The Mechanism Of Selenium In Alleviating Neutrophil Damage Induced By High Iodine Through The AMPK-mTOR Pathway

Objective:To explore the mechanism of NETs release from neutrophils induced by high iodine.The effects of high iodine on the function of neutrophils were observed from oxidative stress,autophagy and apoptosis.To study the mechanism of selenium in maintaining the stability of neutrophil function under the injury induced by high iodine,and to provide a new theoretical basis for selenium preparation in the treatment of Hashimoto’s thyroiditis.Method:1.Human promyelocytic leukemia(HL-60)cells were induced to differentiate into neutrophils by using 70 m M dimethylformamide(DMF)for 5 days.The co-expression of neutrophil surface markers CD11b~+and CD66b~+was detected by flow cytometry,and the double positive rate>99%was considered as successful neutrophil induction,which was used for subsequent experiments.2.Effect of different iodine concentrations on neutrophil function:CCK8 screened the optimal KI concentration and grouped them for subsequent experiments.Compared with the control group,the expression of NETs marker Cit H3 in the high iodine group and the low iodine group was significantly increased,and the expression of Cit H3 showed an upward trend with the increase of KI concentration.Flow cytometry showed that compared with the control group,the ROS levels in the high iodine group and the low iodine group increased(P<0.05).Western Blot results showed that compared with the control group,the expressions of AMPK,LC3-Ⅱ,and Bax proteins in the low and high iodine groups were significantly increased(P<0.01),and the expressions of mTOR and Bcl-2 proteins were significantly decreased(P<0.05).Compared with the low iodine group,the ROS level in the high iodine group had no significant difference(P>0.05).Western Blot results showed that compared with the low iodine group,the expression of AMPK,mTOR and Bax protein in the high iodine group had no significant difference(P>0.05).However,the expression of LC3-Ⅱprotein was increased(P<0.01),and the expression of anti-apoptotic protein Bcl-2 protein was decreased(P<0.05).3.Selenium reduced the damage of neutrophils induced by high iodine:the optimal selenium concentration was screened by flow cytometry and grouped for subsequent experiments.Compared with the high iodine group,the expression of NETs marker Cit H3in the high iodine+low selenium group and the high iodine+high selenium group gradually decreased,and with the increase of selenium concentration,the expression of Cit H3 gradually decreased.ROS levels were significantly decreased in the low and high selenium groups(P<0.0001).The m RNA expressions of ampk,lc3-Ⅱand bax in the low and high selenium groups were decreased(P<0.0001),and the m RNA expressions of mTOR and bcl-2 were increased(P<0.001).Western Blot results showed that compared with the high iodine group,the expressions of AMPK,p-AMPK,LC3-Ⅱand BAX proteins in the low and high selenium groups were decreased(P<0.01),and the expressions of m-TOR and BCL-2 proteins were increased(P<0.001).Compared with the low selenium+high iodine group,the ROS level in the high selenium+high iodine group was significantly decreased(P<0.01),the m RNA expression of ampk,lc3-Ⅱand bax was decreased(P<0.01),and the m RNA expression of mTOR and bcl-2 was increased(P<0.05).The protein expression levels of AMPK,p-AMPK,mTOR and BCL-2 in the high selenium+high iodine group were not significantly different from those in the low selenium+high iodine group(P>0.05),but the protein expression levels of BAX and LC3-Ⅱin the high selenium+high iodine group were significantly decreased(P<0.01).Results:1.Neutrophils were successfully induced.The results of flow cytometry showed that the double positive rate of cell surface markers CD11b~+and CD66b~+was>99%(P<0.0001),indicating that neutrophils were successfully induced,which could be used for subsequent experiments.2.Effect of different iodine concentrations on neutrophil function.CCK8 screened the optimal KI concentrations and grouped them for subsequent experiments.The expression of NETs marker Cit H3 was significantly increased after KI stimulation of neutrophils compared with the control group,and the expression of Cit H3 tended to increase with increasing KI concentration.Western Blot results showed that the expression of AMPK,LC3-II and Bax proteins were significantly increased(P<0.01)and the expression of mTOR and Bcl-2 proteins were significantly decreased(P<0.05)in the low and high-iodine groups compared with the control group.Compared with the low-iodine group,there was no statistical difference in ROS levels in the high-iodine group(P>0.05),and the Western Blot results showed no difference in AMPK,mTOR,and Bax protein expression in the high-iodine group compared with the low-iodine group(P>0.05),but an increase in LC3-II protein expression(P<0.01)and a decrease in anti-apoptotic protein Bcl-2 protein expression(P<0.05).3.Selenium reduced the damage of neutrophils induced by high iodine:Compared with the high-iodine group,the expression of NETs marker Cit H3 gradually decreased,and the expression of NETs marker Cit H3 gradually decreased with the increase of selenium concentration;the level of ROS significantly decreased in the low and high-selenium groups(P<0.0001);the m RNA expression of AMPK,LC3-II and Bax decreased in the low and high-selenium groups(P<0.0001),and the mTOR and Bcl-2 m RNA expression increased(P<0.001);Western Blot results showed that AMPK,p-AMPK,LC3-II,Bax protein expression decreased(P<0.01)and m-TOR,Bcl-2 protein expression increased(P<0.001)in the low and high-selenium groups compared with the high-iodine group.Compared with the low-selenium+high-iodine group,the high-selenium+high-iodine group showed a significant decrease in ROS level(P<0.01),a decrease in m RNA expression of AMPK,LC3-II,and Bax(P<0.01),and an increase in mTOR and Bcl-2m RNA expression(P<0.05).The protein expression of AMPK,p-AMPK,mTOR,and Bcl-2 in the high-selenium+high-iodine group was not significantly different from that in the low-selenium+high-iodine group(P>0.05),but the protein expression of Bax and LC3-II was significantly decreased(P<0.01).Conclusion:1.High iodine stimulates neutrophils to release ROS,activate the downstream AMPK pathway,promote autophagy to release NETs,and promote apoptosis.2.Selenium inhibits the autophagy,NETs and apoptosis of neutrophils induced by high iodine through AMPK-mTOR pathway.