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Effect Of Zearalenone On Lactate Metabolism Of Rat Sertoli Cells And Its Mechanism

Posted on:2021-02-06Degree:MasterType:Thesis
Country:ChinaCandidate:P R CaiFull Text:PDF
GTID:2493306311475494Subject:Clinical Veterinary Medicine
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Zearalenone(ZEA)is a fungal metabolite widely distributed in crops.It has been proved that ZEA can cause damage to animal reproductive system.Sertoli cells(SCs)are nurse cells in testis,which is an important process to maintain the energy homeostasis in testis.To reveal the effect of ZEA on male reproductive toxicity and its mechanism,the model of ZEA poisoning in vitro was established by primary SCs of rats.Western Blot,RT-PCR and immunofluorescence were used to study the effect of ZEA on lactate metabolism of SCs and the role of AMPK and SIRT1 in the effect of ZEA on lactate metabolism of SCs.1.The effect of ZEA on the metabolize of lactic acid in SCs.Different concentrations of ZEA(0.5,10,20 μmol/L)were applied to primary SCs of rats at different times(0,6,12,24,36,48 h).Lactate and pyruvate kits were used to detect the level of lactate and pyruvate in SCs;Western Blot was used to detect the expression of proteins related to lactate production;Laser confocal was used to detect the fluorescence intensity of LDHA,a key protein of lactate production;RT-PCR was used to detect the expression of glycolysis key genes.The results showed that,the activity of SCs decreased with the increase of ZEA concentration;The concentration of lactate and pyruvate in cells decreased showed concentration-dependent and time-dependent decline manner;The related protein of lactate production GLUT1,LDHA and MCT4 showed a significant or extremely significant decline;The fluorescence intensity of LDHA decreased gradually,and the expression of key genes of glycolysis PFKP,Gpi1,HK1 and Pgam1 decreased significantly.2.The role of AMPK in the effect of ZEA on lactate metabolize of SCs.Through primary SCs of rats exposed to different concentrations of ZEA(0,5,10,20 μmol/L),Western Blot was used to detect the expression of AMPK.20 μmol/L ZEA combined with AMPK inhibitor,5 μmol/L compound C,or AMPK activator,50 μmol/L AICAR,were used to act on SCs.Lactate and pyruvate kits were used to detect the level of lactate and pyruvate in cells;Western Blot was used to detect the expression of lactate production related protein;RT-PCR was used to detect the expression of glycolysis key genes.The results showed that ZEA activated the AMPK protein and inhibited the ATP level in SCs.When ZEA combined with AMPK inhibitor Compound C acted on SCs for 24 h,compared with ZEA group,the level of pyruvate and lactate in ZEA+Compound C co-treated group showed a significant increase;The related protein of lactate production GLUT1,LDHA and MCT4 showed a significant or extremely significant increased;The key genes of glycolysis,HK1 and Gpil,showed a significant increase.When ZEA and AMPK activator AICAR work together on SCs,compared with ZEA group,the concentration of lactate and pyruvate in ZEA+AICAR co-treatment group shown a significant downward trend;The protein expression of GLUT1,LDHA and MCT4 shows a significant downward trend;The key genes of glycolysis,PFKP,Pgam1,Gpi1,and HK1 shown significant downward trend,3.The role of SIRT1 in the effect of ZEA on lactate metabolize of SCs.Through primary SCs of rats exposed to different concentrations of ZEA(0,5,10,20 μmol/L),Western Blot was used to detect SIRT1 expression;20 μmol/L ZEA combined with 2.5μmol/L RSV were used to act on SCs,lactate and pyruvate kits were used to detect intracellular lactate and pyruvate levels;Western Blot was used to detect lactate production related protein expression;RT-PCR was used to detect glycolysis key gene expression.The results showed that the expression of SIRT1 protein decreased significantly compared with the control group;When ZEA combined with SIRT1 inhibitor RSV acted on SCs for 24 h,compared with ZEA group,the level of pyruvate and lactate in ZEA+RSV co-treatment group increased significantly:The related protein of lactate production GLUT1,LDHA and MCT4;The key genes of glycolysis,PFKP and Pgam1 shown increased trend,HK1 shown increased significantly.Conclusion:ZEA can decrease the glycolysis level of SCs and the production of pyruvate and lactate in a dose-dependent and time-dependent manner.The reason why ZEA interferes with the spermatogenesis of male animals is related to the decrease of the supply of lactate to germ cells by SCs.AMPK has a positive regulatory effect on the decrease of SCs lactate induced by ZEA,while SIRT1 has a negative regulatory effect.
Keywords/Search Tags:Zearalenone, Sertoli cells, Lactate, AMPK, SIRT1
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