Polyphenol extract from Tagetes erecta L. flowers stimulates osteogenesis via β-catenin activation.

Osteoporosis, a prevalent bone disorder, results in reduced bone mineral density and mass. With minimal side effects, medicinal plant-based natural remedies are increasingly explored for osteoporosis. However, the osteogenic potential of Tagetes erecta L. flower, traditionally used for cardiovascular and renal diseases, has not yet been studied. This study investigates the osteogenic effects of the polyphenol-enriched extract from T. erecta L. flowers (TE) and its main components on osteoblast differentiation, with an emphasis on anti-osteoporotic activity. The osteogenic activity of TE was assessed in MC3T3-E1 preosteoblast cells, analyzing osteogenic alkaline phosphatase (ALP) activity via a colorimetric assay and mineralization through Alizarin Red S staining over 14 d. Expression levels of osteogenic markers-transcription factor osterix (SP7), runt-related transcription factor 2 (RUNX2), and ALP-were quantified through quantitative reverse transcription-polymerase chain reaction and western blotting. In vivo effects were evaluated using zebrafish larvae for bone formation and anti-osteoporotic properties. Vertebral development was visualized by staining mineralized structures with calcein or Alizarin Red S. Prednisolone (PDS) was administered to zebrafish larvae to model osteoporosis. Furthermore, molecular docking simulations were conducted to assess the binding affinity of TE components to the ATP-binding pocket of glycogen synthase kinase-3β (GSK-3β), and their inhibitory potential on GSK-3β kinase activity was quantified by in vitro kinase assays. Cellular thermal shift assay (CETSA) was performed to monitor direct bindings of TE and its main components to GSK3-3β. TE promoted vertebral and cranial bone formation in zebrafish larvae, elevating key osteogenic genes, such as sp7, runx2a, runx2b, and alpl. Among TE components, kaempferol and patuletin significantly enhanced vertebral formation, while isorhamnetin showed moderate effects. Patulitrin and quercetagetin did not increased vertebral formation. In MC3T3-E1 cells, TE increased ALP activity, mineralization, and the expression of SP7, RUNX2, and ALP. It also induced GSK-3β phosphorylation at serine 9 and promoted β-catenin nuclear translocation. Inhibition of β-catenin signaling reversed TE-induced osteogenic effects. Molecular docking suggested strong GSK-3β binding by TE components, with patuletin showing notable inhibition GSK-3β activity (half-maximal inhibitory concentration = 379.3 ng/mL) and enhancing vertebral formation. CETSA confirmed that TE and its main components, kaempferol and patuletin, degrades GSK-3β. Additionally, TE alleviated PDS-induced osteoporosis in both cellular and zebrafish models. By targeting GSK-3β and activating β-catenin-mediated pathways, TE shows promise as a novel anti-osteoporotic agent. This study highlights the potential of TE for therapeutic use in bone health, warranting further clinical trials to confirm its applicability.

Sanjaya SS ，Park J ，Choi YH ，Park HS ，Sadanaga T ，Jung MJ ，Kim GY ... -  《-》

MiR-147b-3p promotes osteogenesis by targeting NDUFA4 and PI3K/AKT pathway.

Osteoporosis (OP) is a progressive metabolic bone disease characterized by impaired bone microarchitecture, decreased bone strength, and dysregulated bone remodeling, leading to an increased risk of fractures. Among osteoporotic fractures, osteoporotic vertebral compression fractures (OVCF) are the most common and can significantly impact patients' quality of life. Growing evidence suggests that microRNAs (miRNAs) play a crucial role in bone homeostasis by regulating osteoblast differentiation, bone metabolism, and remodeling processes. Notably, miR-147b-3p has been found to be downregulated in OVCF; however, its specific role in osteogenic regulation remains largely unknown. Therefore, further investigation is warranted to elucidate the function and underlying mechanism of miR-147b-3p in osteogenic differentiation. The GSE93883 and GSE74209 datasets were retrieved from the Gene Expression Omnibus (GEO) database to investigate specific microRNAs involved in the regulation of osteogenesis. Differential expression of miR-147b-3p and NDUFA4 was assessed between healthy controls and patients with osteoporotic vertebral compression fractures (OVCF) using real-time quantitative PCR.To modulate the expression levels of miR-147b-3p in MC3T3-E1 cells, both the miR-147b-3p mimic and inhibitor were utilized. Cell viability was evaluated via the CCK-8 assay to assess the impact of miR-147b-3p on MC3T3-E1 cell proliferation. Real-time PCR and Western blot analysis were conducted to quantify the expression levels of osteogenic markers across different experimental groups. Alizarin red staining (ARS) was employed to examine the effect of miR-147b-3p on the mineralization capacity of MC3T3-E1 cells. In vivo experiments were performed to evaluate the functional role of miR-147b-3p. Bioinformatics databases were used to predict the potential target gene of miR-147b-3p (NDUFA4), and the predictions were validated by a dual luciferase reporter gene assay.To investigate the regulatory role of the miR-147b-3p/NDUFA4 axis in osteogenic differentiation, MC3T3-E1 cells were transfected with the NDUFA4 overexpression plasmid and miR-147b-3p mimic. Western blot analysis was performed to assess the phosphorylation levels of PI3K and AKT, in order to explore whether the miR-147b-3p/NDUFA4 axis regulates osteogenic differentiation through the PI3K/AKT signaling pathway. Our results indicated a significant downregulation of miR-147b-3p and a concurrent upregulation of NDUFA4 in patients with osteoporotic vertebral compression fractures (OVCF). A luciferase reporter assay confirmed that NDUFA4 is a direct target gene of miR-147b-3p.To examine the functional role of miR-147b-3p, both in vitro and in vivo experiments were conducted.The experimental findings revealed that the miR-147b-3p mimic significantly enhanced cell viability, increased protein expressions of Alkaline Phosphatase (ALP) and Runt-related Transcription Factor 2 (RUNX2), and promoted mineralization as evidenced by Alizarin Red S staining. Conversely, treatment with the miR-147b-3p inhibitor or overexpression plasmid for NDUFA4 (pNDUFA4) produced opposite effects.Furthermore, the miR-147b-3p/NDUFA4 axis was found to regulate the PI3K/AKT signaling pathway.The miR-147b-3p mimic significantly increased the phosphorylation levels of PI3K (p-PI3K) and AKT (p-AKT), whereas pNDUFA4 led to their reduction. This study demonstrated that miR-147b-3p plays a crucial role in promoting osteogenic differentiation in osteoporotic vertebral compression fractures (OVCF) by targeting NDUFA4 and modulating the PI3K/AKT signaling pathway. These findings provide new insights into the molecular mechanisms underlying the progression of osteoporotic vertebral fractures.

Guo Y ，Shen K ，Li Z ，Niu C ，Luo Y ... -  《Journal of Orthopaedic Surgery and Research》

Anthocyanin-enriched polyphenols from Hibiscus syriacus L. (Malvaceae) exert anti-osteoporosis effects by inhibiting GSK-3β and subsequently activating β-catenin.

The bark and petal of Hibiscus syriacus L. (Malvaceae) have been used to relieve pain in traditional Korean medicine. Recently, we identified anthocyanin-enriched polyphenols from the petal of H. syriacus L. (AHs) and determined its anti-melanogenic, anti-inflammatory, and anti-oxidative properties. Nevertheless, the osteogenic potential of AHs remains unknown. This study was aimed to investigating the effect of AHs on osteoblast differentiation and osteogenesis in osteoblastic cell lines and zebrafish larvae. Furthermore, we investigated whether AHs ameliorates prednisolone (PDS)-induced osteoporosis. Cell viability was assessed by cellular morphology, MTT assay, and flow cytometry analysis, and osteoblast differentiation was measured alizarin red staining, alkaline phosphatase (ALP) activity, and osteoblast-specific marker expression. Osteogenic and anti-osteoporotic effects of AHs were determined in zebrafish larvae. AHs enhanced calcification and ALP activity concomitant with the increased expression of osterix (OSX), runt-related transcription factor 2 (RUNX2), and ALP in MC3T3-E1 preosteoblast and MG-63 osteosarcoma cells. Additionally, AHs accelerated vertebral formation and mineralization in zebrafish larvae, concurrent with the increased expression of OSX, RUNX2a, and ALP. Furthermore, PDS-induced loss of osteogenic activity and vertebral formation were restored by treatment with AHs, accompanied by a significant recovery of calcification, ALP activity, and osteogenic marker expression. Molecular docking studies showed that 16 components in AHs fit to glucagon synthase kinase-3β (GSK-3β); particularly, isovitexin-4'-O-glucoside most strongly binds to the peptide backbone of GSK-3β at GLY47(O), GLY47(N), and ASN361(O), with a binding score of -7.3. Subsequently, AHs phosphorylated GSK-3β at SER9 (an inactive form) and released β-catenin into the nucleus. Pretreatment with FH535, a Wnt/β-catenin inhibitor, significantly inhibited AH-induced vertebral formation in zebrafish larvae. AHs stimulate osteogenic activities through the inhibition of GSK-3β and subsequent activation of β-catenin, leading to anti-osteoporosis effects.

Karunarathne WAHM ，Molagoda IMN ，Lee KT ，Choi YH ，Jin CY ，Kim GY ... -  《-》

Fufang Zhenshu Tiaozhi capsule enhances bone formation and safeguards against glucocorticoid-induced osteoporosis through innovative Mekk2-mediated β-catenin deubiquitination.

Bone homeostasis depends on the regulation of β-catenin in osteoblasts. Glucocorticoids (GCs) are known to diminish β-catenin activity via Wnt pathway signaling, leading to osteoporosis. Conversely, activating β-catenin in osteoblasts through mitogen-activated protein kinase kinase kinase 2 (Mekk2) offers an innovative approach to combat GC-induced osteoporosis (GIOP). Fufang Zhenshu Tiaozhi (FTZ) capsules have shown effectiveness in treating GIOP, but the mechanisms behind this are still unclear. In this study, Mekk2 knockout mice (Mekk2-/-) was generated by CRISPR/Cas9. These mice were then subjected to Alcian Blue-Alizarin Red staining and immunofluorescence to assess their bone and cartilage development. To establish models of GIOP, both Mekk2-/- and wild-type (WT) mice were treated with dexamethasone (DXMS) and subsequently given FTZ capsules. We analyzed the resulting phenotypic changes in these mice using Micro-CT scans and histomorphological studies. Primary osteoblasts, isolated from both Mekk2-/- and WT mice, underwent qRT-PCR to measure key osteogenesis markers, including Runx2, Sp7, Bgalp, Col1a1 and Alp. Cells were then exposed to treatments with either FTZ or Wnt3a and the phosphorylation levels of β-catenin and Mekk2, along with the protein expression of Runx2, were evaluated using Western blotting and immunoprecipitation. Additionally, C3H10T1/2 cells transfected with TOPflash-luciferase and Renilla luciferase reporters were treated with FTZ and Wnt3a to measure β-catenin activity. In our study, administering FTZ in vivo effectively prevented bone loss typically induced by GCs. However, it's important to note that this protective effect was substantially reduced in mice lacking Mekk2. Additionally, FTZ showed a significant ability to enhance osteogenic differentiation in primary osteoblasts, doing so by altering the expression of Mekk2. Intriguingly, the impact of FTZ on Mekk2 appears to function through a pathway separate from the traditional Wnt signaling route. Furthermore, our findings indicate that FTZ also promotes the deubiquitination of β-catenin, contributing further to its positive effects on bone health. This study suggests that FTZ plays a significant role in protecting bone mass in cases of GIOP. The mechanism through which FTZ confers this benefit involves the activation of Mekk2/β-catenin signaling pathways, which represents a promising alternative strategy to counteract the deleterious effects of GIOP by augmenting osteoblastogenesis.

Hong G ，Tang L ，Zhou T ，Xie Y ，Wang J ，Ge D ，Dong Q ，Sun P ... -  《-》

Britannin inhibits hepatocellular carcinoma development and metastasis through the GSK-3β/β-catenin signaling pathway.

Hepatocellular carcinoma (HCC) stands out as a significant contributor to cancer-related death. Traditional Chinese Medicine (TCM) offers several advantages in the treatment of HCC. Britannin, a pivotal compound in Inulae Flos, has demonstrated pharmacological effects against various cancers, yet research on its specific anti-HCC effects remains limited. This study aims to explore the anti-HCC effects of britannin and its underlying mechanism. MTT assay, clone formation assay and flow cytometry were utilized to detect the cell activity, proliferation ability and apoptosis of britannin against HCC cell lines. Cell migration and invasion abilities of HCC cell lines treated with britannin were evaluated by wound-healing assay and transwell migration and invasion assay. H22 xenografted tumor mouse model was constructed and britannin treatment was performed to observe the effect of britannin on HCC tumors. The expression levels of liver cancer biomarkers AFP, AFP-L3, APT and TGF-β were detected by Elisa, and the histopathology was observed by HE staining. Network pharmacology and molecular docking were used to predict the possible signaling pathway of anti-HCC effect of britannin. The surface plasmon resonance (SPR) experiment was used to verify the interaction between britannin and proteins. The cell kinase activity function experiment was employed to detect the effect of britannin on enzyme activity. RT-qPCR and Western-Blot were used to verify the effect of britannin on the mRNA expressions of key genes and protein levels related to GSK-3β/β-catenin pathway in HCC cells and tumor tissues in mice. In vitro experiments showed that britannin could inhibit the activity, proliferation, migration and invasion abilities of HCC cells, while promoting their apoptosis. In vivo experiments revealed that britannin exerted inhibitory effects on the growth of transplanted liver cancer tumors, reducing the inflammatory infiltration and the expression levels of AFP, AFP-L3, APT and TGF-β of liver cancer markers in transplanted mice. Network pharmacology and molecular docking predicted that cell adhesion factors and GSK-3β/β-catenin pathway might be the related signaling pathway and had potential docking activity with key proteins. The SPR experiments elucidated the molecular interaction between britannin and GSK-3β. Enzyme activity assays indicated that britannin could modulate the functional activity of GSK-3β kinase. RT-qPCR suggested britannin could regulate the mRNA expressions of β-catenin, GSK-3β, E-cadherin and NCadherin. Western-Blot further verified that britannin could significantly up-regulate the expression of GSK-3β and down-regulate the expression of p-GSK-3β and β-catenin. At the same time, the expression of E-cadherin increased and NCadherin decreased, thereby reducing the occurrence of EMT and inhibiting the metastasis of HCC. In conclusion, britannin could inhibit the growth, development and metastasis of HCC, and its mechanism may be related to the regulation of GSK-3β/β-catenin signaling pathway to inhibit epithelial-mesenchymal transition of HCC.

Lu Q ，Zhu J ，Teng L ，Chen C ，Bi L ，Chen W ... -  《-》