Research On Sodium Selenite Inhibition GPx1 Knockdown Porcine Splenic Lymphocytes Injury Induced By Deoxynivalenol In Vitro Culture

The pollution of deoxynivalenol is serious in feed and feed materials.DON can act on the immune system,which can cause oxidative damage to immune cells.Selenium participates in the antioxidant stress of the body.This research building GPx1 knockdown porcine spleen lymphocytes by RNAi technology,through adding DON or/and Na₂SeO₃,explore the effects of DON for GPx1 knockdown porcine spleen lymphocytes to oxidative damage,the protective effect of Na₂SeO₃ on the oxidative damage of GPx1 silcncing porcine spleen lymphocytes,further explore whether selenium is antagonistic to DON toxicity through the GPx1.Methods:?1?The establishion of GPx1 knockdown porcine splenic lymphocytes:RNAi technology?flow cytometry?qpcr and wsetern blot were used to establish GPx1knockdown porcine splenic lymphocytes.?2?Detection of antioxidant index:The experiment was divided into 11 groups,including D1?DON 824 ng/mL?,D2?DON 412 ng/mL?,D3?DON 206 ng/mL?,D4?DON 103 ng/mL?,S?Na₂SeO₃ 2?mol/L?,SD1?Na₂SeO₃ 2?mol/L+DON 824ng/mL?,SD2?Na₂SeO₃ 2?mol/L+DON 412 ng/mL?,SD3?Na₂SeO₃ 2?mol/L+DON206 ng/mL?,SD4?Na₂SeO₃ 2?mol/L+DON 103 ng/mL?,P?porcine splenic lymphocytes?,M?GPx1 knockdown porcine splenic lymphocytes?respectively,detected the activities of SOD,CAT,the levels of GSH,H₂O₂,MDA,T-AOC and the ability of inhibiting hydroxyl free radical when DON or/and Na₂SeO₃ acting on GPx1 knockdown porcine splenic lymphocytes 6 h,12 h and 24 h;the ROS content was detected when DON or/and Na₂SeO₃ acting on GPx1 knockdown porcine splenic lymphocytes 24 h.The change rate of all antioxidant indices between SD group and group D was calculated and be compared with the change rate of all antioxidant indices of porcine splenic lymphocytes between SD group and group D in zhao's master's thesis.Results:?1?The establishion of GPx1 knockdown porcine splenic lymphocytes:Flow cytometry detection of siRNA transfection efficiency was up to 92.5%.The GPx1 siRNA used in the experiment could inhibit the expression of GPx1 mRNA in the porcine splenic lymphocytes,and the expression quantity was 28.4%in the control group,and the difference was significant?P<0.05?.Western Blot results showed that the expression of GPx1 protein in knockdown group was inhibited,and the expression quantity was 36.9%in the control group,with significant difference?P<0.05?.The results indicated that GPx1 knockdown porcine splenic lymphocytes was successful in the establishment.?2?Detection of antioxidant index.After knockdown GPx1,compared with normal porcine spleen lymphocytes,GPx1knockdown porcine splenic lymphocytes?M group?of H₂O₂ and MDA content were significantly higher than normal pig spleen lymphocytes?P group??P<0.01?,but the content of GSH,T-AOC,ROS content inhibit the ability of the hydroxyl free radical were significantly lower than that of P group?P<0.05 or P<0.01?,even cultivate a relatively short time?6 h?,SOD activity,CAT activity and lower than P group.DON separate canister GPx1 knockdown porcine splenic lymphocytes,6,12 and 24h respectively,all of groups D cells of H₂O₂ and MDA content were significantly higher than that of group M?P<0.01?,SOD and CAT activities,GSH content and cell T-AOC and inhibit the ability of hydroxyl free radical were significantly lower than M group?P<0.01?.When cultured for 24 h,except for D4 group,the ROS content in the rest group was significantly higher than that in M group?P<0.01?.Add Na₂SeO₃ alone to GPx1 knockdown porcine splenic lymphocytes function 6,12and 24 h,cells of H₂O₂ and MDA content in Se group were significantly lower than M group?P<0.01?,SOD and CAT activities,GSH content and cell T-AOC and inhibiting the ability of hydroxyl free radical were significantly higher than that of group M?P<0.05 or P<0.01?.When cultured for 24 h,the ROS content in Se group was significantly lower than that in M group?P<0.01?.Combined with DON and Na₂SeO₃ at GPx1 knockdown porcine splenic lymphocytes function 6,12 and 24 h,cells of H₂O₂ and MDA content in SD group were significantly higher than that of group D?P<0.01?,the activities of SOD,CAT,the levels of GSH,cell T-AOC and inhibit the ability of the hydroxyl free radical were significantly lower than that of group D mostly?P<0.05 or P<0.01?.When cultured for 24 h,the content of ROS in group SD was significantly lower than that in group D?P<0.01?,except SD1.SD in the test group and the rate of change of D group were lower than Zhao Chuanping SD group and D group of master degree theses of master of the rate of change,that knockdown GPx1 before adding Na₂SeO₃,reduce the proportion of H₂O₂ and MDA content and SOD,CAT activity,GSH content and cell T-AOC and inhibit the ability of the hydroxyl free radicals increases the proportion of all below the normal cell,show that in silent for GPx1 add Na₂SeO₃,Na₂SeO₃ to DON the antagonism between cell oxidative damage induced by reduced.Conclusions:?1?The GPx1 knockdown porcine splenic lymphocytes was successfully established.?2?DON acts on GPx1 knockdown porcine splenic lymphocytes lonely,which can cause greater oxidative damage than normal porcine splenic lymphocytes,it showed that when the expression of selenium protein GPx1 was decreased,the resistance of lymphocytes to DON was decreased.Combined with Na₂SeO₃ and DON,Na₂SeO₃ can antagonise the oxidative damage of the lymphocytes of the GPx1 knockdown porcine splenic lymphocytes,but the antagonistic ability is drastically reduced,it is further indicated that Na₂SeO₃ should be used for the toxicity of antagonistic DON through the selenium protein GPx1.