肿节风总黄酮调节MAPK/JNK信号通路促进巨核细胞分化成熟  

Flavonoids from Sarcandrae glabra Promote Megakaryocyte Differentiation and Maturation Through Regulation of MAPK/JNK Signaling Pathway

作　　者：尚广彬[1,2] 陈中 张钟康[1,2] 卢震 路千里 严小军 卢晓南[4] SHANG Guangbin;CHEN Zhong;ZHANG Zhongkang;LU Zhen;LU Qianli;YAN Xiaojun;LU Xiaonan(Research Center for Differentiation and Development of Basic Theory of TCM,Jiangxi University of Chinese Medicine,Nanchang 330004 Jiangxi,China;Jiangxi Province Key Laboratory of TCM Etiopathogenisis,Nanchang 330004 Jiangxi,China;Clinical Medical School,Jiangxi University of Chinese Medicine,Nanchang 330004 Jiangxi,China;School of Traditional Chinese Medicine,Jiangxi University of Chinese Medicine,Nanchang 330004 Jiangxi,China)

机构地区：[1]江西中医药大学中医基础理论分化发展中心,江西南昌330004 [2]江西省中医病因生物学重点实验室,江西南昌330004 [3]江西中医药大学临床医学院,江西南昌330004 [4]江西中医药大学中医学院,江西南昌330004

出　　处：《中药新药与临床药理》2023年第4期459-465,共7页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：国家自然科学基金项目(81960743,82260798);江西省自然科学基金项目(20192BAB205108);江西中医药大学研究生专项创新基金项目(JZYC20S32);江西省中医药管理局科技项目(2022B1063)。

摘　　要：目的 基于丝裂原活化蛋白激酶(MAPK)/c-Jun N-末端激酶(JNK)信号通路探讨肿节风总黄酮对体外巨核细胞分化成熟障碍模型的影响及作用机制。方法 利用终浓度为5 ng·mL^(-1)的佛波醇12-十四酸酯13-乙酸酯(PMA)和体积分数为1%的抗血小板血清(APS)联合作用于Dami细胞,构建体外巨核细胞分化成熟障碍模型。将细胞分为空白对照组、PMA诱导组(5 ng·mL^(-1))、APS模型组(5 ng·mL^(-1)PMA+1%APS)以及肿节风总黄酮高、中、低剂量组(15.6、7.8、3.9μg·mL^(-1)肿节风总黄酮+5 ng·mL^(-1)PMA+1%APS)。分别于培养48、72、96 h后,采用化学发光法检测细胞增殖活力。培养96 h后,采用流式细胞术检测各组Dami细胞的巨核细胞表面标志物CD41a、CD42b和CD61表达情况;采用Western Blot法检测Dami细胞MAPK/JNK信号通路关键蛋白p-JNK、JNK、p-c-Jun、c-Jun的表达情况。结果 (1)与空白对照组相比,PMA诱导组不同时间点的细胞活力均显著降低(P<0.01);与PMA诱导组相比,APS模型组48 h的细胞活力明显下降(P<0.05),但随时间延长抑制作用减弱,72、96 h的差异无统计学意义(P>0.05);与APS模型组比较,肿节风总黄酮中、高剂量组48、72 h的细胞活力均受到明显抑制(P<0.05,P<0.01),肿节风总黄酮高剂量组96 h的细胞活力受到显著抑制(P<0.01)。(2)与空白对照组比较,PMA诱导组Dami细胞表面的CD41a、CD42b和CD61表达水平均显著升高(P<0.01)。与PMA诱导组比较,APS模型组Dami细胞表面的CD41a、CD42b和CD61表达水平均显著降低(P<0.01);p-JNK/JNK及p-c-Jun/c-Jun蛋白表达显著上调(P<0.01)。与APS模型组比较,肿节风总黄酮高、中剂量组Dami细胞表面的CD41a、CD42b和CD61表达水平均显著升高(P<0.01),细胞p-JNK/JNK蛋白表达明显下调(P<0.05,P<0.01);肿节风总黄酮低、中、高剂量组Dami细胞p-c-Jun/c-Jun蛋白表达显著下调(P<0.01)。结论 肿节风总黄酮可能通过调节MAPK/JNK信号通路来改善巨核细胞分化成熟�Objective To investigate the effects and the mechanisms of flavonoids from Sarcandrae glabra on the differentiation and maturation of megakaryocytes in vitro based on the mitogen activated protein kinase(MAPK)/c-Jun N-terminal kinase(JNK)signaling pathway.Methods An in vitro model of impaired megakaryocyte differentiation and maturation was constructed using a final concentration of 5 ng·mL^(-1)foponol 12-tetradecanoate 13-acetic acid ester(PMA)and a volume fraction of 1% anti-platelet serum(APS)in combination with Dami cell.The cells were divided into a blank control group,a PMA induction group(5 ng·mL^(-1)),an APS model group(5 ng·mL^(-1)PMA+1%APS)and high-,medium-and low-dose groups of flavonoids from Sarcandrae glabra(15.6,7.8 and 3.9μg·mL^(-1) flavonoids from Sarcandrae glabra+5 ng·mL^(-1)PMA+1%APS).After 48,72 and 96 hours respectively,cell proliferation activity was detected by chemiluminescence assay.After 96 hours of culture,the expressions of the megakaryocyte surface markers CD41a,CD42b and CD61 in each group of Dami cell were detected by flow cytometry;the expressions of key proteins p-JNK,JNK,p-c-Jun and c-Jun of MAPK/JNK signaling pathway in Dami cell were detected by Western Blot.Results (1)Compared with the blank control group,the cell activity of the PMA induction group was significantly reduced at different time points(P>0.01);compared with the PMA induction group,the cell activity of the APS model group was significantly reduced at 48 hours(P<0.05),but the inhibitory effect diminished with time extension,and the differences at 72 hours and 96 hours were not statistically significant(P<0.05);compared with the APS model group,the cell viability was significantly inhibited in both the medium-and high-dose groups of flavonoids from Sarcandrae glabra at 48 hours and 72 hours(P<0.05,P<0.01),and significantly inhibited in the high-dose group at 96 hours(P<0.01).(2)The expression levels of CD41a,CD42b and CD61 on the surface of Dami cell were significantly increased in the PMA-induced group compa

关 键 词：肿节风总黄酮 巨核细胞分化成熟 免疫性血小板减少症 MAPK/JNK信号通路 Dami细胞 

分 类 号：R285.5[医药卫生—中药学]