EZH2 is associated with cartilage degeneration in osteoarthritis by promoting SDC1 expression via histone methylation of the microRNA-138 promoter.

Cartilage degeneration has been reported to deteriorate osteoarthritis (OA), a prevalent joint disease caused by intrinsic and epigenetic factors. This study aimed to examine the molecular mechanism of enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2)/microRNA-138 (miR-138)/syndecan 1 (SDC1) and its epigenetic regulation in cartilage degeneration in OA. An OA cell model was induced by stimulating chondrocytes with interleukin (IL)-1ß at a final concentration of 10 ng/mL, followed by alterations in EZH2 and miR-138 expression. Afterwards, cell apoptosis was analyzed using flow cytometry. The expression patterns of cartilage catabolism-related factors (MMP-13, ADAMTS-4, and ADAMTS-5) were determined using RT-qPCR and western blot analyses. The EZH2 and H3K27me3 enrichment at the miR-138 promoter region were determined using ChIP-qPCR. Finally, an OA mouse model was constructed to verify the function of EZH2 in vivo. EZH2 was expressed at high levels in OA models. EZH2 depletion ameliorated OA, as evidenced by reduced cell apoptosis in IL-1ß-treated chondrocytes and decreased levels of cartilage catabolism-related factors. Moreover, EZH2 promoted histone methylation at the miR-138 promoter to suppress miR-138 expression, thereby upregulating the expression of SDC1, a target gene of miR-138. Changes in this pathway increased the expression of cartilage catabolism-related factors in vitro while promoting cartilage degeneration in vivo. Our data provided evidence that EZH2 inhibits miR-138 expression by promoting the histone methylation of its promoter, which induces cartilage degeneration in OA models by upregulating SDC1 expression, suggesting a novel mechanistic strategy for OA treatment.

Therapeutic potential of hyaluronic acid/chitosan nanoparticles for the delivery of curcuminoid in knee osteoarthritis and an in vitro evaluation in chondrocytes.

Knee osteoarthritis (OA) is the main cause of leg pain in middle­aged and elderly individuals. Hyaluronic acid (HA), as well as curcuminoid, has been used in the treatment of knee OA. In the present study, HA/chitosan nanoparticles (CNPs) were prepared for the delivery of curcuminoid, in order to investigate whether HA and curcuminoid can act synergistically as a better treatment option. The knee OA model was established by the Hulth method, and a knee OA chondrocyte model was constructed by the co­induction of interleukin­1ß and tumor necrosis factor (TNF)­α. The drug loading capacity of HA/CNP for the delivery of curcuminoid was measured by an ultraviolet assay, and the cytotoxicity to chondrocytes was measured by an MTT assay. Collagen II was detected by immunofluorescence, and the expression levels of nuclear factor (NF)­κB and inflammation­related genes in cartilage tissue and chondrocytes were detected. Chondrocyte proliferation was determined by an EdU assay, and chondrocyte apoptosis was determined by flow cytometry. The Mankin pathological score of the Outerbridge classification was obtained. The results demonstrated that the optimum drug loading capacity of HA/CNP for the delivery of curcuminoid was 38.44%, with a good sustained release function. HA/CNP treatment resulted in inhibition of the NF­κB pathway, as well as the expression of matrix metalloproteinase (MMP)­1 and MMP­13, but it increased collagen II expression. HA/CNP for the delivery of curcuminoid significantly decreased the Outerbridge classification and Mankin pathological scores to close to normal until the 4th week. Furthermore, it was also observed that all the effects of HA/CNP on the delivery of curcuminoid were more prominent compared with the effects of HA or curcuminoid treatment individually. Taken together, these findings demonstrated that HA/CNP for the delivery of curcuminoid may suppress inflammation and chondrocyte apoptosis in knee OA via repression of the NF­κB pathway.

[EXPRESSION AND FUNCTION OF HIGH MOBILITY GROUP BOX CHROMOSOMAL PROTEIN 1 IN SYNOVIOCYTES OF PATIENTS WITH OSTEOARTHRITIS].

OBJECTIVE: To explore the pathological role of high mobility group box chromosomal protein 1 (HMGB1) in osteoarthritis (OA) by comparing the difference of HMGB1 in the synoviocytes between OA and normal knees. METHODS: Synoviocyte lines from OA and normal knees were collected and cultured. Immunohistochemistry and Western blot were applied to identify the difference of HMGB1 between the OA and normal synoviocyte lines. The eukaryotic expression vector containing human Pgenesil-1/HMGB1 small interfering RNA (siRNA) were constructed and identified. The synoviocyte lines were transfected with the eukaryotic expression vector of Pgenesil-1/HMGB1 siRNA (Pgenesil-1/HMGB1 siRNA group) and with Pgenesil-1 plasmid (Pgenesil-1 group) and were not transfected as a control (untransfected group). Western blot was applied to identify the difference of HMGB1 among groups, and the levels of interleukin 1ß (IL-1ß) and tumor necrosis factor α (TNF-α) protein synthesis in the supernatants were measured by ELISA. RESULTS: Primary knee synoviocytes cultured in vitro were fibroblast-like cells with long- spindle shape. The immunohistochemistry and immunofluorescence results showed positive staining for HMGB1 in cytoplasm and weak positive staining in the nucleus in the OA synoviocyte line, but positive staining for HMGB1 in the nucleus and weak positive staining in the cytoplasm in the synoviocyte line of normal knee. The level of HMGB1 in the OA synoviocytes (0.687 ± 0.025) was significantly higher than that of normal synoviocytes (0.172 ± 0.030) (t = 32.159, P = 0.000) by Western blot. The recombinant plasmid Pgenesil-1/HMGB1 siRNA was successfully constructed. The expression of HMGB1 protein in Pgenesil-1/HMGB1 siRNA group (0.134 ± 0.048) was significantly lower than that of Pgenesil-1 group (0.581± 0.032) and untransfected group (0.514 ± 0.069) (P < 0.05). ELISA results showed that IL-1ß and TNF-α in supernatants of Pgenesil-1/HMGB1 siRNA group were significantly lower than those of Pgenesil-1 group and untransfected group (P < 0.05). CONCLUSION: The up-regulated expression and expressed location (from nucleus to cytoplasm) of HMGB1 in the synoviocyte are closely related to CA. The siRNA targeting inhibition of HMGB1 gene expression can obviously inhibit II-lp and TNF-a in supernatants of the CA synoviocyte line and delayed the inflammation of CA.

[Applications of myo-periosteal fibular bone bridging for traumatic transtibial amputation].

OBJECTIVE: To compare the effectiveness between the myo-periosteal fibular bone bridging and traditional transtibial amputation in the treatment of amputation below knee so as to provide theoretical basis for choosing transtibial amputation in clinical application. METHODS: Between November 2001 and November 2011, 38 patients with mangled lower extremity were treated by transtibial amputation. Among 38 patients, 17 (group A) underwent myo-periosteal fibular bone bridging (the operation techniques of an attached peroneal muscle myo-periosteal fibular strut bridge between the end of the tibia and fibula below knee amputation), and other 21 (group B) underwent traditional transtibial amputation. There was no significant difference in age, gender, injury cause, amputation cause, side, and disease duration between 2 groups (P > 0.05). The quality of life (QOL) was analyzed using 36-item short form health survey (SF-36), and prosthesis satisfaction by Trinity amputation and prosthesis experience scale (TAPES). RESULTS: Healing of incision by first intention was obtained in all patients of 2 groups; no necrosis, infection, or poor stumps was observed. The mean follow-up time was 22 months (range, 14-30 months) in group A, and 26 months (range, 15-30 months) in group B. The patients achieved good healing of bone bridging, no bone nonunion occurred. The healing time was (5.1 +/- 1.1) months in group A and (3.3 +/- 0.6) months in group B, showing significant difference between 2 groups (t=9.82, P=-0.00). Spur occurred at the distal fibula in an 11-year-old boy of group B after 2 years of operation, which blocked use of prosthesis; prosthesis was well used in the other patients. After 12 months of operation, SF-36 score was 55.84 +/- 14.01 in group A and 49.93 +/- 12.78 in group B, showing significant difference (P < 0. 05); the physical functioning, social functioning, role-physical, vitality, body pain, general health scores in group A were significantly higher than those in group B (P < 0.05), but no significant difference was found in role-emotional and mental health scores between 2 groups (P > 0.05). TAPES score was 12.12 +/- 2.23 in group A and 10.10 +/- 2.00 in group B, showing significant difference (t=2.891, P=0.006). CONCLUSION: It is a very effective method to treat traumatic amputation using an attached myo- periosteal fibular bone bridging between the end of the tibia and fibula below knee, which can afford better quality of life and prosthesis satisfaction.

[Differential proteomic analysis and function study of human prostate carcinoma cells with different osseous metastatic tendency].

OBJECTIVE: To investigate the differential protein expression profiles of human prostatic carcinoma cells with different metastatic tendency and to screen the osseous metastasis associated proteins and investigate their function. METHODS: Proteomics and Western blotting were applied to screen and identify the differentially expressed proteins in the prostatic carcinoma cells of different lines: line T3B with high osseous metastasis potential, line P2-4 with high lymphatic metastasis potential, and their common parent cell line PC-3. The eukaryotic expression vector carrying human Pgenesil-1/HMGB1 siRNA was constructed and transfected into T3B cells by Lipofectamine 2000 and the positive clones was screened by G418. Pgenesil-1/HMGB1 siRNA/T3B, Pgenesil-1/T3B, and T3B cells were inoculated into the left ventricles of nude mice. Twelve weeks later the mice were killed. The number of osseous metastatic nodules and osseous metastasis inhibition rate were calculated. The mice metastatic tumor cells were identified by immunohistochemistry. RESULT: Six differential expressed proteins, correlated with cytoskeleton, transcriptional control, cellular metabolism, and phosphorylation were identified by proteomics and Western blotting. The recombinant plasmid Pgenesil-1/HMGB1 siRNA was successfully constructed. The HMGB1 expression of the T3B cells transfected with Pgenesil-1/HMGB1 siRNA was significantly lower than those of the other 2 groups (both P <0.05). The number of osseous metastatic nodules of the mice inoculated with Pgenesil-1/HMGB1 siRNA/T3B was significantly less than those of the other 2 groups (both P < 0.05). The metastatic osseous tumor cells were identified as the human prostatic carcinoma cells. CONCLUSION: Osseous metastasis associated proteins exist in prostatic carcinoma cells of the line with high osseous metastasis potential. HMGB1 is closely related to the osseous metastasis of human prostatic carcinoma cells. siRNA targeting HMGB1 specifically suppresses the expression of HMGB1 gene in the human prostatic carcinoma cells with high osseous metastasis potential and effectively inhibits the osseous metastasis.

Immunological purification of rat precartilaginous stem cells and construction of the immortalized cell strain.

Precartilaginous stem cells (PCSC) are adult stem cells that control limb growth of animals and can differentiate directionally. In a previous study, PCSC was reported to begin differentiating at the fifth passage; therefore, sufficient uni-phenotype PCSC cannot be harvested from primary cell culture. The purpose of this study was to examine whether simian virus 40 large T antigen gene (SV40Tag) could induce rat PCSC to immortalize. Immunomagnetic separation was used to isolate PCSC labeled with fibroblast growth factor receptor-3 (FGFR-3). Plasmid pCMVSV40T/PUR containing SV40Tag was transfected into PCSC by liposome transfection method. One anti-puromycin cell clone was obtained, which was confirmed as FGFR-3 positive, and expanded to immortalized cell strain. Results from RT-PCR and immunocytochemistry demonstrated that SV40Tag was highly expressed at both mRNA and protein levels after stable transfection. The cells transfected with SV40Tag were expanded to immortalized cell strain, which could maintain its characteristics for 30 passages, named immortalized precartilaginous stem cells (IPCSC). IPCSC were short fusiform or triangular cells with two or three short axons. Immunocytochemistry results of FGFR-3 and Collagen II demonstrated that IPCSC retained the characteristics of PCSC and the high proliferation capability of IPCSC was confirmed by methyl thiazolyl tetrazolium assay. Therefore, we concluded that rat precartilaginous stem cells were purified and immortalized precartilaginous stem cell strain was established. It may provide a stable cell resource for basic research and cell transplantation therapies.