Mechanisms Of Aptamers On Relieving Apoptosis And Oxidative Damage Induced By Fumonisin B1 In Mice

Fumonisin B1(FB1)was a kind of mycotoxins produced by Fusarium moniliforme Sheld fungi,which contaminated various animal feedstuffs,such as corn and wheat.It caused many diseases such as porcine pulmonary edema,equine leucoencephalomalacia,endangering the health of animals and causing huge economic losses.There was currently no effective method to clearing FB1 in vivo.In order to find out a fast and efficient elimination or adsorption method of FB1 and explore its protective effects in vitro and in vivo,high affinity and specificity aptamer F102 was screened by the use of immobilization-free selection and the affinity effect between streptomycin magnetic beads and biotin probes in our researches.The affinity of it was 42.0±13.7 nmol/L which was determined by ELISA and it's specific with low cross-reactivity with other mycotoxins such as Zeralone(ZEN),T-2 and Aflatoxin B1(AFB1).The binding mechanism of aptamers and FB1 was deduced by molecular modeling.Detailed analysis showed that the carbonyl “O” atoms of the fumonisin formed four hydrogen bond interactions with the F102's A-8,A-39,A-40 and A-42.In addition,one of the carboxy groups of the fumonisin formed a hydrogen bond interaction with the nucleotide U-12.FB1 was anchored to the binding site of the aptamer by that.Mouse's kidney cells were primary cultured to detect every indexs of it.The results showed that after 24 h treatment with different concentrations of FB1,the cell survival rate decreased significantly and showed a dose-dependence.The minimum damage concentration of FB1 was2,500 ng/mL.When the concentration of aptamer was 1 ?mol/L,the cell survival rate was max and decreased with the increase of concentration.It was showed that cells were atrophy and even death with statistical difference(P<0.05);the content of Reactive oxygen species(ROS)increased significantly(P<0.05),Superoxide dismutase(SOD)enzyme activity significantly decreased(P<0.05);the expression of antiapoptotic gene Bcl-2 was significantly lower(P<0.05)while proapoptotic gene Caspase 9 and 3 significantly increased(P<0.05)in the FB1 treatment group for24 h.Compared with the FB1 treatment group,the aptamer F102 and F39 could significantly improve the change of the above index,and the effect of F102 was better than that of F39(P<0.05).Mice were divided into 5 groups:the normal control group,the solvent control group,the FB1 treatment group,the F102 treatment group,the F39 treatment group,and were treated by the abdominal cavity subcutaneous injection.Each treatment group was sampled at 24 h and 48 h,respectively.Results showed that compared with the FB1 treatment group,FB1 residues decreased significantly(P<0.05);liver and kidney tissues necrosis significantly reduced;no significant difference between the Viscera index(P>0.05);blood urea nitrogen increased significantly(P<0.05),aspartate aminotransferase significantly decreased(P<0.05);total cholesterol increased significantly after 48 h treatment(P<0.05),alanine aminotransferase decreased significantly after24 h treatment(P<0.05);SOD and Glutathione peroxidase(GSH-Px)enzyme activity were significantly increased(P<0.05),with the prolonging of enzyme activity decreased but still significantly increased(P<0.05);MDA content was significantly decreased(P<0.05),with the prolonging of the content of MDA increases in tissue but still significantly decreased(P<0.05);the expression of antiapoptotic gene Bcl-2 was significantly increased(P<0.05)while proapoptotic gene Caspase 9 and 3 significantly decreased(P<0.05)which was time dependent after aptamer treated mice for 24 h and 48 h.And the treatment effect of F102 was better than that of F39.Above all,this study obtained FB1 specific aptamer by SELEX screening,which was verified that it can alleviate the damage induced by FB1 in mice.And it's possible that it could directly scavenge ROS,improve the antioxidant capacity of SOD and GSH-Px enzymes,increase the expression of apoptosis gene Bcl-2 and decrease that of Caspase 3 and 9.This provides ideas for the research and development of mycotoxin adsorbents,provides a basis for the application of aptamers in vivo and for the effective prevention and control of experimental animals.