Role and mechanism of endoplasmic reticulum stress and Ca2+ overload in pulmonary endothelial cell damage induced by heat stress / 解放军医学杂志

ABSTRACT

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 Ca2+, 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 Ca2+ 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 Ca2+, 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 Ca2+ 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.