CircSPI1_005 ameliorates osteoarthritis by sponging miR-370-3p to regulate the expression of MAP3K9.

BACKGROUND: Osteoarthritis (OA), caused by the destruction of joint cartilage, is the most prevalent form of arthritis, causing pain and stiffness in joints among millions of patients worldwide. Increasing evidence suggests that non-coding RNAs, including circular RNAs, play important roles in the pathogenesis of OA, but the precise signaling pathway is still unclear. METHODS: To study OA, we established a mouse model by the destabilized medial meniscus (DMM) surgery and used IL-1ß stimulated human cell line C28/I2 as an in vitro study. To further study the role of circSPI1_005 in regulating cell proliferation and apoptosis, EdU staining and FACS-based (fluorescence-activated cell sorting) apoptosis examination were performed after the manipulation of the expression of circSPI1_005. Also, bioinformatics predictions were conducted to analyze the downstream microRNAs of circSPI1_005 and the protein regulated by circSPI1_005. The luciferase assay and the RNA immunoprecipitation (RIP) assay were used to confirm the binding between circSPI1_005 and the predicted microRNA. To verify the role of circSPI1_005 in regulating OA in vivo, we also over-expressed circSPI1_005 by injecting AAV into previously injured knees to improve the OA symptoms. RESULTS: In this study, we found that circSPI1_005 was significantly down-regulated in IL-1ß treated chondrocyte cell lines and cartilage tissues of the OA mouse model. Overexpression of circSPI1_005 ameliorated OA by increasing proliferation and inhibiting apoptosis, and knockdown of circSPI1_005 in chondrocytes mimicked OA phenotypes. Bioinformatics study showed circSPI1_005 could sponge to miR-370-3p, and mechanistic studies confirmed the functional binding between circSPI1_005 and miR-370-3p. Furthermore, we conducted a TargetScan analysis and found that MAP3K9 (mitogen-activated protein kinase kinase kinase 9) could be the downstream protein effector. The expression level of MAP3K9 was regulated by miR-370-3p and overexpression of MAP3K9 could efficiently ameliorate OA. Also, we over-expressed circSPI1_005 in vivo and found that the cartilage surface in the OA mouse model was improved with overexpression of circSPI1_005. CONCLUSIONS: Collectively, circSPI1_005 could sponge to miR-370-3p to regulate the expression of MAP3K9, ameliorating the progression of osteoarthritis.

Circular RNA circANKRD36 regulates Casz1 by targeting miR-599 to prevent osteoarthritis chondrocyte apoptosis and inflammation.

Osteoarthritis (OA) is an ageing-related disease characterized by articular cartilage degradation and joint inflammation. circRNA has been known to involve in the regulation of multiple inflammatory diseases including OA. However, the mechanism underlying how circRNA regulates OA remains to be elucidated. Here, we report circANKRD36 prevents OA chondrocyte apoptosis and inflammation by targeting miR-599, which specifically degrades Casz1. We performed circRNA sequencing in normal and OA tissues and found the expression of circANKRD36 is decreased in OA tissues. circANKRD36 is also reduced in IL-1ß-treated human chondrocytes. FACS analysis and Western blot showed that the knockdown of circANKRD36 promotes the apoptosis and inflammation of chondrocytes in IL-1ß stress. We then found miR-599 to be the target of circANKRD36 and correlate well with circANKRD36 both in vitro and in vivo. By database analysis and luciferase assay, Casz1 was found to be the direct target of miR-599. Casz1 helps to prevent apoptosis and inflammation of chondrocytes in response to IL-1ß. In conclusion, our results proved circANKRD36 sponge miR-599 to up-regulate the expression of Casz1 and thus prevent apoptosis and inflammation in OA.

Hsa-let-7g promotes osteosarcoma by reducing HOXB1 to activate NF-kB pathway.

MicroRNA (miRNA) is known to be involved in regulating the proliferation, migration and apoptosis of cancer cells in osteosarcoma. In this study, We aim to explore the expression of hsa-let-7 g and its role in pathogenesis of osteosarcoma. By analyzing clinical data. We found high expression of hsa-let-7 g in patients with osteosarcoma. The patients with higher expression of hsa-let-7 g showed poorer prognosis and lower survival rate. After downregulation of hsa-let-7 g in cell model and animal model, we found that with downregulation of hsa-let-7 g, the proliferation of osteosarcoma cells was significantly reduced, the level of migration and invasion was down-regulated, the cell cycle was inhibited, and cell apoptosis was increased. Through Dual Luciferase Reporter, immunohistochemistry, western blot and other experiments, it was found that hsa-let-7 g down-regulated HOXB1 gene and activated NF-kB pathway to promote the development of osteosarcoma. In conclusion, hsa-let-7 g is highly expressed in osteosarcoma tissues, and high expression of hsa-let-7 g can promote the occurrence of osteosarcoma by down-regulating HOXB1 and activating NF-kB pathway.

Local intra­articular injection of vascular endothelial growth factor accelerates articular cartilage degeneration in rat osteoarthritis model.

In the pathophysiology of osteoarthritis (OA), articular cartilage degeneration exhibits a significant role. Vascular endothelial growth factor (VEGF) is considered to be an effective angiogenic factor and a crucial regulator of articular cartilage degeneration in the development of OA. Therefore, the present study aimed to investigate the underlying influences of exogenous VEGF on articular cartilage degeneration in OA model rat. A total of 24 male Sprague­Dawley rats were randomly allocated into 3 groups. In the normal saline (NS) and VEGF groups, animals received bilateral anterior cruciate ligament (ACL) transection to establish the OA model; at 4 weeks post­surgery, the rats received local intra­articular injections of 100 µl NS or VEGF solution, respectively, every week for 4 weeks. The Control group received neither surgery nor injections. All animals were sacrificed at 12 weeks following surgery. Prominent cartilage degeneration was observed in rats in the NS­ and VEGF­injected groups. The extent and the grade of cartilage damage in the VEGF­injected group were notably more severe compared with those in the NS­treated group. Western blotting results demonstrated that the expression levels of aggrecan and type II collagen were significantly reduced in OA model rats that were treated with VEGF. In addition, the expression levels of matrix metalloproteinase (MMP)­3, MMP­9, MMP­13, a disintegrin and metalloproteinase with thrombospondin motifs (a disintegrin and metalloproteinase; ADAMTS)­4, ­5 and ­12, type III collagen and transforming growth factor­ß1 were significantly increased following VEGF administration. Results from the present study indicated that VEGF may exhibit a promoting role in the development of OA by destroying articular cartilage matrix.

Sesamin Protects the Femoral Head From Osteonecrosis by Inhibiting ROS-Induced Osteoblast Apoptosis in Rat Model.

Glucocorticoids intake has become the most common pathogenic factor for osteonecrosis of the femoral head (ONFH). Annually, tens of millions of patients suffer from pain related to ONFH. Researchers have proposed several underlying mechanisms of ONFH, including osteocyte apoptosis, cell differentiation disorder, and angiogenesis hindrance. Sesamin, isolated from Sesamum indicum seeds, was reported could affect osteocyte inflammation and differentiation in osteoarthritis and osteoporosis. We investigated the underlying influence of sesamin on ONFH rat model. Fifteen male Sprague-Dawley rats were randomly divided into three groups. The ONFH model group only received the methylprednisolone (MPS) and lipopolysaccharide (LPS) injection to promote the development of ONFH. The sesamin treatment group was injected with sesamin, MPS, and LPS. The control group was untreated. Rats from above groups were sacrificed 4 weeks later. The effect of sesamin on ONFH rats was validated by H&E staining. TUNEL staining showed that femoral head necrosis was attenuated by sesamin. Furthermore, the phosphorylation of Akt was increased and the downstream cellular apoptosis signal pathway was inhibited. Intracellular ROS level was decreased after sesamin treatment. In conclusion, our findings suggest that sesamin protects the femoral head from osteonecrosis by inhibiting ROS-induced osteoblast apoptosis.

Comparison study of the effect of fusion and non-fusion fixation on the movement of injured lumbar spine / 中国组织工程研究

BACKGROUND: Degenerative change in lumbar spine is the natural physiological and pathological process with age increasing, which seriously affects the patients' quality of life. Conventional surgical treatments may bring about some complications. Interspinous stabilization Coflex demonstrates good clinical outcomes, while there is lack of experimental evidence.OBJECTIVE: To investigate the effect of fixation methods on the movement of lumbar vertebrae, and to provide reference for clinical operation from the aspect of biomechanics.METHODS: Five fresh specimens of pig cadaveric lumbar spine were selected and divided into five groups: blank control group (group A), injury group (group B), L3-L4 Coflex fixed (group C), L3-L4 Coflex fixed plus decompression (group D),and L3-L4 intervertebral fusion fixed (group E). The biomechanical performance of the five lumbar specimens was tested in six loading directions of flexion/extension, left/right lateral bending, and left/right axial rotation driven by a robot system.The motion range of the operation segments (L3-L4) and the whole lumbar spine was observed by a multi-directional torque sensor combined with a three-dimensional motion capture system. Finally, the influence of lumber fixation methods on the spinal movement was explored.RESULTS AND CONCLUSION: (1) For the range of three-dimensional angle at the operation segments (L3-L4), it was found that groups C and D were similar to group A, and groups B and E were quite different from group A. (2) Group E could restore the motion range of the injured segments, but the motion angle was much smaller than that in the groups C and D. (3) These results indicate that the motion angle of the injured spine treated with Coflex can restore to the normal state, with long-term stability. For unstable lumbar spine, interspinous Coflex is better than transforaminal lumbar interbody fusion, which can induce similar biomechanical properties to normal lumbar spine after operation.

The Relationship between HIF-2α and VEGF with Radiographic Severity in the Primary Osteoarthritic Knee

PURPOSE: The aim of this study was to determine the relationship of hypoxia-inducible factor-2 (HIF-2α) and vascular endothelial growth factor (VEGF) with radiographic severity in primary osteoarthritis (OA) of the knee. Expression of these two factors in cartilage samples from OA knee joints was examined at mRNA and protein levels. MATERIALS AND METHODS: Knee joints were examined using plain radiographs, and OA severity was assessed using the Kellgren and Lawrence (KL) grading system. Specimens were collected from 29 patients (31 knees) who underwent total knee replacement because of severe medial OA of the knee (KL grades 3 and 4), 16 patients who underwent knee arthroscopy (KL grade 2), and 5 patients with traumatic knees (KL grade 0). HIF-2α and VEGF expression was quantified by real-time polymerase chain reaction and western blotting. RESULTS: Cartilage degeneration correlated with the radiographic severity grade. OA severity, determined using the Mankin scale, correlated positively with the KL grade (r=0.8790, p<0.01), and HIF-2α and VEGF levels with the radiographic severity of knee OA (r=0.7001, p<0.05; r=0.6647, p<0.05). CONCLUSION: In OA cartilage, HIF-2α and VEGF mRNA and protein levels were significantly and positively correlated. The expression of both factors correlated positively with the KL grade. HIF-2α and VEGF, therefore, may serve as biochemical markers as well as potential therapeutic targets in knee OA.

Pulsed electromagnetic fields protect the balance between adipogenesis and osteogenesis on steroid-induced osteonecrosis of femoral head at the pre-collapse stage in rats.

This study was designed to investigate the effects of pulsed electromagnetic fields (PEMF) on the balance of adipogenesis and osteogenesis on steroid-induced osteonecrosis of the femoral head (OFH) in rats. Forty-two rats were divided into three groups: Steroid group (S, n = 16); Steroid + PEMF group (S + P, n = 16); and Control group (C, n = 10). For groups S and S + P, all rats were first intravenously given 10 µg/kg lipopolysaccharide on day 1, and then intramuscularly injected with 20 mg/kg methylprednisolone acetate on days 2, 3, and 4, with an interval of 24 h. After 4 weeks, the S + P group was treated with PEMF (4.5-ms square pulse, repeated at 15 Hz, with a peak of 1.2 mT) for 4 h a day for the next 8 weeks. Group S was not exposed to PEMF. Group C was chosen as the control group, without steroid use and exposure to PEMF. After 8 weeks of treatment, the histological changes, and mRNA and protein expressions of PPAR-γ2 and Runx2 were measured and analyzed. Compared with the S group, lower incidence of osteonecrosis (31% vs. 69%, P < 0.05) and empty osteocyte lacuna rate (36.16 ± 15.34 vs. 59.55 ± 21.70, P < 0.01) was observed in the S + P group. Furthermore, PEMF suppressed the expressions of PPAR-γ2 and improved the expressions of Runx2 in the femoral head (P < 0.05). All data suggest that PEMF is an effective physiotherapy in the treatment of steroid-induced ONFH, and the possible underlying mechanisms include protecting the balance between adipogenesis and osteogenesis.

PKCa Agonists Enhance the Protective Effect of Hyaluronic Acid on Nitric Oxide-Induced Apoptosis of Articular Chondrocytes in Vitro.

OBJECTIVE(S): Protein kinase C (PKCα) is involved in modulating articular chondrocytes apoptosis induced by nitric oxide (NO). Hyaluronic acid (HA) inhibits nitric oxide-induced apoptosis of articular chondrocytes by protecting PKCα, but the mechanism remains unclear. The present study was performed to investigate the effects and mechanisms of PKCα regulate protective effect of hyaluronic acid. Materials and Methods The ratio of apoptotic cell and cell viability was surveyed by PCNA and MTT assay. The expression of caspase-3 was determined by real-time PCR and western blot. RESULTS: It was showed that HA was able to reduce the nuclei fragment and PCNA expression, and NO-induced articular apoptosis blocked by HA, pretreated chondrocytes with PMA, HA significantly inhibits the activation of caspase-3 induced by NO, but pretrement with CHR, HA significantly incresed the expression of caspase-3. CONCLUSION: The results may be showed that PKCa regulate the expresion of caspase-3, which contribute to the apoptosis of chondrocytes induced by NO. PKC α agonists enhance the protective effect of hyaluronic acid on nitric oxide-induced articular chondrocytes apoptosis.

Pulsed electromagnetic fields stimulation prevents steroid-induced osteonecrosis in rats.

BACKGROUND: Pulsed electromagnetic fields (PEMF) stimulation has been used successfully to treat nonunion fractures and femoral head osteonecrosis, but relatively little is known about its effects on preventing steroid-induced osteonecrosis. The purpose of the study was to investigate the effects of PEMF stimulation on the prevention of steroid-induced osteonecrosis in rats and explore the underlying mechanisms. METHODS: Seventy-two male adult Wistar rats were divided into three groups and treated as follows. (1) PEMF stimulation group (PEMF group, n = 24): intravenously injected with lipopolysaccharide (LPS, 10 µg/kg) on day 0 and intramuscularly injected with methylprednisolone acetate (MPSL, 20 mg/kg) on days 1, 2 and 3, then subjected to PEMF stimulation 4 h per day for 1 to 8 weeks. (2) Methylprednisolone-treated group (MPSL group, n = 24): injected the same dose of LPS and MPSL as the PEMF group but without exposure to PEMF. (3) Control group (PS group, n = 24): injected 0.9% saline in the same mode at the same time points. The incidence of osteonecrosis, serum lipid levels and the mRNA and protein expression of transforming growth factor ß1 (TGF-ß1) in the proximal femur were measured 1, 2, 4 and 8 weeks after the last MPSL (or saline) injection. RESULTS: The incidence of osteonecrosis in the PEMF group (29%) was significantly lower than that observed in the MPSL group (75%), while no osteonecrosis was observed in the PS group. The serum lipid levels were significantly lower in the PEMF and PS groups than in the MPSL group. Compared with the MPSL and PS groups, the mRNA expression of TGF-ß1 increased, reaching a peak 1 week after PEMF treatment, and remained high for 4 weeks, then declined at 8 weeks, whereas the protein expression of TGF-ß1 increased, reaching a peak at 2 weeks after PEMF treatment, and remained high for 8 weeks. CONCLUSIONS: PEMF stimulation can prevent steroid-induced osteonecrosis in rats, and the underlying mechanisms involve decreased serum lipid levels and increased expression of TGF-ß1.