北京中医药大学学报2025，Vol.48Issue(1)：68-79,12.DOI:10.3969/j.issn.1006-2157.2025.01.009
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
In vitro studies of the anti-inflammatory activity of micheliolide on myeloproliferative neoplasm cell lines
摘要
Abstract
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.关键词
含笑内酯/骨髓增殖性肿瘤/细胞因子/信号传导和转录激活因子3/核转录因子-κBKey words
micheliolide/myeloproliferative neoplasms/cytokine/signal transducer and activator of transcription 3/nuclear factor-kappa B分类
医药卫生引用本文复制引用
陈猛,刘晋琴,张莹,史哲新,肖志坚..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[J].北京中医药大学学报,2025,48(1):68-79,12.基金项目
国家自然科学基金项目(No.82104785) （No.82104785）
天津市名中医传承工作室建设项目(No.tjmzy2406) National Natural Science Foundation of China(No.82104785) （No.tjmzy2406）
