肺热普清散对低氧诱导斑马鱼运动损伤的保护作用及对Hif1α的影响

Protective effect of Feirepuqing Powder on hypoxia-induced sports injury in zebrafish model by inhibiting Hif1α

探讨藏药复方肺热普清散对低氧环境下斑马鱼模型的保护作用及其对低氧诱导因子1α(Hif1α)的影响。建立斑马鱼低氧运动损伤模型,分别检测低氧条件下斑马鱼行为学和整体乳酸水平,以受精后72 h AB型斑马鱼幼鱼为实验对象,空白对照组采用正常溶氧100%(8.4 mg/L),低氧组采用纯氮气置换获得溶氧50%(4.2 mg/L)培养水,50%低氧水加不同浓度肺热普清散处理胚胎24 h。采用斑马鱼行为学分析仪记录30 min游泳轨迹,使用Zeblab软件导出30 min内运动距离和运动时间;分别对实验后各组斑马鱼进行整体匀浆并测定组织乳酸含量和乳酸脱氢酶(lactate dehydrogenase,LDH)活力;利用蛋白印迹(western blot,WB)分析各组斑马鱼Hif1α的表达水平变化;使用免疫组织化学方法对各组鱼尾鳍部Hif1α表达进行比较。结果表明,与低氧对照组比较,肺热普清散10~30 mg/L剂量组可显著增加低氧诱导引起的运动距离和运动时间(p<0.05),并且相对于低氧引起的乳酸升高(p<0.001),10~30 mg/L组的乳酸含量明显降低(p<0.05);同时LDH与低氧组活力比较,肺热普清散30 mg/L组可显著降低LDH活力(p<0.05);WB实验结果显示,与低氧组比较,肺热普清散可抑制Hif1α表达(p<0.05);同时免疫组化结果显示10 mg/L肺热普清散可减少Hif1α表达。由此肺热普清散对急性斑马鱼低氧损伤模型引起的运动损伤有保护作用,其耐低氧机制与Hif1α表达有关。

This study aimed to investigate the protective effects of the Tibetan medicinal compound Feirepuqing Powder on a zebrafish model in a hypoxic environment and its effect on the hypoxia-inducible factor 1α(Hif1α).Through establishing a zebrafish hypoxic injury model,behavioral and overall lactate changes under hypoxic conditions were investigated.Experiments were conducted using AB zebrafish larvae 72 h after fertilization.In the control group,100%standard dissolved oxygen(8.4 mg/L)was used,while in the hypoxia group,pure nitrogen(N 2)gas replacement was used to obtain 50%dissolved oxygen(4.2 mg/L)culture water,and the embryos were treated with 50%hypoxic water add different concentrations of Feirepuqing Powder for 24 h.The zebrafish behavioral analyzer was used to record the swimming trajectory for 30 min,and the Zeblab software was used to derive the movement distance and time during the 30 min.Following the experiment,all the zebrafish from both the groups were homogenized and the lactic acid and lactate dehydrogenase(LDH)content in their tissue were determined.The changes in the Hif1αexpression level of the zebrafish in each group were analyzed via western blot(WB)analysis,and the expression of Hif1αin the caudal fin of each group of fish was compared using immunohistochemistry.The results revealed that compared with the hypoxic control group,10 to 30 mg/L Feirepuqing Powder group significantly increased the hypoxia-induced exercise distance and exercise time(p<0.05),and relative to the hypoxia-induced lactic acid increase(p<0.001),the lactic acid content of the 30 mg/L Feirepuqing Powder group significantly reduced.When LDH activity was compared with that of the hypoxic group,LDH activity was significantly reduced in the 30 mg/L Feirepuqing Powder group(p<0.05).The WB results revealed that when compared with the hypoxia group,Feirepuqing Powder inhibited the expression of Hif1α(p<0.05).Finally,the immunohistochemical results showed that 10 mg/L of Feirepuqing Powder reduced the expression of Hif1α.The results of this study show that Feirepuqing Powder exhibits a protective effect on induced exercise injuries in an acute zebrafish hypoxic injury model and that its hypoxia-tolerance mechanism is related to the expression of Hif1α.