| ObjectiveSchistosome-derived molecule is considered a promising candidate for the treatment of autoimmune and inflammatory diseases.SJMHE1 is an HSP60-derived peptide from Schistosoma japonicum and has been demonstrated to possess anti-inflammatory effects in many tissues and organs.Recent studies have indicated that pyroptosis plays a critical role in the pathogenesis of Hashimoto ’s thyroiditis(HT).However,the specific effects of SJMHE1 on pyroptosis in HT remain unclear.This study aimed to elucidate the effect of SJMHE1 on pyroptosis in thyroid follicular epithelial cells(TFCs)induced by excessive iodine and reveal the potential mechanism.Methods1.To evaluate the cytotoxicity of SJMHE1 on Nthy-ori 3-1 cells,Nthy-ori 3-1 cells were treated with different concentrations(0,0.1,0.5 and 1.0 μg/m L)SJMHE1 for 24 h,the cell viability was assessed by CCK-8 assays,the cytotoxicity was evaluated by Annexin V-FITC and PI staining for flow cytometry analysis,and Western blotting(WB)was used to detect the cleaved Caspase 3/Caspase 3 ratio to assess the effect of SJMHE1 on apoptosis.2.To selecte the appropriate concentration of SJMHE1,Nthy-ori 3-1 cells were treated with different concentrations(0,0.1,0.5 and 1.0 μg/m L)of SJMHE1 in the presence of Na I(50 m M),and Control(Con)values were obtained in the absence of Na I.After 24 h,CCK-8 assay was used to detect cell viability.3.To verify the effect of SJMHE1 on pyroptosis,Nthy-ori 3-1 cells were treated with Na I(50 m M)and different concentrations(0,0.5 and 1.0 μg/m L)of SJMHE1 for 24 h,the concentration of pyroptosis downstream product IL-1β was measured by ELISA,the levels of pyroptosis-related proteins,including NLRP3,GSDMD-N and C-caspase-1 were detected by WB.4.The mean fluorescence intensity(MFI)of intracellular ROS in Nthy-ori 3-1 cells treated with various concentrations of SJMHE1(0,0.5 and 1.0 μg/m L)and /or 50 m M Na I for 24 h was analyzed by flow cytometry.5.To investigate the possible signaling pathway involved in the SJMHE1-regulated anti-pyroptosis,WB was used to determine the levels of MAPK(p38 MAPK,JNK and ERK1/2),IκBα and NF-κB p65 phosphorylation in Nthy-ori 3-1 cells when the cells were treated with various concentrations of SJMHE1(0,0.5 and 1.0 μg/m L)and/or 50 m M Na I for 24 h.6.TLR2 and TLR4 expression in Nthy-ori 3-1 cells treated with the indicated concentration of SJMHE1 was evaluated in the presence of Na I(50 m M)for 24 h using WB.7.Nthy-ori 3-1 cells were preincubated with anti-TLR2(10 μg/m L),anti-TLR4(10μg/m L)or anti-Ig G antibodies(10 μg/m L)for 1 h and then treated with Na I(50 m M)with or without SJMHE1(1.0 μg/m L)for 24 h,IL-1 β levels were detected by ELISA.8.Nthy-ori 3-1 cells were pretreated with blocking antibodies(TLR2,TLR4 and isotype Ig G)before stimulation with Na I(50 m M)and SJMHE1(1 μg/m L),pyroptosis-related proteins,including NLRP3,GSDMD-N and C-caspase-1 were detected by WB.Results1.Compared with the control,gradient concentrations of SJMHE1 groups(0.1,0.5and 1.0 μg/m L)had no significant effect on cell viability,there was no difference in the percentage of apoptotic cells as measured by flow cytometry for each group,SJMHE1 did not alter the cleaved-Caspase 3/Caspase 3 ratio relative to the control.Together,these results suggested that SJMHE1 did not show any cytotoxicity on Nthy-ori 3-1 at the indicated concentrations.2.The cell viability of Nthy-ori 3-1 cells was significantly decreased by Na I treatment group,while the cell viability of Nthy-ori 3-1 cells was increased in the SJMHE1co-treatment groups in a concentration-dependent manner.3.Excessive iodine treatment stimulated the generation of IL-1β,which was significantly decreased in the SJMHE1 co-treatment groups.4.Compared with the control group,excessive iodine treatment stimulated the expressions of NLRP3,GSDMD-N and C-caspase-1 proteins while the expression levels of NLRP3,GSDMD-N and C-caspase-1 were significantly decreased in the SJMHE1 co-treatment groups.5.Compared with the control group,excessive iodine treatment increased the level of ROS.Compared with the excessive iodine single treatment group,the MFI of ROS in the SJMHE1 co-treatment groups was significantly reduced.6.Compared with the control group,excessive iodine treatment activated the MAPK and NF-κB signaling pathways.Compared with the excessive iodine treatment group,the SJMHE1 co-treatment group reduced the phosphorylation of p38 MAPK,JNK,and ERK1/2.On the other hand,SJMHE1 co-treatment group markedly suppressed the phosphorylation of IκBα and NF-κB p65.7.Nthy-ori 3-1 cells expressed TLR2 and TLR4,and excessive iodine or SJMHE1 treatment had no effect on the expression of TLR2 or TLR4.8.SJMHE1 exhibited a potent antipyroptotic effect in Nthy-ori 3-1 cells,whereas the antipyroptotic effect of SJMHE1 was weakened in Nthy-ori 3-1 cells when blocked with anti-TLR2 antibodies.In contrast,there were no significant differences in SJMHE1-mediated IL-1β production or pyroptosis-related protein expression between pretreatment with anti-TLR4 and anti-Ig G control antibodies.ConclusionThe present study suggested a protective role of SJMHE1 in excessive iodine-induced pyroptosis in Nthy-ori 3-1 cells,the possible molecular mechanisms of SJMHE1 may be attributed to its suppression of ROS/MAPK/NF-κB signaling pathways with a TLR2-dependent mechanism,which may pave the road to new the rapeutic modalities for the treatment of HT. |
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