The Effect Of Selenium On NF-κB Pathway On Macrophage Polarization And Its Involvement In Hashimoto’s Thyroiditis

Objective To investigate the M1 and M2 polarization phenotypes of peripheral blood macrophages in Hashimoto’s thyroiditis(HT)patients.The macrophage model and the co-culture system of macrophages and thyroid follicular epithelial cells were constructed in the cell experiment,and the mechanism of the effect of selenium on the polarization of macrophages in HT was discussed.thyroid follicular epithelial cells,and to clarify the mechanism of the effect of selenium on Hashimoto’s thyroiditis.Methods 1.Proportion of peripheral blood macrophages in different subsets of HT patients were selected from 38 HT patients admitted to endocrinology outpatient department of our hospital(the First Affiliated Hospital of Bengbu Medical College)from March 2021 to March 2022,and 19 healthy subjects were selected as control group.Fasting venous blood of patients and healthy control group(HC)was collected,serum was separated by centrifugal method,and thyroid function indexes such as TSH,TT3,TT4 and thyroid autoantibodies(TPOAb,Tg Ab)in serum of each sample were detected by chemiluminescence immunoassay.In addition,peripheral venous blood was collected and flow cytometry was used to detect the proportion of M1 type and M2 type macrophage subsets in peripheral blood of the subjects,so as to statistically analyze the difference of experimental data between the patients and the control group.2.The effect of different concentrations of sodium selenite(SSe)on the polarization of macrophages The macrophage model was established with THP-1 cells.The suspended THP-1 cells were induced into macrophages(M0)by adding 150 n M PMA(Fobo ester),and then 100ng/ml LPS(lipopolysaccharide)to induce the polarization of macrophages.The above macrophages were grouped and treated with300 n M SSe,400 n M SSe and 500 n M SSe to interfere with macrophage polarization,respectively.CCK8 was used to detect the effect of SSe on macrophage activity and screen out the appropriate selenium concentration.Flow cytometry was used to detect the proportion of M1-type macrophages under different conditions.The experiment was divided into 5 groups: PMA group(blank control group),PMA+LPS group,PMA+LPS+300n M SSe(low selenium group),PMA+LPS+400n M SSe(medium selenium group),PMA+LPS+500n M SSe(high selenium group).3.The effect and mechanism of sodium selenite on the co-culture system of macrophages and thyroid follicular cells were co-cultured with thyroid follicular epithelial cells(Nthy-ori3-1)in the above 5 groups.CCK8 was used to detect the effect of macrophages on the proliferation of Nthy-ori3-1,ROS levels in macrophages were detected by reactive oxygen species kit,and apoptosis levels of thyroid follicular epithelial cells were detected by apoptosis kit.The expression level of GPX4 in thyroid follicular epithelial cells and NF-κB in macrophages was detected by q PCR and WB.Results 1.Flow cytometry was used to detect the proportion of M1 and M2 macrophages in peripheral blood of HT patients and healthy control group.The proportion of M1 macrophages in HT patients was higher than that in healthy control group,and there was a difference between the two groups(P < 0.05).There was no difference in M2 macrophages between HT group and control group(P > 0.05).2.The effect of sodium selenite on macrophage polarization was observed by adding different concentrations of sodium selenite.(1)CCK8 results showed that SSe concentration at 400 nm and 500 n M had inhibitory effect on M1 macrophages.(2)Compared with PMA(blank control group),the expression of M1 macrophage markers in PMA+LPS group and PMA+LPS+SSe(low selenium,medium selenium,high selenium)groups was increased(P < 0.01).Compared with PMA+LPS group,the expression of M1-type macrophage markers in PMA+LPS+ SSe(low selenium,medium selenium and high selenium)groups was decreased(P < 0.001),and the expression of M1-type macrophage markers decreased significantly with the increase of selenium concentration.3.Effects of different concentrations of sodium selenite on macrophages and Nthyori3-1 oxidative stress and apoptosis in co-culture system: SSe can reduce ROS content,inhibit NF-κB pathway,reduce M1 macrophage polarization and reduce Nthy-ori3-1apoptosis.The results of CCK8 showed that M1 macrophages had an inhibitory effect on Nthy-ori3-1,and the inhibitory effect of macrophages on Nthy-ori3-1 was decreased after adding SSe.(1)Expression of ROS levels and factors related to NF-κB pathway in macrophages:(1)ROS levels in ROS: PMA+LPS group and PMA+LPS+SSe(low selenium,medium selenium and high selenium)groups were higher than those in blank group(P < 0.01);ROS levels in PMA+LPS+SSe(low selenium,medium selenium and high selenium)groups were lower than those in PMA+LPS groups(P < 0.01),and ROS levels were decreased with the increase of selenium concentration.(2)The m RNA expression of NF-κB pathway in macrophages was increased after addition of LPS compared with blank group(P < 0.05).The mrna expression of NF-κB pathway in M1 macrophages was decreased compared with PMA+LPS group after intervention with different concentrations of selenium(P < 0.05),and the expression decreased with the increase of selenium concentration.The results of WB expression were consistent with those of q PCR.(2)Apoptosis of Nthy-ori3-1 and GPX4 gene expression:(1)Apoptosis: The apoptosis level in PMA+LPS group and PMA+LPS+SSe(low selenium,medium selenium and high selenium)groups was higher than that in blank group(P < 0.05);Apoptosis was decreased in PMA+LPS+SSe(medium selenium and high selenium)groups compared with PMA+LPS groups(P < 0.05),but was not significantly decreased in low selenium group(P > 0.05),and apoptosis was decreased with the increase of selenium concentration.(2)Compared with blank group,GPX4 m RNA expression in Nthy-ori3-1 was decreased under LPS treatment(P < 0.05),and increased in PMA+LPS+SSe(low selenium,medium selenium and high selenium)groups compared with PMA+LPS(P < 0.05).The results of WB expression were consistent with those of q PCR.Conclusion 1.The proportion of M1-type macrophages in peripheral blood of Hashimoto’s thyroiditis patients increased,suggesting that M1-type macrophages may be involved in the occurrence of HT.2.Selenium can reduce the polarization of M1-type macrophages and reduce the damage to thyroid follicular epithelial cells by alleviating oxidative stress and inhibiting NF-κB signaling pathway in macrophages.The mechanism may become a new intervention pathway for Hashimoto’s thyroiditis.