Osteoarthritis (OA) is driven in part by chondrocyte senescence, mitochondrial dysfunction and chronic inflammation, yet disease-modifying therapies that directly target these ageing mechanisms are lacking. Here, we identify the grape-seed-derived polyphenol procyanidin C1 (PCC1) as a senotherapeutic candidate that preserves chondrocyte mitochondrial homoeostasis and attenuates senescence via activation of the Nrf2/HO-1 axis. In tert-butyl hydroperoxide-induced senescent chondrocytes, PCC1 reduced senescence-associated β-galactosidase activity, dampened secretion of senescence-associated secretory phenotype factors, restored mitochondrial membrane potential and dynamics, limited extracellular matrix degradation and suppressed NF-κB activation. Network pharmacology and transcriptomic analyses converged on Nrf2 as a potential molecular target of PCC1, which we validated using cellular thermal shift and drug affinity responsive target stability assays. Genetic silencing of Nrf2 abrogated PCC1-mediated protection in vitro, confirming that Nrf2 is required for the anti-senescent and mitochondrial effects of PCC1. In a surgically induced anterior cruciate ligament transection model of OA, PCC1 administration reduced cartilage erosion, preserved matrix organisation and subchondral bone structure, and mitigated synovial inflammation in Nrf2-sufficient mice, whereas Nrf2 deficiency abolished these benefits. Together, these findings establish a mechanistic link between PCC1, Nrf2/HO-1 activation and chondrocyte mitochondrial homoeostasis in osteoarthritis, and support Nrf2-directed senotherapies as a regenerative strategy to slow OA progression.

Several researchers have focused on understanding the pathogenesis and treatment strategies for osteoarthritis (OA). Gastrodin (GAS) is a potential anti-inflammatory agent. In this study, we constructed an in vitro OA chondrocyte model by treating chondrocytes with IL-1β. Next, we determined the expression of aging-related markers and mitochondrial functions in chondrocytes treated with GAS. Further, we constructed a "drug-component-target-pathway-disease" interactive network and determined the effect of GAS on the functions and pathways related to OA. Finally, we constructed the OA rat model by removing the medial meniscus of the right knee and transection of the anterior cruciate ligament. The results revealed that GAS reduced senescence and improved mitochondrial functions in OA chondrocytes. We used network pharmacology and bioinformatics to screen for key molecules Sirt3 and the PI3K-AKT pathway involved in regulating the effect of GAS on OA. Further studies showed an increase in SIRT3 expression and reduced chondrocyte aging, mitochondrial damage, and the phosphorylation of the PI3K-AKT pathway. The results showed that GAS ameliorates pathological changes related to aging, increases SIRT3 expression, and protects the ECM in the OA rat model. These results were consistent with our bioinformatics results and previous studies. In summary, GAS slows down the aging of chondrocytes and mitochondrial damage in OA by regulating the phosphorylation of the PI3K-AKT pathway via SIRT3.

Abstract The centrosome, a vital component in mitosis in eukaryotes, plays a pivotal role in cancer progression by influencing the proliferation and differentiation of malignant cells, making it a significant therapeutic target. We collected genes associated with centrosomes from existing literature and established a prognostic model for 85 osteosarcoma patients from the TARGET database. Genes associated with prognosis were identified through univariate Cox regression. We then mitigated overfitting by addressing collinearity using LASSO regression. Ultimately, a set of five genes was selected for the model through multivariable Cox regression. Model performance was assessed using ROC curves, which yielded a training set AUC of 0.965 and a validation set AUC of 0.770, indicating satisfactory model performance. We further identified genes with differential expression in high and low-risk groups and conducted functional enrichment analysis using KEGG, GO, Progeny, GSVA, and GSEA. Results revealed significant variances in various immune-related pathways between high and low-risk cohorts. Analysis of the immune microenvironment using ssGSEA and ESTIMATE indicated that individuals with unfavorable prognoses had lower immune scores, stromal scores, and ESTIMATE scores, coupled with higher tumor purity. This suggests that high-risk individuals have compromised immune microenvironments, potentially contributing to their unfavorable prognoses. Additionally, drug sensitivity and molecular docking analysis revealed increased responsiveness to paclitaxel in high-risk individuals, implying its prognostic value. The JTB-encoded protein exhibited a negative binding energy of -5.5 kcal/mol when interacting with paclitaxel, indicating its potential to enhance the patient's immune microenvironment. This framework enables patient prognosis prediction and sheds light on paclitaxel's mechanism in osteosarcoma treatment, facilitating personalized treatment approaches.

OBJECTIVE:Osteoarthritis (OA) is characterized by cartilage degeneration and inflammation. Procyanidin B2 (PCB2), a natural flavonoid compound, exhibits potential anti-inflammatory and anti-oxidative effects against several diseases. However, its curative effects on OA remain unclear. PURPOSE:Herein, we explored the anti-arthritic effects of PCB2 on OA onset and progress and its potential mechanism. METHODS:CCK-8 assays and EdU staining were used to assess the cytotoxic effects and cell proliferation activity of PCB2. Flow cytometry was used to detect apoptosis in chondrocytes. ELISA, qPCR, and western blotting, were applied to explore the expression of apoptosis and senescence-associated secretion phenotype (SASP) factors. The Nrf2/NF-κB signaling cascade was explored using immunofluorescence and western blotting. Additionally, we silenced the Nrf2 gene using siRNAs to verify its function in PCB2 regulation of senescence and apoptosis phenotypes. Safranin O-Fast Green (SO) and immunohistochemical staining were used to explore the effects of PCB2 on OA model rats. RESULTS:PCB2 dampened interleukin (IL)-1β-triggered expression of SASP factors in vitro. Additionally, PCB2 diminished IL-1β-triggered destruction of the extracellular matrix (ECM) via downregulating the expression of MMPs, while upregulating the expression of collagen II and aggrecan. In addition, PCB2 treatment reduced IL-1β-induced apoptosis of chondrocytes. Mechanistically, PCB2 could attenuated chondrocyte senescence in vitro via the Nrf2/NF-κB pathway. Moreover, PCB2 exhibited anti-apoptotic properties via the Nrf2/BAX/Bcl-2 pathway. PCB2 alleviated knee cartilage degeneration in an OA rat model. CONCLUSIONS:Our results suggest that PCB2 may be used as a therapeutic agent for OA.

Background Steroid-induced osteonecrosis of the femoral head (SONFH) is a disorder that causes severe disability in patients and has a high incidence worldwide. Although glucocorticoid (GC)-induced apoptosis of osteoblasts is an important cytological basis of SONFH, the detailed mechanism underlying SONFH pathogenesis remains elusive. PI3K/AKT signaling pathway was reported to involve in cell survival and apoptosis. Objective We explored the role of PI3K/AKT/FOXO1 signaling pathway and its downstream targets during glucocorticoid -induced osteonecrosis of the femoral head. Methods We obtained gene expression profile of osteoblasts subjected to dexamethasone (Dex) treatment from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were screened out and functional enrichment analysis were conducted by bioinformatics analysis. In vitro , we analyzed Dex-induced apoptosis in MC3T3-E1 cells and explored the role of PI3K/AKT/FOXO1 signaling pathway in this phenomenon by employing siRNA-FOXO1 and IGF-1(PI3K/AKT agonist). Finally, we verified our results in a rat model of SONFH. Results In Dex-treated osteoblasts, DEGs were mainly enriched in the FOXO signaling pathway. Dex inhibited MC3T3-E1 cell viability in a dose-dependent effect and induced apoptosis by increasing the expression levels of FOXO1, Bax, cleaved-Caspase-3, and cleaved-Caspase-9, while reducing the expression of Bcl-2. Notably, these results were reversed by siRNA-FOXO1 treatment. Dex inhibited PI3K/AKT signaling pathway, upregulated FOXO1 expression and increased FOXO1 nuclear translocation, which were reversed by IGF-1. Compared to normal rats, the femoral head of SONFH showed increased expression of FOXO1, increased number of apoptotic cells, and empty osteocytic lacunas, as well as decreased bone tissue content and femoral head integrity. Significantly, the effects of GC-induced SONFH were alleviated following IGF-1 treatment. Conclusion Dex induces osteoblast apoptosis via the PI3K/AKT/FOXO1 signaling pathway. Our research offers new insights into the underlying molecular mechanisms of glucocorticoid-induced osteonecrosis in SONFH and proposes FOXO1 as a therapeutic target for this disease.

Sphingolipid metabolism (SM) fuels tumorigenesis and the malignant progression of osteosarcoma (OS), which leads to an unfavorable prognosis. Elucidating the molecular mechanisms underlying SM in osteosarcoma and developing a SM-based prognostic signature could be beneficial in the clinical setting. This study included 88 frozen OS samples to recognize the vital SM-relevant genes in the development of OS utilizing univariate Cox regression. The Least Absolute Shrinkage and Selection Operator (LASSO) regression analysis was conducted on the SM- relevant genes to minimize the risk of overfitting. The prognostic signature was generate utilizing the multivariable Cox regression analysis and was verified in the validation cohort. Moreover, cellular and molecular mechanisms associated with SM have an unfavorable prognosis for OS patients and have been widely studied. Resultantly, an SM-based prognostic risk model was established according to critical prognostic genes (CBS, GLB1, and HACD1), which had an excellent ability to predict the prognosis of OS patients (AUC for the train cohort was 0.887 and AUC for validation cohort was 0.737). The high-risk OS patients identified based on this prognostic signature had significantly poor immune microenvironment, indicated by significantly low immune score (mean=216.290 ± 662.463), reduced infiltrations of 25 immune cells, including NK cells (LogFC= -0.3597), CD8+T cells ((LogFC=-0.2346), Cytolytic activity ((LogFC=-0.1998), etc. The immunosuppressive microenvironment could be due to dysregulated SM of glycolipids. Further, a nomogram was constructed by integrating the SM-based prognostic signature and clinical paraments to facilitate clinical application. The nomogram could accurately predict the prognosis of OS invalids. Collectively, this study clarified the function of SM in the development of OS and helped develop a tool for risk stratification based on SM-related genes with application in clinical settings. The results of our study will aid in identifying high-risk patients and provide individualized treatments.

Objective:To investigate the effect of hypoxia preconditioning on the cell function of bone marrow mesenchymal stem cells (BMSCs) and the preventive and therapeutic effects of hypoxia preconditioning BMSCs in steroid-induced femoral head necrosis (SONFH) in rats after intravenous transplantation.Methods:BMSCs were isolated from the bone marrow cavity of the femur of rats. Flow cytometry identification of surface antigens (CD34, CD44, CD45 and CD90). Evaluation of cell function of BMSCs in normoxia group (No) and hypoxia group (Hp) by cell counting kit-8 (CCK-8) analysis, scratch test, alizarin red staining and alkaline phosphatase staining. A total of 32 SD rats were randomly divided into four groups ( n=8): (a) a control group, (b) a model group, (c) a normoxic group, and(d)a hypoxic group. Model group used lipopolysaccharide [2 mg/(kg·d)] combined with methylprednisolone [20 mg/(kg·d)] to construct the SONFH model. After SONFH model was constructed, the normoxic-rat bone marrow mesenchymal stem cells (rBMSCs) group and hypoxic-rBMSCs group were injected with 10 7 normoxic or hypoxia preconditioned rBMSCs through the tail vein. All rats were sacrificed and the femoral heads were collected and evaluated by Micro CT. After femoral heads decalcification, the slices were stained with hematoxylin-eosin to observe and count the incidence of osteonecrosis. Results:The results of flow cytometric identification of rBMSCs showed that rBMSCs expressed high expression of CD44 (100%) and CD90 (99.4%), and low expression of CD34 (0.48%) and CD45 (0.39%). The CCK-8 analysis showed that hypoxia preconditioning can enhance the proliferation ability of rBMSCs ( t=7.199, P<0.05). The scratch test analysis showed that compared with No group, Hp group enhanced rBMSCs′ migration ability at 12 h ( t=2.462, P<0.05), 24 h ( t=2.471, P<0.05) and 36 h ( t=3.434, P<0.01). The alizarin red staining showed hypoxia precondition significantly enhanced the mineralization ability of rBMSCs in vitro ( t=6.722, P<0.05). The alkaline phosphatase (ALP) staining showed that hypoxia pretreatment can significantly enhance the osteogenic ability of rBMSCs in vitro ( t=13.37, P<0.05). In animal experiments, the incidence of osteonecrosis in group C, group M, group N, and group H were 0% (0/16), 87.5% (14/16), 25.0% (4/16), 12.5% (2/16). The hypoxia precondition of rBMSCs can reduce the incidence of SONFH. The empty bone lacuna rates of HE staining in group C, M, N, and H were (2.765±0.423)%, (30.817±1.203)%, (14.724±1.312)% and (8.487±1.671)%, indicated the incidence of empty bone lacuna in the rBMSCs treatment groups were significantly reduced ( F=286.3, P<0.05); The Micro CT scan measurement showed that BV/TV, Tb. Th and Tb. N were significantly increased after rBMSCs treatment ( F=197.6, 290.7, 68.7, P<0.05), while Tb. Sp was significantly reduced ( F=123.8, P<0.05). It is proved that intravenous transplantation of rBMSCs can improve the microstructure of the femoral head, and the improvement effect of rBMSCs after hypoxia preconditioning is more significant ( P<0.01). Conclusion:Hypoxic precondition can significantly improve the proliferation, migration and osteogenic ability of rBMSCs in vitro, which is beneficial to enhance the preventive and therapeutic effects of rBMSCs on SONFH in rats.

Objective:To observe the effects of β-elemonic acid (β-EA) on proliferation, migration and apoptosis of rat chondrocytes induced by interleukin-1β (IL-1β).Methods:Chondrocytes of primary rats were extracted and identified by toluidine blue staining, and divided into control group, IL-1β group, low and high dose β-EA group by random number table. Cell counting kit-8 (CCK-8) assay was used to detect the cell viability. The proliferation of Chondrocytes was detected by 5-Ethynyl-2′-deoxyuridine (EdU) assay. Transwell experiment was used to evaluate the cell migration. The apoptosis of chondrocytes was detected by flow cytometry. Western blotting was used to detect the protein levels of apoptosisrelated genes cysteinyl aspartate-specific protease (Caspase)-3, Caspase-9, B cell lymphoma/leukemia-2 associated X protein (bax) and B cell lymphoma/leukemia-2 (bcl-2) in chondrocytes. One-way analysis of variance was used for data processing.Results:After treating normal chondrocytes with different concentrations of β-EA, the cell viability in control group, β-EA low-dose group and β-EA high-dose group was 97%, 97.36% and 95.80% respectively. The viability of chondrocytes in control group, IL-1β group, β-EA low-dose group and β-EA high-dose group was 97.22%, 49.57%, 60.20% and 79.95% respectively. The percentage of proliferating cells was 28.38%, 9.16%, 13.62% and 24.56% respectively. The number of migrating cells was 354.67, 86.67, 136.67 and 192.67 respectively. The apoptosis rates were 12.18%, 36.74%, 24.41% and 16.98% respectively. These results showed that β-EA had no significant toxicity to normal chondrocytes ( F=0.438, P>0.05) and promoted the recovery of inhibited chondrocytes induced by IL-1β ( F=379.200, P<0.05). As compared with the control group, the number of proliferating and migrating cells in IL-1β group was significantly decreased ( F=84.920, 117.100, P< 0.05). As compared with IL-1β group, the proliferation and migration of cells in low-dose and high-dose β-EA groups were significantly increased ( F=84.920, 117.100, P<0.05). IL-1β significantly induced the apoptosis of rat chondrocytes ( F=169.600, P<0.05). After treatment with high and low doses of β-EA, the apoptosis of rat chondrocytes induced by IL-1β was significantly inhibited ( F=169.600, P<0.05). The expression of apoptosis-related proteins including Caspase-3, Caspase-9 and bax was significantly decreased ( F=111.600, 50.830, 132.500, P<0.05), and that of bcl-2 was significantly increased in the β-EA treated group ( F=59.850, P<0.05). Conclusion:β-EA can inhibit IL-1β-mediated chondrocytes apoptosis, promotes chondrocytes proliferation and migration and protects chondrocytes to some extent.

Objective:To investigate the role of Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signal transduction pathway in dexamethasone induced osteoblasts MC3T3-E1 apoptosis.Methods:The osteoblasts MC3T3-E1 were divided into three groups: control group, DEX group and DEX+ AG490 group. The control group was treated with routine medium. DEX group was treated with 200 μmol/L DEX to culture the cells. DEX+ AG490 group was treated with 50 μmol/L AG490 in advance and then treated with 200 μmol/L DEX to culture the cells. Cell counting kit-8 (CCK-8)assay was used to detect the cell viability. The apoptosis of osteoblasts was detected by flow cytometry. Western blotting was used to detect the protein levels of pathways related genes and apoptosis related genes. Univariate anova was used to compare the mean values. T test was used for comparison between groups. Results:The cell viability of control group, DEX group and DEX+ 12.5, 25, 50, 75 μmol/L AG490 group were (97.74±1.45)%, (52.05±5.50)%, (54.98±3.77)%, (70.99±4.15)%, (81.53±6.43)% and (79.16±7.35)% respectively. These results showed that DEX significantly decreased the cell viability of osteoblasts MC3T3-E1 ( t=11.364, P<0.001).50 μmol/L JAK2/STAT3 pathway inhibitor (AG490) significantly inhibited the decreased cell viability of osteoblasts MC3T3-E1 induced by DEX ( t=4.928, P<0.01). The apoptosis rates of the control group, DEX group and DEX+ AG490 group were (6.067±0.545)%, (26.233±2.631)% and (8.463±1.179)% respectively. These results showed that DEX significantly induced apoptosis of osteoblasts MC3T3-E1 ( t=12.999, P<0.001). After treated with AG490, the apoptosis of osteoblasts MC3T3-E1 induced by DEX was significantly inhibited ( t=10.675, P<0.001). Western blotting showed the expression of p-JAK2 protein in control group, DEX group and DEX+ AG490 group was 1.000±0.323, 2.839±0.640 and 0.286±0.068, that of p-STAT3 protein was 1.000±0.245, 3.471±0.157, 0.618±0.078, that of bax protein was 1.000±0.083, 6.571±0.405, 3.048±0.905, that of bcl-2 protein was 1.000±0.086, 0.207±0.040, 0.563±0.083, and that of cleaved Caspase-3 protein was 1.000±0.192, 5.685±0.699, 3.411±0.247. Compared with control group, the expression of p-Jak2, p-STAT3, cleaved Caspase-3, and bax were significantly increased, and that of bcl-2 was significantly decreased in DEX group ( t=4.429, 14.912, 11.100, 23.561, 14.565, P<0.05). Compared with DEX group, the expression of p-JAK2, p-STAT3, cleaved Caspase-3, and bax were significantly decreased, and that of bcl-2 significantly was increased in AG490+ DEX group ( t=6.860, 28.404, 5.262, 6.185, 6.721, P<0.01). Conclusion:DEX regulate cleaved Caspase-3, bax, and bcl-2 expression through JAK2/STAT3 signal transduction pathway, inducing apoptosis of osteoblasts MC3T3-E1.