Ai Guo

A low ratio of n-6/n-3 polyunsaturated fatty acids suppresses matrix metalloproteinase 13 expression and reduces adjuvant-induced arthritis in rats

Increased expression of matrix metalloproteinase 13 (MMP13) in chondrocytes contributes to the development of osteoarthritis. The hypothesis of this study was that diet with a low ratio of n-6/n-3 polyunsaturated fatty acids (PUFAs) is associated with reduced MMP13 expression in inflammatory chondrocytes in vitro and in vivo. Human chondrocytes were cultured with different ratios of linoleic acid (LA, n-6 PUFA) to α-linolenic acid (n-3 PUFA) from 1:1 to 10:1. Proliferation of chondrocytes, MMP13 protein and mRNA levels were detected, respectively. Sprague-Dawley rats (n=30) were fed diets containing different ratios of n-6/n-3 PUFA. Freunds complete adjuvant was injected to make the model of arthritis. Paw swelling rate was measured and all rats were euthanized after 6 weeks of treatment. Serum MMP13 and IL-1 were measured by enzyme-linked immunosorbent assay. Joint histological sections were stained with safranin-O Fast Green to evaluate cartilage damage. Low ratio of LA/α-linolenic acid decreased the mRNA and protein levels of MMP13 but did not affect chondrocytes proliferation. Ratios of PUFA such as 1:1 and 2:1 significantly reduced paw swelling rate, and serum MMP13 and IL-1 levels in a rat model. Histological staining showed that ratios of 1:1 and 2:1 PUFA significantly alleviated cartilage damage in adjuvant-induced arthritis. A ratio of n-6/n-3 PUFA of 1:1 showed the strongest inhibitory effect on MMP13. Our results indicate that a low ratio of n-6/n-3 PUFA at 1:1 significantly suppressed MMP13 expression both in vitro and in vivo and reduced adjuvant-induced arthritis in rats could be a means to control and reduce the symptoms of osteoarthritis.

Role of autophagy in the progression of osteoarthritis: The autophagy inhibitor, 3-methyladenine, aggravates the severity of experimental osteoarthritis

Accumulating evidence suggests that autophagy is closely related to the pathogenesis of osteoarthritis (OA). The aim of this study was to determine the changes in autophagy during the progression of OA and to elucidate the specific role of autophagy in OA. For this purpose, a cellular model of OA was generated by stimulating SW1353 cells with interleukin (IL)-1β and a rabbit model of OA was also established by an intra-articular injection of collagenase, followed by treatment with the autophagy specific inhibitor, 3-methyladenine (3-MA). Cell viability was analyzed by MTS assay, and the mRNA expression levels of matrix metalloproteinases (MMP)-13 and tissue inhibitor of metalloproteinase (TIMP)-1 were determined by RT-qPCR. Cartilage degeneration was examined under a light microscope, and autophagosome and chondrocyte degeneration was observed by transmission electron microscopy. The protein expression of Beclin-1 and light chain 3 (LC3)B was evaluated by western blot analysis and immunofluorescence staining. We found that the autophagy was enhanced during the early stages and was weakened during the late stages of experimental OA. The inhibition of autophagy by 3-MA significantly aggravated the degeneration of chondrocytes and cartilage in experimental OA. Our results thus determine the changes in autophagy during different stages of OA, as well as the role of impaired autophagy in the development of OA. Our data suggest that the regulation of autophagy may be a potential therapeutic strategy with which to attenuate OA.

Intra-articular injection of Torin 1 reduces degeneration of articular cartilage in a rabbit osteoarthritis model

Objectives Recent studies have shown that systemic injection of rapamycin can prevent the development of osteoarthritis (OA)-like changes in human chondrocytes and reduce the severity of experimental OA. However, the systemic injection of rapamycin leads to many side effects. The purpose of this study was to determine the effects of intra-articular injection of Torin 1, which as a specific inhibitor of mTOR which can cause induction of autophagy, is similar to rapamycin, on articular cartilage degeneration in a rabbit osteoarthritis model and to investigate the mechanism of Torin 1’s effects on experimental OA. Methods Collagenase (type II) was injected twice into both knees of three-month-old rabbits to induce OA, combined with two intra–articular injections of Torin 1 (400 nM). Degeneration of articular cartilage was evaluated by histology using the Mankin scoring system at eight weeks after injection. Chondrocyte degeneration and autophagosomes were observed by transmission electron microscopy. Matrix metallopeptidase-13 (MMP-13) and vascular endothelial growth factor (VEGF) expression were analysed by quantitative RT-PCR (qPCR).Beclin-1 and light chain 3 (LC3) expression were examined by Western blotting. Results Intra-articular injection of Torin 1 significantly reduced degeneration of the articular cartilage after induction of OA. Autophagosomes andBeclin-1 and LC3 expression were increased in the chondrocytes from Torin 1-treated rabbits. Torin 1 treatment also reduced MMP-13 and VEGF expression at eight weeks after collagenase injection. Conclusion Our results demonstrate that intra-articular injection of Torin 1 reduces degeneration of articular cartilage in collagenase-induced OA, at least partially by autophagy activation, suggesting a novel therapeutic approach for preventing cartilage degeneration and treating OA. Cite this article: N-T. Cheng, A. Guo, Y-P. Cui. Intra-articular injection of Torin 1 reduces degeneration of articular cartilage in a rabbit osteoarthritis model. Bone Joint Res 2016;5:218–224. DOI: 10.1302/2046-3758.56.BJR-2015-0001.

Inhibitory effects of evodiamine on human osteosarcoma cell proliferation and apoptosis

Osteosarcoma is a primary malignancy of bone, which is characterized by the proliferation of malignant mesenchymal cells, particularly in children and adolescents. Evodiamine is extracted from a variety of traditional Chinese medicines, which has been reported to induce apoptosis in certain tumors, including cervical, prostate and breast cancer, however, its effect on oestosarcoma cells remains unclear. The aim of the present study was to investigate the effect of evodiamine on osteosarcoma cell proliferation and apoptosis, and explore the associated underlying molecular mechanism. A Cell Counting Kit 8 assay was performed to detect the effects of evodiamine on the proliferation of human osteosarcoma U2OS cells. Annexin V-fluorescein isothiocyanate/propidium iodide staining was performed to analyze the apoptotic rate of the cells. The effect of evodiamine on the protein expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), caspase-3 and survivin were detected by performing western blot analysis. Evodiamine inhibited the growth of human osteosarcoma U2OS cells by inhibiting cell proliferation and inducing cell apoptosis. Western blotting demonstrated that evodiamine downregulated the expression of Bcl-2, caspase-3 and survivin, and upregulated the expression of Bax in human osteosarcoma cells. Evodiamine effectively inhibited proliferation and induced apoptosis of osteosarcoma cells in a dose-dependent manner via downregulation of Bcl-2, caspase-3 and survivin protein expression levels and upregulation of Bax protein expression levels.

Docosahexenoic acid treatment ameliorates cartilage degeneration via a p38 MAPK-dependent mechanism

Osteoarthritis (OA) is a common chronic inflammatory disease, characterized by cartilage degradation. The aberrant expression of matrix metalloproteinase-13 (MMP-13) plays a vital role in the pathogenesis of OA. The anti-inflammatory property of docosahexenoic acid (DHA) was previously revealed and showed that DHA retards the progress of many types of inflammatory disease. To evaluate the prophylactic function of DHA in OA, the effect of DHA on cartilage degeneration was assessed in interleukin-1β (IL-1β) stimulated human chondrosarcoma SW1353 cells or a rat model of adjuvant-induced arthritis (AIA). The safe concentration range (0–50 µg/ml in vitro) of DHA was determined by flow cytometry and MTT assay. The inhibitory effects of DHA on MMP-13 mRNA and protein expression were confirmed by RT-qPCR, ELISA and western blotting. Furthermore, findings of an in vivo study showed that DHA can increase the thickness of articular cartilage and decrease MMP-13 expression in cartilage matrix in a rat AIA model. We also revealed the mechanism by which DHA ameliorates cartilage degeneration from OA. The DHA-mediated inhibition of MMP-13 expression was partially attributed to the inactivation of the p38 mitogen-activated protein kinases pathway by suppressing p-p38 in IL-1β-stimulated SW1353 cells and a rat AIA model. Our findings suggested that DHA is a promising therapeutic agent that may be used for the prevention and treatment of OA.

Identification of aberrantly expressed long non-coding RNAs in postmenopausal osteoporosis

Postmenopausal osteoporosis (PMOP) is a common skeletal disorder in postmenopausal women. The present study aimed to identify the key long non-coding RNAs (lncRNAs) in PMOP through RNA sequencing. RNA sequencing was performed to obtain the expression profile of lncRNAs and mRNAs in blood samples of patients with PMOP and normal controls (NCs). Following the identification of differentially expressed mRNAs (DEmRNAs) and differentially expressed lncRNAs (DElncRNAs), the DElncRNA-DEmRNA co-expression network was constructed. A search was performed for the DEGs transcribed within a 100-kb window upstream or downstream of DElncRNAs, which served as nearby DEmRNAs of DElncRNAs. Functional annotation of the DEmRNAs co-expressed with DElncRNAs was performed. The GSE56815 dataset was used to verify the expression of selected DEmRNAs and DElncRNAs. Three blood samples from patients with PMOP and two blood samples from NCs were used for RNA sequencing. Compared with the NC group, a total of 185 DEmRNAs and 51 DElncRNAs were obtained in PMOP. A total of 3,057 co-expression DElncRNA-DEmRNA pairs and 97 DElncRNA-nearby DEmRNA pairs were obtained. Six DEmRNAs [diacylglycerol O-acyltransferase 2, potassium voltage-gated channel subfamily S member 1, peptidase inhibitor 3, secretory leukocyte peptidase inhibitor, galectin-related protein and alkaline phosphatase, liver/bone/kidney (ALPL)] were nearby co-expressed genes of four DElncRNAs, including LOC105376834, LOC101929866, LOC105374771 and LOC100506113. Three PMOP-associated DEmRNAs, including ALPL, suppressor of cytokine signaling 3 and adrenomedullin, were co-expressed with the hub DElncRNAs (LINC00963, LOC105378415, LOC105377067, HCG27, LOC101928143 and LINC01094) of the positively and negatively co-expressed DElncRNA-DEmRNA interaction network. The expression of selected DEmRNAs and DElncRNAs was consistent with the RNA-sequencing results. In conclusion, the present study identified the key DEmRNAs and DElncRNAs in PMOP, which may provide clues for understanding the mechanism and developing novel biomarkers for PMOP.

Long-term outcomes and prognostic analysis of modified open-door laminoplasty with lateral mass screw fusion in treatment of cervical spondylotic myelopathy

Objectives The purpose of the present study was to explore and analyze the long-term outcomes and factors that affect the prognosis of expansive open-door laminoplasty with lateral mass screw fusion in treatment of cervical spondylotic myelopathy (CSM). Methods We retrospectively reviewed 49 patients with multilevel CSM who had undergone expansive open-door laminoplasty with lateral mass screws fixation and fusion in our hospital between February 2008 and February 2012. The average follow-up period was 4.6 years. The clinical data of patients, including age, sex, operation records, pre- and postoperation Japanese Orthopedic Association (JOA) scores, cervical spine canal stenosis, and cervical curvature, were collected. Increased signal intensity (ISI) on T2-weighted magnetic resonance imaging and ossification of the posterior longitudinal ligament were also observed. Paired t-test was used to analyze the treatment effectiveness and recovery of neuronal function. The prognostic factors were analyzed with multivariable linear regression model. Results Forty-nine patients with CSM with a mean age of 59.44 years were enrolled in this study. The average of preoperative JOA score was 9.14±2.25, and postoperative JOA score was 15.31±1.73. There was significant difference between the pre- and postoperative JOA scores. The clinical improvement rate was 80.27%. On follow-up, five patients had complaints of neck and shoulder pain, but no evidence of C5 nerve palsy was found. Developmental cervical spine canal stenosis was present in all patients before surgery. Before surgery, ISI was observed in eight patients, while ossification of the posterior longitudinal ligament was found in 12 patients. The average of preoperative cervical curvature was 21.27°±8.37° and postoperative cervical curvature was 20.09°±1.29°, and there was no significant difference between the pre- and postoperative cervical curvatures. Multivariable linear regression analysis results showed that the postoperation JOA scores were significantly affected by age, preoperative JOA scores, and preoperative ISI. Except one case of epidural hematoma, there were no complications associated with the surgery. Conclusion Treatment of CSM with posterior open-door laminoplasty with lateral mass screw fusion is effective with few complications. In addition, the normal cervical lordosis was well maintained. Age, preoperative JOA scores, and preoperative ISI were the independent factors that significantly affect disease prognosis and surgical outcomes.

BFH-OSTM, a new predictive screening tool for identifying osteoporosis in elderly Han Chinese males

Purpose To develop and validate a new clinical screening tool to identify primary osteoporosis by dual-energy X-ray absorptiometry (DXA) in two elderly Han Chinese male populations. Methods A cross-sectional study was conducted, enrolling 1,870 community-dwelling and 574 hospital-checkup elderly Han Chinese males aged ≥50 years. All subjects completed a structured questionnaire and had their bone mineral density (BMD) measured using DXA. Using logistic regression analysis in the 1,870 community-dwelling males, we assessed the ability of numerous potential clinical risk factors to identify male with osteoporosis. Multiple variable regression analysis and item reduction yielded a final tool named the Beijing Friendship Hospital Osteoporosis Self-assessment Tool for Elderly Male (BFH-OSTM). Receiver operating characteristic (ROC) curve was generated to compare the validation of the BFH-OSTM and Osteoporosis Self-assessment Tool for Asians (OSTA) for identifying elderly male at increased the risk of primary osteoporosis in the 574 hospital-checkup males. Results In screening the 1,870 community-dwelling subjects with DXA, 14.2% (266/1,870) had osteoporosis, and a further 51.8% (969/1,870) had osteopenia. Of the items screened in the questionnaire, weight, height and previous history of fragility fracture were predictive of osteoporosis. A final tool (BFH-OSTM) was based on body weight and fragility fracture history only. The BFH-OSTM index (cutoff =70) had a sensitivity of 85% and specificity of 53% for identifying osteoporosis according to the WHO criteria, with an area under the ROC curve of 0.763. The predictive value of BFH-OSTM was validated in the 574 hospital-checkup population, which performed better than OSTA. Conclusion The BFH-OSTM may perform well for identifying elderly male at increased risk for osteoporosis and applying it would result in more prudent use of BMD measurement by DXA, especially for Han Chinese male.