目的 研究色氨酸犬尿氨酸代谢途径对高热诱导的小鼠肝损伤的影响。方法 （1）构建小鼠热习服（heat acclimation,HA）模型。36只雄性C57BL/6J小鼠随机分为4组：对照（Ctrl）组、Ctrl +热应激（heat stress, HS）组、HA组、HA+HS组,每周称重并监测核心体温（肛温）,4 w后收集小鼠血液和肝脏样本,检测血清谷丙转氨酶（alanine aminotransferase,ALT）和谷草转氨酶（aspartate aminotransferase,AST）的活性,并观察肝脏切片病理变化及高热条件下生存时间,利用非靶向代谢组学对Ctrl组和HA组小鼠的肝脏组织样本进行分析,检测肝脏犬尿氨酸途径（kynurenine pathway, KP）中犬尿喹啉酸（kynurenic acid,KYNA）等代谢物的含量,实时荧光定量检测小鼠肝脏KP中关键酶的表达水平,蛋白免疫印迹检测相应蛋白水平;（2）构建小鼠HS模型,36只雄性C57BL/6J小鼠随机分为4组：Ctrl组、Ctrl+HS组、Kyat2抑制剂PF-04859989干预（PF）组、PF+HS组。PF和PF+HS组腹腔注射PF-04859989 10 mg/kg,其余两组腹腔注射等体积DMSO稀释液,干预1h后热应激或直接取材,热应激恢复6h后收集小鼠血液和肝脏样本,检测ALT、AST和乳酸脱氢酶（lactate dehydrogenase,LDH）的活性并观察小鼠高热条件下生存时间,通过实时荧光定量和蛋白免疫印迹检测Kyat2表达水平。结果 （1）相较于Ctrl+HS组,HA+HS组小鼠血清ALT、AST水平明显降低,小鼠生存时间显著延长;与Ctrl组小鼠相比,HA小鼠肝脏中KP代谢产物3-羟基邻氨基苯甲酸（3-HAA）、吡啶甲酸（PA）含量明显升高,而KYNA、L-色氨酸（L-Trp）、L-犬尿氨酸（L-kyn）含量明显降低,肝脏中Afmid、Kynu、Kmo酶的表达水平显著升高,而Kyat2酶的表达显著降低;（2）相较于Ctrl组,PF组小鼠ALT、AST、LDH和生存时间均无显著差异;与Ctrl+HS组相比,PF+HS组明显延长了小鼠的生存时间,且ALT、AST、LDH明显降低。结论 HA增强小鼠肝脏中的色氨酸KP代谢,但KYNA含量明显降低,且抑制KYNA的生成可有效减轻高热诱导的小鼠肝损伤。

Objective To investigate the protective effect of heat acclimation on hyperthermia-induced liver injury in mice and the associated changes in the kynurenine pathway in the liver. Methods (1) The heat acclimation (HA) model was constructed. Thirty-six male C57BL/6J mice were randomly divided into four groups control group(Ctrl, not subjected to heatstroke), HA group ( subjected to heat acclimation but not to heatstroke), Ctrl +heat stress (HS )group ( only subjected to HS ) and HA + HS group ( subjected to HA and HS ). Core body temperature was monitored once a week. Blood and liver specimens were collected after four weeks. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured. The pathological changes in the liver and survival time under hyperthermia conditions were observated. Untargeted metabolomics was used to analyze hepatic metabolic changes. Kynurenic acid (KYNA) and other metabolites in the hepatic kynurenine pathway (KP) were measured. Real-time quantitative PCR (Q-PCR) was used to detect the mRNA expressions of the key enzymes in the hepatic KP. Protein immunoblotting was used to detect the corresponding protein levels. (2) The HS model after Kyat2 inhibition was constructed. Thirty-six male C57BL/6J micewere randomly divided into four groups: Ctrl group, Ctrl+HS group, Kyat2 inhibitor PF-04859989 intervention (PF) group, PF+ HS group. The PF and PF+HS groups were injected intraperitoneally with PF-04859989 10mg/kg, and the remaining two groups were injected intraperitoneally with an equal volume of DMSO solution. HS or sampling was initiated 1 h after intervention. Blood and liver specimens were collected from mice 6 h after recovery from HS, and the activities of ALT, AST and lactate dehydrogenase (LDH) were detected. The survival time under hyperthermia conditions was observated. The expression level of Kyat2 in the liver was detected by Q-PCR. The corresponding protein level was detected by protein immunoblotting. Results (1) Compared with the Ctrl+HS group, serum ALT and AST levels of the HA+HS group were significantly lower, and the survival time was significantly prolonged. Compared with the Ctrl group, the levels of KYNA in the serum and KP metabolites 3-hydroxy-phthalamidobenzoic acid （3-HAA） and pyridinecarboxylic acid（PA） in the liver were significantly higher in the HA group, whereas the levels of KYNA, L-Trp, and L-kyn in the liver were significantly lower. The expressions of Afmid、Kynu and Kmo were higher than Ctrl, whereas the expression of Kyat2 enzyme in the liver was significantly lower. (2) Compared with the Ctrl group, there were no significant differences in serum ALT, AST, LDH and survival time in the PF group. Compared with the Ctrl+HS group, the PF+HS group displayed significantly prolonged survival time, and serum ALT, AST, and LDH were significantly reduced. Conclusion Tryptophan metabolism through KP was enhanced in the liver of heat-acclimated mice. Inhibition of kynurenic acid production attenuated hyperthermia-induced liver injury in mice.