The Role Of Autophagy In Aflatoxin B1-mediated Impairment Of Lactate Secretion In Dairy Goat Sertoli Cells

Aflatoxin B1(AFB1)is a secondary metabolite produced by molds and is widely detected in many aspects of the transportation and storage of crops such as wheat,corn,soybeans,nuts and their by-products.In the process of livestock and poultry breeding,male livestock and poultry feeding aflatoxin can cause damage to the reproductive system of livestock and poultry in many aspects,of which male livestock and poultry performance can cause a reduction in semen quality and,male fertility,seriously limiting the development of animal husbandry.The testicular Sertoli cells(SCs)play an important role in maintaining and regulating the normal spermatogenesis process,and the stability of their lactate secretion is the key to the normal development of germ cells.AFB1 can cause testicular damage and spermatogenesis disorder,however,the effect of AFB1 on testicular SCs and the potential mechanism is not clear yet.Therefore,in this study,we investigated the effects of AFB1 on lactate secretion in SCs by examining the changes in glucose utilization,pyruvate content,lactate dehydrogenase activity,and lactate secretion in SCs after AFB1 treatment.Secondly,cellular autophagy was examined by transmission electron microscopy,Western blot,Compound C,and AICAR pretreatment to investigate the effect of AFB1 on autophagy in SCs.After that,to clarify the role of autophagy in the AFB1-mediated decrease of lactate secretion in SCs,SCs were pretreated with chloroquine(CQ)and rapamycin(Rapa),and the changes in lactate secretion were examined.Finally,based on the results of previous laboratory studies,the addition of broussoflavone to SCs medium was investigated for its effects on AFB1-induced impairment of lactate secretion and autophagy,thus clarifying the possibility of regulating autophagy to relieve AFB1-mediated impairment of lactate secretion in dairy goat SCs and providing a theoretical basis for alleviating aflatoxin-induced reproductive toxicity in dairy goats.The main results of this study were obtained as follows:1.Primary SCs were obtained by a two-step enzymatic digestion from the testes of2～3month old dairy goats.SCs were stained for the specific markers WT1 and SOX9 by immunofluorescence,and the staining results were further analyzed using flow cytometry.the results showed that the percentages of WT1-CY3~+and SOX9-FITC~+cells were 94.7%±1.2%and 92.0%±1.8%;cell viability was measured by CCK-8 after 24 h of AFB1treatment of SCs.The results demonstrated that when the concentration of AFB1 was higher than 5μM,the viability of SCs showed a significant dose-dependent inhibition(P<0.001);in addition,when the concentration of AFB1 exceeded 20μM,its cytotoxicity increased significantly with the increase of AFB1 concentration(P<0.01);furthermore,compared with the control group,AFB1 treatment was able to induce a significant increase in glucose uptake,lactate In addition,AFB1 treatment caused a concentration-dependent decrease in glucose uptake,lactate secretion level,lactate dehydrogenase activity,and pyruvate level in SCs compared with the control group(P<0.01);Western blot showed that the expression of GLUT1,GLUT3,LDHA,and MCT4,key proteins involved in lactate secretion process,decreased significantly after AFB1 treatment(P<0.01).of lactate secretion.2.AFB1 was added to the dairy goat Sertoli cells medium for 24 h.The changes in intracellular autophagy-like structures were observed using transmission electron microscopy,and the results showed that 50μM AFB1 treatment increased intracellular autophagy-like structures;Western blot results showed that the level of LC3-II/I was significantly increased compared with the control group and the level of p62 was significantly decreased(P<0.05);in addition,the phosphorylation of AMPK and the phosphorylation level of ULK1 were significantly enhanced by AFB1(P<0.01);pretreatment of AFB1-treated SCs using 50μM AICAR and 10μM compound C.Western blot results showed that in the presence of compound C pretreatment,the protein of LC3-II in AFB1-treated cells levels was reduced(P<0.01)and p62 expression was significantly increased(P<0.01)in AFB1-treated cells;AICAR treatment further induced a significant upregulation of autophagic flux in cells(P<0.01);pretreatment of AFB1-treated SCs using CQ and Rapa revealed that inhibition of autophagy by CQ further led to glucose depletion,pyruvate production,lactate Western blotting showed that inhibition of autophagy significantly reduced the expression of GLUT1,GLUT3,LDHA,and MCT4 in the AFB1-treated group(P<0.01),while enhanced autophagy significantly reversed the reduction in the expression of these proteins(P<0.01).The reduction in the expression of these proteins was significantly reversed by enhanced autophagy(P<0.05).These results suggest that AFB1 can enhance autophagic flux within SCs via AMPK/ULK1 flux,and the enhanced autophagy can,to some extent,attenuate the AFB1-induced reduction in lactate secretion from SCs.3.The cell viability of SCs treated with broussoflavone and AFB1 in dairy goat SCs medium for 24 h was measured by CCK-8,and it was found that 10μM and 25μM of broussoflavone treatment significantly inhibited the decrease in viability of SCs induced by AFB1(P<0.05);In addition,broussoflavone treatment significantly alleviated the decrease in glucose uptake,lactate dehydrogenase activity,pyruvate level and lactate secretion level(P<0.01);compared with the AFB1-treated group,broussoflavone significantly increased the protein expression of GLUT1,GLUT3,LDHA,and MCT4(P<0.05);meanwhile,broussoflavone highly significantly enhanced the ratio of LC3-II/I and the degradation table of p62 protein(P<0.01).The above results suggest that broussoflavone can alleviate the AFB1-induced decrease in lactate secretion in SCs by enhancing autophagy.In conclusion,this study found that AFB1 at doses above 10μM reduced could reduce glucose uptake and lactate secretion in SCs and inhibit the expression of lactate metabolism-related proteins GLUT1,GLUT3,LDHA,and MCT4.In addition,AFB1induced autophagy through the AMPK/ULK1 signaling pathway,which in turn resisted its mediated decrease in lactate secretion from SCs.25μM of broussoflavone could further enhance autophagy to alleviate the AFB1-induced decrease in lactate secretion from SCs.This study provides a new idea to alleviate aflatoxin-induced reproductive toxicity in dairy goats and is important to further explore the regulatory role of autophagy in reproductive damage in males.