The analysis of Modified Qing' E Formula on the differential expression of exosomal miRNAs in the femoral head bone tissue of mice with steroid-induced ischemic necrosis of femoral head.
The analysis of Modified Qing' E Formula on the differential expression of exosomal miRNAs in the femoral head bone tissue of mice with steroid-induced ischemic necrosis of femoral head.
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DOI：
10.3389/fendo.2022.954778
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发表时间：
2022
影响因子：
5.2
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中科院分区：
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目的探讨清莪方加味对激素性股骨头缺血性坏死（INFH）骨髓组织中exosomal miRNAs表达的影响。采用激素诱导股骨头缺血性坏死模型，并给予补肾益气方治疗。建模成功后，分离股骨组织外泌体进行miRNA测序，获得股骨组织外泌体差异miRNA。通过GO分析和KEGG分析两组差异基因，确定MQEF发挥抗INFH作用的主要外泌体miRNAs以及主要的信号通路。接下来，对MQEF进行了具有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RT-qPCR对测序结果进行验证。通过网络药理学和RT-qPCR双重检测验证miRNA测序结果，阐明MQEF抗INFH作用调控的外泌体miRNA及其作用的特异性信号通路。在两组小鼠的exosomes中共预测了65，389个靶基因，获得了18个显著差异表达的miRNAs，其中14个上调，4个下调。GO富集分析表明，这些预测的靶基因在12371个生物过程、1727个细胞组分和4112个分子功能中富集。KEGG分析显示，预测的miRNA靶基因共注释了342条信号通路，其中与骨代谢密切相关的信号通路高度富集，分别为PI 3 K-Akt信号通路、MAPK信号通路和Wnt信号通路。上调最明显的是miR-185- 3 p和miR-1b-5 p，下调最明显的是miR-129 b-5 p和miR-223- 5 p，其中靶基因与PI 3 K-Akt信号通路密切相关。MQEF水煎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PI 3 K-Akt信号通路密切相关。实时定量PCR验证显示，治疗组miR-185- 3 p上调7.2倍，miR-129 b-5 p下调2.2倍，差异有统计学意义（P < 0.05）。MQEF可调控激素诱导的INFH模型中exosomal miRNA的表达，miR-185- 3 p或miR-129 b-5 p/PI 3 K-Akt信号轴可能是MQEF抗激素诱导INFH的部分机制。
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.