Mechanisms of Liuwei Dihuang Pills in a rat model of postmenopausal osteoporosis

doi:10.12307/2026.867

Abstract

Abstract: BACKGROUND: Liuwei Dihuang Pill has been widely used in the treatment of osteoporosis with liver and kidney yin deficiency syndrome, but its therapeutic targets are still unclear and need further research.
OBJECTIVE: To analyze the mechanism of Liuwei Dihuang Pills in the treatment of postmenopausal osteoporosis using network pharmacology and single-cell sequencing.
METHODS: (1) The active ingredients and targets of each traditional Chinese medicine component of Liuwei Dihuang Pills were analyzed using the TCMSP and ETCM databases. The genes related to osteoporosis were retrieved from the GeneCards, OMIM, Disgenet, PharmGkb, and TTD databases. The drug-disease-target network was constructed and the core genes were screened by using analytical tools such as Cytoscape and STRING. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed by core genes. (2) Nine Sprague-Dawley rats were randomly divided into sham group (n=3), model group (n=3) and Liuwei Dihuang Pill group (n=3). Sham group did not undergo modeling, while the model and Liuwei Dihuang Pill group were modeled by bilateral oophorectomy. Four weeks after modeling, the Liuwei Dihuang Pill group was administered by gavage with a suspension prepared by Liuwei Dihuang Pills, while the sham group and the model group were administered by gavage with the same amount of normal saline. Administration in each group was given twice daily, for 12 consecutive weeks. After the final administration, peripheral blood was collected for single-cell sequencing and network pharmacology analyses.
RESULTS AND CONCLUSION: (1) There were a total of 116 intersection targets between Liuwei Dihuang Pills and osteoporosis. The "drug-disease-target" network diagram and protein-protein interaction analysis indicated that 15 genes such as B-cell lymphoma 2, Fos proto-oncogene 1, estrogen receptor 1, and interleukin-10 might be its core targets. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses indicated that the therapeutic mechanism of Liuwei Dihuang Pills might be related to osteoclast differentiation, cell senescence and apoptosis, steroid hormone regulation and inflammatory response. (2) Single-cell sequencing indicated that monocytes might be the key cell population in the treatment of postmenopausal osteoporosis with Liuwei Dihuang Pills. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses indicated that mononuclear macrophage populations were closely related to immunity, apoptosis, reactive oxygen species metabolism, etc. The combined analysis of single-cell sequencing and network pharmacology indicated that Liuwei Dihuang Pills for postmenopausal osteoporosis was mainly achieved through osteoclast and apoptosis-related pathways. To conclude, Liuwei Dihuang Pills treat postmenopausal osteoporosis through multiple targets, multiple effects and multiple mechanisms, and its main mechanism is related to the differentiation and apoptosis of osteoclasts. The CD16+ monocyte subset is the key cell subset responsible for this effect.

Key words: postmenopausal osteoporosis, network pharmacology, single-cell sequencing, Liuwei Dihuang Pill, CD16+ monocyte

CLC Number:

Xu Pengchao, Xie Lihua, Huang Jingwen, Chen Juan, Chen Xuan, Li Shengqiang, Ge Jirong. Mechanisms of Liuwei Dihuang Pills in a rat model of postmenopausal osteoporosis[J]. Chinese Journal of Tissue Engineering Research, 2026, 30(36): 9402-9412.

Figures/Tables 12

2.1.3 “药物-疾病-靶点”网络图构建与蛋白质互作网络分析使用Venn包获得交集靶点116个(图2)，将其通过STRING数据库进行蛋白质互作网络分析，构建蛋白质互作网络图(图3)，并导出靶点互作文件。将数据导入Cytoscape构建“药物-疾病-靶点”网络图(图4)，不同颜色和形状的节点有不同信息，绿色六边形为中药，橙色椭圆表示药物有效成分，紫色椭圆代表疾病靶点，各色椭圆的大小根据该靶点的度值确定，浅灰色连线表明各节点间相互作用关系。使用Cytoscape的CytoNCA插件计算靶点网络相关性指标中间中心性、接近中心性、度中心性、特征向量中心性、局部平均连通性、网络中心性以鉴定核心基因。通过计算上述指标，取其中位数进行2轮筛选后确定15个核心基因(表2，图5)。 2.1.4 交集靶点的GO与KEGG富集分析使用ClusterProfiler包对交集基因进行GO和KEGG富集分析。GO富集分析获得生物过程数据2 329条，细胞组分数据48条，分子功能数据134条，KEGG富集到174条相关通路(P < 0.1)。基于P值、Count数等信息筛选富集信息，使用R软件中的ggplot2包对富集分析结果进行可视化，各富集分析结果见图6。GO_生物过程分析表明，交集靶点与细胞对营养水平、氧化应激、激素、凋亡、炎症反应等生物过程关系密切；GO_细胞组分分析表明，交集靶点与线粒体、内质网、RNA聚合酶、肽酶抑制剂等细胞成分有关；GO_分子功能分析表明，交集靶点与核受体、转录因子、类固醇受体、细胞因子等功能相关。KEGG富集分析显示，交集基因主要富集到激素、炎症、氧化应激、破骨细胞分化、细胞衰老与凋亡等多条相关通路。 2.2 动物实验结果 2.2.1 实验动物数量分析 9只大鼠全部进入结果分析。 2.2.2 各组大鼠骨密度情况末次给药结束后，假手术组、模型组、六味地黄丸组大鼠骨密度分别为0.279，0.247，0.268 g/cm2，3组间整体比较差异有显著性意义(F=14.217，P=0.005)，假手术组、六味地黄丸组大鼠骨密度高于模型组(P < 0.05)，说明六味地黄丸可改善绝经后骨质疏松症大鼠的骨密度。 2.2.3 单细胞测序数据的降维聚类基于单细胞测序数据高维度且复杂的特性，使用Seurat包中的LogNormalize标准化方法进"

2.2.5 单核巨噬细胞富集分析为进一步分析单核巨噬细胞的功能情况，对单核巨噬细胞群进行了GO、KEGG富集分析(图11)。GO富集分析结果表明，单核巨噬细胞群主要富集到细胞凋亡相关功能(淋巴细胞凋亡过程、外源性凋亡信号通路的正调控和活性氧代谢过程的正调控)、淋巴细胞分化功能(α-β T细胞活化、分化的正调控、T细胞分化的正调控)等功能，此外与免疫系统功能也关系密切。KEGG富集分析表明，与单核巨噬细胞群关系密切的有凋亡与免疫相关功能，值得注意的是，单核巨噬细胞群与破骨细胞分化也有密切联系(Rich number=43，Rich Factor=0.506，P < 0.000 1)。针对富集分析结果，进一步使用富集弦图的方式观察每个富集功能对应到哪些基因与这些基因的上调、下调情况(图12)。KEGG富集弦图表明，CD16表达上调、Fos基因表达下调等与破骨细胞分化关系密切。 2.3 网络药理学-单细胞测序联合分析结果 2.3.1 单核巨噬细胞的核心基因表达量分析此次测序鉴定为单核巨噬细胞的细胞总数为12 702个，CD16+ 细胞总计4 463个，CD16+ 细胞占单核巨噬细胞比例为35.14%(表5)。为进一步探索六味地黄丸治疗绝经后骨质疏松症的机制，基于网络药理学分析所获得的核心基因，提取了此次测序单核巨噬细胞的相关基因表达量进行进一步分析(表6)。结果表明，核心基因中仅有MYC、MMP9、IL6组间比较差异无显著性意义，其余基因的表达均发生了明显变化。在组间有差异的核心基因中，ESR1、IL10、TNF、PTGS2、EGFR等核心基因的本底表达较低，而BCL2本底表达高且组间变化明显。作为单核巨噬细胞亚群Marker的CD16表达在模型组最高，在六味地黄丸组、假手术组中下降。 2.3.2 核心基因与单核巨噬细胞的联合富集分析见图13。"

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