| Objective: To investigate whether the sperm quality and testicular injury induced by acrylonitrile(ACN)would be intervented by apigenin(AP)and its possible mechanisms and provide a certain basis for the protection of reproductive toxicity of ACN and the use of AP.Methods: Sixty of SPF level male rats were randomly divided into the control group(corn oil),ACN group(46 mg/kg ACN),low-dose AP-intervention group(46 mg/kg ACN+117 mg/kg AP),middle-dose AP-intervention group(46 mg/kg ACN+234 mg/kg AP)and high-dose AP-intervention group(46 mg/kg ACN+351 mg/kg AP),12 rats in each group.These rats were administered by intragastric administration,once a day,6 days a week.After 28 days,all rats were killed.(1)The bilateral testis and epididymis were taken and weighed.The organ coefficients were calculated;(2)Sperm density,viability,motility and sperm motility parameters were analyzed by computer-assisted sperm analysis(CASA)system;eosin staining smears were prepared to observe sperm morphology;acridine orange staining smears were prepared to observe sperm DNA integrity;(3)The histopathological sections of testicular tissue were prepared to evaluate the histological changes of testicular tissue;(4)The activity of alkaline phosphatase(AKP),acid phosphatase(ACP),succinate dehydrogenase(SDH)and lactate dehydrogenase(LDH)in testicular tissue were detected by kit;(5)The content of malondialdehyde(MDA),glutathione(GSH),the activity of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),catalase(CAT)and the total antioxidant capacity(T-AOC)in testicular tissue were detected by kit.(6)The relative expression of ASK1,MKK7,JNK,p38,Cyt c,Caspase-9,Caspase-3,Bax and Bcl-2 mRNA were detected by qRT-PCR;(7)The expression of ASK1,p-ASK1,MKK7,p-MKK7,JNK,p-JNK,p38,p-p38,Cyt c,Caspase-9,Caspase-3,Bax and Bcl-2 protein in testis tissue were detected by Western Blot;(8)TUNEL staining sections were prepared to detect the apoptotic level of testicular tissues.Results:(1)Body weight and organ coefficient: Compared with the control group,the wet weight and organ coefficient of epididymis in the ACN group decreased significantly(P<0.05).Compared with the ACN group,the wet weight and organ coefficient of epididymis in the low-dose AP-intervention group were significantly increased(P<0.05).(2)Sperm quality: Compared with the control group,the percentage of grade a and b sperm of rats in the ACN group increased significantly,and the percentage of grade d decreased significantly(P<0.05);The sperm viability and motility increased significantly(P<0.05);VCL,VSL,VAP and MAD increased significantly,BCF decreased significantly(P<0.05).Compared with the control group,the rate of sperm deformation,head deformation and tail deformation in the ACN group increased significantly(P<0.05).Compared with the ACN group,the rate of sperm deformation and head deformation in the low-dose and middle-dose AP-intervention group decreased significantly(P<0.05).Compared with the control group,the sperm DFI index of rats in the ACN group increased significantly(P<0.05).Compared with the ACN group,the sperm DFI index of rats in the AP-intervention group decreased significantly(P<0.05).(3)Testicular tissue structure: The seminiferous tubules in the ACN group were atrophied and deformed.The diameter of tubules became smaller and the interstitium became wider.Seminiferous cells decreased and arranged disorderly.The number of mature sperm in the lumen was very small.While in the low-dose and middle-dose AP-intervention group,seminiferous tubules were completed and arranged regularly.Spermatogenic cells and mature sperm can be seen in the lumen.(4)Testicular marker enzyme: Compared with the control group,the activity of AKP and SDH in testicular tissue of rats in the ACN group decreased significantly(P<0.05).Compared with the ACN group,the activity of LDH in the testicular tissue of rats in the AP-intervention group decreased significantly,and the activity of AKP increased significantly(P<0.05).(5)Indicators of oxidative stress in testicular tissues: Compared with the control group,the activity of GSH-Px activity and T-AOC in testicular tissues of rats in the ACN group decreased significantly(P<0.05);Compared with the ACN group,the content of GSH and T-AOC in testicular tissues of rats in the low-dose AP-intervention group increased significantly(P<0.05);Compared with the ACN group,the GSH-Px of rats in the high-dose AP-intervention group decreased significantly(P<0.05).(6)Genetic test results: Compared with the control group,the relative expression of ASK1,MKK7,JNK,p38,Caspase-9 and Bax mRNA in testicular tissues of rats in the ACN group increased,and the relative expression of Bcl-2 mRNA decreased(P<0.05).Compared with the ACN group,the relative expression of MKK7,JNK,p38 and Bax mRNA in testicular tissues of rats in the low-dose AP-intervention group decreased,and the relative expression of Bcl-2 mRNA increased(P<0.05);The relative expression of JNK and Bax mRNA in testicular tissues of rats in the middle-dose AP-intervention group decreased,and the relative expression of Bcl-2 mRNA increased(P<0.05);The relative expression of Bax mRNA in testicular tissues of rats in the high-dose AP-intervention group decreased(P<0.05).(7)Protein detection results: Compared with the control group,The expression of ASK1,p-ASK1,MKK7,p-MKK7,JNK,p-JNK,p38,p-p38,Cyt c,Caspase-9,Caspase-3 and Bax protein in testicular tissues of rats in the ACN group increased,the expression of Bcl-2 protein decreased(P<0.05),the ratio of p-JNK/JNK,p-p38/p38 increased,and the ratio of Bcl-2/Bax decreased(P<0.05).Compared with the ACN group,the expression of ASK1,p-ASK1,MKK7,p-MKK7,JNK,p-JNK,p38,p-p38,Caspase-9,Caspase-3 and Bax protein in the testicular tissues of rats in the low-dose AP-intervention group decreased,the expression of Bcl-2 protein increased(P<0.05),the ratio of p-JNK/JNK,p-p38/p38 decreased,and the ratio of Bcl-2/Bax increased(P<0.05);The expression of ASK1,p-ASK1,MKK7,p-MKK7,JNK,p-JNK,p38,p-p38,Caspase-9,Caspase-3 and Bax protein in the testicular tissues of rats in the middle-dose AP-intervention group decreased(P<0.05),the expression of Bcl-2 protein increased,the ratio of p-ASK1/ASK1,p-JNK/JNK decreased,and the ratio of Bcl-2/Bax increased(P<0.05).The expression of Caspase-9,Caspase-3 and Bax protein in the testicular tissues of rats in the high-dose AP-intervention group decreased,the expression of Bcl-2 protein increased(P<0.05),the ratio of p-ASK1/ASK1,p-JNK/JNK decreased,and the ratio of Bcl-2/Bax increased(P<0.05).(8)TUNEL staining sections: Compared with the control group,the number of TUNEL-positive cells in each seminiferous tubule of rats in the ACN group increased(P<0.05);Compared with the ACN group,the number of TUNEL-positive cells in each seminiferous tubule of rats in the AP-intervention group decreased(P<0.05).Conclusion:(1)AP has a protective effect on sperm deformity and DNA damage induced by ACN,and can alleviate the pathological damage of testicular tissues induced by ACN and improve the changes of testicular marker enzymes.(2)AP can play a protective role by reducing oxidative stress of testis induced by ACN,inhibiting the activation of ASK1-JNK/p38 signaling pathway and mitochondrial-mediated apoptosis. |
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