2.2 Induction of the HPMEC under heat stress combined with LPS in vitro
Pretreatment before molding Heat stress and lipopolysaccharide (HS+LPS) were employed to construct the in vitro models of heat stroke in our previous study. 15. All the HPMEC were categorized into 4 groups with 8 wells in each group: control group (CON group), heat stroke group (HS group), heparanase group (HPSE group), and unfractionated heparin group (UFH group). HPSE was known to aggravate endothelial glycocalyx degradation as the only endonuclease.16, 17 UFH was known to attenuate glycocalyx degradation via non-covalent binding with glycocalyx.13 After conventional culture for 72 hours, the cell in the CON group was cultured in normal conditions, without receiving heat stress and LPS (Sigma, America). No pretreatment was performed on the HS group before modeling. The cell in the HPSE group was subjected to a 0.2 U/ ml Heparanase (Sigma, America) culture medium and incubated at 37 °C for 30 minutes. The UFH group was subjected to a medium containing 10 U/ ml unfractionated heparin (Sigma, America) and incubated 12 hours before modeling. 18, 1920
Modeling Vitro Briefly, except for CON group, HPMEC in HS, HPSE, and UFH groups were subjected to heat stress at 43 °C for 30 minutes, and then moved to a 5% CO2, 37 °C incubators, stimulated with LPS at a concentration of 500 ng/ml for 12 hours.