ObjectiveTo investigate the protective effect human umbilical cord mesenchymal stem cells (hUC-MSCs) have on Dexamethasone (Dex)-induced apoptosis in osteogenesis via the Nrf2-ARE signaling pathway.MethodsGlucocorticoid-induced osteonecrosis of the femoral head (GC-ONFH) was developed in rats through the administration of lipopolysaccharide and methylprednisolone. The incidence of femoral head necrosis, cavity notch, apoptosis of osteoblasts, and bone density were observed by HE staining, TUNEL staining, and Micro-CT. HUC-MSCs were co-cultured with mouse pre-osteoblast MC3T3-E1. The survival rate of osteoblasts was determined by CCK8, and apoptosis and ROS levels of osteoblasts were determined by flow cytometer. The viability of antioxidant enzymes SOD, GSH-Px, and CAT was analyzed by biochemistry. Nrf2 expression levels and those of its downstream proteins and apoptosis-related proteins were analyzed by Western blotting.ResultsIn rats, hUC-MSCs can reduce the rates of empty bone lacuna and osteoblast apoptosis that are induced by glucocorticoids (GCs), while reducing the incidence of GC-ONFH. hUC-MSCs can significantly improve the survival rate and antioxidant SOD, GSH-Px, and CAT activity of MC3T3-E1 cells caused by Dex, and inhibit apoptosis and oxidative stress levels. In addition, hUC-MSCs can up-regulate the expression of osteoblast antioxidant protein Nrf2 and its downstream protein HO-1, NQO-1, GCLC, GCLM, and apoptosis-related protein bcl-2, while also down-regulating the expression of apoptosis-related protein bax, cleaved caspase-3, cleaved caspase-9, and cytochrome C in MC3T3-E1 cells. hUC-MSCs improve the ability of MC3T3-E1 cells to mineralize to osteogenesis. However, the promoting effects of hUC-MSCs were abolished following the blocking of the Nrf2-ARE signaling pathway for osteoblasts.ConclusionThe results reveal that hUC-MSCs can reduce Dex-induced apoptosis in osteoblasts via the Nrf2-ARE signaling pathway.

Extracellular vesicles (EVs) secreted by human umbilical cord mesenchymal stem cells (hucMSC) have excellent therapeutic potential for many diseases. The aim of this study was to define the role of hucMSC-EVs in the prevention and treatment of steroid-induced avascular necrosis of the femoral head (SANFH). After establishing the SANFH rat model, the effects of hucMSC-EVs were assessed by measuring the microstructure of the femoral head using HE staining, micro-computed tomography (micro-CT), and TUNEL staining. The administration of hucMSC-EVs caused a significant reduction to glucocorticoids (GCs)-induced osteoblast apoptosis and empty lacuna of the femoral head, while effectively improving the microstructure. HucMSC-EVs rescued the deacti-vation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway induced by GCs, and reversed the proliferation and migration of osteoblasts inhibited by GCs. In addition, hucMSC-EVs attenuated the inhibitory effects of GCs on rat osteoblast osteogenesis, angiogenesis of endothelial cells, and prevented osteoblast apoptosis. However, the promoting effects of hucMSC-EVs were abolished following the blockade of PI3K/AKT on osteoblasts. hucMSC-EVs were found to prevent glucocorticoid-induced femoral head necrosis in rats through the PI3K/AKT pathway.

To study the influence of membrane fatty acid composition on the formation of prostanoids and hydroxy fatty acids by rat peritoneal mast cells (MC), animals were fed three different types of fatty acids: mackerel oil (MO), abundant in n - 3 fatty acids; sunflower seed oil (SO), rich in linoleic acid; and hydrogenated coconut oil (HCO), mainly containing saturated fatty acids. The presence of n - 3 fatty acids in the diet resulted in the incorporation of 20: 5(n - 3), 22: 5(n - 3) and 22: 6(n - 3) in MC phospholipids. A decrease of arachidonic acid, 20:4(n - 6), was observed in MC-phospholipids of the MO-fed animals. Furthermore, increasing the relative amounts of 18:2(n - 6) in the diet (SO group) led to an increased incorporation of linoleic acid, 18:2(n - 6) in MC phospholipids when compared to both other dietary groups. The changes in MC phospholipid fatty acid composition were (partly) reflected in the formation of prostanoids and hydroxy fatty acids upon stimulation with the calcium ionophore A23187. The decrease in arachidonic acid content in MC phospholipids of MO-fed rats resulted in a decreased formation of PGD2 when compared to both other groups. Also, the increased amounts of 18:2(n - 6) in MC phospholipids of SO-fed rats resulted in an increased formation of 9-and 13-HODE upon stimulation. The results show that modifications in the fatty acid composition of the diet influences MC membrane fatty acid composition which ultimately results in changes in prostanoid and hydroxy fatty acid synthesis by MC upon stimulation with the calcium ionophore A23187.

Background Osteoarthritis (OA) and rheumatoid arthritis (RA) are well-known cause of joint disability. Although they have shown the analogous clinical features involving chronic synovitis that progresses to cartilage and bone destruction, the pathogenesis that initiates and perpetuates synovial lesions between RA and OA remains elusive. Objective This study is aimed at identifying disease-specific hub genes, exploring immune cell infiltration, and elucidating the underlying mechanisms associated with RA and OA synovial lesion. Methods Gene expression profiles (GSE55235, GSE55457, GSE55584, and GSE12021) were selected from Gene Expression Omnibus for analysis. Differentially expressed genes (DEGs) were identified by the “LIMMA” package in Bioconductor. The DEGs were identified by Gene Ontology (GO) and KEGG pathway analysis. A protein-protein interaction network was constructed to identify candidate hub genes by using STRING and Cytoscape. Hub genes were identified by validating from GSE12021. Furthermore, we employed the CIBERSORT website to assess immune cell infiltration between OA and RA. Finally, we explored the correlation between the levels of hub genes and relative proportion of immune cells in OA and RA. Results We identified 68 DEGs which were mainly enriched in immune response and chemokine signaling pathway. Six hub genes with a cutoff of AUC > 0.80 by ROC analysis and relative expression of P < 0.05 were identified successfully. Compared with OA, the RA synovial tissues consisted of a higher proportion of 7 immune cells, whereas 4 immune cells were found in relatively lower proportion (P < 0.05). In addition, the levels of 6 hub genes were closely associated with relative proportion of 11 immune cells in OA and RA. Conclusions We used bioinformatics analysis to identify hub genes and explored immune cell infiltration of immune microenvironment in synovial tissues. Our results should offer insights into the underlying molecular mechanisms of synovial lesion and provide potential target for immune-based therapies of OA and RA.

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.

长期过度饮酒是引起酒精性股骨头坏死(ONFH)的重要原因之一.饮酒与ONFH的发生有显著的量效关系.酒精性ONFH具有高致残率,是国内外骨科领域难治性疾病之一.酒精性ONFH的发病机制与脂质代谢紊乱、氧化应激、遗传易感性等因素有关,但确切的发病机制目前仍不明确.目前治疗ONFH的方法主要包括髓芯减压、肌骨瓣移植等,但总体效果不理想,且疗效仍存在争议.近年来,利用干细胞治疗酒精性ONFH的临床研究已取得突破性进展,成为今后发展的重要方向.

Osteonecrosis of the femoral head (ONFH) is a common clinical disease with a high disability rate. Apoptosis of osteoblasts caused by high‐dose short‐term or low‐dose long‐term glucocorticoid (GC) administration is the biological basis of steroid‐induced avascular necrosis of the femoral head (SANFH). The pathogenesis of SANFH has not yet been fully elucidated, and there is currently a lack of effective clinical treatments. Here, we investigated the role of the reactive oxygen species (ROS)/JNK/c‐Jun signaling pathway in SANFH. Dexamethasone (Dex) was used to induce apoptosis in osteoblasts, and this resulted in a significant increase in levels of p‐JNK, p‐c‐Jun, Bax, caspase‐3, caspase‐9, cytochrome C, Beclin‐1, and LC3, and a decrease in levels of P62 and Bcl‐2. In addition, intracellular ROS levels were increased and mitochondrial membrane potential was decreased. Administration of 3‐MA, an autophagy inhibitor, attenuated Dex‐mediated changes in autophagy and apoptosis. A rat model of ONFH exhibited severe bone trabecular hollow bone pits along with a significant increase in femoral head cell apoptosis compared with the control group. Additionally, micro‐CT analysis showed that both bone tissue content and femoral head integrity were significantly reduced in the ONFH group. Furthermore, 3‐MA treatment decreased the effect of Dex on GC‐induced ONFH and osteoblast apoptosis in rats and could counteract microstructure destruction due to femoral head necrosis. In summary, our data suggest that GC can induce osteoblast apoptosis and autophagy through the ROS/JNK/c‐Jun signaling pathway, which contributes to ONFH.

Objective: To explore the role and functional mechanism of crocin in glucocorticoids induced osteonecrosis of the femoral head (ONFH). Methods: Primary osteoblasts from rat were stimulated with dexamethasone and treated with crocin in different doses and reactive oxygen species (ROS) inhibitor in vitro. Cell viability and cell apoptosis rate were assayed by cell counting kit-8 and flow cytometry, respectively. The level of alkaline phosphatase activity, ROS and mitochondrial membrane potential were detected by ELISA. The expression of related proteins was detected via Western blot. As for in vivo study, 30 rats were equally grouped into control group, ONFH model group (steroid-induced ONFH) and crocin treatment group. The occurrence and histopathological changes of ONFH, bone tissue cell apoptosis, and bone microstructure and loss were analyzed. Results: In vitro experiments, crocin and ROS inhibitor could reverse the reduction of cell viability, differentiation and mitochondrial membrane potential content, as well as the increase of ROS production, apoptosis rate and the expression levels of p-JNK and p-c-Jun in primary osteoblasts caused by dexamethasone. In vivo experiments, compared with rats in the control group, ONFH model rats had decreased amount of femoral head bone and the integrity of bone tissues, but aggravated apoptosis rate of femoral head cells and empty trabecular; however, crocin treatment significantly improved these microstructures of femoral head bone. Conclusion: Crocin had a favorable therapeutic effect on steroid-induced ONFH in rats by ROS/JNK/c-Jun pathway.

Nowadays, the cumulative intake of glucocorticoids has become the most common pathogenic factor for non-traumatic osteonecrosis of the femoral head (ONFH). Apoptosis of osteoblasts is considered as the main reason of ONFH at the molecular level. Glycogen synthase kinase 3β (GSK3β) is an important regulator of cellular differentiation and apoptosis pathway, which can modulate the balance between osteoblasts and osteoclasts. Several studies have reported about its function in osteoporosis, but little is known about it in osteonecrosis. In our study, lipopolysaccharide and methylprednisolone were utilized to establish a rat ONFH model. The phosphorylation of GSK3β Ser-9 was decreased in the model. Western blotting examination of β-catenin, Bcl-2, Bax and caspase-3 revealed that the osteoblasts were apoptotic. In dexamethasone (Dex)-incubated primary osteoblasts, the expression profile of GSK3β phosphorylation and apoptotic factors were consistent with those in the rat ONFH model. To further investigate the regulation of osteonecrosis caused by GSK3β, the expression and function of GSK3β were inhibited in Dex-incubated primary osteoblasts. The knockdown of GSK3β by siRNA decreased the expression of Bax and cleaved caspase-3, but increased Bcl-2 and β-catenin. On the other hand, selective inhibition of GSK3β function by LiCl counteracted the activation of caspase-3 induced by Dex. Our work is the first study about the GSK3β phosphorylation in ONFH, and provides evidence for further therapeutic methods.

OBJECTIVE:Glucocorticoids (GCs)-induced osteoblast apoptosis has been identified as an important cause of GCs related osteonecrosis of the femoral head (ONFH). Glycogen synthase kinase 3β (GSK3β) has been proved to mediate dexamethasone (Dex)-induced osteoblast apoptosis. This study aimed to investigate the underlying mechanism of GSK3β in Dex-induced osteoblast apoptosis. METHODS:Osteoblast cells were transfected with lentivirus expressing GSK3β-shRNA, and a DNA microarray was performed to analyze gene expression after Dex treatment with or without GSK3β-shRNA. Some differentially expressed genes were further validated by quantitative real-time-PCR (qRT-PCR). RESULTS:460 genes were up-regulated (at least 2-fold) with Dex treatment but down-regulated (at least 2-fold) with GSK3β-shRNA treatment. In addition, 315 genes were down-regulated (at least 2-fold) with Dex treatment but up-regulated (at least 2-fold) with GSK3β-shRNA treatment. Among these genes, the apoptosis-related genes Hoxb8, Kif18a, Dock8, Dlk1, Tnfsf14, Casq2, Bcl2l14 and mechanosensation-related gene Piezo2 were selected for further qRT-PCR analysis. 7 of 8 genes (Piezo2, Hoxb8, Kif18a, Dlk1, Tnfsf14, Casq2, Bcl2l14) showed the same tendency between gene chip results and qRT-PCR results. The microarray data also showed that apoptotic pathway, MAPK pathway, TGFβ pathway and Wnt pathway might be related to the mechanism of GSK3β in Dex-induced osteoblast apoptosis. CONCLUSION:Our findings indicate that GSK3β-shRNA treatment can alter various genes expression levels and change diverse signaling pathways involved in Dex-induced osteoblast apoptosis. Furthermore, Piezo2, Hoxb8, Kif18a, Dlk1, Tnfsf14, Casq2 and Bcl2l14 genes may play an important role in the GSK3β-mediated osteoblast apoptosis process.