第一部分：淫羊藿苷对早期激素性股骨头坏死兔骨组织形态和显微结构的影响

目的： 长期或大量使用糖皮质激素（glucocorticoid, GC）已经成为非创伤性股骨 头坏死（osteonecrosis of the femoral head, ONFH）的首要致病原因。该病发生机 制复杂，临床诊疗十分棘手，目前尚缺乏特异性的治疗手段。淫羊藿苷（icariin, ICA）为 8-异戊烯基黄酮苷类化合物，是中药淫羊藿的主要活性成分。现代药理 学研究证实 ICA 具有调节骨代谢、增强机体免疫、促进骨生长等多种药理活性。 本研究旨在利用 GC 诱导的早期股骨头坏死兔模型观察 ICA 对骨组织形态和显微 结构的影响，评估 ICA 对早期激素性 ONFH 的干预效果。 方法： 选取 50 只成年新西兰兔（体质量 2.5～3.0 kg），随机分为对照组（n=10）、 激素组（n=20）及淫羊藿苷组（n=20）。 激素组和淫羊藿苷组动物采用脂多糖 （lipopolysaccharide, LPS）和甲泼尼龙（methylprednisolone, MPS）序贯注射法制 备激素性 ONFH 动物模型。淫羊藿苷组在首次注射甲泼尼龙时开始灌喂淫羊藿 苷药液，每日 1 次，连续 6 周。对照组及激素组灌喂等量生理盐水，连续 6 周。 6 周后处死动物，取动物双侧股骨头标本进行研究：①肉眼观察各组股骨头标本 外观形态差异；②使用 Micro-CT 对各组标本进行连续扫描和三维重建，观察骨 组织形态变化，并定量分析松质骨显微结构参数，主要包括骨小梁数量（trabecular number, Tb.N）、骨小梁相对体积（bone volume to total volume, BV/TV）、骨小梁 厚度（trabecular thickness, Tb.Tn）和骨小梁分离度（trabecular separation，Tb.Sp）； ③通过 HE 染色观察各组股骨头标本组织病理学特征，骨细胞病理改变，并计算 空骨陷窝率；④利用同步辐射相位衬度纳米微观成像及重建技术，分析观察各组 标本单根骨小梁显微结构变化。 结果： 实验过程中 43 只动物（对照组 9 只；激素组 16 只；淫羊藿苷组 18 只） 纳入结果分析。①肉眼观察，三组动物标本在股骨头轮廓外形、塌陷与否及软骨 面色泽方面均有明显差异。②骨组织形态 Micro-CT 扫描重建观察，激素组股骨 头标本塌陷明显，骨小梁稀疏、断裂、排列紊乱；淫羊藿苷组股骨头标本塌陷不 明显，骨小梁结构轻度退变，而对照组无变化。松质骨显微结构参数测量分析， 激素组和实验组 Tb.N、Tb.Tn、BV/TV 低于对照组（P<0.05），Tb.Sp 高于对照组 （P<0.05）；实验组 Tb.N、Tb.Tn、BV/TV 高于激素组（P<0.05），Tb.Sp 低于对 照组（P<0.05）。③组织病理特征观察，激素组骨细胞凋亡和空骨陷窝数增加， 骨小梁间可见脂肪细胞堆积，部分呈囊状融合；淫羊藿苷组骨小梁形态较激素组 规则完整，骨细胞坏死数量少，脂肪细胞肥大不明显。对照组动物标本无 ONFH发生，激素组标本 ONFH 发生率为 81.3%（13/16），实验组为 66.7%（12/18）， 差异无统计学意义（P=0.448）；激素组空骨陷窝率（33.1%±1.4%）高于淫羊藿 苷组（18.9%±0.8%, P<0.05），两者均高于对照组（12.7%±1.5%, P<0.05）。④同 步辐射相位衬度成像观察显示，激素组骨小梁变薄、断裂，结构破坏严重，淫羊 藿苷组骨小梁体积厚度正常，结构相对完整。 结论： 在激素诱导的早期股骨头坏死兔子模型中，淫羊藿苷能够缓解激素对骨组 织结构的破坏作用，改善骨组织形态，稳定骨显微结构，促进损伤骨组织自我修 复，同时减少骨细胞坏死、凋亡，降低空骨陷窝率，延缓病情进展。

第二部分：淫羊藿苷和糖皮质激素对大鼠骨髓来源的巨噬细胞 mRNA 表达的影响

目的： 巨噬细胞在激素性股骨头坏死发生和进展过程中发挥重要的调控作用。激 素诱导骨细胞和骨髓组织损伤后吸引大量巨噬细胞迁移至坏死区，后者分泌大量 促炎因子，进一步扩大炎性反应，组织破坏加重，导致骨坏死病情进展。淫羊藿 苷能够通过转换巨噬细胞的功能状态控制炎症反应，抑制骨吸收，延缓病情进展， 但具体发生机制仍不清楚。该实验旨在研究淫羊藿苷和糖皮质激素对大鼠骨髓源 性巨噬细胞（bone marrow-derived macrophages，BMMs）mRNA 表达的影响，并 利用生物信息学方法分析差异表达基因(differentially expressed genes，DEGs) 对 巨噬细胞功能的影响。 方法： 体外分离提取大鼠骨髓源性巨噬细胞， 使用巨噬细胞集落刺激因子 （M-CSF）进行诱导分化 7 天，通过形态学观察和细胞免疫组化进行鉴定。将细 胞随机分为对照组（n=3）、激素组（n=3）和淫羊藿苷组（n=3），后两组分别经 地塞米松（dexamethasone，DEXA）和地塞米松＋淫羊藿苷（Icariin, ICA）干预 24h，CCK-8 法检测各组细胞活性。使用高通量测序分析三组细胞 mRNA 表达谱， 采用基因表达差异显著性分析方法筛选 DEGs，对 DEGs 进行基因功能 GO (gene ontology，GO) 与信号通路富集 KEGG (kyoto encycopedia of genes and genomes, KEGG)分析。使用 STRING 数据库构建 DEGs 蛋白质-蛋白质互作网络（PPI）， 鉴别关键基因（hub gene），筛选 PPI 网络基因模块，建立 miRNA-Gene 调控网络。 结果： 激素组和淫羊藿苷组大鼠 BMMs 活性均低于对照组，而淫羊藿苷组细胞活 性高于激素组。经高通量测序，激素组和对照组大鼠 BMMs 样本共筛选出了 641 个 DEGs，其中 DEGs 上调 358 个，下调 283 个。GO 和 KEGG 功能富集分析表明这 些 DEGs 主要参与应激、脂质代谢、蛋白质分解等生物学过程，并涉及与细胞间 紧密连接、氮素代谢相关的信号通路；基于 PPI 网络数据筛选出了 6 个关键基因，分别是 Rp126-ps1、Ghr1、LOC500594、LOC688462、LOC683961、Gng4； miRNA-Gene 调控网络显示 mo-miR-125b-5p、mo-miR-9a-5p 和 mo-miR-9a-5p 与 DEGs 具有交互作用。激素组和淫羊藿苷组大鼠 BMMs 样本共筛选出了 629 个 DEGs，其中 DEGs 上调 300 个，下调 329 个。GO 和 KEGG 功能富集分析表明 这些 DEGs 主要参与细胞黏附、离子跨膜转运、细胞膜筏极化等生物学过程，并 涉及钙离子信号通路、神经活性配体-受体相互作用通路相关的信号通路；基于 PPI 网络数据筛选出了 7 个关键基因， 分别是 LOC688462、LOC683961、 Rp126-ps1、LOC500594、Rp128、Gng4、Ghrl；miRNA-Gene 调控网络显示 mo-miR-125b-5p、mo-miR-9a-5p、mo-miR-128-3p、mo-miR-290 和 mo-miR-382 与 DEGs 具有交互作用。 结论： 激素对大鼠 BMMs 活性具有抑制作用，而淫羊藿苷能够缓解激素对细胞活 性的抑制作用。经激素和淫羊藿苷干预后，大鼠 BMMs 的基因表达发生显著变化， 差异表达的基因在巨噬细胞多个生物学过程和和信号通路中具有潜在作用，有助 于理解巨噬细胞在不同环境下功能变化的分子机制及其在激素诱导股骨头坏死 发生发展中的作用。

Part one: Effect of icariin on bone tissue morphology and microstructure in rabbit with early steroid-induced osteonecrosis of the femoral head

Objective: Long-term or high dosage of glucocorticoid (GC) has become the major inducement of non-traumatic osteonecrosis of the femoral head (ONFH). However, the mechanism of this disease is complicated. Clinical diagnosis and treatment are still a challenge without specific and effective treatment methods. Icariin (ICA) is an 8-isopentenyl flavonoid glycoside, and it is the main active component of herba epimedii. Modern pharmacological studies have confirmed that ICA has a variety of pharmacological activities, such as enhancing immunity, regulating bone metabolism, promoting bone growth and so on. The purpose of this study is to observe the effects of ICA on bone tissue morphology and microstructure using the rabbit model of early steroid-induced ONFH.

Methods: Fifty mature New Zealand rabbits (weighing, 2.5-3.0 kg) were randomly divided into control group (n=10), GC group (n=20), and ICA group (n=20). The rabbits of GC and ICA groups were injected with lipopolysaccharide and methylprednisolone, modelling early steroid-induced ONFH. The rabbits in ICA group were gavaged by icariin solution once a day for 6 weeks since the first injection of methylprednisolone, whereas the rabbits of control and GC groups were given normal saline at the same time. Six weeks later, the animals were sacrificed and the femoral head specimens were collected for study: ① The morphology of the femoral head specimens of each group were observed with naked eyes; ② Bone tissue morphological changes were observed by Micro-CT scan and reconstruction technique. Quantitative analysis was performed to analyze the microstructure parameters of cancellous bone, and the main parameters include trabecular bone volume to total volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Tn), and trabecular separation (Tb.Sp); ③ The histopathological characteristics of the femoral head specimens of each group were observed by HE staining, and the empty bone lacuna rate was calculated; ④ The microstructural changes of a single bone trabeculae in each group were analyzed by synchrotron radiation phase contrast nano-imaging and reconstruction technique.

Results: Finally, 9, 16, and 18 rabbits in the control group, GC group, and ICA group

were enrolled the analysis respectively. ① The gross observation showed that there were significant differences in the contour of femoral head, collapse, and cartilage surface color among the three groups. ② Micro-CT scanning and reconstruction of bone histomorphology showed that the femoral head in the GC group collapsed obviously and the trabeculae were sparse, incomplete and misaligned. While in the icariin group, the collapse was not obvious, the trabecular structure was slightly degenerated and there was no change in the control group. According to the measurement and analysis of the microstructure parameters of cancellous bone, the Tb.N, Tb.Tn and BV/TV in the GC group and ICA group were lower than those in the control group (P<0.05), while the Tb.Sp was higher than that in the control group (P<0.05). The Tb.N, Tb.Tn and BV/TV in the ICA group were higher than those in the GC group (P<0.05), and the Tb.Sp was lower than that in the control group (P<0.05). ③ In the GC group, HE staining showed that the number of osteocytes decreased, while empty lacunae increased. The adipocytes accumulated between trabeculae, some mashed together into a cyst. In the ICA group, the trabecular structure was relatively intact compared with GC group and no obvious apoptosis of osteocytes was observed. The size and number of adipocytes were almost normal. No ONFH occurred in control group based on the criteria for pathological diagnosis. The incidence was 81.3% (13/16) in the GC group and 66.7% (12/18) in the ICA group, and the difference was not significant (P=0.448). The rate of empty lacunae of osteonecrotic femoral heads in the GC group was 33.1%±1.4%, which was higher than that in ICA group (18.9%±0.8%) and control group (12.7%±1.5%, P<0.05). ④The observation of synchrotron radiation phase contrast imaging showed that the bone trabeculae became thinner and broken in the GC group, and the bone trabecular structure was badly destroyed in the GC group. In the ICA group, the trabecular volume thickness and structural morphology were normal.

Conclusion: In the rabbit model of early steroid-induced ONFH, icariin can alleviate the destructive effect of GC on bone tissue structure, maintain bone tissue morphology, stabilize bone microstructure, promote self-repair of injured bone tissue, and reduce osteocyte apoptosis and the rate of empty bone lacunae, delaying the progress of the disease.

Part two: Effects of Icariin and glucocorticoid on mRNA expression of rat bone marrow-derived macrophages

Objective: Macrophages play an important role in the occurrence and progression of steroid-induced osteonecrosis of the femoral head. A large number of macrophages will migrate to the necrotic lesion after osteocyte and bone marrow tissue injury. Then, the macrophages will secrete large amounts of pro-inflammatory factors, which further expand the inflammatory reaction and aggravate tissue destruction, leading to the progression of disease. By changing the macrophage function, Icariin could effectively control inflammation and inhibit bone resorption, delaying the progression of osteonecrosis. However, the exact mechanism is still unclear. The purpose of this study is to evaluate the effects of Icariin and glucocorticoid on mRNA expression of rat bone marrow-derived macrophages (BMMs), and to analyze the effects of differentially expressed gene (DEGs) on the function of macrophages through bioinformatics analysis.

Methods: Rat BMMs were extracted in vitro and induced by macrophage colony-stimulating factor (M-CSF) for 7 days. It was identified by morphological observation and cellular immunohistochemistry. The cells were randomly divided into control group (n = 3), dexamethasone (DEXA) group (n = 3) and icariin (ICA) group (n = 3). The DEXA and ICA group were treated by dexamethasone and dexamethasone plus icariin for 24h, respectively. Cell viability was determined with CCK-8 method. The mRNA expression profile of the three groups was analyzed using high-throughput sequencing and DEGs were selected. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of DEGs were performed. A protein-protein interaction (PPI) network was established using STRING database followed by identification of key genes. A miRNA-Gene regulatory network was established finally.

Results: The BMMs cell activity in the DEXA group and ICA group was lower than that in the control group, while the cell activity in the ICA group was higher than that in the DEXA group. Differentially expressed analysis resulted in a total of 641 DEGs between DEXA and control group, of which 358 were up-regulated and 283 were down-regulated. GO and KEGG functional enrichment analysis showed that these DEGs were involved in heat stress, lipid metabolism, Ubiquitin-dependent protein catabolic process and other biological process, and mainly regulating renin secretion pathways, tight junction, etc. Based on PPI network, 6 hub genes were selected:

Rp126-ps1, Ghr1, LOC500594, LOC688462, LOC683961, Gng4. MiRNA-Gene regulatory network showed that mo-miR-125b-5p, mo-miR-9a-5p, mo-miR-128-3p, mo-miR-290 and mo-miR-382 interacted with DEGs. Differentially expressed analysis resulted in a total of 629 DEGs between DEXA and ICA group, of which 300 were up-regulated and 329 were down-regulated. GO and KEGG functional enrichment analysis showed that these DEGs were involved in cell adhesion, regulation of ion transmembrane transport, Membrane raft polarization process and other biological process, and mainly regulating Calcium signaling pathway, Neuroactive ligand-receptor interaction, etc. Based on PPI network, 7 hub genes were selected: LOC688462, LOC683961, Rp126-ps1, LOC500594, Rp128, Gng4, Ghrl. MiRNA-Gene regulatory network showed that mo-miR-125b-5p, mo-miR-9a-5p, mo-miR-128-3p, mo-miR-290 and mo-miR-382 interacted with DEGs.

Conclusions: The DEXA can inhibit the cell activity of BMMs, while ICA can alleviate the inhibitory effect. After DEXA and ICA intervention, the gene expression of BMMs changed significantly, and the DEGs played a role in many biological processes and signal pathways of macrophages. It contributes to understand the molecular mechanism of functional changes of macrophages in different micro -environments and its effects in the occurrence and progression of steroid-induced osteonecrosis of the femoral head.