Molecular Mechanism Of AFB₁-induced Testosterone Synthesis Disorder In Mice And The Regulation Of ROS/AMPK Signaling Pathway

AFB₁ is one of the most toxic mycotoxins.It is widely present in grain food and feed.It is a great harm to human and animal health,animal and plant food safety,and economic and trade development.Studies have shown that exposure to AFB₁ can inhibit testosterone（T）synthesis and cause male reproductive disorders,but the mechanism is not yet clear.AMPK signaling pathway is a key pathway for regulating T.When there is a lack of energy in the cell or external stimuli such as ROS,the AMPK signaling pathway is activated to inhibit T synthesis.Oxidative stress has been proven to be the main toxic mechanism of AFB₁.Exposure to AFB₁ can trigger oxidative stress and increase ROS levels.However,whether AFB₁ activates ROS-mediated AMPK signaling pathway and causes T synthesis disorders is unclear.Therefore,this study takes the relationship between ROS,AMPK signaling pathway and T synthesis as the starting point,and"AFB₁ inhibits T synthesis by activating ROS-mediated AMPK signaling pathway"as the theoretical hypothesis,and intends to expand the following four aspects of research.（1）First,male Kunming mice were taken as subjects,and 0 mg/kg BW（control group,CG）,0.375 mg/kg BW（low-dose group,LG）,0.75 mg/kg BW（medium-dose group,MG）and 1.5 mg/kg BW（high-dose group,HG）AFB₁ for 30 days,establish an animal model of subchronic AFB₁-induced T synthesis disorder,and detect mouse growth and development（mental state;weight）,testicular damage（testicular coefficient and volume,testicular microstructure and ultrastructure;sperm concentration,vitality and deformity rate）,serum T content,oxidative stress（testicular tissue ROS and MDA content and CAT and GSH-Px activities）,mitochondrial damage（testicular tissue cell mitochondrial ultrastructure,MMP level,ATP content）,AMPK signaling pathway（p-AMPK,t-AMPK,p53 and Nur77 protein expressions）,apoptosis（TUNEL staining,Bax and Bcl-2 m RNA and protein expressions）and the key enzymes of T synthesis（St AR,P450scc,3β-HSD,P450c17 and 17β-HSD m RNA and protein expressions）are designed to explore the inhibitory effect of AFB₁ on T synthesis and the influence of ROS-mediated AMPK signaling pathway.（2）Then,using TM3（mouse testicular stromal cell line）as the test subject,add 0μM,2.5μM(1/30 IC₅₀),5μM(1/15 IC₅₀)and 10μM(2/15 IC₅₀)of AFB₁,cultured for 24 hours,established AFB₁ poisoning cell model,and detected cell damage（cell viability,cell morphology and ultrastructure）,T content of cell culture supernatant,oxidative stress,mitochondrial damage,AMPK signaling pathway,apoptosis and key enzymes of T synthesis,confirming the inhibitory effect of AFB₁ on T synthesis in TM3 cells and ROS-mediated AMPK signaling pathway and analyze the dose-effect relationship between AFB₁ and T content and AMPK signaling pathway,and screen out the appropriate dose of AFB₁ in subsequent intervention experiments.（3）Compound C（AMPK inhibitor）was used to treat TM3 cells exposed to AFB₁,and the experiment was divided into the control group（0μM Compound C+0μM AFB₁）,AFB₁ poisoning group（5μM AFB₁）,AMPK intervention Group（10μM Compound C+5μM AFB₁）and AMPK intervention control group（10μM Compound C）,cultured for 24 hours,detected the T content of cell culture supernatant,AMPK signal pathway,cell apoptosis and the key to T synthesis enzymes（St AR,3β-HSD,and P450c17 m RNA and protein expressions）to verify the role of AMPK signaling pathway in AFB₁-induced T synthesis disorders.（4）Finally,NAC（ROS scavenger）was used to treat TM3 cells exposed to AFB₁,and the experiment was divided into the control group（0μM NAC+0μM AFB₁）,AFB₁ poisoning group（5μM AFB₁）,ROS intervention group（5 m M NAC+5μM AFB₁）and AMPK intervention control group（5 m M NAC）,cultured for 24 hours,and the AMPK signaling pathway（p-AMPK and t-AMPK protein expressions）were detected to verify whether AFB₁ activates the AMPK signaling pathway by promoting ROS production.Comprehensive analysis of the results of experiments to clarify the molecular mechanism of AFB₁-induced T synthesis disorder and the regulation of ROS/AMPK signaling pathway.The research results may screen out the target of AFB₁male reproductive dysfunction and provide new ideas for finding target drugs or defense measures for prevention and control of AFB₁ reproductive toxicity,and can also offer reference for comparative medicine and public health safety evaluation.The experimental results are as follows:（1）Test results of AFB₁ treatment of mice:1)The mice in AFB₁ treatment group were depressed and slow to respond;The MG and HG was significantly lower than that of the CG（P<0.05）,indicating that AFB₁ inhibited the growth and development of mice.2)The testicular coefficient in the MG and HG was significantly lower than the CG（P<0.05;P<0.01）;the testicular volume in AFB₁ treatment groups was significantly lower than the CG（P<0.05;P<0.01）;the microscopic and ultrastructure of mice testis tissue in the AFB₁ treatment groups showed obvious damage;the sperm density of the mice epididymis in the MG and HG was significantly lower than that of the CG（P<0.05,P<0.01）,mice epididymal sperm abnormality rate in was significantly higher than the CG（P<0.01）;mice epididymal sperm viability was significantly lower than the CG（P<0.05,P<0.01）,indicating that AFB₁ damaged the structure and function of testicular tissue and inhibited spermatogenesis.3)The T content of the mice serum in AFB₁ treatment groups was significantly lower than the CG（P<0.01）,indicating that AFB₁ caused T synthesis disorder in mice testicular tissue.4)The content of ROS of mice testis tissue in AFB₁ treatment groups was significantly higher than that of the CG（P<0.01）,and the activities of CAT and GSH-Px were significantly lower than the CG（P<0.01）;the content of MDA of mice testis tissue in the MG and HG was significantly higher than the CG（P<0.01）,indicating that AFB₁ caused ROS accumulation and oxidative stress in testis tissue.5)The mitochondrial ultrastructure of spermatogenic cells and Leydig cells of mice testis tissue in AFB₁ treatment groups was damaged;the MMP level of mice testis tissue in AFB₁ treatment groups was significantly lower than the CG（P<0.05,P<0.01）;the ATP content of mice testis tissue in the MG and HG was significantly lower than that in the CG（P<0.01）,indicating that AFB₁ caused mitochondrial damage in testicular tissue cells.6)The expressions of p-AMPK and p53 protein of mouse testis in AFB₁ treatment groups were significantly higher than the CG（P<0.05,P<0.01）;the Nur77 protein expression of mice testis tissue in the MG and HG was significantly lower than that of the CG（P<0.05,P<0.01）,indicating that AFB₁ can activate AMPK signaling pathway and its downstream target proteins.7)TUNEL staining showed that spermatogenic cells and Leydig cells of mice testis tissue in AFB₁ treatment groups undergo apoptosis;the Bax m RNA expression of mice testis tissue in AFB₁treatment groups was significantly higher than that of the CG（P<0.01）,the m RNA and protein expressions of Bcl-2 were significantly lower than the CG（P<0.01）;the Bax protein expression and the ratio of Bax and Bcl-2 of mice testis tissue in the MG and HG was significantly higher than the CG（P<0.01;P<0.05）,indicating that AFB₁ caused cell apoptosis in testicular tissue.8)The m RNA expressions of St AR,P450scc,3β-HSD,P450c17 and 17β-HSD of mice testis in AFB₁ treatment groups were significantly lower than the CG（P<0.01）,the protein expressions of P450scc,3β-HSD,P450c17 and 17β-HSD were significantly lower than the CG（P<0.05,P<0.01）;the St AR protein expression of mice testis in the MG and HG was significantly lower than the CG（P<0.01）,indicating that AFB₁ inhibited the expression of key enzymes for T synthesis in testicular tissue.（2）Test results of the AFB₁ treatment of TM3 cells:1)The IC₅₀ of AFB₁ to TM3 cells is 74.58μM.2)TM3 cells activity in AFB₁ treatment groups was significantly lower than the 0μM group（P<0.05;P<0.01）;the microstructure and ultrastructure of TM3 cells in AFB₁ treatment groups were destroyed,indicating that AFB₁ caused TM3 cell damage.3)The T content of TM3 cells culture supernatant in the 5μM and 10μM AFB₁ treatment groups were significantly lower than the 0μM group（P<0.01）,indicating that AFB₁ caused T synthesis disorder in TM3 cells.4)The ROS and MDA contents of TM3 cells in AFB₁ treatment groups were significantly higher than the 0μM group（P<0.01）;CAT and GSH-Px activities of TM3 cells in AFB₁ treatment groups were significantly lower than the 0μM group（P<0.01）,indicating that AFB₁ caused ROS accumulation and oxidative stress in TM3 cells.5)The mitochondrial ultrastructure of TM3 cells in AFB₁ treatment groups was damaged;the MMP level and ATP content of TM3 cells in AFB₁ treatment groups were significantly lower than the 0μM group（P<0.01）,indicating that AFB₁ caused mitochondrial damage in TM3 cells.6)The p-AMPK expression of TM3 cells in AFB₁ treatment groups was significantly higher than that in the 0μM group（P<0.05;P<0.01）;the p53 protein expression in the 5μM and 10μM AFB₁ treatment groups was significantly higher than that in the 0μM group（P<0.01）;the Nur77protein expression of TM3 cells in AFB₁ treatment groups was significantly lower than the 0μM group（P<0.05;P<0.01）,indicating that AFB₁ activated AMPK signaling pathway and its downstream target proteins.7)The apoptotic rate of TM3 cells in AFB₁ treatment groups was significantly higher than the0μM group（P<0.05,P<0.01）;the m RNA and protein expressions of Bax and the ratio of Bax and Bcl-2 in TM3 cells were significantly higher than the 0μM group（P<0.05;P<0.01）;the m RNA and protein expressions of Bcl-2 in TM3 cells were significantly lower than the 0μM group（P<0.01）,indicating that AFB₁ caused TM3 cell apoptosis.8)The m RNA expressions of St AR,P450scc,3β-HSD,P450c17 and 17β-HSD in TM3 cells of AFB₁ treatment groups were all significantly lower than the 0μM group（P<0.01）;the protein expressions of St AR,P450scc,3β-HSD and P450c17 of TM3 cells in AFB₁ treatment group were significantly lower than those of the 0μM group（P<0.05;P<0.01）;the 17β-HSD protein expression of TM3 cells in the 5μM and 10μM AFB₁ treatment groups was significantly lower than that in the0μM group（P<0.01）,indicating that AFB₁ inhibited the expression of key T synthesis enzymes in TM3 cells.（3）Test results of Compound C intervention in AFB₁ treatment TM3 cells:1)10μM Compound C significantly alleviated the decrease of T content in TM3 cell culture supernatant induced by AFB₁（P<0.05;P<0.01）,indicating that inhibition of AMPK signaling pathway can alleviate AFB₁-induced T synthesis disorder.2)10μM Compound C significantly alleviated the increased protein expression of p-AMPK and p53 and the decreased protein expression of Nur77 in TM3 cells induced by AFB₁（P<0.05;P<0.01）.3)10μM Compound C significantly alleviated the increase of apoptosis rate,Bax m RNA and protein expression,and the ratio of Bax and Bcl-2（P<0.01）,and the decrease of Bcl-2 m RNA and protein expression（P<0.01）in of TM3 cells induced by AFB₁,which indicated that inhibition of AMPK signaling pathway could alleviate apoptosis induced by AFB₁.4)10μM Compound C significantly alleviated the decrease in m RNA and protein expression of key T synthesis enzymes St AR,3β-HSD and P450c17 in TM3 cells induced by AFB₁（P<0.05;P<0.01）.（4）Test results of NAC intervention in AFB₁ treatment of TM3 cells:1)5 m M NAC significantly alleviated ROS accumulation in TM3 cells induced by AFB₁（P<0.01）.2)5 m M NAC significantly alleviated the increase of p-AMPK protein expression in TM3 cells induced by AFB₁（P<0.01）,suggesting that ROS intervention could inhibit the activation of AMPK signaling pathway induced by AFB₁.In summary,AFB₁ can cause testicular tissue and TM3 cells damage,reduce T content,induce oxidative stress,damage mitochondria of testicular tissue and TM3 cells,and activate ROS-mediated AMPK signaling pathway.AFB₁ promoted the apoptosis of testicular Leydig cells and inhibited the expression of key enzymes in T synthesis by activating the ROS/AMPK signaling pathway,which led to the disorder of T synthesis.