黄芩素通过调控cAMP-PKA-NF-κB/CREB通路改善人脑小胶质细胞的炎性反应  

Baicalin improves inflammatory response of human microglia by regulating cAMP-PKA-NF-κB/CREB pathway

作　　者：郑晓宇 张业昊 宋文婷[1] 刘光宇[1] 丁昭 刘建勋[1] ZHENG Xiao-yu;ZHANG Ye-hao;SONG Wen-ting;LIU Guang-yu;DING Zhao;LIU Jian-xun(Beijing Key Laboratory of Pharmacology of Chinese Materia Medica,Institute of Basic Medical Sciences of Xiyuan Hospital,China Academy of Chinese Medical Sciences,Beijing 100091,China)

机构地区：[1]中国中医科学院西苑医院基础医学研究所中药药理北京市重点实验室,北京100091

出　　处：《中国中药杂志》2023年第21期5863-5870,共8页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金重点项目(8203000944);中国中医科学院科技创新工程项目(CI2021A01414,CI2021A01304)。

摘　　要：该文旨在研究黄芩素(baicalein,BAI)对脂多糖(lipopolysaccharide,LPS)诱导的人脑小胶质细胞(HMC3)的影响,从抑制炎性反应的角度,并基于调控cAMP-PKA-NF-κB/CREB通路,阐释BAI用于治疗缺血性脑卒中的潜在作用机制,对中药治疗脑缺血疾病具有借鉴意义。该实验首先筛选了BAI的安全用药浓度,再采用LPS诱导HMC3细胞24 h的方法制作细胞炎性模型,将分组设定为control组,model组,BAI高、中、低剂量(5、2.5、1.25μmol·L^(-1))组。分别测定细胞提取物中超氧化物歧化酶(superoxide dismutase,SOD)和丙二醛(malondialdehyde,MDA)的含量以及细胞上清液中白细胞介素(interleukin,IL)-1β、IL-6、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、环磷酸腺苷(cyclic adenosine monophosphate,cAMP)的含量,用Wes-tern blot法观察蛋白激酶A(protein kinase A,PKA)、磷酸化环磷腺苷效应元件结合蛋白(phosphory cAMP-response element binding protein,p-CREB)、核因子-κB p65(nuclear factor kappaB p65,NF-κB p65)的表达,用Hoechst 33342/PI染色法观察LPS诱导后细胞凋亡的情况。在机制探讨过程中采用BAI高、低剂量作为给药组。实验结果显示,BAI在10μmol·L^(-1)及以下的浓度对正常培养的HMC3细胞无影响;200 ng·mL^(-1)的LPS诱导24 h后可以降低HMC3细胞的SOD酶活力并提高MDA的含量,BAI在5、2.5、1.25μmol·L^(-1)的浓度下均能显著提高SOD酶活力,并在5μmol·L^(-1)的浓度下均能显著降低MDA含量;细胞形态结果显示,BAI可以改善因LPS诱导所致的HMC3细胞向M1型转变;ELISA检测的结果显示,BAI能使细胞上清液中TNF-α、IL-1β、IL-6、cAMP的含量显著降低;Western blot结果显示,BAI能够提高PKA和p-CREB的蛋白表达,降低NF-κB p65的表达;Hoechst 33342/PI染色结果显示,BAI能够减少HMC3细胞的凋亡情况。以上结果表明,BAI对LPS诱导的HMC3细胞具有保护作用,能够抑制炎性反应和改善细胞凋亡的情况,这可能与调控HMC3细胞上cAMP-PKA-NF-This study aims to investigate the effects of baicalein(BAI) on lipopolysaccharide(LPS)-induced human microglial clone 3(HMC3) cells,with a focus on suppressing inflammatory responses and elucidating the potential mechanism underlying the therapeutic effects of BAI on ischemic stroke via modulating the cAMP-PKA-NF-κB/CREB pathway.The findings have significant implications for the application of traditional Chinese medicine in treating cerebral ischemic diseases.First,the safe dosage of BAI was screened,and then an inflammation model was established with HMC3 cells by induction with LPS for 24 h.The cells were assigned into a control group,a model group,and high-,medium-,and low-dose(5,2.5,and 1.25 μmol·L~(-1),respectively) BAI groups.The levels of superoxide dismutase(SOD) and malondialdehyde(MDA) in cell extracts,as well as the levels of interleukin-1β(IL-1β),IL-6,tumor necrosis factor-α(TNF-α),and cyclic adenosine monophosphate(cAMP) in the cell supernatant,were measured.Western blot was performed to determine the expression of protein kinase A(PKA),phosphorylated cAMP-response element binding protein(p-CREB),and nuclear factor-kappa B p65(NF-κB p65).Hoechst 33342/PI staining was employed to assess cell apoptosis.High and low doses of BAI were used for treatment in the research on the mechanism.The results revealed that BAI at the concentrations of 10 μmol·L~(-1) and below had no impact on normally cultured HMC3 cells.LPS induction at 200 ng·mL~(-1) for 24 h reduced the SOD activity and increased the MDA content in HMC3 cells.However,5,2.5,and 1.25 μmol·L~(-1) BAI significantly increased the SOD activity and 5 μmol·L~(-1) BAI significantly decreased the MDA content.In addition,BAI ameliorated the M1 polarization of HMC3 cells induced by LPS,as indicated by cellular morphology.The results of ELISA demonstrated that BAI significantly lowered the levels of TNF-α,IL-1β,IL-6,and cAMP in the cell supernatant.Western blot revealed that BAI up-regulated the protein levels of PKA and p-CREB while down-r

关 键 词：黄芩素 小胶质细胞 炎性反应 脂多糖 脑缺血 

分 类 号：R285[医药卫生—中药学]