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.
目的: 研究羟基红花黄色素A（HSYA）是否在重症中暑肺损伤中起保护作用及其可能的作用机制。
方法: 使用不同浓度（1.125、2.25、4.5 mg/kg）HSYA腹腔注射预处理小鼠，建立重症中暑（sHS）小鼠模型，分为低、中、高剂量HSYA中暑组、单纯中暑组及正常对照组，12只/组。初步观察及比较各组热耐受的情况，以确定HSYA最佳治疗剂量；后使用中剂量HSYA及RIP1活化抑制剂Nec-1预处理小鼠，分组为HSYA+HS组、Nec-1+HS组、HS组及正常对照组，8只/组，观察72 h恢复期核心体温变化特征，比较热耐受情况及生存情况。给予相同处理因素处理小鼠分组为正常对照组，HS组，HSYA+HS组及Nec-1+HS组，正常对照组6只，其余18只/组，分别于重症中暑恢复期不同阶段（0、2、6、12、24 h）处死小鼠，每个时间点处死3只小鼠，收集小鼠的肺组织、肺泡灌洗液及血液样本，取肺组织行HE染色，并进行病理评分，检测肺湿干重比，肺含水量，肺泡灌洗液中白细胞、中性粒细胞、蛋白含量；ELISA法检测肺泡灌洗液中HMGB1水平及血清中TNF-α、IL-6及HMGB1水平；Western blotting检测恢复期（2、6、12 h）肺组织中RIP1、RIP3、MLKL-s358、MLKL表达水平，及经HSYA预处理后MLKL-s358蛋白水平。
结果: 中剂量及高剂量HSYA预处理可明显改善小鼠热耐受能力，中剂量与高剂量无显著差异，后续药物预处理以中剂量（2.25 mg/kg）作为标准剂量；与HS组相比，HSYA+HS组和Nec-1+HS组小鼠热耐受程度均增加（P<0.05），HSYA+HS组和Nec-1+HS组无明显差异。HSYA及Nec-1预处理组小鼠生存率增加（P<0.05），肺组织病理评分、TNF-α、IL-6及HMGB1水平降低（P<0.05），肺湿干重比，肺含水量，肺泡灌洗液中白细胞、中性粒细胞、蛋白含量及HMGB1水平降低（P<0.05），HS小鼠恢复期肺组织RIP1水平及MLKL-s358磷酸化水平升高（P<0.05），与HS组相比，HSYA+HS组MLKL-s358磷酸化水平降低。
结论: 重症中暑小鼠肺组织可发生程序性坏死，HSYA可通过抑制程序性坏死发挥肺保护作用。
Keywords: MLKL-s358; RIP1; hydroxysafflor yellow A; necroptosis; severe heat stroke.