Mechanism Of Arginine And Its Metabolites On Heat Stress-induced Apoptosis Of Sertoli Cells In Immature Boar

Heat stress greatly damages the reproductive capacity of male animals.Sertoli cells are a type of somatic cells with important functions in testis,which are also sensitive to heat stress.As a functional non-essential amino acid,arginine is essential in promoting the growth and development of piglets.Arginine and its metabolites play important roles in cell processes and signal transduction.However,the protective role of arginine in heat stress-induced apoptosis of Sertoli cells and its mechanism are still unclear.The purpose of this study was to explore whether arginine is involved in heat stress-induced apoptosis of Sertoli cells,and to clarity the mechanism of arginine and its metabolites on heat stress-induced apoptosis of Sertoli cells.In this experiment,Sertoli cells treated by heat stress in vitro was used to detect amino acid metabolites by mass spectrometry multiple reaction monitoring(MRM)to find the amino acids with significant differences.Metabolic analysis of different amino acids was used to predict the possible relationship between amino acids and apoptosis.The metabolic enzymes and metabolites of related amino acids were detected by QRT PCR,ELISA,Western blotting and flow cytometry.Combined with the metabolites detected by mass spectrometry,the metabolism of related amino acids in Sertoli cells was determined.Finally,the effects of amino acid and their metabolites was verified by adding metabolites to heat-treated Sertoli cells.The resu Lts are as follows:1.After heat treatment,the activity of Sertoli cells decreased by 42%(P < 0.01),and the apoptosis rate increased by 134%.2.After heat treatment,amino acid metabolites in Sertoli cells changed significantly,including glutamate,creatine,aminoethanesulfonic acid and aminoadipic acid(P <0.01),of which the concentration of arginine decreased by 54.9%(P < 0.01),and metabolites of arginine also changed significantly.The concentration of ornithine and putrescine increased by 70%,103% respectively(P < 0.01)wheras the concentration of citrulline decreased by 39.8%(P < 0.05).3.After heat treatment,the expression of i NOS protein expression increased,and concentration of NO in Sertoli cells increased by 57%(P < 0.01).In arginine-NO metabolism pathway,heat stress decreased the concentration of citrulline by 39.8%(P <0.05).In arginine-spermine metabolism pathway,heat stress increased the expression of arg1,arg2 by 20 times and 1.8 times,respectively(P < 0.01),and the concentration of ornithine,putrescine and the expression of ODC and SSAT protein also increased.However,the concentration of the spermine did not change.4.0.05mmol/l arginine increased the viability of Sertoli cells significantly,and decreased the apoptosis rate by 9.7%5.Compared with heat stress,adding 0.05mmol/l arginine to Sertoli cells decreased the expression of i NOS protein and the content of metabolite NO by 15.3%(P < 0.01),while citrulline remained unchanged.In arginine spermine metabolism pathway,the expression of arg1 protein decreased by 23%(P < 0.01),but the corresponding metabolite ornithine remained unchanged,while the expression of ODC protein increased by 41.5%(P < 0.01),the expression of SSAT protein remained unchanged.Furthermore,the concentration of metabolite spermine increased by 10.2%(P < 0.01).6.5μM spermine decreased the concentration of NO,apoptosis rate and Caspase3 protein expression by 24.3%(P < 0.01),44.9%,and 21.7%(P < 0.05).In addditon,Bax protein expression decreased by 30.9%(P < 0.05),and Bcl-2 protein expression increased by 16.8%(P < 0.05).Based on the above results,the following conclusions are drawn.1.Heat stress induces Sertoli cell apoptosis by activating endogenous and exogenous pathways,decreasing the concentration of arginine,while increasing the concentration of NO,but unchang the concentration of spermine.2.Exogenous arginine can alleviate heat stress-induced apoptosis,decrease the concentration of NO,increase the concentration of spermine,but has no effect of the content of citrulline.3.Exogenous spermine at the concentration of 5μM can reduce the concentration of NO and alleviate the apoptosis induced by heat stress.In conclusion,heat stress can induce apoptosis of Sertoli cells by enhancing arginine-No metabolism pathway,activating Endogenous and exogenous pathways.Exogenous arginine can change the pathway of arginine metabolism,and decrease the concentration of NO and the concentration of spermine increased.Exogenous arginine and spermine reduces NO production and heat stress-induced apoptosis.