Zhu Wei, Zhang Faxue, Lu Junjie, Ma Chen, Shen Lin, Hu Desheng, Xu Xiaojuan, Shuai Bo
Department of Integrated Traditional Chinese and Western Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Department of Occupational and Environmental Health, School of Public Health, Wuhan University, Wuhan, China.
Front Endocrinol (Lausanne). 2022 Aug 10;13:954778. doi: 10.3389/fendo.2022.954778. eCollection 2022.
To investigate the differential expression of exosomal miRNAs in the bone marrow tissue of Modified Qing' E Formula (MQEF) on steroid-induced ischemic necrosis of the femoral head (INFH) model.
Steroid hormones were used to establish the INFH model and treated with MQEF. After successful modeling, femoral tissue exosomes were isolated for miRNA sequencing to obtain femoral tissue exosomal differential miRNAs. By GO analysis and KEGG analysis of the differential genes in both groups, the major exosomal miRNAs of MQEF exerting anti-INFH as well as the major signaling pathways were identified. Next, a quantitative metabolomic validation of MQEF with broad targeting was performed to obtain the main active components of MQEF and to perform biological analysis and signaling pathway prediction of the active components by network pharmacology. Finally, the sequencing results were validated by using RT-qPCR. The results of miRNA sequencing were verified by double examination of network pharmacology and RT-qPCR, and the exosomal miRNAs regulated by the anti-INFH effect of MQEF and the specific signaling pathway of the effect were clarified.
A total of 65,389 target genes were predicted in the exosomes of two groups of mice, and 18 significant differentially expressed miRNAs were obtained, of which 14 were up-regulated and 4 down-regulated. GO enrichment analysis showed that these predicted target genes were enriched in 12371 biological processes, 1727 cell components, and 4112 molecular functions. KEGG analysis showed that the predicted miRNA target genes were annotated to 342 signal pathways, in which the highly enriched pathways closely related to bone metabolism were PI3K-Akt signal pathway, MAPK signal pathway, and Wnt signal pathway. The most significantly up-regulated miRNAs were miR-185-3p and miR-1b-5p and the most significantly down-regulated miRNAs were miR-129b-5p and miR-223-5p, of which the targeted genes were closely related to the PI3K-Akt signal pathway. MQEF aqueous decoction extract targeted metabolomics quantitatively combined with network pharmacology predicted targets also closely related to PI3K-Akt signaling pathway. Real-time quantitative PCR validation showed that miR-185-3p was up-regulated 7.2-fold and miR-129b-5p was down-regulated 2.2-fold in the treatment group, and the difference was significant (P < 0.05).
MQEF can regulate exosomal miRNA expression in steroid-induced INFH models, miR-185-3p or miR-129b-5p/PI3K-Akt signal axis may be part of the mechanism of MQEF against steroid-induced INFH.
探讨加味青娥方（MQEF）对激素性股骨头缺血性坏死（INFH）模型骨髓组织中外泌体微小RNA（miRNA）的差异表达。
采用类固醇激素建立INFH模型并给予MQEF治疗。建模成功后，分离股骨组织外泌体进行miRNA测序，以获得股骨组织外泌体差异miRNA。通过对两组差异基因进行基因本体（GO）分析和京都基因与基因组百科全书（KEGG）分析，确定MQEF发挥抗INFH作用的主要外泌体miRNA及其主要信号通路。接下来，对MQEF进行广泛靶向的定量代谢组学验证，以获得MQEF的主要活性成分，并通过网络药理学对活性成分进行生物学分析和信号通路预测。最后，采用逆转录-定量聚合酶链反应（RT-qPCR）对测序结果进行验证。通过网络药理学和RT-qPCR双重检验验证miRNA测序结果，阐明MQEF抗INFH作用所调控的外泌体miRNA及其作用的具体信号通路。
两组小鼠外泌体共预测到65389个靶基因，获得18个显著差异表达的miRNA，其中14个上调，4个下调。GO富集分析显示，这些预测的靶基因富集于12371个生物学过程、1727个细胞成分和4112个分子功能。KEGG分析显示，预测的miRNA靶基因注释到342条信号通路，其中与骨代谢密切相关的高度富集通路为磷脂酰肌醇-3激酶（PI3K）-蛋白激酶B（Akt）信号通路、丝裂原活化蛋白激酶（MAPK）信号通路和Wnt信号通路。上调最显著的miRNA为miR-185-3p和miR-1b-5p，下调最显著的miRNA为miR-129b-5p和miR-223-5p，其靶向基因与PI3K-Akt信号通路密切相关。MQEF水煎液提取物靶向代谢组学定量结合网络药理学预测的靶点也与PI3K-Akt信号通路密切相关。实时定量PCR验证显示，治疗组miR-185-3p上调7.2倍，miR-129b-5p下调2.2倍，差异有统计学意义（P<0.05）。
MQEF可调节激素性INFH模型中外泌体miRNA表达，miR-185-3p或miR-129b-5p/PI3K-Akt信号轴可能是MQEF抗激素性INFH作用机制的一部分。
