increased and apoptosis occurred, and it was the most obvious in the 43℃ heat stress group, and the difference was statistically significant （P〈0.05）. Pretreatment with Ca2＋ inhibitors promoted the recovery of the MPTP hole, mitochondrial membrane potential and cell permeability, and reduced the occurrence of apoptosis. While pretreatment with the mitochondrial protective agent did not reduce the release of Ca^2＋, but it could promote the recovery of cell permeability and reduce the occurrence of apoptosis. Conclusion Heat stress activates endoplasmic reticulum stress response, induces intracellular Ca^2＋ overload mediated cell and mitochondrial damages in PMVEC cells, which may be one of the important mechanisms of endothelial cell injury induced by heat stress.