PGRMC2 influences the onset of postmenopausal osteoporosis through disulfidptosis in monocytes: Evidence from experimental validation and Mendelian randomization.

This study explores the role of disulfidptosis in monocytes and its relation to postmenopausal osteoporosis (PMOP). Using single-cell RNA sequencing and microarray assays, we identified key genes: LONRF1, ACAP2, IPO9, and PGRMC2. Through differential analysis, Weighted Gene Co-expression Network Analysis (WGCNA), and machine learning, these genes were linked to PMOP. Functional enrichment and ROC curve analysis demonstrated their effectiveness in distinguishing postmenopausal fracture patients from healthy individuals. Notably, PGRMC2 exhibited significant expression differences, highlighted by a notable Area Under the Curve (AUC) value of 0.665. Further investigation involved Western blotting and immunohistochemical assays, revealing decreased PGRMC2 expression in ovariectomized (OVX) mice. This decrease was consistent across both experimental methods, emphasizing PGRMC2's role in PMOP. Moreover, PGRMC2 was predominantly present in macrophages compared to monocytes within bone tissue and was significantly located in bone marrow mesenchymal stem cells (BM-MSCs) in PMOP patients. It was also abundantly found in osteoblasts and adipocytes. Additionally, a Mendelian randomization analysis using the TwoSampleMR R package, with data from decode and GWAS databases, was conducted. This analysis showed a significant impact of PGRMC2 on osteoporosis risk (p = 0.0048, OR = 0.6836), suggesting a potential protective effect against the disease. Our results suggest that PGRMC2 may facilitate the differentiation of monocytes into macrophages in bone tissue, influencing the behavior of BM-MSCs. This, in turn, could impact the progression and severity of PMOP. The study provides new insights into the molecular mechanisms underlying postmenopausal osteoporosis and highlights the potential of PGRMC2 as a therapeutic target or biomarker for this condition.

Sirt1: An Increasingly Interesting Molecule with a Potential Role in Bone Metabolism and Osteoporosis.

Osteoporosis (OP) is a common metabolic bone disease characterized by low bone mass, decreased bone mineral density, and degradation of bone tissue microarchitecture. However, our understanding of the mechanisms of bone remodeling and factors affecting bone mass remains incomplete. Sirtuin1 (SIRT1) is a nicotinamide adenine dinucleotide-dependent deacetylase that regulates a variety of cellular metabolisms, including inflammation, tumorigenesis, and bone metabolism. Recent studies have emphasized the important role of SIRT1 in bone homeostasis. This article reviews the role of SIRT1 in bone metabolism and OP and also discusses therapeutic strategies and future research directions for targeting SIRT1.

Transcriptome analysis of the diseased intervertebral disc tissue in patients with spinal tuberculosis.

OBJECTIVE: To investigate the differential expression genes (DEGs) in spinal tuberculosis using transcriptomics, with the aim of identifying novel therapeutic targets and prognostic indicators for the clinical management of spinal tuberculosis. METHODS: Patients who visited the Department of Orthopedics at the Second Hospital, Lanzhou University from January 2021 to May 2023 were enrolled. Based on the inclusion and exclusion criteria, there were 5 patients in the test group and 5 patients in the control group. Total RNA was extracted and paired-end sequencing was conducted on the sequencing platform. After processing the sequencing data with clean reads and annotating the reference genome, FPKM normalization and differential expression analysis were performed. The DEGs and long non-coding RNAs (LncRNAs) were analyzed for Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment. The cis-regulation of differentially expressed mRNAs (DE mRNAs) by LncRNAs was predicted and analyzed to establish a co-expression network. RESULTS: This study identified 2366 DEGs, with 974 genes significantly upregulated and 1392 genes significantly downregulated. The upregulated genes are associated with cytokine-cytokine receptor interactions, tuberculosis, and TNF-α signaling pathways, primarily enriched in biological processes such as immunity and inflammation. The downregulated genes are related to muscle development, contraction, fungal defense response, and collagen metabolism processes. Analysis of LncRNAs from bone tuberculosis RNA-seq data detected a total of 3652 LncRNAs, with 356 significantly upregulated and 184 significantly downregulated. Further analysis identified 311 significantly different LncRNAs that could cis-regulate 777 target genes, enriched in pathways such as muscle contraction, inflammatory response, and immune response, closely related to bone tuberculosis. There are 51 genes enriched in the immune response pathway regulated by cis-acting LncRNAs. LncRNAs that regulate immune response-related genes, such as upregulated RP11-451G4.2, RP11-701P16.5, AC079767.4, AC017002.1, LINC01094, CTA-384D8.35, and AC092484.1, as well as downregulated RP11-2C24.7, may serve as potential prognostic and therapeutic targets. CONCLUSION: The DE mRNAs and LncRNAs in spinal tuberculosis are both associated with immune regulatory pathways. These pathways promote or inhibit the tuberculosis infection and development at the mechanistic level and play an important role in the process of tuberculosis transferring to bone tissue.

Unraveling Key m6A Modification Regulators Signatures in Postmenopausal Osteoporosis through Bioinformatics and Experimental Verification.

OBJECTIVE: Bone marrow mesenchymal stem cells (BMSCs) show significant potential for osteogenic differentiation. However, the underlying mechanisms of osteogenic capability in osteoporosis-derived BMSCs (OP-BMSCs) remain unclear. This study aims to explore the impact of YTHDF3 (YTH N6-methyladenosine RNA binding protein 3) on the osteogenic traits of OP-BMSCs and identify potential therapeutic targets to boost their bone formation ability. METHODS: We examined microarray datasets (GSE35956 and GSE35958) from the Gene Expression Omnibus (GEO) to identify potential m6A regulators in osteoporosis (OP). Employing differential, protein interaction, and machine learning analyses, we pinpointed critical hub genes linked to OP. We further probed the relationship between these genes and OP using single-cell analysis, immune infiltration assessment, and Mendelian randomization. Our in vivo and in vitro experiments validated the expression and functionality of the key hub gene. RESULTS: Differential analysis revealed seven key hub genes related to OP, with YTHDF3 as a central player, supported by protein interaction analysis and machine learning methodologies. Subsequent single-cell, immune infiltration, and Mendelian randomization studies consistently validated YTHDF3's significant link to osteoporosis. YTHDF3 levels are significantly reduced in femoral head tissue from postmenopausal osteoporosis (PMOP) patients and femoral bone tissue from PMOP mice. Additionally, silencing YTHDF3 in OP-BMSCs substantially impedes their proliferation and differentiation. CONCLUSION: YTHDF3 may be implicated in the pathogenesis of OP by regulating the proliferation and osteogenic differentiation of OP-BMSCs.

METTL3-mediated m6A modification increases Hspa1a stability to inhibit osteoblast aging.

Senile osteoporosis is mainly caused by osteoblasts attenuation, which results in reduced bone mass and disrupted bone remodeling. Numerous studies have focused on the regulatory role of m6A modification in osteoporosis; however, most of the studies have investigated the differentiation of bone marrow mesenchymal stem cells (BMSCs), while the direct regulatory mechanism of m6A on osteoblasts remains unknown. This study revealed that the progression of senile osteoporosis is closely related to the downregulation of m6A modification and methyltransferase-like 3 (METTL3). Overexpression of METTL3 inhibits osteoblast aging. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) revealed that METTL3 upregulates the stability of Hspa1a mRNA, thereby inhibiting osteoblast aging. Moreover, the results demonstrated that METTL3 enhances the stability of Hspa1a mRNA via m6A modification to regulate osteoblast aging. Notably, YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) participates in stabilizing Hspa1a mRNA in the METTL3-mediated m6A modification process, rather than the well-known degradation function. Mechanistically, METTL3 increases the stability of Hspa1a mRNA in a YTHDF2-dependent manner to inhibit osteoblast aging. Our results confirmed the significant role of METTL3 in osteoblast aging and suggested that METTL3 could be a potential therapeutic target for senile osteoporosis.

METTL3-mediated m6A modification of SOX4 regulates osteoblast proliferation and differentiation via YTHDF3 recognition.

N6-methyladenosine (m6A), the most prevalent internal modification in mRNA, is related to the pathogenesis of osteoporosis (OP). Although methyltransferase Like-3 (METTL3), an m6A transferase, has been shown to mitigate OP progression, the mechanisms of METTL3-mediated m6A modification in osteoblast function remain unclear. Here, fluid shear stress (FSS) induced osteoblast proliferation and differentiation, resulting in elevated levels of METTL3 expression and m6A modification. Through Methylated RNA Immunoprecipitation Sequencing (MeRIP-seq) and Transcriptomic RNA Sequencing (RNA-seq), SRY (Sex Determining Region Y)-box 4 (SOX4) was screened as a target of METTL3, whose m6A-modified coding sequence (CDS) regions exhibited binding affinity towards METTL3. Further functional experiments demonstrated that knockdown of METTL3 and SOX4 hampered osteogenesis, and METTL3 knockdown compromised SOX4 mRNA stability. Via RNA immunoprecipitation (RIP) assays, we further confirmed the direct interaction between METTL3 and SOX4. YTH N6-Methyladenosine RNA Binding Protein 3 (YTHDF3) was identified as the m6A reader responsible for modulating SOX4 mRNA and protein levels by affecting its degradation. Furthermore, in vivo experiments demonstrated that bone loss in an ovariectomized (OVX) mouse model was reversed through the overexpression of SOX4 mediated by adeno-associated virus serotype 2 (AAV2). In conclusion, our research demonstrates that METTL3-mediated m6A modification of SOX4 plays a crucial role in regulating osteoblast proliferation and differentiation through its recognition by YTHDF3. Our research confirms METTL3-m6A-SOX4-YTHDF3 as an essential axis and potential mechanism in OP.

A novel lncRNA GM15416 regulates osteoblast apoptosis and differentiation through the c-Fos/Fas axis and mitigates osteoporosis.

Osteoporosis (OP) is a common systemic bone disorder, and the programmed cell death of osteoblasts is closely linked to the development of osteoporosis. Previous studies have shown that c-fos can cause osteoblast apoptosis. Furthermore, it has been demonstrated that long non-coding RNA (lncRNA) plays a pervasive role in regulating the biology of osteoblasts. Nevertheless, the precise role and mechanism of long non-coding RNA (lncRNA) in relation to c-Fos at the transcriptional level in osteoblast cell death remain uncertain. Compared with normal osteoblasts, serum deprivation resulted in significant upregulation of the transcription factor c-Fos and apoptosis-related Fas proteins in osteoblasts. In addition, the expression of lncRNA GM15416 related to c-Fos was significantly increased. The results showed that overexpression of c-Fos leads to an increase in downstream Fas protein, which subsequently leads to osteoblast apoptosis and hinders osteogenesis. On the contrary, a decrease in lncRNA GM15416 expression leads to a decrease in c-Fos/Fas expression, which hinders osteoblast apoptosis and promotes osteogenesis. Our results suggest that lncRNA GM15416 exerts inhibitory effects on osteoblast apoptosis and acts as a preventive factor against osteoporosis. As a result, GM15416 emerges as an important lncRNA associated with osteoporosis and holds potential as a future therapeutic target.

Apply Anticoagulants after Discharge not Benefit to the Incidence of Symptomatic Thrombus in High-Altitude Areas: A Retrospective Study.

OBJECTIVE: Many guidelines indicate that continuous use of anticoagulant drugs reduces the incidence of venous thrombus (VT), but no studies show the effect on the incidence of symptomatic venous thrombus (SVT) in total knee arthroplasty (TKA) patients after discharge. This study aimed to investigate whether it is necessary to apply anticoagulants to TKA patients after discharge. METHODS: Patients who met the exclusion criteria requirement, underwent TKA by the same surgical team and received anticoagulant therapy after the operation were eligible for the study. Finally, a total of 567 TKA patients were recruited as participants. The patients were divided into two groups. The patients in group A were taken low molecular heparin for 5-10 days after surgery, which included but was not limited to low molecular weight heparin calcium injection (0.4 mL, ih, Qd), calcium dioxin injection (0.6 mL, ih, Qd), or enoxaparin sodium injection (0.4 mL, ih, Qd), and the patients needed to continue oral anticoagulant drug (10 mg, po, Qd) for 7-21 days after discharge. The patients in group B only took low molecular heparin 5-10 days after surgery and no treatment after discharge. The baseline characteristics of patients, total complications of SVT include lower limb vascular pain (LLVP), lower limb vascular pain no fester (LLVPNF), lower limbs swelling (LLS), lower limb fester (LLF), and death by thrombosis (DT), bleeding and mortality following discharged were compared between two groups. RESULTS: The study showed that the incidence of SVT patients had no significant difference between the two groups (p = 0.489). Moreover, the incidence of LLVP (p = 0.265), LLS (p = 0.84), LLVPNF (p = 0.213), LLF (p = 0.907), DT (p = 0.907), death from other causes, and bleeding (p = 0.323) had no significant differences between the two groups. However, the incidence of SVT in patients with smoking (p = 0.0001 or 0.0011) or drinking (p = 0.0002 or 0.0001) was significantly increased. CONCLUSION: There is not enough evidence showing that the TKA patients given anticoagulants after discharge had benefits in decreasing the risk of SVT. Furthermore, smoking and drinking would significantly increase the risk of SVT in TKA patients.

Screening and identification of potential hub genes and immune cell infiltration in the synovial tissue of rheumatoid arthritis by bioinformatic approach.

Background: Rheumatoid arthritis (RA) is an autoimmune disease that affects individuals of all ages. The basic pathological manifestations are synovial inflammation, pannus formation, and erosion of articular cartilage, bone destruction will eventually lead to joint deformities and loss of function. However, the specific molecular mechanisms of synovitis tissue in RA are still unclear. Therefore, this study aimed to screen and explore the potential hub genes and immune cell infiltration in RA. Methods: Three microarray datasets (GSE12021, GSE55457, and GSE55235), from the Gene Expression Omnibus (GEO) database, have been analyzed to explore the potential hub genes and immune cell infiltration in RA. First, the LIMMA package was used to screen the differentially expression genes (DEGs) after removing the batch effect. Then the clusterProfiler package was used to perform functional enrichment analyses. Second, through weighted coexpression network analysis (WGCNA), the key module was identified in the coexpression network of the gene set. Third, the protein-protein interaction (PPI) network was constructed through STRING website and the module analysis was performed using Cytoscape software. Fourth, the CIBERSORT and ssGSEA algorithm were used to analyze the immune status of RA and healthy synovial tissue, and the associations between immune cell infiltration and RA-related diagnostic biomarkers were evaluated. Fifth, we used the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to validate the expression levels of the hub genes, and ROC curve analysis of hub genes for discriminating between RA and healthy tissue. Finally, the gene-drug interaction network was constructed using DrugCentral database, and identification of drug molecules based on hub genes using the Drug Signature Database (DSigDB) by Enrichr. Results: A total of 679 DEGs were identified, containing 270 downregulated genes and 409 upregulated genes. DEGs were primarily enriched in immune response and chemokine signaling pathways, according to functional enrichment analysis of DEGs. WGCNA explored the co-expression network of the gene set and identified key modules, the blue module was selected as the key module associated with RA. Seven hub genes are identified when PPI network and WGCNA core modules are intersected. Immune infiltration analysis using CIBERSORT and ssGSEA algorithms revealed that multiple types of immune infiltration were found to be upregulated in RA tissue compared to normal tissue. Furthermore, the levels of 7 hub genes were closely related to the relative proportions of multiple immune cells in RA. The results of the qRT-PCR demonstrated that the relative expression levels of 6 hub genes (CD27, LCK, CD2, GZMB, IL7R, and IL2RG) were up-regulated in RA synovial tissue, compared with normal tissue. Simultaneously, ROC curves indicated that the above 6 hub genes had strong biomarker potential for RA (AUC >0.8). Conclusions: Through bioinformatics analysis and qRT-PCR experiment, our study ultimately discovered 6 hub genes (CD27, LCK, CD2, GZMB, IL7R, and IL2RG) that closely related to RA. These findings may provide valuable direction for future RA clinical diagnosis, treatment, and associated research.

Application of Triggered EMG in the Intraoperative Neurophysiological Monitoring of Posterior Percutaneous Endoscopic Cervical Discectomy.

OBJECTIVE: To describe the rationale and application of triggered EMG (T-EMG) in intraoperative neurophysiological monitoring, and to explore the efficacy and safety of posterior percutaneous endoscopic cervical discectomy (PPECD) in the treatment of cervical spondylotic radiculopathy (CSR) under multimodal intraoperative neurophysiological monitoring (IOM). METHODS: This study was a retrospective cohort control study. The clinical data of 74 patients with single-segment CSR from June 2015 to August 2018 were analyzed retrospectively, of whom 35 underwent IOM-assisted PPECD with triggered EMG (T-EMG group), while 39 were subjected to IOM-assisted PPECD alone (IOM group). Operation time, hospital stay, and complications were recorded for both groups. The curative effect was evaluated according to the Visual Analog Scale (VAS) of neck and arm pain, Japanese Orthopaedic Association (JOA) score, and modified MacNab scale. RESULTS: Operations were successful and all patients were followed up for at least 24 (average 31.77 ± 9.51) months with no patient lost to follow-up. No significant difference was found in preoperative baseline data between the T-EMG and the IOM group (P > 0.05). Also, no significant difference was found in the operation time between the T-EMG (108.29 ± 11.44 min) and the IOM (110.13 ± 12.70 min) (P > 0.05) group, but the difference in hospital stay (T-EMG: 5.66 ± 0.99 days; IOM: 7.10 ± 1.43 days) was statistically significant (P < 0.05). The VAS for the neck and upper limbs in the two groups at 1 month post-operation (T-EMG: 2.09 ± 1.07, 2.26 ± 0.92; IOM:2.18 ± 1.05, 2.31 ± 0.77) and the last follow-up (T-EMG: 0.83 ± 0.62, 0.86 ± 0.55; IOM: 0.90 ± 0.50, 0.87 ± 0.61) were significantly different from the preoperative scores (T-EMG: 6.14 ± 1.09, 7.17 ± 1.04; IOM: 6.18 ± 1.28, 7.15 ± 1.23) (P < 0.05). However, no significant difference was found between the two groups (P > 0.05). The 1-month postoperative JOA scores for the two groups (12.69 ± 0.76; 12.59 ± 0.82) and those at the last follow-up (14.60 ± 0.77; 14.36 ± 0.78) were significantly different from the preoperative scores (11.09 ± 0.98; 11.05 ± 0.89) (P < 0.05), but the difference between the two groups was not significant (P > 0.05). One patient in the T-EMG group developed a transient aggravation of symptoms on the first day after surgery. In the IOM group, three patients had intraoperative cerebrospinal fluid leakage, and symptoms of C5 nerve root paralysis were presented in four patients following surgery. Compared with the IOM group, the T-EMG group had fewer complications (1/35; 7/39, P < 0.05). At the last follow-up, the modified MacNab criteria were 91.43% (32/35) and 89.7% (35/39) for the T-EMG group and IOM group, respectively. CONCLUSIONS: Triggered EMG prevents the occurrence of neurological complications, which not only aids PPECD for CSR treatment in achieving satisfactory results, but also reduces average hospital stay and complication rates.

α-Mangostin promotes apoptosis of human rheumatoid arthritis fibroblast-like synoviocytes by reactive oxygen species-dependent activation of ERK1/2 mitogen-activated protein kinase.

α-Mangostin (α-M) is a commonly used traditional medicine with various biological and pharmacological activities. Our study aimed to explore the effects and mechanism of α-M in regulating apoptosis of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). α-M of 10 to 100 µM was used to treat RA-FLS for 24 hours, followed by measuring cell viability and apoptosis. The involvement of reactive oxygen species (ROS) and mitogen-activated protein kinases was detected. Treatment of α-M promoted apoptosis and reduced viability of RA-FLS in a dose-dependent manner. The mitochondrial membrane potential in RA-FLS was remarkably reduced by α-M treatment, accompanied by the cytochrome c accumulation in the cytosol and increased activities of caspase-3 and caspase-9. Moreover, we found that α-M treatment promoted ROS production and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. The proapoptotic activity of α-M in RA-FLS was markedly reversed by the co-induction with the ERK1/2 inhibitor LY3214996 or ROS scavenger N-acetyl-l-cysteine. In conclusion, our studies found that α-M had remarkable proapoptotic activities in RA-FLS, which is regulated by the induction of ROS accumulation and ERK1/2 phosphorylation. α-M may thus have potential therapeutic effects for rheumatoid arthritis.

MRI analysis of tibial PCL attachment in a large population of adult patients: reference data for anatomic PCL reconstruction.

BACKGROUND: Consistent reference data used for anatomic posterior cruciate ligament (PCL) reconstruction is not well defined. Quantitative guidelines defining the location of PCL attachment would aid in performing anatomic PCL reconstruction. The purpose was to characterize anatomic parameters of the PCL tibial attachment based on magnetic resonance imaging (MRI) in a large population of adult knees. METHODS: The PCL tibial attachment site was examined in 736 adult knees with an intact PCL using 3.0-T proton density-weighted sagittal MRI. The outcomes measured were the anterior-posterior diameter (APD) of the tibial plateau; angle between the tibial plateau and the posterior tibial 'shelf' (the slope where the PCL tibial attachment site was) (PTS); length of the PTS; proximal, central, and distal PCL attachment positions as well as the width of the PCL attachment site; and vertical dimension of the PCL attachment site inferior from the tibial plateau. RESULTS: The average APD of the tibia plateau was 33.6 ± 3.5 mm, yielding significant differences between males (35.5 ± 3.0 mm) and females (31.6 ± 2.7 mm), P <.05, and there was a significantly decreasing trend with increasing age in males (P <.05). Mean angle between the tibial plateau and the PTS was 122.4° ± 8.1°, and subgroup analysis showed that the young group had a differently smaller angle (120.9° ± 7.5°) than the middle-aged (123.7° ± 8.2°) and the old (123.4° ± 7.7°) in males population, while there were no significant differences between sexes (P >.05). The proximal, central positions and width of the PCL attachment site were 13.4 ± 3.0 mm, 17.8 ± 3.0 mm and 9.6 ± 2.4 mm along the PTS, with significant differences between males and females (P <.05), and accounted for 60.0 % ± 9.1 %, 80.0 % ± 4.6 % and 43.3 % ± 9.7 % of the PTS respectively, with no significant differences between sexes and among age groups (all P >.05). CONCLUSIONS: This study provides reference data of the tibial PCL attachment based on MRI in the sagittal orientation. In analysis of retrospective data from a large population of adult patients, the quantitative values can be used as references to define the inserted angle and depth of the drill guide, and the exact position and size of the tibial PCL tunnel for performing arthroscopic anatomic PCL reconstruction.

Structural characterization and stimulating effect on osteoblast differentiation of a purified heteropolysaccharide isolated from Hedysarum polybotrys.

Radix Hedysari polysaccharides (HPS) is the principal active fraction of Radix Hedysari (RH). The information about HPS3d, the main fraction of HPS3, and its effect on bone is still unknown. In the present study, the purified HPS3d was obtained by anion-exchange column. It consisted of 94.38% polysaccharide, 3.40% protein and 13.30% uronic acid. The molecular weight was measured to be 84.6kDa. The backbone consisted of galactopyranose and galacturonopyranose, and the side chains were composed of glucopyranose, rhamnopyranose and arabinofuranose. The FT-IR and elemental analysis showed that HPS3d was the sulfated polysaccharide. HPS3d upregulated alkaline phosphatase (ALP) activity and the expression of other osteogenic marker genes in osteoblast. In addition, HPS3d increased the expression and transcriptional activity of Runt-related transcription factor 2 (Runx-2) and Osterix, the two master genes of osteoblast differentiation. These findings suggest that HPS3d stimulates osteoblast differentiation by activation of Runx-2 and Osterix.

Cyclic fluid shear stress promotes osteoblastic cells proliferation through ERK5 signaling pathway.

Fluid shear stress plays an important role in bone remodeling, however, the mechanism of mechanotransduction in bone tissue remains unclear. Recently, ERK5 has been found to be involved in multiple cellular processes. This study was designed to investigate the potential involvement of ERK5 in the proliferative response of osteoblastic cells to cyclic fluid shear stress. We reported here that cyclic fluid shear stress promoted ERK5 phosphorylation in MC3T3-E1 cells. Inhibition of ERK5 phosphorylation attenuated the increased expression of AP-1 and cyclin D1 and cell proliferation induced by cyclic fluid flow, but promoted p-16 expression. Further more, we found that cyclic fluid shear stress was a better stimuli for ERK5 activation and cyclin D1 expression compared with continuous fluid shear stress. Moreover, the pharmacological ERK5 inhibitor, BIX02189, which inhibited ERK5 phosphorylation in a time-dependent manner and the suppression lasted for at least 4 h. Taken together, we demonstrate that ERK5/AP-1/cyclin D1 pathway is involved in the mechanism of osteoblasts proliferation induced by cyclic fluid shear stress, which is superior in promoting cellular proliferation compared with continuous fluid shear stress.