Heat stress combined with lipopolysaccharide induces pulmonary microvascular endothelial cell glycocalyx inflammatory damage in vitro.

Heat stroke is a life-threatening disease with high mortality and complications. Endothelial glycocalyx (EGCX) is essential for maintaining endothelial cell structure and function as well as preventing the adhesion of inflammatory cells. Potential relationship that underlies the imbalance in inflammation and coagulation remains elusive. Moreover, the role of EGCX in heat stroke-induced organ injury remained unclear. Therefore, the current study aimed to illustrate if EGCX aggravates apoptosis, inflammation, and oxidative damage in human pulmonary microvascular endothelial cells (HPMEC). Heat stress and lipopolysaccharide (LPS) were employed to construct in vitro models to study the changes of glycocalyx structure and function, as well as levels of heparansulfate proteoglycan (HSPG), syndecan-1 (SDC-1), heparansulfate (HS), tumor necrosis factor-α (TNF-α), interleukin (IL)-6, Von Willebrand factor (vWF), endothelin-1 (ET-1), occludin, E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and reactive oxygen species (ROS). Here, we showed that heat stress and LPS devastated EGCX structure, activated EGCX degradation, and triggered oxidative damage and apoptosis in HPMEC. Stimulation of heat stress and LPS decreased expression of HSPG, increased levels of SDC-1 and HS in culture supernatant, promoted the production and release of proinflammation cytokines (TNF-α and IL-6,) and coagulative factors (vWF and ET-1) in HPMEC. Furthermore, Expressions of E-selection, VCAM-1, and ROS were upregulated, while that of occludin was downregulated. These changes could be deteriorated by heparanase, whereas they meliorated by unfractionated heparin. This study indicated that EGCX may contribute to apoptosis and heat stroke-induced coagulopathy, and these effects may have been due to the decrease in the shedding of EGCX.

PULMONARY VASCULAR ENDOTHELIAL GLYCOCALYX DEGRADATION CONTRIBUTES TO ACUTE LUNG INJURY IN EXPERIENCING HEATSTROKE.

ABSTRACT: Objectives: This study investigated the role and potential involvement of pulmonary vascular glycocalyx degradation in acute lung injury in rats with severe heatstroke (HS). Methods: Rats in an established HS model were exposed to a heated environment for 60 min in an incubator (temperature, 40°C ± 2°C; humidity, 65% ± 5%). Following pretreatment with heparanase III (HPSE III) or heparin, pathological lung injury, arterial blood gas, alveolar barrier disruption, and hemodynamic changes were evaluated. The vascular endothelial structures of the lungs were examined using electron microscopy. The concentration of Evans blue dye in the lungs and arterial blood gas were assessed. An enzyme-linked immunosorbent assay was used to quantify the plasma concentration of heparan sulfate proteoglycan. The expression of glypican-1 and syndecan-1 in pulmonary vessels was measured using immunofluorescence. Western blots were used to detect the expression of TNF-α, IL-6, and vascular endothelial biomarkers in the rat lungs. Pulmonary apoptosis was assessed using a TUNEL (terminal dUTP nick end labeling) assay, and the concentrations of malondialdehyde were measured. Results: Glycocalyx shedding aggravated lung injuries. Severe histopathological damage was observed, and indexes of lung function deviated from abnormal ranges. In addition, pulmonary vascular endothelial cells were disrupted. Compared with the HS group, the plasma concentration of heparan sulfate proteoglycan significantly increased in the HPSE group ( P < 0.05). The expression of glypican-1 and syndecan-1 decreased, and the extravasation of Evans blue dye increased ( P < 0.01). Endothelial biomarker expression increased in the lung tissue, whereas occludin expression decreased. Moreover, TNF-α and IL-6 were overexpressed following heat stress. Furthermore, apoptosis of pulmonary tissues and the concentration of malondialdehyde in rat lungs increased in the HS and HPSE groups. Conclusions : Heatstroke induced pulmonary glycocalyx degradation, which increased vascular permeability and aggravated vascular endothelial dysfunction, contributing to apoptosis, inflammation, and oxidation in the pulmonary tissues.

Heat stress combined with lipopolysaccharide alter the activity and superficial molecules of peripheral monocytes.

The purpose of this study was to focus on the underlying relationship between the hyperactivity for the peripheral monocytes and heat stroke by investigating the inflammatory oxidative activity of and the expression of superficial molecules. Peripheral blood samples were collected from 10 healthy adult volunteers. Human blood monocytes were isolated by density gradient centrifugation and sequent adherent culture. The objectives were divided into four groups: 43°C heat stress combined with lipopolysaccharide (LPS) group, 43°C heat stress group, LPS group, and control group. There were 10 cases in each group. An enzyme-linked immunosorbent assay (ELISA) test was used to measure the concentrations of supernatant inflammatory mediators (tumor necrosis factor alpha (TNF-α), interleukin-1ß (IL-1ß) and interleukin-10 (IL-10)). After loaded by 2,7-Dichlorodi-hydrofluorescein-diacetate (DCFHDA) fluorescent probe, intracellular reactive oxygen species (ROS) levels were determined by a flow cytometry. After fluorescent microspheres incubation, the phagocytosis of monocytes was observed under a fluorescent microscope. Respectively, the flow cytometry and Western blot were used to evaluate the level of triggering receptor expressed on myeloid cells-1 (TREM-1) and Toll-like receptor-4 (TLR-4) on the monocytes. Furthermore, the mRNA expression of TREM-1 and TLR-4 was detected by real-time polymerase chain reaction (RT-PCR). The heat stress combined with LPS stimulation promoted the peripheral monocytes to produce inflammatory mediators (TNF-α, IL-1ß, and IL-10) and release ROS. Otherwise, such complex strike significantly suppressed the phagocytic activity of monocytes in peripheral blood. Moreover, the expression of TREM-1, TLR-4 and CD86 was measured by the flow cytometry on peripheral monocytes which were respectively promoted by the union of heat stress and LPS. The results of Western blot and RT-PCR demonstrated the similar kinetics on these superficial molecules (TREM-1, TLR-4, and CD86) stimulated by the combination of heat stress and LPS. The underlying mechanism of the dysfunction for the peripheral monocytes may be related to the abnormal expression of superficial molecules TREM-1, TLR-4, and CD86 on the monocytes induced by heat stress and LPS.

Microcirculatory Disorders and Protective Role of Xuebijing in Severe Heat Stroke.

This study was conducted to explore underlying mechanism of microcirculation dysfunction and protectiverole of Xuebijing in heat stroke. Forty rats were divided into: control, vehicle + heat stress (HS), superoxide dismutase (SOD) + HS, and Xuebijing + HS groups. Rats in heat stress groups were subjected to continuous heat stress in infant incubator 1 h after tail vein injection of the tested compound and spinotrapezius preparation. Velocity of blood flow through micro-vessels and vascular diameter were detected in real time. Another 27 rats were divided into: vehicle, SOD, and Xuebijing groups, then further divided into three subgroups each: control, Tcore = 38 °C, Tcore = 41 °C. Rats were sacrificed, and spinotrapezius single-cell suspensions were prepared for detecting SOD and reactive oxygen species (ROS). The results showed that heat stress decreased SOD activity, increased ROS levels, and reduced the blood flow rate. Xuebijing increased SOD activity, decreased ROS levels and exhibited a protective effect in terms of blood flow rate but was less protective than SOD. The survival time in Xuebijing + HS group was longer than that in vehicle group but shorter than that in SOD + HS group. The results suggested Xuebijing could decrease ROS levels and have protective effects in severe heat stroke.