三七总皂苷调控破骨细胞外泌体中差异miRNA表达抑制成骨细胞铁死亡  

Panax notoginseng saponins regulate differential miRNA expression in osteoclast exosomes and inhibit ferroptosis in osteoblasts

作　　者：陶红成 曾平[2,3] 刘金富 田照 丁强 李晁辉 韦建杰 李豪 Tao Hongcheng;Zeng Ping;Liu Jinfu;Tian Zhao;Ding Qiang;Li Chaohui;Wei Jianjie;Li Hao(Guangxi University of Chinese Medicine,Nanning 530200,Guangxi Zhuang Autonomous Region,China;The First Affiliated Hospital of Guangxi University of Chinese Medicine,Nanning 530023,Guangxi Zhuang Autonomous Region,China;Guangxi Key Laboratory of Chinese Medicine Foundation Research,Nanning 530200,Guangxi Zhuang Autonomous Region,China)

机构地区：[1]广西中医药大学,广西壮族自治区南宁市530200 [2]广西中医药大学第一附属医院,广西壮族自治区南宁市530023 [3]广西中医基础研究重点实验室,广西壮族自治区南宁市530200

出　　处：《中国组织工程研究》2025年第19期4011-4021,共11页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金项目(82160913,81960876),项目负责人:曾平;广西壮族自治区博士研究生创新项目(YCBZ2022125),项目负责人:田照。

摘　　要：背景:激素性股骨头坏死多是由于长期大量使用激素所致,但具体的发病机制尚未明确,有待于进一步研究。目的:筛选出三七总皂苷干预破骨细胞来源外泌体中的差异miRNA,在此基础上构建成骨相关铁死亡调控网络,以探索激素性股骨头坏死发生的潜在机制及研究方向。方法:MTT实验检测不同浓度地塞米松和不同质量浓度三七总皂苷对Raw264.7细胞系的毒性作用;抗酒石酸酸性磷酸酶染色和TUNEL染色检测三七总皂苷对破骨细胞抑制和凋亡的影响;从三七总皂苷干预的破骨细胞中提取外泌体,对外泌体进行测序找出差异表达miRNA,通过CytoScape 3.9.1构建并可视化差异表达的miRNA与mRNA间的调控网络;通过GO分析和KEGG分析筛选候选mRNA;最后筛选出铁死亡相关的差异基因,构建铁死亡相关基因的调控网络。结果与结论:①通过MTT实验确定了适合干预Raw264.7细胞的地塞米松浓度(0.1μmol/L)和三七总皂苷质量浓度(1736.85μg/mL);②三七总皂苷对破骨细胞有抑制作用并能促进其凋亡;③从破骨细胞来源外泌体样本中鉴定出20个差异miRNA,通过靶标mRNA预测出11种成骨相关的差异miRNA,并构建了4个上调的差异表达miRNA对应于155个下调的候选mRNA以及7个下调的差异表达miRNA对应于238个上调的候选mRNA的调控网络;④在差异基因中筛选出与铁死亡相关的基因24个,最终构建了12个网络(miR-98-5p/PTGS2,miR-23b-3p/PTGS2,miR-425-5p/TFRC,miR-133a-3p/TFRC,miR-185-5p/TFRC,miR-23b-3p/NFE2L2,miR-23b-3p/LAMP2,miR-98-5p/LAMP2,miR-182-5p/LAMP2,miR-182-5p/TLR4,miR-23b-3p/ZFP36,miR-182-5p/ZFP36)。这些结果表明三七总皂苷可能通过调控破骨细胞外泌体来源miRNA的表达来调节成骨细胞铁死亡,为激素性股骨头坏死的机制研究提供新的思路。BACKGROUND:Steroid-induced femoral head necrosis is mostly caused by long-term and extensive use of hormones,but its specific pathogenesis is not yet clear and needs further study.OBJECTIVE:To screen out the differential miRNAs in osteoclast exosomes after the intervention of Panax notoginseng saponins,and on this basis,to further construct an osteogenic-related ferroptosis regulatory network to explore the potential mechanism and research direction of steroid-induced osteonecrosis of the femoral head.METHODS:MTT assay was used to detect the toxic effects of different concentrations of dexamethasone and different mass concentrations of Panax notoginseng saponins on Raw264.7 cell line.Tartrate resistant acid phosphatase staining and TUNEL assay were used to detect the effects of Panax notoginseng saponins on osteoclast inhibition and apoptosis.Exosomes were extracted from cultured osteoclasts with Panax notoginseng saponins intervention.Exosomes from different groups were sequenced to identify differentially expressed miRNAs.CytoScape 3.9.1 was used to construct and visualize the regulatory network between differentially expressed miRNAs and mRNAs.Candidate mRNAs were screened by GO analysis and KEGG analysis.Finally,the differential genes related to ferroptosis were screened out,and the regulatory network of ferroptosis-related genes was constructed.RESULTS AND CONCLUSION:(1)The concentration of dexamethasone(0.1μmol/L)and Panax notoginseng saponins(1736.85μg/mL)suitable for intervention of Raw264.7 cells was determined by MTT assay.(2)Panax notoginseng saponins had an inhibitory effect on osteoclasts and could promote their apoptosis.(3)Totally 20 differentially expressed miRNAs were identified from osteoclast-derived exosome samples,and 11 differentially expressed miRNAs related to osteogenesis were predicted by target mRNAs.The regulatory networks of 4 up-regulated differentially expressed miRNAs corresponding to 155 downregulated candidate mRNAs and 7 down-regulated differentially expressed miRNAs correspon

关 键 词：激素性股骨头坏死 三七总皂苷 破骨细胞 外泌体 铁死亡 调控网络 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R274.9