Peimine suppresses interleukin­1ß­induced inflammation via MAPK downregulation in chondrocytes.

Osteoarthritis (OA) is the most common type of degenerative joint disease and secreted inflammatory molecules serve a pivotal role in it. Peimine has been reported to have anti­inflammatory activity. In order to investigate the potential therapeutic role of Peimine in OA, mouse articular chondrocytes were treated with IL­1ß and different doses of Peimine in vitro. The data revealed that Peimine not only suppressed IL­1ß­induced production of nitric oxide (NO) and prostaglandin E2, but also reduced the protein levels of inducible NO synthase (iNOS) and cyclooxygenase­2 (COX­2). In addition, Peimine inhibited the IL­1ß­induced mRNA expression of matrix metalloproteinase (MMP)­1, MMP­3, MMP­9, MMP­13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)­4 and ADAMTS­5. Furthermore, Peimine inhibited IL­1ß­induced activation of the mitogen­activated protein kinase (MAPK) pathway. The protective effect of Peimine on IL­1ß­treated chondrocytes was attenuated following activation of the MAPK pathway, as demonstrated by the increased expression levels of MMP­3, MMP­13, ADAMTS­5, iNOS and COX­2 compared with the Peimine group. The in vivo data suggested that Peimine limited the development of OA in the mouse model. In general, the data indicate that Peimine suppresses IL­1ß­induced inflammation in mouse chondrocytes by inhibiting the MAPK pathway, suggesting a promising therapeutic role for Peimine in the treatment of OA.

Necrostatin-1 Attenuates Trauma-Induced Mouse Osteoarthritis and IL-1ß Induced Apoptosis via HMGB1/TLR4/SDF-1 in Primary Mouse Chondrocytes.

Necrostatin-1 (Nec-1) is a specific small molecule inhibitor of receptor-interacting protein kinase 1 (RIPK1) that specifically inhibits phosphorylation of RIPK1. RIPK1 regulates inflammation and cell death by interacting with receptor-interacting serine/threonine protein kinases 3(RIPK3). We hypothesized that Nec-1 may have anti-inflammatory efficacy in patients with osteoarthritis (OA), as the pathophysiology of OA involves the activation of inflammation-related signaling pathways and apoptosis. In this study, we explored the effects of Nec-1 on interleukin (IL)-1ß-induced inflammation in mouse chondrocytes and the destabilised medial meniscus (DMM) mouse model. Inhibiting RIPK1 with Nec-1 dramatically suppressed catabolism both in vivo and in vitro, but did not inhibit changes in subchondral bone. Nec-1 abolished the in vitro increases in matrix metalloproteinase (MMP) and ADAM metallopeptidase with thrombospondin type 1 motif 5 (ADAMTs5) expression induced by IL-1ß. However, adding high-mobility group box 1 (HMGB1) partially abrogated this effect, indicating the essential role of HMGB1 and Nec-1 in the protection of primary chondrocytes. Furthermore, Nec-1 decreased the expression of Toll-like receptor 4 (TLR4) and stromal cell-derived factor-1 (SDF-1), and attenuated the interaction between TLR4 and HMGB1. Western blot results suggested that Nec-1 significantly suppressed IL-1ß-induced NF-κB transcriptional activity, but not MAPK pathway. Micro-computed tomography, immunohistochemical staining, and Safranin O/Fast Green staining were used in vivo to assess the degree of destruction of OA cartilage. The results show that NEC-1 can significantly reduce the degree of destruction of OA cartilage. Therefore, Nec-1 may be a novel therapeutic candidate to treat OA.

CD146+ skeletal stem cells from growth plate exhibit specific chondrogenic differentiation capacity in vitro.

Skeletal stem cells (SSCs) are a population of progenitor cells which give rise to postnatal skeletal tissues including bone, cartilage and bone marrow stroma, however not to adipose, haematopoietic or muscle tissue. Growth plate chondrocytes exhibit the ability of continuous proliferation and differentiation, which contributes to the continuous physiological growth. The growth plate has been hypothesized to contain SSCs which exhibit a desirable differentiation capacity to generate bone and cartilage. Due to the heterogeneity of the growth plate chondrocytes, SSCs in the growth plate are not well studied. The present study used cluster of differentiation (CD)146 and CD105 as markers to isolate purified SSCs. CD105+ SSCs and CD146+ SSCs were isolated using a magnetic activated cell sorting method. To quantitatively investigate the proliferation and differentiation ability, the colony-forming efficiency (CFE) and multi­lineage differentiation capacity of CD105+ SSCs and CD146+ SSCs were compared with unsorted cells and adipose-derived stem cells (ASCs). It was revealed that CD105+ and CD146+ subpopulations represented subsets of SSCs which generated chondrocytes and osteocytes, however not adipocytes. Compared with CD105+ subpopulations and ASCs, the CD146+ subpopulation exhibited a greater CFE and continuous high chondrogenic differentiation capacity in vitro. Therefore, the present study suggested that the CD146+ subpopulation represented a chondrolineage­restricted subpopulation of SSCs and may therefore act as a valuable cell source for cartilage regeneration.

Iguratimod prevents ovariectomy­induced bone loss and suppresses osteoclastogenesis via inhibition of peroxisome proliferator­activated receptor­Î³.

Iguratimod is known for its anti­inflammatory activities and therapeutic effects in patients with rheumatoid arthritis. It has previously been demonstrated that iguratimod attenuates bone destruction and osteoclast formation in the Walker 256 rat mammary gland carcinoma cell­induced bone cancer pain model. Therefore, it was hypothesized that iguratimod may additionally exhibit therapeutic effects on benign osteoclast­associated diseases including postmenopausal osteoporosis. In the present study, ovariectomized mice were used to investigate the effects of iguratimod in vivo. Bone marrow mononuclear cells were cultured to detect the effects of iguratimod on receptor activator of nuclear factor­κB ligand (RANKL)­induced osteoclastogenesis in vitro and the molecular mechanisms involved. It was demonstrated that iguratimod may prevent ovariectomy­induced bone loss by suppressing osteoclast activity in vivo. Consistently, iguratimod may inhibit RANKL­induced osteoclastogenesis and bone resorption in primary bone marrow mononuclear cells. At the molecular level, peroxisome proliferator­activated receptor­Î³ (PPAR­Î³)/c­Fos pathway, which is essential in RANKL­induced osteoclast differentiation, was suppressed by iguratimod. Subsequently, iguratimod decreased the expression of nuclear factor of activated T cells c1 and downstream osteoclast marker genes. The results of the present study demonstrated that iguratimod may inhibit ovariectomy­induced bone loss and osteoclastogenesis by modulating RANKL signaling. Therefore, iguratimod may act as a novel therapeutic to prevent postmenopausal osteoporosis.

Effect of lentivirus-mediated uPA silencing on the proliferation and apoptosis of chondrocytes and the expression of MMPs.

The lentivirus-mediated uPA interference in the proliferation, apoptosis, and secretion of osteoarthritic chondrocytes was examined in this study. Cells were obtained from the cartilage tissues of New Zealand white rabbits. They were cultured with interleukin (IL)-1ß (10 ng/mL) for 24 h and then divided into three groups: uPA-siRNA group (cells transfected with uPA-siRNA lentiviruses), blank control group (untreated cells), and negative control group (cells transfected with empty vectors). Western blotting and real-time quantitative reverse transcription-PCR (RT-QPCR) were performed to detect the protein and mRNA expression levels of uPA, MMP-1, MMP-3, MMP-9, MMP-10, MMP-13 and MMP-14 in osteoarthritic chondrocytes. Cell Counting Kit-8, flow cytometry, and colony formation assay were used to examine the proliferation and apoptosis of chondrocytes. The results showed that after uPA-siRNA transfection, the protein and mRNA expression levels of uPA, MMP-1, MMP-3, MMP-9, MMP-10, MMP-13, and MMP-14 were significantly decreased (P<0.05 for MMP-1, MMP-9, MMP-10 and MMP-14, P<0.01 for uPA, MMP-3 and MMP-13). Cell proliferation and colony formation rate were significantly higher and the cell apoptosis rate was significantly lower in uPA-siRNA group than in control groups (P<0.01). The proportion of cells in G0/G1 phase was markedly increased and that in the S phase decreased, and the cell cycle was arrested at the G1/S phase in the control group. In the uPA-siRNA group, the proportion of cells in the S phase was significantly increased, resulting in a different proportion of cells in cell cycle phase (P<0.01). It was suggested that the down-regulation of uPA gene could inhibit the expression of MMPs protein and cell apoptosis, increase the proliferation and colony formation of osteoarthritic chondrocytes.

Construction and verification of the targeted uPA-shRNA lentiviral vector and evaluation of the transfection and silencing rate.

Urokinase-type plasminogen activator (uPA) receptors, which are released by the synovial tissue, are responsible for the activation of cartilage-breakdown proteases and play critical roles in cartilage degradation during the progression of osteoarthritis (OA). RNA interference (RNAi) technology has emerged as a potent tool to generate cellular knockdown phenotypes of a desired gene. The aims of the present study were to investigate the effect of siRNA specific to the uPA gene on chondrocytes and to investigate the possible mechanisms of OA. Firstly, four types of small hairpin RNA (shRNA) sequence (P1, P2, P3 and P4) were obtained from the targeted uPA gene of the New Zealand rabbit, based on siRNA theory. The sequences were designed, constructed and subjected to restriction enzyme digestion, transformation, polymerase chain reaction (PCR) identification, positive clone sequencing and lentivirus packaging. Secondly, primary culturing cartilage cells from the New Zealand rabbit were transfected with P1, P2, P3 or P4 to observe the transfection rate under a fluorescence microscope. The mRNA expression levels of uPA were analyzed in cartilage cells using quantitative PCR, while protein expression levels were analyzed in the cartilage cells using western blot technology. Four types of uPA-shRNA lentiviral vectors were constructed successfully, which were all able to be transfected into the primary culturing cartilage cells. The transfection rate was as high as 85% when the multiplicity of infection was 100, which demonstrated that P1, P2, P3 and P4 were all capable of inhibiting the mRNA and protein expression of uPA in cartilage cells. In addition, among the four sequences, the P2 sequence exhibited the highest silencing rate of 70%. Statistical significance (P<0.05) was observed when analyzing the silencing rate of P2 compared to the other three groups. The most efficient targeted uPA-shRNA sequence was identified following screening. The results strongly verified that siRNA lentiviral vectors can be transfected into cartilage cells to further inhibit the expression of the uPA gene efficiently and steadily. Thus, the results provide the foundation for further research on the role of uPA in the pathogenesis of OA.

Peroxisome proliferator­activated receptor γ agonist rosiglitazone inhibits migration and invasion of prostate cancer cells through inhibition of the CXCR4/CXCL12 axis.

It has been indicated that the C­X­C chemokine receptor type 4/C­X­C chemokine ligand 12 (CXCR4/CXCL12) axis is involved in promoting invasion and metastasis in tumors. Therefore, novel drugs capable of downregulating the CXCR4/CXCL12 axis may demonstrate potential for the treatment of metastatic prostate cancer (PCa). Rosiglitazone (RSG), a thiazolidinedione ligand of the peroxisome proliferator­activated receptor (PPAR) Î³, has been found to inhibit proliferation, induce apoptosis, suppress angiogenesis and inhibit metastasis. However, the precise mechanisms by which RSG regulates CXCR4 gene expression and the consequent effects on prostate cell migration and invasion are not fully understood. In this study, it was observed that RSG is capable of downregulating the expression of CXCR4 in PCa cells in a dose­, time­ and PPARγ­dependent manner. Furthermore, it was observed that the downregulation of CXCR4 expression occurred at a transcriptional level, as indicated by a reduction in CXCR4 mRNA expression. Suppression of CXCR4 expression by RSG further correlated with the inhibition of CXCL12­induced migration and invasion in PCa cells. Analysis of the predominant intracellular signaling pathways that act downstream of the activated CXCR4/CXCL12 axis, namely the phosphatidyl inositol 3­kinase (PI3K)­protein kinase B (Akt) cascades, revealed that RSG rapidly interferes with the phosphorylation/activation of Akt, which mediates CXCL12­stimulated migration and invasion. Overall, the findings of this study suggest that RSG represents a novel inhibitor of CXCR4 expression and, thus, has significant potential as a powerful therapeutic agent for the treatment of metastatic PCa.

Peroxisome proliferator-activated receptor γ ligands inhibit VEGF-mediated vasculogenic mimicry of prostate cancer through the AKT signaling pathway.

Vasculogenic mimicry (VM) describes functional vascular channels composed only of tumor cells and its presence predicts a poor prognosis for patients with prostate cancer. The present study demonstrated that prostate cancer PC-3 cells were able to form a patterned matrix or tubular VM in 3D cultures in vitro and rosiglitazone (RSG), the ligand of peroxisome proliferator-activated receptor Î³ (PPARγ) and effectively inhibited the formation of VM structures in a dose- and PPARγ­dependent manner. In addition, RSG significantly inhibited prostate cancer cell migration and invasion. The inhibitory effect of RSG on VM formation could be at least partially explained by an RSG-driven downregulation of vascular endothelial growth factor (VEGF) levels and phosphorylation of AKT, which is known to be important in VM. Furthermore, the present study highlighted that VEGF and the phosphoinositide 3-kinase/AKT pathway exert a positive feedback regulation in the process of VM formation. These findings reveal new therapeutic potential for PPARγ ligands in anti­cancer therapy.

Inhibitory effects of high glucose/insulin environment on osteoclast formation and resorption in vitro.

Patients with type 2 diabetes mellitus (T2DM) exhibit hyperglycemia and hyperinsulinemia and increased risk of fracture at early stage, but they were found to have normal or even enhanced bone mineral density (BMD). This study was aimed to examine the molecular mechanisms governing changes in bone structure and integrity under both hyperglycemic and hyperinsulinemic conditions. Monocytes were isolated from the bone marrow of the C57BL/6 mice, induced to differentiate into osteoclasts by receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) and exposed to high glucose (33.6 mmol/L), high insulin (1 µmol/L), or a combination of high glucose/high insulin (33.6 mmol/L glucose and 1 µmol/L insulin). Cells cultured in α-MEM alone served as control. After four days of incubation, the cells were harvested and stained for tartrate resistant acid phosphatase (TRAP). Osteoclast-related genes including RANK, cathepsin K and TRAP were determined by using real-time PCR. The resorptive activity of osteoclasts was measured by using a pit formation assay. Osteoclasts that were derived from monocytes were of multinucleated nature and positive for TRAP, a characteristic marker of osteoclasts. Cell counting showed that the number of osteoclasts was much less in high glucose and high glucose/high insulin groups than in normal glucose and high insulin groups. The expression levels of RANK and cathepsin K were significantly decreased in high glucose, high insulin and high glucose/high insulin groups as compared with normal glucose group, and the TRAP activity was substantially inhibited in high glucose environment. The pit formation assay revealed that the resorptive activity of osteoclasts was obviously decreased in high glucose group and high glucose/high insulin group as compared with normal group. It was concluded that osteoclastogenesis is suppressed under hyperglycemic and hyperinsulinemic conditions, suggesting a disruption of the bone metabolism in diabetic patients.

LY294002 induces G0/G1 cell cycle arrest and apoptosis of cancer stem-like cells from human osteosarcoma via down-regulation of PI3K activity.

Osteosarcoma, the most common primary mesenchymal malignant tumor, usually has bad prognosis in man, with cancer stem-like cells (CSCs) considered to play a critical role in tumorigenesis and drug-resistance. It is known that phosphatidylinositol 3-kinase (PI3K) is involved in regulation of tumor cell fates, such as proliferation, cell cycling, survival and apoptosis. Whether and how PI3K and inhibitors might cooperate in human osteosarcoma CSCs is still unknown. We therefore evaluated the effects of LY294002, a PI3K inhibitor, on the cell cycle and apoptosis of osteosarcoma CSCs in vitro. LY294002 prevented phosphorylation of protein kinase B (PKB/Akt) by inhibition of PI3K phosphorylation activity, thereby inducing G0/G1 cell cycle arrest and apoptosis in osteosarcoma CSCs. Further studies also demonstrated that apoptosis induction by LY294002 is accompanied by activation of caspase-9, caspase-3 and PARP, which are involved in the mitochondrial apoptosis pathway. Therefore, our results indicate PI3K inhibitors may represent a potential strategy for managing human osteosarcoma via affecting CSCs.

[Effect of short-hairpin RNA expression vector-mediated osteopontin RNA interference on proliferation and invasion of prostate cancer PC-3 cells].

BACKGROUND AND OBJECTIVE: Studies showed that osteopontin (OPN) regulates cell migration and invasion in a variety of cancers, which associates with the activities of matrix metalloproteinase (MMP)-2 and MMP-9. This study was to investigate the role of OPN in the proliferation and invasion of human prostate cancer PC-3 cells and the possible functions of IgammaB kinase (IKK) in nuclear factor kappa B (NF-kappaB)-mediated signaling pathways. METHODS: OPN short-hairpin RNA (shRNA) recombinant plasmids were transfected into PC-3 cells and different concentrations of IKK inhibitors were used to inhibit the activities of IKKalpha and IKKbeta. The mRNA and protein expression levers of OPN, MMP-2, and MMP-9 were detected by real-time polymerase chain reaction (PCR) and Western blot. Cell cycle was detected by flow cytometry, cell proliferation by MTT assay, and cell invasion by Transwell chamber assay. RESULTS: Compared with untreated cells, the protein levers of OPN, MMP-2, and MMP-9 in OPN shRNA-transfected PC-3 cells were reduced by 55.22%, 51.71%, and 28.35%, respectively, and the ability of cell migration and invasion were decreased by 45.48% and 51.96%, respectively (P<0.05). Moreover, the inhibition of IKKbeta inhibited the expressions of MMP-2 and MMP-9. CONCLUSION: A shRNA expression vector-mediated OPN gene silencing can inhibit the malignant biological behaviors of PC-3 cells. IKKbeta may play a crucial role in the OPN-induced activation of MMP-2 and MMP-9 via NF-kappaB-mediated IkappaB/IKKbeta pathways.

Sensory and sympathetic innervation of cervical facet joint in rats.

OBJECTIVE: To explore the patterns of innervation of cervical facet joints and determine the pathways from facet joints to dorsal root ganglions (DRGs) in order to clarify the causes of diffuse neck pain, headache, and shoulder pain. METHODS: Forty-two male-Sprague-Dawley rats, weighing 250-300 g, were randomly divided into three groups: Group A (n=18), Group B (n=18), and Group C (n=6). Under anesthesia with intraperitoneal pentobarbital sodium (45 mg/kg body weight), a midline dorsal longitudinal incision was made over the cervical spine to expose the left cervical facet joint capsule of all the rats under a microscope. The rats in Group A underwent sympathectomy, but the rats in Group B and Group C did not undergo sympathectomy. Then 0.6 microlitre 5% bisbenzimide (Bb) were injected into the C1-2, C3-4 and C5-6 facet joints of 6 rats respectively in Group A and Group B. The holes were immediately sealed with mineral wax to prevent leakage of Bb and the fascia and skin were closed. But in Group C, 0.9% normal saline was injected into the corresponding joint capsules. Then under deep re-anesthesia with intraperitoneal pentobarbital sodium (45 mg/kg body weight), C1-C8 left DRGs in all rats and the sympathetic ganglions in Group B were obtained and the number of the labeled neurons was determined. RESULTS: Neurons labeled with Bb were present in C1-C8 DRGs in both Group A and Group B, and sympathetic ganglions in Group B. In the C1-2 and C3-4 subgroups, labeled neurons were present from C1 to C8 DRGs, while in C5-6 subgroups they were from C3 to C8. The number of Bb(+) neurons after sympathectomy was not significantly different in the injected level from that without sympathectomy. But in the other levels, the number of Bb(+) neurons after sympathectomy was significantly less than that without sympathectomy. CONCLUSIONS: The innervation of the cervical facet joints is derived from both sensory and sympathetic nervous system, and DRGs are associated with sympathetic ganglions through nerve fibers outside the central nerve system.

[Regulation of differentiation and proliferation of epiphysis stem cells by Notch1 signaling system].

OBJECTIVE: To investigate the regulation of differentiation and proliferation of epiphysis stem cells by Notch1 signaling system. METHODS: Costocostal cartilage was taken from a SD rat. Epiphysis stem cells were isolated and cultured. Recombinant human nuclear factor-kappaB (rhNF-kappaB), an activator of the Notch signaling system, and gamma-secretase inhibitor (MW167), an inhibitor of the Notch signaling system, were added into the culture medium respectively. The cells cultured in the medium added with phosphate-buffered saline were used as control group. Then the cultured cells were collected. The expression of the homologous Notch receptors and homologous Notch ligands was detected by RT-PCR. Immunohistochemistry was used to detect the levels of collagen II, collagen X, and proliferating cell nuclear antigen (PCNA). MTT method was used to calculate the growth curve. The cell phase was examined by flow cytometry. The level of alkaline phosphatase (AP) was measured. Western blotting was used to detect the protein expression of collagen II, collagen X, and stathmin, a signaling protein of proliferation. RESULTS: Only 2 the expression of the receptor Notch1 and the ligand Jagged1 was found. The expression of PCNA was stronger in the rhNF-kappaB group than in the other 2 groups. rhNF-kappaB remarkably promoted the expression of collagen II and inhibited the expression of collagen X and MW167 remarkably promoted the expression of collagen X and did not remarkably influence the expression of collagen II. MTT method showed that rhNF-kappaB significantly promote the proliferation of the cells (P = 0.027), and MW167 did not significantly promote the cell proliferation (P > 0.05). The percentage of cells at S phase of the rhNF-kappaB group was 26.54%, significantly higher than those of the MW167 group and control group (8.22% and 6.15%). AP was significantly expressed in the MW167 group, and less expressed in the other groups. Western blotting showed a significantly increased expression of collagen X protein and decreased expression of collagen II protein and stathmin. CONCLUSION: When the Notch signaling system is activated the epiphysis stem cells proliferate, and when the Notch signaling system is suppressed the epiphysis stem cells differentiate.