淫羊藿总黄酮微颗粒对肺转移前微环境调控的影响  

Regulatory effect of Epimedium flavonoid microparticles on pulmonary pre-metastatic microenvironment

作　　者：车晓瑜 徐梦婷 李亚芹 陈文静[1,2] 刘玉萍 陈彦[1,2] CHE Xiao-yu;XU Meng-ting;LI Ya-qin;CHEN Wen-jing;LIU Yu-ping;CHEN Yan(Affiliated Hospital of Integrated Traditional Chinese and Western Medicine,Nanjing University of Chinese Medicine,Nanjing 210028,China;Multi-component of Traditional Chinese Medicine and Microecology Research Center,Jiangsu Provincial Academy of Chinese Medicine,Nanjing 210028,China)

机构地区：[1]南京中医药大学附属中西医结合医院,江苏南京210028 [2]江苏省中医药研究院中药组分与微生态研究中心,江苏南京210028

出　　处：《中国中药杂志》2024年第21期5856-5864,共9页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(82374082,81903858);江苏省中医药局科技项目(ZT202111)。

摘　　要：肿瘤转移是临床患者死亡的主要原因,转移前微环境假说的提出为肿瘤转移提供新的研究方向,通过靶向抑制肿瘤细胞衍生微颗粒对干扰素基因刺激因子(STING)信号的激活,有助于减少肿瘤转移。该研究通过构建重组腺相关病毒载体介导短发夹核糖核酸干扰STING(rAAV STING shRNA)肺转移前微环境及肺转移模型,考察STING干扰后对转移前微环境的影响及对淫羊藿总黄酮干预作用的影响。采用小鼠Lewis肺癌细胞(LLC)与淫羊藿总黄酮(EFs)孵育制备含药微颗粒,通过测定平均粒径、多分散系数、电位、释放表征淫羊藿总黄酮微颗粒(EFs-LLC-MPs)。通过Western blot分别考察微颗粒标志性蛋白、淫羊藿总黄酮微颗粒处理小鼠肺泡上皮细胞(MLE-12)后肺转移前微环境的各项指标变化。通过HPLC、流式测定载药量、MLE-12以及小鼠肺泡巨噬细胞(MH-S)对微颗粒的摄取情况。摄取实验结果表明,经鼻滴转染rAAV STING shRNA后,STING表达受到显著抑制,肺转移前微环境指标显著降低。Micro CT结果表明肺转移肿瘤、结节减少,同时淫羊藿总黄酮的抗转移作用受到影响。结果表明,微颗粒为膜结构的囊泡,粒径(373.17±3.18)nm,电位(-35.40±1.08)mV,测得的蛋白质量浓度562.62μg·mL^(-1),每微克蛋白载药0.0609μg,微颗粒可以被MLE-12、MH-S细胞摄取。结果表明,EFs-LLC-MPs处理MLE-12、MH-S细胞后能够降低肺转移前微环境指标纤连蛋白(fibronectin)、赖氨酰氧化酶(LOX)等蛋白的表达。基于以上研究结果,证实STING参与调控肺转移前微环境形成,并成功制备具有干预炎症转移前微环境潜力的淫羊藿总黄酮微颗粒,有望控制肿瘤转移。Tumor metastasis is the main cause of death in clinical patients.The proposal of the pre-metastatic microenvironment hypothesis offers a new research direction for tumor metastasis.Targeting and inhibiting the activation of the stimulator of interferon genes(STING)signals by tumor cell-derived microparticles may help reduce tumor metastasis.This study constructed a pre-metastatic microenvironment and pulmonary metastasis model using recombinant adeno-associated virus vector-mediated short hairpin RNA interference of STING(rAAV STING shRNA)to investigate the effects of STING interference on the pre-metastatic microenvironment and the impact of total Epimedium flavonoids(EFs)as an intervention.Drug-containing microparticles were prepared by incubating mouse Lewis lung cancer(LLC)cells with the total EFs(EFs-LLC-MPs),and EFs-LLC-MPs were characterized by measuring the average particle size,polydispersity index,zeta potential,and release profile.Western blot was used to examine changes in pre-metastatic microenvironment markers in mouse alveolar epithelial cells(MLE-12)after treatment with microparticles or total EFs.Drug loading capacity and the uptake of microparticles by MLE-12 and mouse alveolar macrophages(MH-S)cells were determined using HPLC and flow cytometry.The uptake experiments showed that after nasal administration of rAAV STING shRNA,STING expression was significantly inhibited,and the markers of the pre-metastatic microenvironment were markedly reduced.Micro-CT results indicated a reduction in lung metastases and nodules,and the anti-metastatic effect of total EFs was affected.The results showed that the microparticles were membrane vesicles with a particle size of(373.17±3.18)nm,a Zeta potential of(-35.40±1.08)mV,a protein concentration of 562.62μg·mL^(-1),and a drug loading of 0.0609μg per microgram of protein.These microparticles were effectively taken up by MLE-12 and MH-S cells.Treatment of MLE-12 and MH-S cells with EFs-LLC-MPs reduced the expression of pre-metastatic microenvironment marker

关 键 词：细胞外囊泡 淫羊藿总黄酮 肺转移前微环境 肿瘤转移 干扰素基因刺激因子 

分 类 号：R285.5[医药卫生—中药学]