含笑内酯对骨髓增殖性肿瘤细胞系UKE-1和SET-2中细胞因子表达的作用及机制研究OA北大核心

Objective The effects and molecular mechanisms of micheliolide on cytokine expression in myeloproliferative neoplasm cell lines were explored based on the signal transducer and activator of transcription 3(STAT3)/nuclear factor-kappa B(NF-κB)signaling pathways.Methods The UKE-1 and SET-2 cell lines were investigated,and micheliolide concentrations were screened using the CCK-8 assay.The UKE-1 and SET-2 cells were divided into the control and micheliolide-treated groups at concentrations of 2.5,5.0,and 10.0 μmol/L.Each group received 1 mL of micheliolide solution at final concentrations of 2.5,5.0,and 10.0 μmol/L,respectively,whereas the control group only received an equal volume of culture medium.The inhibition rates of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),and chemokine ligand 2(CCL2)mRNA expression in cells from each group were detected using real-time fluorescent PCR(RT-PCR).Western blotting was used to measure STAT3 and phosphorylated STAT3(p-STAT3)protein expression levels in cells from each group.Reversal experiments with reduced glutathione and dithiothreitol were performed using UKE-1 cells,which were divided into the control group,micheliolide,micheliolide+glutathione,micheliolide+dithiothreitol,and glutathione+dithiothreitol groups.Western blotting was used to detect the STAT3 and p-STAT3 protein expression levels in the cells of each group.UKE-1 cells were stimulated with TNF-α(5 μg/L)to replicate a pathological model of excessive cytokine secretion.Subsequently,UKE-1 cells were divided into the control,model,and three micheliolide-treated groups at concentrations of 2.5,5.0,and 10.0 μmol/L.RT-PCR was used to measure the indicators above.An enzyme-linked immunosorbent assay(ELISA)was used to detect the CCL2 content in the cell culture media of each group.Western blotting was performed to assess the protein expression levels of STAT3,p-STAT3,and proteins related to the NF-κB signaling pathway.Results Compared with the control group,the proliferation inhibition rates of UKE-1 cells at 24,48,and 72 h increased in the micheliolide-treated groups at concentrations of 2.5,5.0,10.0,and 20.0 μmol/L.Similarly,the proliferation inhibition rates of SET-2 at 48 and 72 h increased in the micheliolide-treated groups at concentrations of 5.0,10.0,and 20.0 μmol/L(P<0.05).Concentrations of 2.5,5.0,and 10.0 μmol/L were selected for further studies to exclude the potential influence of high micheliolide concentrations on subsequent result owing to reduced cell numbers.Compared with the control group,the inhibition rates of TNF-α mRNA expression in UKE-1 and SET-2 cells increased in the micheliolide-treated groups at concentrations of 2.5,5.0,and 10.0 μmol/L.Similarly,the inhibition rates of IL-1β mRNA expression in UKE-1 and SET-2 cells also increased in the micheliolide-treated groups at concentrations of 5.0 and 10.0 μmol/L.Additionally,the inhibition rate of CCL2 mRNA expression in UKE-1 and SET-2 cells increased in the micheliolide-treated group at a concentration of 10 μmol/L(P<0.05).Compared with the model group,the inhibition rates of TNF-α,IL-1β,and CCL2 mRNA expression in UKE-1 cells increased in the micheliolide-treated groups at concentrations of 2.5,5.0,and 10.0 μmol/L after stimulation with TNF-α(P<0.05).ELISA showed that compared with the control group,the CCL2 content in UKE-1 cells increased in the model group.Compared with the model group,the CCL2 content in UKE-1 cells decreased in the micheliolide-treated groups at concentrations of 2.5,5.0,and 10.0 μmol/L(P<0.05).Western blotting showed that compared with the control group,the p-STAT3 protein expression levels in UKE-1 and SET-2 cells were downregulated in the micheliolide-treated groups at concentrations of 5.0 and 10.0 μmol/L,and the protein expression level of STAT3 in SET-2 was also downregulated(P<0.05).Compared with the control group,the p-STAT3 expression level in UKE-1 cells decreased in the micheliolide group in the reductive glutathione and dithiothreitol reversal experiments.Compared with the micheliolide group,the p-STAT3 protein expression levels in UKE-1 cells increased in the micheliolide+dithiothreitol and micheliolide+glutathione groups(P<0.05).Compared with the control group,the model group showed increased p-STAT3,p-IκKα/β,p-IκBα,and p-NF-κB p65 protein expression and decreased IκBα protein expression after stimulation with TNF-α.Compared with the model group,the micheliolide-treated groups showed decreased p-IκKα/β,p-IκBα,p-STAT3,and p-NF-κB p65 protein expression at concentrations of 2.5,5.0,and 10.0 μmol/L,whereas the micheliolide-treated groups showed increased IκBα protein expression at concentrations of 5.0 and 10.0 μmol/L(P<0.05).Conclusion Micheliolide potently suppresses IL-1β,TNF-α,and CCL2 mRNA expression in UKE-1 and SET-2 cells,as well as CCL2 secretion by UKE-1 cells,which may be associated with STAT3 phosphorylation suppression and NF-κB signaling pathway activation.