Plumbagin is a NF-κB-inducing kinase inhibitor with dual anabolic and antiresorptive effects that prevents menopausal-related osteoporosis in mice.

Osteoporosis is caused by enhanced bone resorption and relatively reduced bone formation. There is an unmet need to develop new agents with both antiresorptive and anabolic effects to treat osteoporosis, although drugs with either effect alone are available. A small molecular compound, plumbagin, was reported to inhibit receptor activator of nuclear factor kappa-B ligand-induced osteoclast (OC) differentiation by inhibiting IκBα phosphorylation-mediated canonical NF-κB activation. However, the key transcriptional factor RelA/p65 in canonical NF-κB pathway functions to promote OC precursor survival but not terminal OC differentiation. Here, we found that plumbagin inhibited the activity of NF-κB inducing kinase, the key molecule that controls noncanonical NF-κB signaling, in an ATP/ADP-based kinase assay. Consistent with this, plumbagin inhibited processing of NF-κB2 p100 to p52 in the progenitor cells of both OCs and osteoblasts (OBs). Interestingly, plumbagin not only inhibited OC but also stimulated OB differentiation in vitro. Importantly, plumbagin prevented trabecular bone loss in ovariectomized mice. This was associated with decreased OC surfaces on trabecular surface and increased parameters of OBs, including OB surface on trabecular surface, bone formation rate, and level of serum osteocalcin, compared to vehicle-treated mice. In summary, we conclude that plumbagin is a NF-κB-inducing kinase inhibitor with dual anabolic and antiresorptive effects on bone and could represent a new class of agent for the prevention and treatment of osteoporosis.

The emerging role of miR-128 in musculoskeletal diseases.

MicroRNA-128 (miR-128) is associated with cell proliferation, differentiation, migration, apoptosis, and survival. Genetic analysis studies have demonstrated that miR-128 participates in bone metabolism, which involves bone marrow-derived mesenchymal stem cells, osteoblasts, osteoclasts, and adipocytes. miR-128 also participates in regeneration of skeletal muscles by targeting myoblast-associated proteins. The deregulation of miR-128 could lead to a series of musculoskeletal diseases. In this review, we discuss recent findings of miR-128 in relation to bone metabolism and muscle regeneration to determine its potential therapeutic effects in musculoskeletal diseases, and to propose directions for future research in this significant field.

Role of forkhead box gene family in bone metabolism.

Bone metabolism is associated with many bone diseases and regulated by multiple signal pathways. Over the past three decades, the functions of a superfamily of evolutionarily conserved transcriptional regulators, known as forkhead box (Fox) family, has been demonstrated to contribute to the bone metabolism. Genetic analysis studies have demonstrated that Fox gene family participate in bone metabolism and that their expression can be regulated by multiple factors. The deregulation of Fox gene family can lead to a series of bone metabolic diseases. In this manuscript, we sketched the biology of the Foxs family, summarized its function of regulating bone metabolism and maintaining bone homeostasis to estimate its potential therapeutic effects in bone diseases, and suggested directions for future exploration in this important field.

TGFß-induced factor homeobox 2 blocks osteoblastic differentiation through targeting pSmad3/HDAC4/H4ac/Runx2 axis.

TGFß-induced factor homeobox 2 (Tgif2) has been reported as a functional role in cell homeostasis and a key activator of osteoclastogenesis and bone loss, as well. In the present study, we aimed to investigate the potential role of Tgif2 on osteogenic differentiation. Tgif2 expression was assessed during the osteogenic differentiation process of bone marrow-derived mesenchymal stem cells (BMSCs) and primary calvarial osteoblasts (OBs). The expression of Tgif2 in BMSCs and OBs increased by using lentivirus-mediated gene overexpression (OE). The effect of Tgif2 on osteogenic differentiation was compared between Tgif2 negative control (Tgif2-NC) and Tgif2-OE group in BMSCs/OBs via performing alkaline phosphatase (ALP) assay, mineralization assay, and gene expression analysis of some osteogenic markers. To investigate the molecular mechanism, the direct interaction of histone deacetylase 4 (HDAC4) and pSmad3, acetylated histone H4 (H4ac), and Runx2-binding site of the Ocn promoter was confirmed by performing co-immunoprecipitation (CoIP) and chromatin immunoprecipitation (ChIP) assay, respectively. The results showed that Tgif2 abundantly expressed in BMSCs and primary calvarial OBs, but decreased after osteogenic induction. In vitro, osteogenic differentiation was significantly inhibited with Tgif2 overexpression in both BMSCs and OBs, as well as the expression levels of osteogenic markers (Runx2, Sp7, Alp, and Ocn). Moreover, we found that Tgif2 overexpression significantly promoted the interaction of pSmad3 with HDAC4 in differentiated OBs, and sequentially decreased the abundance of H4ac at the Runx2-binding site of the Ocn promoter. These findings indicated that Tgif2 might block osteoblastic differentiation in vitro through targeting pSmad3/HDAC4/H4ac/Runx2 axis.

Autophagy as a target for glucocorticoid-induced osteoporosis therapy.

Autophagy takes part in regulating the eukaryotic cells function and the progression of numerous diseases, but its clinical utility has not been fully developed yet. Recently, mounting evidences highlight an important correlation between autophagy and bone homeostasis, mediated by osteoclasts, osteocytes, bone marrow mesenchymal stem cells, and osteoblasts, and autophagy plays a vital role in the pathogenesis of glucocorticoid-induced osteoporosis (GIOP). The combinations of autophagy activators/inhibitors with anti-GIOP first-line drugs or some new autophagy-based manipulators, such as regulation of B cell lymphoma 2 family proteins and caspase-dependent clearance of autophagy-related gene proteins, are likely to be the promising approaches for GIOP clinical treatments. In view of the important role of autophagy in the pathogenesis of GIOP, here we review the potential mechanisms about the impacts of autophagy in GIOP and its association with GIOP therapy.

Mammalian target of rapamycin as a therapeutic target in osteoporosis.

The mechanistic target of rapamycin (mTOR) plays a key role in sensing and integrating large amounts of environmental cues to regulate organismal growth, homeostasis, and many major cellular processes. Recently, mounting evidences highlight its roles in regulating bone homeostasis, which sheds light on the pathogenesis of osteoporosis. The activation/inhibition of mTOR signaling is reported to positively/negatively regulate bone marrow mesenchymal stem cells (BMSCs)/osteoblasts-mediated bone formation, adipogenic differentiation, osteocytes homeostasis, and osteoclasts-mediated bone resorption, which result in the changes of bone homeostasis, thereby resulting in or protect against osteoporosis. Given the likely importance of mTOR signaling in the pathogenesis of osteoporosis, here we discuss the detailed mechanisms in mTOR machinery and its association with osteoporosis therapy.

Therapeutic potential of microRNAs in osteoporosis function by regulating the biology of cells related to bone homeostasis.

MicroRNAs (miRNAs) are novel regulatory factors that play important roles in numerous cellular processes through the posttranscriptional regulation of gene expression. Recently, deregulation of the miRNA-mediated mechanism has emerged as an important pathological factor in osteoporosis. However, a detailed molecular mechanism between miRNAs and osteoporosis is still not available. In this review, the roles of miRNAs in the regulation of cells related to bone homeostasis as well as miRNAs that deregulate in human or animal are discussed. Moreover, the miRNAs that act as clusters in the biology of cells in the bone microenvironment and the difference of some important miRNAs for bone homeostasis between bone and other organs are mentioned. Overall, miRNAs that contribute to the pathogenesis of osteoporosis and their therapeutic potential are considered.

Plastrum Testudinis Extracts Promote BMSC Proliferation and Osteogenic Differentiation by Regulating Let-7f-5p and the TNFR2/PI3K/AKT Signaling Pathway.

BACKGROUND/AIMS: Plastrum testudinis extracts (PTE) show osteoprotective effects on glucocorticoid-induced osteoporosis in vivo and in vitro. However, the underlying molecular mechanism of PTE in promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is unclear. METHODS: BMSC proliferation was investigated using the Cell Counting Kit-8 assay. BMSC differentiation and osteogenic mineralization were assayed using alkaline phosphatase and Alizarin red staining, respectively. The mRNA expression levels of Let-7f-5p, Tnfr2, Traf2, Pi3k, Akt, ß-catenin, Gsk3ß, Runx2, and Ocn were measured using real time quantitative polymerase chain reaction. Protein levels of TNFR2, TRAF2, p-PI3K, p-AKT, p-ß-CATENIN, and p-GSK3ß were analyzed by western blotting. The functional relationship of Let-7f-5p and Tnfr2 was determined by luciferase reporter assays. RESULTS: The optimum concentration for PTE was 30 µg/ml. PTE significantly promoted BMSC osteogenic differentiation and mineralization after 7 and 14 days in culture, respectively. The combination of PTE and osteogenic induction exhibited significant synergy. PTE upregulated Let-7f-5p, ß-catenin, Runx2, and Ocn mRNA expression, and downregulated Tnfr2, Traf2, Pi3k, Akt, and Gsk3ß mRNA expression. PTE inhibited TNFR2, TRAF2, and p-ß-CATENIN protein expression, and promoted p-PI3K, p-AKT, and p-GSK3ß protein expression. In addition, Tnfr2 was a functional target of Let-7f-5p in 293T cells. CONCLUSIONS: Our results suggested that PTE may promote BMSC proliferation and osteogenic differentiation via a mechanism associated with the regulation of Let-7f-5p and the TNFR2/PI3K/AKT signaling pathway.

Effect of glucocorticoid withdrawal on glucocorticoid inducing bone impairment.

Glucocorticoid (GC) withdrawal after a short-term use was common in clinical practice like immediate post-transplant period. However, previous studies without setting age-control group failed to determine whether the BMD recovery was sufficient and whether it is necessary to accept anti-osteoporosis therapy after GC withdrawal. The aim of this study was to investigate the effect of GC withdrawal on bone impairment in glucocorticoid-induced osteoporosis (GIOP) rats. Twenty-four female Sprague-Dawley rats (3 months' old) were randomly divided into two treatment groups: an untreated age-control group (Con, n = 12); another group receiving a dexamethasone injection (DEXA, n = 12). Animals in the Con group were euthanized at 3rd month (M3) and 6th month (M6), respectively. Six rats in the DEXA group were euthanized at 3rd month (M3), whereas GC intervention was withdrew in the remaining animals of DEXA group, which were euthanized at the end of 6th month (M6). Bone mass, bone microarchitecture, biomechanical properties of vertebrae, morphology, serum levels of PINP and ß-CTX were evaluated. Compared with the Con(M3) group, the Con(M6) group showed significantly better bone quantity, morphology and quality. Compared with the Con(M3) group, the DEXA (M3) group showed significantly lower BMC, BMD, BS/TV, BV/TV, Tb.N, Tb.Th, vBMD, bone strength, compressive displacement, energy absorption capacity, PINP levels, ß-CTX levels, and damaged trabecular morphology. And the same change trend was observed in the comparison between the Con(M6) group and DEXA (M6) group. Compared with the DEXA (M3) group, the DEXA (M6) group showed significantly higher BMC, BMD and AREA, but no significant difference in BS/TV, BV/TV, SMI, Tb.N, Tb.Th, Tb.Sp, vBMD, bone strength, bone stiffness, compressive displacement, energy absorption capacity, PINP levels, ß-CTX levels, and improvement in trabecular morphology was observed. These results indicate that the reverse effect of GC withdrawal for 3 months on bone impairment in GIOP rats was insufficient, which implied that related anti-osteoporosis treatment might be still necessitated after GC withdrawal in clinical setting.

Implications of the Interaction Between miRNAs and Autophagy in Osteoporosis.

Imbalances between bone formation and resorption are the primary cause of osteoporosis. However, currently, a detailed molecular mechanism of osteoporosis is not available. Autophagy is the conserved process characterized by degrading and recycling aggregated proteins, intracellular pathogens, and damaged organelles. MicroRNAs (miRNAs) are novel regulatory factors that play important roles in numerous cellular processes, including autophagy, through the posttranscriptional regulation of gene expression. Conversely, autophagy plays a role in the regulation of miRNA homeostasis. Recent advances have revealed that both autophagy and miRNAs are involved in the maintenance of bone homoeostasis, whereas the role of the interaction of miRNAs with autophagy in osteoporosis remains unclear. In this paper, we review previous reports on autophagy, miRNAs, and their interaction in osteoporosis.

Correlation of Hounsfield Units with Bone Mineral Density and T-Score in Chinese Adults.

OBJECTIVE: To examine the association between vertebral cancellous Hounsfield units (HUs), age, bone mineral density, and T-score in a sample of Chinese adults. METHODS: The study included a sample of 739 participants. Age, bone mineral density, and T-score of each participant were recorded, and HUs were measured in the L1-L4 vertebrae. RESULTS: Data analysis revealed that HUs of vertebral cancellous bone across the pedicle level decreased with age, with women having higher values than men up to age 50 and vice versa thereafter. Furthermore, a positive correlation was found between HUs of vertebral cancellous bone across the pedicle level and bone mineral density/T-score in the L1-L4 vertebrae, but with a weaker correlation in the L4 vertebrae. Additionally, HU values for participants with osteoporosis were significantly lower than HU values for participants with osteopenia and normal bone health. CONCLUSIONS: From the findings of this study, it can be concluded that HUs may be a potential predictor of bone health, with implications for presurgical assessment of the quality of bone-screw interfaces for spinal surgery.

Restoring bone-fat equilibrium: Baicalin's impact on P38 MAPK pathway for treating diabetic osteoporosis.

Diabetes can lead to a disorder of bone-fat balance, a significant cause of osteoporosis due to changes in environmental factors. Baicalin (Bai), an active ingredient of Scutellaria baicalensis, has been confirmed to possess antioxidant, hypoglycemic, and anti-osteoporotic effects. However, a comprehensive understanding of Bai's influence on diabetic osteoporosis (DOP), including its effects and underlying mechanisms, remains elusive. This study investigated Bai's impact on the bone-fat equilibrium in rats with DOP. The results indicated that Bai alleviated bone damage in DOP by promoting osteogenesis and inhibiting adipogenesis. Concurrently, through bioinformatics analysis, it was suggested that Bai's mechanism of action might involve the P38-MAPK pathway. In vitro, Bai was found to enhance the development of bone marrow mesenchymal stem cells (BMSCs) towards osteogenic lineages while suppressing their differentiation towards adipogenic lineages. It was discovered that Bai's promotion of BMSC osteogenic differentiation depends on the P38-MAPK pathway. Additionally, the synergistic effect mediated by Bai and P38-MAPK inhibitor suppressed BMSC adipogenic differentiation. Our research indicates that the P38-MAPK pathway play a role in Bai's effects on the osteogenic-adipogenic differentiation of BMSCs, showcasing the potential for DOP treatment. This study highlights Bai's ability to regulate the equilibrium between bone and fat, presenting a novel approach to adressing DOP.

Dry and wet experiments reveal diagnostic clustering and immune landscapes of cuproptosis patterns in patients with ankylosing spondylitis.

Cuproptosis is a new manner of mitochondrial cell death induced by copper. There is evidence that serum copper has a crucial impact on ankylosing spondylitis (AS) by copper-induced inflammatory response. However, the molecular mechanisms of cuproptosis modulators in AS remain unknown. We aimed to use a bioinformatics-based method to comprehensively investigate cuproptosis-related subtype identification and immune microenvironment infiltration of AS. Additionally, we further verified the results by in vitro experiments, in which peripheral blood and fibroblast cells from AS patients were used to evaluate the functions of significant cuproptosis modulators on AS. Finally, eight significant cuproptosis modulators were identified by analysis of differences between controls and AS cases from GSE73754 dataset. Eight prognostic cuproptosis modulators (LIPT1, DLD, PDHA1, PDHB, SLC31A1, ATP7A, MTF1, CDKN2A) were identified using a random forest model for prediction of AS risk. A nomogram model of the 8 prognostic cuproptosis modulators was then constructed; the model could be beneficial in clinical settings, as indicated by decision curve analysis. Consensus clustering analysis was used to divide AS patients into two cuproptosis subtypes (clusterA & B) according to significant cuproptosis modulators. The cuproptosis score of each sample was calculated by principal component analysis to quantify cuproptosis subtypes. The cuproptosis scores were higher in clusterB than in clusterA. Additionally, cases in clusterA were closely associated with the immunity of activated B cells, Activated CD4 T cell, Type17 T helper cell and Type2 T helper cell, while cases in clusterB were linked to Mast cell, Neutrophil, Plasmacytoid dendritic cell immunity, indicating that clusterB may be more correlated with AS. Notably, key cuproptosis genes including ATP7A, MTF1, SLC31A1 detected by RT-qPCR with peripheral blood exhibited significantly higher expression levels in AS cases than controls; LIPT1 showed the opposite results; High MTF1 expression is correlated with increased osteogenic capacity. In general, this study of cuproptosis patterns may provide promising biomarkers and immunotherapeutic strategies for future AS treatment.

Endoscopic Resection and Decompression for Lateral Displacement of Cage after Oblique Lumbar Interbody Fusion: A Case Report from a Single Center.

BACKGROUND: Lateral displacement of cage is a rarely seen complication of oblique lumbar interbody fusion (OLIF). To the best of our knowledge, this complication has always been revised with posterior open surgery. However, open surgery often associates with large trauma and long period of recovery. CASE PRESENTATION: In the case presented, a 64-year-old male patient with lateral displacement of cage which consequently caused neurological symptoms after OLIF, was reported and surgically revised with an endoscopic resection and decompression technique. The surgery was performed through a posterolateral approach which was similar to transforaminal approach, with estimated blood loss of 45mL and whole operation time of 70 min. Neurological symptoms were disappeared after operation immediately and the patient was discharged 2 days later. He reported no symptoms other than mild weakness of the lower back at the last follow-up of 12 months. CONCLUSION: Endoscopic decompression technique may be an effective alternative to surgically treat lateral displacement of cage after OLIF with advantages of minimal invasion and quick recovery.

Plastrum testudinis Ameliorates Oxidative Stress in Nucleus Pulposus Cells via Downregulating the TNF-α Signaling Pathway.

BackgroundPlastrum testudinis (PT), a widely used traditional Chinese medicine, exerts protective effects against bone diseases such as intervertebral disc degeneration (IDD). Despite its effectiveness, the molecular mechanisms underlying the effects of PT on IDD remain unclear. Methods In this study, we used a comprehensive strategy combining bioinformatic analysis with experimental verification to investigate the possible molecular mechanisms of PT against IDD. We retrieved targets for PT and IDD, and then used their overlapped targets for protein-protein interaction (PPI) analysis. In addition, we used Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses to investigate the anti-IDD mechanisms of PT. Moreover, in vivo and in vitro experiment validations including hematoxylin-eosin (HE) and safranine O-green staining, senescence-associated ß-galactosidase (SA-ß-gal) assay, cell immunofluorescence staining, intracellular ROS measurement and Western blot analysis were performed to verify bioinformatics findings. Results We identified 342 and 872 PT- and IDD-related targets (32 overlapping targets). GO enrichment analysis yielded 450 terms related to oxidative stress and inflammatory response regulation. KEGG analysis identified 48 signaling pathways, 10 of which were significant; the TNF-α signaling pathway had the highest p-value, and prostaglandin G/H synthase 2 (PTGS2), endothelin-1 (EDN1), TNF-α, JUN and FOS were enriched in this pathway. Histopathological results and safranin O/green staining demonstrated that PT attenuated IDD, and SA-ß-gal assay showed that PT ameliorated nucleus pulposus cell (NPC) senescence. An ROS probe was adopted to confirm the protective effect of PT against oxidative stress. Western blot analyses confirmed that PT downregulated the protein expression of PTGS2, EDN1, TNF-α, JUN and FOS in the TNF-α signaling pathway as well as cellular senescence marker p16, proinflammatory cytokine interleukin-6 (IL6), while PT upregulated the expression of NPC-specific markers including COL2A1 and ACAN in a concentration-dependent manner. Conclusions To the best of our knowledge, this study is the first to report that PT alleviates IDD by downregulating the protein expression of PTGS2, EDN1, TNF-α, JUN and FOS in the TNF-α signaling pathway and upregulating that of COL2A1 and ACAN, thus suppressing inflammatory responses and oxidative stress in NPCs.

Unravelling diagnostic clusters and immune landscapes of cuproptosis patterns in intervertebral disc degeneration through dry and wet experiments.

Cuproptosis is a manner of mitochondrial cell death induced by copper. However, cuproptosis modulators' molecular processes in intervertebral disc degeneration (IDD) are still unclear. To better understand the processes of cuproptosis regulators in IDD, a thorough analysis of cuproptosis regulators in the diagnostic biomarkers and subtype determination of IDD was conducted. Then we collected clinical IDD samples and successfully established IDD model in vivo and in vitro, and carried out real-time quantitative polymerase chain reaction (RT-qPCR) validation of significant cuproptosis modulators. Totally we identified 8 crucial cuproptosis regulators in the present research. Using a random forest model, we isolated 8 diagnostic cuproptosis modulators for the prediction of IDD risk. Then, based on our following decision curve analysis, we selected the five diagnostic cuproptosis regulators with importance scores greater than two and built a nomogram model. Using a consensus clustering method, we divided IDD patients into two cuproptosis clusters (clusterA and clusterB) based on the important cuproptosis regulators. Additionally, each sample's cuproptosis value was evaluated using principal component analysis in order to quantify the cuproptosis clusters. Patients in clusterB had higher cuproptosis scores than patients in clusterA. Moreover, we found that clusterB was involved in the immunity of natural killer cell, while clusterA was related to activated CD4 T cell, activated B cell, etc. Notably, cuproptosis modulators detected by RT-qPCR showed generally consistent expression levels with the bioinformatics results. To sum up, cuproptosis modulators play a crucial role in the pathogenic process of IDD, providing biomarkers and immunotherapeutic approaches for IDD.

Unearths IFNB1 immune infiltrates in SOP-related ossification of ligamentum flavum pathogenesis.

Background: Ossification of ligamentum flavum (OLF) is a hidden, indolent disease condition with variable unexplained etiology and pathology. Growing evidences show a correlation between senile osteoporosis (SOP) and OLF, but the fundamental relationship between SOP and OLF remains unclear. Therefore, the purpose of this work is to investigate unique SOP-related genes and their potential functions in OLF. Methods: Gene Expression Omnibus (GEO) database was utilized to gather the mRNA expression data (GSE106253) and then analyzed by R software. A variety of methods, including ssGSEA, machine learning (LASSO and SVM-RFE), GO and KEGG enrichment, PPI network, transcription factor enrichment analysis (TFEA), GSEA and xCells were employed to verified the critical genes and signaling pathways. Furthermore, ligamentum flavum cells were cultured and used in vitro to identify the expression of the core genes. Results: The preliminary identification of 236 SODEGs revealed their involvement in BP pathways associated with ossification, inflammation, and immune response, including the TNF signaling pathway, PI3K/AKT signaling pathway and osteoclast differentiation. Four down-regulated genes (SERPINE1, SOCS3, AKT1, CCL2) and one up-regulated gene (IFNB1) were among the five hub SODEGs that were validated. Additionally, they were performed by ssGSEA and xCell to show the relationship of immune cells infiltrating in OLF. The most fundamental gene, IFNB1, which was only found in the classical ossification- and inflammation-related pathways, suggested that it may affect OLF via regulating the inflammatory response. In vitro experiment, we found that IFNB1 expression was dramatically higher in cells cocultured with osteogenic induction than in controls. Conclusion: As far as we are concerned, this is the first observation using transcriptome data mining to reveal distinct SOP-related gene profiles between OLF and normal controls. Five hub SODEGs were ultimately found using bioinformatics algorithms and experimental verification. These genes may mediate intricate inflammatory/immune responses or signaling pathways in the pathogenesis of OLF, according to the thorough functional annotations. Since IFNB1 was discovered to be a key gene and was connected to numerous immune infiltrates in OLF, it is possible that IFNB1 expression has a substantial impact on the pathogenesis of OLF. Our research will give rise to new possibilities for potential therapeutics that target SOP reverent genes and immune-associated pathways in OLF.

Bioinformatics identification and experimental validation of m6A-related diagnostic biomarkers in the subtype classification of blood monocytes from postmenopausal osteoporosis patients.

Background: Postmenopausal osteoporosis (PMOP) is a common bone disorder. Existing study has confirmed the role of exosome in regulating RNA N6-methyladenosine (m6A) methylation as therapies in osteoporosis. However, it still stays unclear on the roles of m6A modulators derived from serum exosome in PMOP. A comprehensive evaluation on the roles of m6A modulators in the diagnostic biomarkers and subtype identification of PMOP on the basis of GSE56815 and GSE2208 datasets was carried out to investigate the molecular mechanisms of m6A modulators in PMOP. Methods: We carried out a series of bioinformatics analyses including difference analysis to identify significant m6A modulators, m6A model construction of random forest, support vector machine and nomogram, m6A subtype consensus clustering, GO and KEGG enrichment analysis of differentially expressed genes (DEGs) between different m6A patterns, principal component analysis, and single sample gene set enrichment analysis (ssGSEA) for evaluation of immune cell infiltration, experimental validation of significant m6A modulators by real-time quantitative polymerase chain reaction (RT-qPCR), etc. Results: In the current study, we authenticated 7 significant m6A modulators via difference analysis between normal and PMOP patients from GSE56815 and GSE2208 datasets. In order to predict the risk of PMOP, we adopted random forest model to identify 7 diagnostic m6A modulators, including FTO, FMR1, YTHDC2, HNRNPC, RBM15, RBM15B and WTAP. Then we selected the 7 diagnostic m6A modulators to construct a nomogram model, which could provide benefit with patients according to our subsequent decision curve analysis. We classified PMOP patients into 2 m6A subtypes (clusterA and clusterB) on the basis of the significant m6A modulators via a consensus clustering approach. In addition, principal component analysis was utilized to evaluate the m6A score of each sample for quantification of the m6A subgroups. The m6A scores of patients in clusterB were higher than those of patients in clusterA. Moreover, we observed that the patients in clusterA had close correlation with immature B cell and gamma delta T cell immunity while clusterB was linked to monocyte, neutrophil, CD56dim natural killer cell, and regulatory T cell immunity, which has close connection with osteoclast differentiation. Notably, m6A modulators detected by RT-qPCR showed generally consistent expression levels with the bioinformatics results. Conclusion: In general, m6A modulators exert integral function in the pathological process of PMOP. Our study of m6A patterns may provide diagnostic biomarkers and immunotherapeutic strategies for future PMOP treatment.

Zuo-Gui-Wan Aqueous Extract Ameliorates Glucocorticoid-Induced Spinal Osteoporosis of Rats by Regulating let-7f and Autophagy.

Objective: This study proposes to explore the protective effect of Zuo-Gui-Wan (ZGW) aqueous extract on spinal glucocorticoid-induced osteoporosis (GIOP) in vivo and in vitro, and the underlying mechanisms of ZGW in GIOP and osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) were conducted. Methods: In vivo, SD rats were randomly divided into three groups: control group (CON), dexamethasone (DEXM) group, and ZGW group, which were given vehicle, DEXM injection, and ZGW intragastric administration at the same time. Vertebral bone microarchitecture, biomechanics, histomorphology, serum AKP activity, and the autophagosome of osteoblasts were examined. The mRNA expressions of let-7f, autophagy-associated genes (mTORC1, Beclin-1, ATG12, ATG5, and LC3), Runx2, and CTSK were examined. In vitro, the let-7f overexpression/silencing vector was constructed and transfected to evaluate the osteogenic differentiation of BMSCs. Western blot was employed to detect the expression of autophagy-associated proteins (ULK2, ATG5, ATG12, Beclin-1, LC3). Results: In vivo, ZGW promoted the bone quantity, quality, and strength; alleviated histological damage; increased the serum AKP activity; and reduced the autophagosome number in osteoblasts. Moreover, ZGW increased the let-7f, mTORC1, and Runx2 mRNA expressions and reduced the Beclin-1, ATG12, ATG5, LC3, and CTSK mRNA expressions. In vitro, bioinformatics prediction and dual luciferase reporter gene assay verified that let-7f targeted the binding to ULK2 and negatively regulated the ULK2 expression. Furthermore, by let-7f overexpression/silencing, ZGW may promote osteoblast differentiation of BMSCs by regulating let-7f and autophagy as evidenced by Western blot (ULK2, ATG5, ATG12, Beclin-1, LC3). Conclusions: ZGW may ameliorate GC-induced spinal osteoporosis by promoting osteoblast differentiation of BMSCs by activation of let-7f and suppression of autophagy.

Research on the Mechanism of Kaempferol for Treating Senile Osteoporosis by Network Pharmacology and Molecular Docking.

Kaempferol (KP), as a natural anti-inflammatory compound, has been reported to have curative effects on alleviating senile osteoporosis (SOP), which is an inflammation-related musculoskeletal disease, but the molecular mechanisms remain unclear due to scanty relevant studies. We predicted the targets of KP and SOP, and the common targets of them were subsequently used to carry out PPI analysis. Moreover, we adopted GO and KEGG enrichment analysis and molecular docking to explore potential mechanisms of KP against SOP. There were totally 152 KP-related targets and 978 SOP-related targets, and their overlapped targets comprised 68 intersection targets. GO enrichment analysis showed 1529 biological processes (p < 0.05), which involved regulation of inflammatory response, oxidative stress, regulation of bone resorption and remodeling, osteoblast and osteoclast differentiation, etc. Moreover, KEGG analysis revealed 146 items including 44 signaling pathways (p < 0.05), which were closely linked to TNF, IL-17, NF-kappa B, PI3K-Akt, MAPK, estrogen, p53, prolactin, VEGF, and HIF-1 signaling pathways. By means of molecular docking, we found that kaempferol is bound with the key targets' active pockets through some connections such as hydrogen bond, pi-alkyl, pi-sigma, pi-pi Stacked, pi-pi T-shaped, and van der Waals, illustrating that kaempferol has close combination with the key targets. Collectively, various targets and pathways involve in the process of kaempferol treatment against SOP through regulating inflammatory response, oxidative stress, bone homeostasis, etc. Moreover, our study first reported that kaempferol may regulate core targets' expression with involvement of inflammatory response, oxidative stress, and bone homeostasis, thus treating SOP.