HIF-1 Promotes Adaptation Of CIK Cells And Gymnocyprinus Przewalskii To High Ammonia

High concentration of ammonia nitrogen can damage gill tissue of fish,affect gas exchange,increase blood ammonia in fish,cause coma or death of fish.Alkaline environment can inhibit ammonia nitrogen excretion of fish.Qinghai Lake is the largest inland saline lake in China,and Gymnocypris przewalskii is the only economic fish in Qinghai Lake.It has evolved many mechanisms to adapt to saline alkali environment,but the specific molecular mechanism is still unclear.HIF-1（hypoxia inducible factor-1）is a transcription factor widely existing in animals under hypoxia.Its transcription activity mainly depends onαsubunit.Previous studies found that the HIF-1αof Gymnocypris przewalskii in Qinghai Lake has a special P550 variation,which can promote the stability of HIF-1αof Gymnocypris przewalskii,and may help fish adapt to high ammonia environment.In order to explore the possible mechanism of HIF-1 involved in high ammonia adaptation,CCK-8,fluorescence quantitative PCR,Western blot,flow cytometry and metabonomics were used to analyze the samples obtained from kidney cell line CIK and Gymnocypris przewalskii.The main research contents are shown as follows:Part Ⅰ:HIF-1 can promote the proliferation of CIK cells and inhibit the apoptosis of CIK cells in high ammonia environment1.0 mM DMOG treatment for 12 hours could activate HIF-1 pathway in CIK cells most effectively:CIK cells were cultured in the medium containing different concentrations of DMOG（0.2 mM,0.4 mM,0.8 mM,1.0 mM,1.2 mM,1.4 mM,1.6 mM,1.8 mM,2.0 mM）for 12 hours,and the cellular viability of CIK cells was determined by CCK-8 method.CIK cells were treated with 1.0 mM DMOG for different time（0,3,6,12,24,48 hours）.The expression of HIF-1 target gene VEGF mRNA and HIF-1 protein in CIK cells were detected by qPCR and Western blot,respectively.It was found that the HIF-1 pathway was activated most effectively after 12 hours of treatment.DMOG can reduce the toxicity of high ammonia on CIK cells:The appropriate concentration of NH₄Cl（0-160 mM）was determined by CCK-8 method.CIK cells were treated with ammonia（40 mM,80 mM,120 mM NH₄Cl）and ammonia+1mM DMOG,and the proliferation and apoptosis of CIK cells were detected by CCK-8 method and flow cytometry after 12 hours of treatment.It was found that different concentrations of high ammonia treatment could inhibit the proliferation of CIK cells and promote the apoptosis of CIK cells;DMOG could reduce the inhibitory effect of 40 mM NH₄Cl on the proliferation of CIK cells and inhibit the apoptosis induced by 80 mM NH₄Cl and 120 mM NH₄Cl,suggesting that DMOG can help CIK cells adapt to high ammonia environment by promoting the proliferation and inhibiting apoptosis of CIK cells in high ammonia environment.PartⅡ:HIF-1 can recover TCA cycle metabolism which is inhibited by high ammonia in CIK cellsThe contents of TCA cycle related metabolites（glutamine,glutamate and orotate）were detected in CIK cells treated with high ammonia（80 mM NH₄Cl）,1mM DMOG or high ammonia+1 mM DMOG by metabonomics.For metabolic flux analysis,CIK cells were cultured in MEM complete medium containing 1 mM¹³C-glutamine and treated with ammonia,1 mM DMOG,ammonia+1 mM DMOG for 12 hours.The contents of ¹³C labeled orotate,aspartic acid,citric acid,acetyl-coenzyme A and glutamine were detected by metabolic flow technique.The proportions and contents of metabolites in glucose metabolism pathway and glutamine metabolism pathway were calculated and analyzed.qPCR was used to detect the expression of phosphoenolpyruvate carboxykinase（PEPCK）mRNA in CIK cells treated with high ammonia and high ammonia+DMOG.The results show that:（1）High ammonia inhibited the TCA cycle of CIK cells:high ammonia promoted the accumulation of glutamate and aspartate in CIK cells,and decreased the level of citric acid.qPCR analysis showed that high ammonia induced the expression of PEPCK.The increase of mRNA level suggested that high ammonia may reduce the level of intermediate metabolites in TCA cycle and inhibit TCA cycle through PEPCK induced"cataplerosis";（2）DMOG alleviated the inhibition of TCA cycle induced by high ammonia in CIK cells:DMOG inhibited the increase of aspartate and glutamate levels induced by high ammonia in CIK cells,decreased the level of acetyl-coenzyme A,and restored the level of citric acid.qPCR analysis showed that DMOG treatment reduced the excessive accumulation of PEPCK induced by high ammonia,indicating that the HIF-1 stabilizer DMOG could regulate the expression of PEPCKand restored TCA cycle metabolism inhibited by high ammonia;（3）Orotate was not involved in the adaptation of CIK cells to high ammonia environment:although it has been reported that HIF-1 can remove excessive ammonia by promoting the synthesis of orotate in glutamine metabolism pathway in some cells,orotate mainly comes from glucose metabolism in CIK cells,and DMOG treatment has no obvious effect on the process of high ammonia induced accumulation of orotate,so orotate pathway is unrelated with the inhibition effect of DMOG on the ammonia-induced apoptosis of CIK cells.PartⅢ:HIF-1 may eliminate the inhibition of TCA cycle in Gymnocypris przewalskii by inhibiting the over expression of PEPCKAlkaline lake water inhibited TCA cycle of Gymnocypris przewalskii,and long-term alkaline treatment restored it.Metabonomics technology was used to detect the TCA cycle-related metabolites（glutamine,glutamate,orotate）in the serum of Gymnocypris przewalskii which was cultured in the alkaline lake water for12-hour or 24-hour,and freshwater river water for 12-houror 24-hour,and qPCR was used to detect the expression of PEPCK mRNA in Gymnocypris przewalskii was liver.The results showed that compared with river water treatment for 12 hours,river water treatment for 24 hours had little effect on the content of blood metabolites of Gymnocypris przewalskii;alkaline lake water treatment for 12 hours decreased the level of plasma glutamine,increased the level of glutamate,and increased the mRNA level of PEPCK in liver tissues.The results suggested that alkaline environment inhibited ammonia excretion,promoted glutamine metabolism and induced the interruption of TCA cycle through PEPCK.After 24 hours of alkaline lake water treatment,the levels of glutamine and glutamate in plasma and PEPCK mRNA in liver were restored to the control level,suggesting that relatively stable HIF-1αin Gymnocypris przewalskii（induced by P550 variation）is helpful to inhibit the interruption of TCA cycle induced by overexpression of PEPCK in high ammonia environment.In conclusion,DMOG,a stabilizer of HIF-1,can promote CIK cell proliferation,restore TCA cycle,and inhibit apoptosis induced by high ammonia.HIF-1 may reduce the toxicity of high ammonia to fish cells by affecting the expression of PEPCK gene and help fish adapt to high ammonia environment.