Rachel W. Li

Anti-inflammatory activity of Chinese medicinal vine plants

Anti-inflammatory activities of ethanol extracts from nine vine plants used in traditional Chinese medicine to treat inflammatory conditions were evaluated against a panel of key enzymes relating to inflammation. The enzymes included cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), phospholipase A(2) (PLA(2)), 5-lipoxygenase (5-LO) and 12-lipoxygenase (12-LO). The vine plants studied were: the stem of Spatholobus suberectus Dunn, the stem of Trachelospermum jasminoides Lem., the root from Tripterygium wilfordii Hook. f., the stem of Sinomenium acutum Rehder and Wilson, the stem of Piper kadsura (Choisy) Ohwi, the stem of Polygonum multiflorum Thunb., the root and stem from Tinospora sagittata Gagnep., the root of Tinospora sinensis (Lour.) Merrill, and the stem of Clematis chinensis Osbeck. All of the plant extracts showed inhibitory activities against at least one of the enzymes in various percentages depending upon the concentrations. The extract from S. suberectus was found to be active against all enzymes except COX-2. Its IC(50) values were 158, 54, 31 and 35 microg/ml in COX-1, PLA(2), 5-LO and 12-LO assays, respectively. T. jasminoides showed potent inhibitory activities against both COX-1 (IC(50) 35 microg/ml) and PLA(2) (IC(50) 33 microg/ml). The most potent COX-1, COX-2 and 5-LO inhibition was observed in the extract of T. wilfordii with the IC(50) values of 27, 125 and 22 microg/ml, respectively. The findings of this study may partly explain the use of these vine plants in traditional Chinese medicine for the treatment of inflammatory conditions.

A cross-cultural study: anti-inflammatory activity of Australian and Chinese plants.

In this study, in vitro inhibitory effects of 33 ethanol extracts obtained from 24 plant species (representing 11 different families) on cyclooxygenase-1 (COX-1) were evaluated. The plant materials selected for this study have been used in aboriginal medicine in Australia and traditional medicine in China for the treatment of various diseases that are considered as inflammation in nature, e.g. asthma, arthritis, rheumatism, fever, edema, infections, snakebite and related inflammatory diseases. All of the selected plants, with one exception, showed inhibitory activity against COX-1, which supports their traditional uses. The most potent COX-1 inhibition were observed from the extracts of Acacia ancistrocarpa leaves (IC(50)=23 microg/ml). Ficus racemosa bark, Clematis pickeringii stem, Acacia adsurgens leaves, Tinospora smilacina stem and Morinda citrifolia fruit powder exhibited inhibition of COX-1 with the IC(50) of 100, 141, 144, 158 and 163 microg/ml, respectively. Aspirin and indomethacin used as the reference COX-1 inhibitors in this study inhibited COX-1 with IC(50) of 241 and 1.2 microg/ml, respectively. The findings of this study may explain at least in part why these plants have been traditionally used for the treatment of inflammatory conditions in Australian aboriginal medicine and traditional Chinese medicine.

Arthritogenic alphaviral infection perturbs osteoblast function and triggers pathologic bone loss

Significance Persistent polyarthritis, which occurs in 30–40% of alphavirus-infected patients, has been proposed to be caused by proinflammatory mediators such as IL-6. In the present study we investigated the susceptibility and response of primary human osteoblasts to Ross River virus (RRV) infection and determined whether infection could result in bone pathology. RRV infection of osteoblasts resulted in increased receptor activator of nuclear factor-kappaB ligand (RANKL) but decreased osteoprotegerin (OPG). We are the first to report that alphavirus infection results in bone loss in an established RRV murine model and that this bone loss is prevented by IL-6 inhibition. These findings reveal that RRV can disrupt bone homeostasis and that osteoblasts play an important role in alphavirus-induced arthritis by regulating IL-6 and contribute to bone loss by disrupting the RANKL/OPG balance. Arthritogenic alphaviruses including Ross River virus (RRV), Sindbis virus, and chikungunya virus cause worldwide outbreaks of musculoskeletal disease. The ability of alphaviruses to induce bone pathologies remains poorly defined. Here we show that primary human osteoblasts (hOBs) can be productively infected by RRV. RRV-infected hOBs produced high levels of inflammatory cytokine including IL-6. The RANKL/OPG ratio was disrupted in the synovial fluid of RRV patients, and this was accompanied by an increase in serum Tartrate-resistant acid phosphatase 5b (TRAP5b) levels. Infection of bone cells with RRV was validated using an established RRV murine model. In wild-type mice, infectious virus was detected in the femur, tibia, patella, and foot, together with reduced bone volume in the tibial epiphysis and vertebrae detected by microcomputed tomographic (µCT) analysis. The RANKL/OPG ratio was also disrupted in mice infected with RRV; both this effect and the bone loss were blocked by treatment with an IL-6 neutralizing antibody. Collectively, these findings provide previously unidentified evidence that alphavirus infection induces bone loss and that OBs are capable of producing proinflammatory mediators during alphavirus-induced arthralgia. The perturbed RANKL/OPG ratio in RRV-infected OBs may therefore contribute to bone loss in alphavirus infection.

Dramatic regulation of heparanase activity and angiogenesis gene expression in synovium from patients with rheumatoid arthritis.

OBJECTIVE Although heparanase is recognized as a proangiogenic factor, the involvement of heparanase in rheumatoid arthritis (RA) is unclear. In this study, we assessed heparanase activity in synovial fluid (SF) and synovial tissue (ST) from patients with RA or osteoarthritis (OA), and analyzed the expression of angiogenic pathway-focused genes in ST from RA and OA patients. METHODS SF and ST were obtained from the knees of patients with either RA or OA and from asymptomatic donors with no documented history of degenerative or inflammatory joint diseases. Heparanase activity was determined by an enzymatic assay using a radiolabeled substrate, and the presence of heparanase in ST was demonstrated by Western blotting. The expression of angiogenesis genes, including heparanase, in ST was analyzed by real-time quantitative polymerase chain reaction. RESULTS Heparanase activity was dramatically higher (>100-fold) in SF and ST from RA patients than in SF and ST from OA patients and asymptomatic donors. Active heparanase enzyme was detected and heparanase messenger RNA was up-regulated in ST from RA patients. We also found that angiogenesis gene expression was significantly regulated in RA synovium, and was correlated with heparanase activity. CONCLUSION These findings are novel and contribute to our understanding of joint destruction in RA, suggesting that heparanase may be a reliable prognostic factor for RA progression and an attractive target for the treatment of RA.

Interleukin 6, RANKL, and Osteoprotegerin Expression by Chikungunya Virus–Infected Human Osteoblasts

TO THE EDITOR—Chow et al [1] recently implicated interleukin 6 (IL-6) in the persistent arthralgia that occurs in some patients following infection with chikungunya virus (CHIKV). They observed that plasma IL-6 concentrations in patients with persistent arthralgia were higher than those in fully recovered patients; the significance of this observation is supported by the known role of IL-6 in causing joint pain [2]. A role for IL-6 in persistent arthralgia is further supported by the finding by Hoarau et al [3] that IL-6 is specifically expressed in the affected joint during chronic chikungunya disease. Nevertheless, the plasma IL-6 concentration in patients with chronic disease is low (interquartile range, 4–40 pg/mL) and close to normal values [1]. IL-6 is expressed by a variety of cell types, including osteoblasts, and the low circulating levels suggest the joint as a potential source of this cytokine. IL-6 stimulates the release of RANKL [4] and inhibits the one of its decoy receptor osteoprotegerin (OPG) [5] by osteoblasts, therefore promoting osteoclastogenesis and bone resorption [6]. The RANKL/ OPG ratio indeed drives osteoclastogenesis and osteoclast activation [7]. This raises the possibility that dysregulation of IL-6, RANKL, and OPG during CHIKV infection may contribute to joint pathology. In this context, we aimed to determine whether osteoblasts may be involved in CHIKV induced chronic rheumatic syndromes. First, we tested whether primary human osteoblasts are susceptible to CHIKV infection in vitro. Osteoblast cultures were prepared from bone samples obtained from a healthy male subject during a knee operation for a cause unrelated to arthritis. The patient’s medical history indicated no autoimmune disorders, metabolic diseases, intake of immune suppressant/stimulating drugs, or immunotherapy for 3 months before surgery. Bone fragments were cultured in α minimum essential medium supplemented with 10% fetal calf serum, 100 mM ascorbic acid, 20 mM HEPES, and 2 mM L-glutamine. After 2 weeks, confluent cells surrounding fragments were collected and subcultured. Osteoblast characterization by osteocalcin staining and measurement of alkaline phosphatase activity showed >98% purity [8]. Cell monolayers were infected with CHIKV at a multiplicity of infection of 0.1 for 1 hour at 37°C, washed, and fed with fresh media. As shown in Figure 1A, CHIKV replicated in osteoblasts between days 1 and 20 after infection, at levels comparable to that described for macrophages [9]. The decrease in virus production with

The influence of biodegradable magnesium alloys on the osteogenic differentiation of human mesenchymal stem cells

The postdegradation effect of pure Mg, Mg-1Y, Mg-5Al, and Mg-2Ca alloys on the differentiation, proliferation and gene expression of human mesenchymal stem cells (hMSCs) was investigated. It was revealed that that Mg(2+) ions result in an increase in cell proliferation. However, we observed a maximum concentration (approximately 8.0 × 10(-4) M) that was favourable to ATP production, above which ATP production began to decrease. In contrast to proliferation, no maximum concentration for osteogenic differentiation was observed, with increasing concentration of Mg(2+) ions resulting in an increase in osteogenic differentiation across the entire tested range. Interestingly, the Mg-2Ca alloy had minimal effect on osteogenic differentiation, with Mg-1Y and pure Mg having a superior effect on the proliferation and differentiation of hMSCs. This was also observed from gene expression data, where these alloys upregulated TGFβ-1, SMAD4, FGF-2, FGF-10, and BMP-2, while SOX-2, SOX-9, and TNF-α were downregulated. Increased expression of TGFβ-1, SMAD4, BMPs, and COLIA1 protein provided further evidence to support osteogenic differentiation and that the influence of the alloying extracts on differentiation may be via the SMAD signaling pathway.

Heparanase in Primary Human Osteoblasts

Heparanase (HPSE) is known to be involved in fracture repair in mice, but its presence and function in human bone formation remains unclear. Our aim was to determine the expression of HPSE in human bone forming osteoblasts and to better understand its role in osteogenesis. HPSE protein expression and enzymatic activity were demonstrated in osteoblasts isolated from trabecular bone specimens of patients with osteoporosis (OP) and from healthy subjects, although the levels differed markedly. Thus, low levels of HPSE expression were observed in osteoporotic osteoblasts, including in the nucleus compared to those from healthy subjects. Notably, HPSE gene expression was associated with alkaline phosphatase (ALP) activity, the bone turnover marker. Gene profile studies demonstrated that osteogenic genes were downregulated in osteoporotic osteoblasts. We further exposed osteoblasts to exogenous HPSE and found that the level of histone H3 phosphorylation was increased. We provide evidence, for the first time, demonstrating that HPSE expresses and functions in human osteoblasts. Our data suggest that previously undescribed function of HPSE‐mediated osteoblastogenesis through regulation of osteogenic gene expression and histone H3 modification. HPSE upregulation may be a novel therapeutic approach in the prevention and treatment of OP. Published by Wiley Periodicals, Inc. J Orthop Res 28:1315–1322, 2010

Monitoring and Controlling Intramedullary Pressure Increase in Long Bone Instrumentation : A Study on Sheep

Intramedullary reamed nailing causes elevation in intramedullary pressure and extravazation of intramedullary contents into the venous blood system. This study investigated the effect of an intramedullary suction system, recently developed in our laboratory, on the pressure and fat extravazation in isolated bovine bone and a sheep model. During reaming, the pressure with and without suction was recorded at each step of the procedure. Hemodynamic parameters of mean arterial blood pressure, pulmonary artery pressure, pulmonary arterial CO2 (PaCO2), heart rate, and oxygen saturation were monitored. Blood and lung tissue samples were collected for the examination of medullary fat intravazation. The increases of intramedullary pressure were dramatically reduced in the suction group (p < 0.05) in both in vitro and in vivo experiments. PaCO2 was significantly lower in the suction group than nonsuction group (32 vs. 40 mmHg, respectively, p = 0.02), while oxygen saturation was higher in the suction group (99 vs. 91 mmHg, respectively, p = 0.009). Histological data revealed a significant higher count of fat emboli in sheep lung tissue in the nonsuction group. Total lipids in lung specimens was lower in the suction group (7.6 mg/g tissue) than in the nonsuction group (13.6 mg/g, p = 0.04). The suction system appears to control the surge in intramedullary pressure and therefore prevent fat embolism.

Nano-osteoimmunology as an important consideration in the design of future implants.

The size of wear particles emanating from a prosthesis at interfaces is critical to the interfacial properties of the joint replacement and responses from the biological environment. Nanoscale particles in particular require investigation. This project aimed to evaluate the osteoimmunological response to nanoscale ultrahigh molecular weight polyethylene (UHMWPE) wear particles in vitro, including dendritic cells (DCs), macrophages, osteoclasts (OCs), cytokine secretion, and co-cultured OCs and osteoblasts (OBs). The wear particles generated from a constant-load knee prosthesis actuator were profiled using atomic force microscopy and fractionated into sizes of 0.05-0.2, 0.2-0.8, 0.8-1, 1-5 and 5-10 μm. The fractions were exposed to DCs isolated from mice spleen, human OCs, and co-cultured human OBs and OCs, and the effects of the particles on the cells were determined. Results revealed that exposure to nanoscale UHMWPE wear particles induced significant DC activation (p<0.05) and consequently increased cytokine interleukin (IL)-6 and IL-1β secretion (p<0.05). Exposure to nanoscale particles promoted OC maturation, resulting in the suppression of OB proliferation in OB and OC co-cultures. Therefore, the results of this study could contribute to a more mechanistic understanding of wear-debris-associated prosthesis failure. Furthermore, nanoscale UHMWPE wear particles should be considered as mediators of periprosthetic inflammation in the future development of biomaterials for joint replacement bearing surfaces.

Specific inhibition of NLRP3 in chikungunya disease reveals a role for inflammasomes in alphavirus-induced inflammation

Mosquito-borne viruses can cause severe inflammatory diseases and there are limited therapeutic solutions targeted specifically at virus-induced inflammation. Chikungunya virus (CHIKV), a re-emerging alphavirus responsible for several outbreaks worldwide in the past decade, causes debilitating joint inflammation and severe pain. Here, we show that CHIKV infection activates the NLRP3 inflammasome in humans and mice. Peripheral blood mononuclear cells isolated from CHIKV-infected patients showed elevated NLRP3, caspase-1 and interleukin-18 messenger RNA expression and, using a mouse model of CHIKV infection, we found that high NLRP3 expression was associated with peak inflammatory symptoms. Inhibition of NLRP3 activation using the small-molecule inhibitor MCC950 resulted in reduced CHIKV-induced inflammation and abrogated osteoclastogenic bone loss and myositis, but did not affect in vivo viral replication. Mice treated with MCC950 displayed lower expression levels of the cytokines interleukin-6, chemokine ligand 2 and tumour necrosis factor in joint tissue. Interestingly, MCC950 treatment abrogated disease signs in mice infected with a related arthritogenic alphavirus, Ross River virus, but not in mice infected with West Nile virus—a flavivirus. Here, using mouse models of alphavirus-induced musculoskeletal disease, we demonstrate that NLRP3 inhibition in vivo can reduce inflammatory pathology and that further development of therapeutic solutions targeting inflammasome function could help treat arboviral diseases.Chikungunya virus infection leads to painful arthritis-like joint inflammation. This study shows that the NLRP3 inflammasome is crucial for alphavirus-induced inflammation and its inhibition is an effective therapeutic strategy.