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Objective To profile the high mobility group protein B1 (HMGB1) in exosomes derived from heat-stressed monocytes and its effect on endothelial cell inflammation. Methods The heatstroke model was established. The rats were divided into control group (n=6) and heatstroke group (n=6). Monocytes from blood were isolated and exosomes were extracted. Isobaric tags for relative and absolute quantification technology were used to identify the exosomal protein components, such as HMGB1, before functional description with annotation databases. Then, Western blotting and enzyme-linked immunosorbent assay (ELISA) were used to verify the changes of monocyte-derived exosomes HMGB1 in rats and patients. Umbilical vein endothelial cells (HUVECs) were treated with exosomes from rats in control group, heatstroke group and heatstorke+ethyl pyruvate group. The expression levels of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) and interleukin-1β (IL-1β) were detected with RT-qPCR and Western blotting. HMGB1, NLRP3 and IL-1β expresion levels in exosomes of patient and healthy donors, were detected with ELISA. Results In heatstroke group, the number of exosomes (×104/monocyte) derived from monocytes significantly increased than that in control group (11.24±2.66 vs. 1.52±0.21, P<0.001). Proteomic analysis showed that the expression of exosome HMGB1 in heatstroke group was up regulated than control group (18.63 times, P<0.001), and these proteins were enriched in NOD-like receptors and other signaling pathways. Western blotting showed that the relative expression of HMGB1 in exosomes of rats in heatstroke group was significantly higher than that in control group (4.13±0.22 vs. 1.00±0.15, P<0.001). ELISA also demonstrated that the level of HMGB1 in exosomes of patients in heatstroke group increased than that in control group [(0.29±0.11) ng/ml vs. (0.12±0.04) ng/ml, P=0.006]. qPCR and Western blotting showed that compared with control group, the mRNA and protein levels of NLRP3 and IL-1β in endothelial cells of heatstroke group were increased, which could be alleviated by the inhibitor of HMGB1, and the difference was statistically significant (P<0.05). Conclusion Heat stress increased the HMGB1 level in the monocyte-derived exosomes and might mediate endothelial cell inflammation via HMGB1.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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