摘要
目的探讨依普利酮(eplerenone,EPL)基于电压门控钾通道1.3(voltage-gated potassium channel 1.3,Kv1.3)/B淋巴细胞瘤-2(B cell lymphoma-2,Bcl-2)/核转录因子-κB(nuclear factor-κB,NF-κB)抑制巨噬细胞M1型极化而缓解小鼠类风湿性关节炎(rheumatoid arthritis,RA)。方法使用生物信息学技术筛选疾病通路与靶点、计算EPL-Kv1.3复合物体系结合亲和力和稳定性。建立RA小鼠模型,灌胃EPL干预42 d,记录并检测反映药物缓解RA的指标;利用RAW264.7细胞模型给予EPL,检测反映药物影响巨噬细胞M1极化的指标,并验证药物介导的相关信号通路上下游关键靶点。结果生物信息学分析发现,疾病靶点主要涉及炎症反应和NF-κB信号通路、EPL-Kv1.3亲和力较高且结合稳定。动物实验发现,检测抗环瓜氨酸肽抗体(CCP-Ab)与小鼠关节评分显示造模成功。与模型组(Model)相比,EPL可减少足趾红肿评分、缓解小鼠足跖红肿、滑膜肿胀、减少纤维增生及炎性细胞浸润,且EPL中、高剂量组降低HE染色评分(P<0.05、P<0.01),EPL高剂量组可降低小鼠血清RF(P<0.01)。细胞实验CCK-8结果显示,EPL低、中、高剂量对RAW264.7巨噬细胞活性没有影响(P>0.05)。与Model组相比,EPL各剂量组明显降低细胞上清液IL-6、TNF-α及NO含量(P<0.01),EPL高剂量组减少NF-κB-p65核移位,EPL中、高剂量组降低细胞M1极化并升高M2极化比例(P<0.01)。EPL各剂量组明显降低细胞相关mRNA含量(P<0.01)。EPL可升高Bcl-2蛋白表达(P<0.01),降低细胞NF-κB通路蛋白表达(P<0.05)。结论EPL可能通过调控Kv1.3/Bcl-2/NF-κB通路,减少巨噬细胞M1型极化,改善巨噬细胞相关炎症反应,发挥免疫调节作用从而缓解小鼠RA。
Aim To investigate the effect of eplerenone(EPL)on the alleviation of rheumatoid arthritis(RA)based on voltage-gated potassium channel 1.3(Kv1.3)/B-cell lymphoma-2(Bcl-2)/nuclear factor-κB(NF-κB)to inhibit macrophage M1 polarization in mice.Methods Bioinformatics technology was used to screen disease pathways and targets,and the binding affinity and stability of EPL-Kv1.3 complex system were calculated.A mouse model of RA was established and treated with EPL by intragastric administration for 42 days.The indicators reflecting drug remission of RA were recorded and detected.RAW264.7 cells were treated with EPL to detect the indicators reflecting the effect of drugs on macrophage M1 polarization,and to verify the upstream and downstream key targets of related signaling pathways mediated by drugs.Results Bioinformatics analysis showed that the disease targets were mainly involved in inflammatory response and NF-κB signaling pathway,and EPL-Kv1.3 had high affinity and stable binding.In animal experiments,the detection of anti-cyclic citrullinated peptide antibody(CCP-Ab)and joint score indicated the successful establishment of the model.Compared with the model group,EPL could reduce the toe redness and swelling score,alleviate the plantar redness and swelling,synovial swelling,and reduce fibrosis and inflammatory cell infiltration in mice.The medium-dose and high-dose EPL groups reduced the HE staining score(P<0.05,P<0.01),and the high-dose EPL group reduced the serum RF in mice(P<0.01).CCK-8 results showed that low,medium and high doses of EPL had No.effect on the activity of RAW264.7 macrophages(P>0.05).Compared with the model group,EPL treatment significantly reduced the contents of IL-6,TNF-αand No.in supernatant of the cells(P<0.01),reduced the nuclear translocation of NF-κB-p65 in the high-dose EPL group,reduced the M1 polarization and increased the proportion of M2 polarization in the medium and high-dose EPL groups(P<0.01).The mRNA levels of MyD88,IκB-α,NF-κB-p65,NF-κB-p50,IL-1βand iNOS were significantly reduced in each dose group of EPL(P<0.01).EPL significantly increased the protein expression of Bcl-2(P<0.01)and decreased the protein expression of Kv1.3,MyD88,p-IκB-α/IκB-α,p-p65/p65,IL-1βand iNOS(P<0.05).Conclusion EPL may play an immunomodulatory role in relieving RA in mice by regulating Kv1.3/Bcl-2/NF-κB pathway,reducing macrophage M1 polarization and ameliorating macrophage-associated inflammatory response.
作者
肖翅
蔡静
王子航
张雍正
杨劲
程路峰
XIAO Chi;CAI Jing;WANG Zi-hang;ZHANG Yong-zheng;YANG Jing;CHENG Lu-feng(School of Pharmacy,Xinjiang Medical University,Urumqi 830017,China;the Second Affiliated Hospital of Xinjiang Medical University,Urumqi 830028,China;School of Basic Medicine and Clinical Pharmacy,China Pharmaceutical University,Nanjing 210009,China;School of Pharmaceutical Sciences,China Pharmaceutical University,Nanjing 210009,China;Xinjiang Key Laboratory of Biopharmaceuticals and Medical Devices,Urumqi 830017,China;Engineering Research Center of Xinjiang and Central Asian Medicine Resources,Ministry of Education,Urumqi 830017,China;Xinjiang Key Laboratory of Natural Medicines Active Components and Drug Release Technology,Urumqi 830017,China)
出处
《中国药理学通报》
2025年第4期726-737,共12页
Chinese Pharmacological Bulletin
基金
新疆维吾尔自治区重大科技专项项目(No.2022A03007)
化学药改良型创新药的研发(No.2022A03007-4)。
