Abstract: Objective To investigate the protective effect of hydroxysafflor yellow A (HSYA) against heat stroke (HS)-induced acute lung injury and its possible mechanism. Methods The optimal dose of HSYA pretreatment via intraperitoneal injection prior to HS was determined in a mice by observing heat tolerance of the mice. C57BL/6J mice were pretreated with HSYA at the optimal dose or with Nec-1 (a RIP1 activation inhibitor) before HS, and the changes in core body temperature and survival of the mice were observed during the 72-h recovery period. At different stages of recovery, lung tissues, bronchoalveolar lavage fluid and blood samples were collected from the mice for assessing lung tissue pathology, wet-to-dry weight ratio and water content of the lungs; leukocyte and neutrophil counts, total protein levels and HMGB1 level in the bronchoalveolar lavage fluid (BLF) were also detected. Serum levels of TNF-α, IL-6 and HMGB1 were detected with ELISA, and the expression levels of RIP1, RIP3, MLKL-s358, MLKL and MLKL-s358 proteins in the lung tissues were detected using Western blotting. Results HSYA pretreatment at the moderate and high doses significantly improved heat tolerance of the mice with comparable effects. At the optimal dose of 2.25 mg/kg, HSYA pretreatment significantly increased heat tolerance of the mice (P<0.05), showing a similar effect with Nec-1 pretreatment. Pretreatment with HSYA and Nec-1 both significantly increased survival rate of the mice (P<0.05), lowered histopathological score and water content of the lungs, and reduced the levels of TNF-α, IL-6 and HMGB1 (P<0.05), leukocyte and neutrophil counts, and total protein and HMGB1 levels in the BLF (P<0.05). The mice during recovery from HS showed significantly increased RIP1 expression and MLKL-s358 phosphorylation level in the lung tissue (P<0.05), which were obviously lowered by HSYA pretreatment of the mice. Conclusion Severe HS results in necroptosis in the lung tissue of mice, which can be alleviated by HSYA pretreatment.

Key words: severe heat stroke; hydroxysafflor yellow A; necroptosis; RIP1; MLKL-s358