3.4 Degradation of endothelial glycocalyx potentially activated oxidative stress injuries in HPMEC
TNF-α and IL-6 were the classical pro-inflammation cytokines to accessing inflammatory activities and prognostic outcomes. We, therefore, evaluated the expression of TNF-α and IL-6 in treated cells. Our results found that heat stress and LPS significantly increased the production of TNF-α and IL-6 in HS, HPSE, and UFH groups, compared with the CON group (P < 0.05) (Figure 4A ). This upregulation was more pronounced in cells treated with HPSE but more modest in those treated with UFH in comparison with those treated with heat stress and LPS alone (P < 0.05) (Figure 4A ). These results indicated that heat stress and LPS promoted inflammatory activities of HPMEC by an endothelial glycocalyx degraded mechanism.
Our previous study demonstrated that generation of ROS was the critical mediator in heat stress-induced apoptosis, 27 which was also known to be a classical marker related to oxidative stress in the tissue. We, thus, detected the changes of ROS level in HPMEC by DCFH-DA marked with green fluorescence, which produces enhanced fluorescence when cells generate ROS. Heat stress and LPS significantly increased the intracellular ROS levels in HPMEC in comparison with the CON group (Figure 4B ) (P < 0.05). In addition, the ROS level was significantly higher in HPSE treated-cells but was lower in UFH treated-cells than HS+LPS-treated cells (P< 0.05) (Figure 4B ). These results suggested that oxidative stress of HPMEC as a result of damaged endothelial glycocalyx induced by heat stroke.