BACKGROUND:Regulation of the survival and differentiation of bone marrow mesenchymal stem cells is an essential consideration in the development of targeted drugs for treatment of osteoporosis.PURPOSE:The present study aimed to evaluate the combined effect of wedelolactone and oleonuezhenide, two compounds from Chinese formula Er-Zhi-Wan, on osteoblastogenesis and the underlying molecular mechanisms.METHODS:MTT assay was taken to evaluate cell proliferation. The alkaline phosphatase (ALP) activity assay was used to determine the activity of ALP. Alizarin red S (ARS) staining was taken to indicate the intensity of the calcium deposits. Quantitative real-time PCR and Western blot were performed to the levels of Runx2, Osteocalcin, and Osterix expression in mouse bone marrow mesenchymal stem cells (BMSCs). Ovariectomized mouse model and bone histomorphometric analysis were also used to research the effects of wedelolactone and oleonuezhenide on bone loss caused by ovariectomy.RESULTS:Wedelolactone combined with oleonuezhenide enhanced osteoblast differentiation and bone mineralization. Osteoblastogenesis-related marker genes including osteocalcin, Runx2, and osteorix were upregulated in the presence of wedelolactone and oleonuezhenide. At the molecular level, oleonuezhenide did not affect GSK-3β phosphorylation induced by wedelolactone, but elevated casein kinase 2-alpha (CK2α) expression, resulting in β-catenin and Runx2 nuclear translocation. In addition, 30 µM wedelolactone-induced cytotoxicity in bone marrow mesenchymal stem cells was relieved by 9 µM oleonuezhenide. These cells were protected by oleonuezhenide and maintained osteoblastic activity. Oleonuezhenide increased Wnt5a and CK2α expression. Wedelolactone-reduced extracellular signal-regulated kinase (ERK) phosphorylation was reversed by oleonuezhenide. In ovariectomized mice, administration of wedelolactone and oleonuezhenide prevented ovariectomy-induced bone loss by enhancing osteoblastic activity.CONCLUSION:These results suggested that oleonuezhenide enhanced the effects of wedelolactone on osteoblastogenesis. These two compounds could be developed as a combined therapeutic agent for osteoporosis.

中药学课程具有较强的专业性与实用性,是中医辨证施治、理法方药的重要环节.为培养具有综合能力的中药学人才,满足中药现代应用发展需要,本文旨在促使中药学顺应时代发展,促进传统中医药理论与现代临床应用深度融合,开展中药学课程教学改革与实践的研究.通过中药学常规教学的现状分析,相关文献的调研,实践运用的研究,总结得到传统中医药理论与现代应用融合的中药学教学方式有助于相关专业学生对中药学全新知识体系架构的建立,对于提高教学质量和人才培养具有重要的意义.

Our previous study showed that wedelolactone, a compound isolated from Ecliptae herba, has the potential to enhance osteoblastogenesis. However, the molecular mechanisms by which wedelolactone promoted osteoblastogenesis from bone marrow mesenchymal stem cells (BMSCs) remain largely unknown. In this study, treatment with wedelolactone (2 μg/mL) for 3, 6, and 9 days resulted in an increase in phosphorylation of extracellular signal-regulated kinases (ERKs), c-Jun N-terminal protein kinase (JNK), and p38. Phosphorylation of mitogen-activated protein kinases (MAPKs), ERK and JNK started to increase on day 3 of treatment, and p38 phosphorylation was increased by day 6 of treatment. Expression of bone morphogenetic protein (BMP2) mRNA and phosphorylation of Smad1/5/8 was enhanced after treatment of cells with wedelolactone for 6 and 9 days. The addition of the JNK inhibitor SP600125, ERK inhibitor PD98059, and p38 inhibitor SB203580 suppressed wedelolactone-induced alkaline-phosphatase activity, bone mineralization, and osteoblastogenesis-related marker genes including Runx2, Bglap, and Sp7. Increased expression of BMP2 mRNA and Smad1/5/8 phosphorylation was blocked by SP600125 and PD98059, but not by SB203580. These results suggested that wedelolactone enhanced osteoblastogenesis through induction of JNK- and ERK-mediated BMP2 expression and Smad1/5/8 phosphorylation.

目的:探讨女贞子提取物及其成分对小鼠骨髓间充质干细胞(BMSCs)成骨分化的影响及其机制.方法:比较实验组(加入成骨诱导剂和女贞子提取物及成分)、成骨诱导组与空白对照组之间碱性磷酸酶(ALP)活性.茜素红染色鉴定钙结节,qRT-PCR检测骨钙素,LRP6和Runx2的基因表达情况.结果:与成骨诱导组比较,10、100μ g/mL的女贞子提取物处理的BMSCs ALP活性分别提高了52.38％和95.23％.20μg/mL特女贞苷、女贞苷G13和油女贞苷处理的BMSCs ALP活性分别提高了59.52％、95.23％和100.04％.20μg/mL 3种成分增加钙化结节数量,提高了骨钙素、Runx2和LRP6 mRNA表达.结论:女贞子提取物及其3种成分可能通过诱导Wnt通路的激活促进BMSCs向成骨细胞分化,是发挥抗骨质疏松活性的主要成分.

依据《国家中长期教育改革和发展规划纲要(2010—2020年)》中提出的大学本科教育"探索多种培养方式,形成各类人才辈出、拔尖创新人才不断涌现的局面"的主旨.本研究针对医学专业创新人才导师制的实施模式进行探索,以科学问题为导向的导师小组制使传统"一对多"的教师—学生模式向"多对一"的创新人才培养模式转变,为导师制培养医学创新人才的实施方法提供了经验支撑.

Regenerative medicine has a high demand for the development of scaffold materials combined with other osteogenic inducers to generate bioactive composite materials for bone replacement therapies. Previously, we reported that wedelolactone promoted osteoblastogenesis of bone marrow mesenchymal stem cells (BMSCs). In this study, the effect of hydroxyapatites (HAps), bone composite materials we prepared, and the combined effect of wedelolactone and HAps on osteoblastogenesis differentiation was first evaluated. Three kinds of HAps constructed by a rod-like shape with particle size of 25 nm (HAp-1), 37 nm (HAp-2), and 33 nm (HAp-3) did not affect BMSC survival, but induced activity of alkaline phosphatase(ALP), a marker enzyme for osteoblastogenesis. HAp-1 treatment resulted in a more significant increase in the number of ALP staining-positive BMSC, and maintained an extended time for the increased number of ALP staining-positive BMSC. Moreover, HAp-1 combined with wedelolactone induced a higher ALP activity for a longer time than HAp-2 and HAp-3, and also increased the bone mineralization level. Osteoblastogenesis-related marker genes expression including osteorix, osteocalcin, and runx2 were increased after BMSC were treated with HAp-1 for 6 days. Although three kinds of HAps treatment for 9 days increased osteorix mRNA expression, osteocalcin, and runx2 mRNA expression levels were upregulated only by HAp-1. Similarly, only HAp-1 enhanced wedelolactone-induced osteocalcin, osteorix, and runx2 mRNA expression after 9 days treatment. Together, these results suggested that HAps with different sizes generated different effect on osteoblastogenesis. HAp-1 combined with wedelolacone can exert an enhanced effect on osteoblastogenesis, which has potential for treating osteoporosis. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 145-153, 2019.

Our previous study showed that wedelolactone, isolated from Ecliptae herba, enhanced osteoblastogenesis but inhibited osteoclastogenesis through Sema3A signaling pathway. This study aims to investigate the role of other semaphorins in wedelolactone-enhanced osteoblastogenesis and -inhibited osteoclastogenesis. Wedelolactone inhibited RANKL-induced Sema4D and Sema7A production, but had no effect on RANKL-reduced Sema6D expression in osteoclastic RAW264.7 cells. In mouse bone marrow mesenchymal stem cells (BMSC), wedelolactone reversed osteogenic medium(OS)-reduced Sema7A expression and OS-enhanced Sema3E mRNA expression, but no effect on OS-reduced Sema3B mRNA expression. Addition of Sema4D antibody promoted wedelolactone-reduced TRAP activity and bone resorption pit formation. Wedelolactone combined with Sema4D antibody inhibited the formation of Sema4D-Plexin B1 complex. In co-culture of BMSC with RAW264.7 cells, Sema7A antibody, similar with Sema 3A antibody, reversed wedelolactone-enhanced ALP activity and mineralization level, but promoted wedelolactone-inhibited TRAP activity. However, Sema3E and Sema3B antibodies had no effect. Further, wedelolactone enhanced the binding of Sema7A with PlexinC1 and Beta1, but addition of Sema7A antibody partially blocked this binding. Our data demonstrated that wedelolactone inhibited Sema4D production and Sema4D-PlexinB1 complex formation in RAW264.7 cells, thereafter inhibiting osteoclastogenesis. At the same time, wedelolactone enhanced osteoblastogenesis through promoting Sema7A production and Sema7A-PlexinC1-Beta1 complex formation in BMSC.

Targeting fibroblast-like synoviocyte (FLS) migration and invasion-mediated bone erosion is a promising clinical strategy for the treatment of rheumatoid arthritis (RA). Drug sensitivity testing is fundamental to this scheme. We designed a microfluidic chip-based, cell co-cultured platform to mimic RA FLS-mediated bone erosion and perform drug-sensitive assay. Human synovium SW982 cells were cultured in the central channel and migrated to flow through matrigel-coated side channels towards cell culture chamber where RANKL-stimulated osteoclastic RAW264.7 and osteogenic medium (OS)-stimulated bone marrow mesenchymal stem cells (BMSC) were cultured in the microfluidic chip device, mimicking FLS migration and invasion-mediated bone erosion in RA. These SW982 cells showed different migration potentials to osteoclasts and BMSC. The migration of SW982 cells with high expression of cadherin-11 was more potent when SW982 cells were connected with the co-culture of RAW264.7 and BMSC. Simultaneously, in the co-cultured chamber, tartrate-resistant acid phosphatase (TRAP) activity of RANKL-stimulated RAW264.7 cells was enhanced, but alkaline phosphatase (ALP) activity was decreased in comparison with mono-cultured chamber. Furthermore, it was confirmed that celastrol, a positive drug for the treatment of RA, inhibited SW982 cell migration as well as TRAP activity in the cell-cultured microfluidic chips. Thus, the migration and invasion to bone-related cells was reconstituted on the microfluidic model. It may provide an effective anti-RA drug screen model for targeting FLS migration-mediated bone erosion.

Ethnopharmacological relevance: Aconiti Sinomontani Radix is frequently used in the treatment of Bi syndrome in traditional Chinese medicine. Several reports indicate that Aconiti Sinomontani Radix has therapeutic effects for rheumatoid arthritis (RA). However, the cellular mode of action is still unclear. To investigate the effect of alkaloid extracts of Aconiti Sinomontani Radix on proliferation and migration of human synovial sarcoma SW982 cells as well as the molecular mechanism underlying. Materials and methods: SW982 cells were examined for proliferation by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) method. Wound scratch assays were performed to assess the migrated rate of SW982 cells. Quantitative real-time PCR was used to measure the mRNA expression levels of Wnt5a, Runx2, MMP3, and Bmp2. Western blotting was used to measure the phosphorylated levels of JNK and NF-kappa B as well as the expression of MMP3. Results: The alkaloid extract from Aconiti Sinomontani Radix (MQA) and MQB, which removed lappaconitine from MQA significantly inhibited the proliferation of SW982 in a dose-dependent manner. The proliferation inhibitory effect of MQB was more potent. Incubation with 10 mu g/ml MQB for 12, 24, and 36 h inhibited the migration of SW982 cells by 83%, 58%, and 42%, respectively. Treatment with different concentrations of MQB for 24 h inhibited mRNA expression of Wnt5a, Runx2, and MMP3, but Bmp2 mRNA expression was elevated by MQB. Further, MQB inhibited phosphorylation of JNK and NF-kappa B p65 as well as MMP3 expression by Western blotting analysis. Conclusion: The results showed that MQB inhibited proliferation and migration of SW982 cells possibly through suppressing Wnt5a-mediated JNK and NF-kappa B pathways. These results indicated that MQB might be an active extract of Aconiti Sinomontani Radix for targeting fibroblast-like synoviocytes (FLS) and be potential for RA therapy.

Bone homeostasis is maintained by osteoclast-mediated bone destruction and osteoblast-mediated bone formation, which are two tightly coupled and controlled processes. Double protection for bones through enhancing osteoblastogenesis but inhibiting osteoclastogenesis becomes a promising strategy for treating osteoporosis. However, there are little small molecular compounds found to regulate bone remodeling balance. We recently examined the effect of wedelolactone, a natural product from  Ecliptae herba , on osteoblastogenesis and osteoclastogenesis in vitro and in vivo. Our results revealed that wedelolactone stimulated osteoblast differentiation and bone mineralization in mouse bone marrow mesenchymal stem cells (BMSC). At the same concentration range, wedelolactone inhibited RANKL-induced preosteoclastic RAW264.7 actin-ring formation and bone resorption pits. The dual functional role of wedelolactone was mediated through inducing Wnt/GSK-3β/β-catenin pathway in BMSC and inhibiting NF-κB/c-fos/NFATc1 pathway in preosteoclastic RAW264.7 cells. Thus, we found that wedelolacone simultaneously regulated bone resorption and bone formation, and could therefore develop a new class of dual-action therapeutic agents for osteoporosis.