红景天苷抑制库普弗细胞的HMGB1产生保护热射病小鼠肝损伤

目的 肝损伤是重症中暑的常见并发症,是导致患者死亡的直接原因。本研究拟探讨红景天苷(salidroside, Sal)对热射病小鼠急性肝损伤的保护作用及其可能机制。方法 动物实验部分,将40只ICR (institute of cancer research)小鼠随机分为空白对照组、Sal对照组、热射病组、Sal干预组。苏木精-伊红染色法(hematoxylin-eosin staining, HE)染色后观察小鼠肝脏损伤的程度。检测肝功能指标丙氨酸氨基转移酶(alanine aminotransferase, ALT)和天冬氨酸氨基转移酶(aspartate aminotransferase, AST)的改变。体外实验部分,分离和培养库普弗细胞(Kupffer cells,KCs),以不同终浓度(80 μmol/L、160 μmol/L及320 μmol/L)的Sal预处理KCs 24 h,43 ℃±0.5 ℃热应激2 h。MTT 3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2-H-tetrazolium bromide]比色法检测细胞存活率。采用相应的试剂盒测定各组细胞上清液中乳酸脱氢酶(lactate dehydrogenase,LDH)、丙二醛(malonaldehyde,MDA)、肿瘤坏死因子-α (tumor necrosis factor-alpha, TNF-α) 和高迁移率族蛋白1 (high mobility group box 1, HMGB1)释放量。采用实时聚合酶链反应(real-time polymerase chain reaction, real-time PCR)评估HMGB1的mRNA表达。应用细胞核/细胞质提取试剂盒提取细胞核和细胞质蛋白,Western印迹法观察HMGB1从细胞核向细胞质转移的变化。结果 本实验条件下,动物实验部分,与空白对照组、Sal对照组相比,热射病小鼠肝脏病理损伤主要表现为肝细胞肿胀、透明或水样变性,伴随肝功能指标ALT和AST的明显升高;与热射病组相比,Sal干预能够缓解其肝细胞肿胀、透明或水样变性等病理改变并降低热射病小鼠血清ALT和AST水平。细胞实验部分,热刺激可诱导KCs上清液中LDH、MDA以及TNF-α释放,而随着Sal剂量的增加,可显著抑制热应激诱导的LDH、MDA以及TNF-α释放,提高细胞的存活率。且Sal显著抑制热应激诱导的HMGB1表达和分泌,以及HMGB1从细胞核向细胞质的易位。结论 Sal保护热射病小鼠肝损伤,可能是通过其抑制KCs的HMGB1表达和分泌,以及HMGB1从细胞核向细胞质的易位发挥作用。

Salidroside protect liver of heatstroke mice by inhibition of production HMGB1 in Kupffer cells

Objective Liver injury is a common complication of heatstroke, and the direct cause of death. The aim of this study is to investigate the protective effect of salidroside (Sal) on acute liver injury induced by heat stroke in mice and its possible mechanism. Methods In animal experiment, 40 ICR (Institute of Cancer Research) mice were randomly divided into the blank group, Sal control group, heatstroke group and Sal intervention group. The degree of liver injury in mice was assessed by hematoxylin-eosin (HE) staining. Meanwhile, the changes of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected to reflect the liver function. In vitro, Kupffer cells (KCs) were isolated and cultured. KCs were pretreated with Sal for 24 hours with different final concentrations (80 μmol/L, 160 μmol/L and 320 μmol/L), and were heat stressed with (43±0.5)℃ for 2 hours. MTT 3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2-H-tetrazolium bromide] colorimetric assay was used to detect cell survival. The corresponding kits were used to determine the release of lactate dehydrogenase (LDH), malondialdehyde (MDA), tumor necrosis factor - alpha (TNF-α) and high mobility group box 1 (HMGB1) in culture supernatant. Real-time polymerase chain reaction (real-time PCR) was used to evaluate the mRNA expression of HMGB1. Nuclei and cytoplasmic proteins were extracted by nuclear/cytoplasmic extraction kit to check the translocation of HMGB1 from nucleus to cytoplasm changes by Western blotting. Results Under our experiment conditions, in vivo, compared with the blank group and Sal control group, liver pathological damage was mainly manifested as hepatocyte swelling, hyaline or water degeneration, accompanied by a marked increase with ALT and AST. Compared with the heatstroke group, Sal could reduce serum ALT and AST levels and improve liver pathological changes in heatstroke mice. In vitro, heatstroke promoted the release of LDH, MDA and TNF-α, and Sal significantly inhibited the release of LDH, MDA and TNF-α induced by heat stress, and increased the cell survival rate. Sal significantly inhibited the expression and secretion of HMGB1 induced by heat stress, and the translocation of HMGB1 from nucleus to cytoplasm. Conclusions Sal can protect liver in heatstroke mice. The possible mechanism is that it can inhibit the expression and secretion of HMGB1 in KCs, and it also can inhibit the translocation of HMGB1 from cytoplasm to cytoplasm.