Formononetin Antagonizes the Interleukin-1ß-Induced Catabolic Effects Through Suppressing Inflammation in Primary Rat Chondrocytes.

In the present study, we demonstrated the anti-catabolic effects of formononetin, a phytoestrogen derived from herbal plants, against interleukin-1ß (IL-1ß)-induced severe catabolic effects in primary rat chondrocytes and articular cartilage. Formononetin did not affect the viability of primary rat chondrocytes in both short- (24 h) and long-term (21 days) treatment periods. Furthermore, formononetin effectively antagonized the IL-1ß-induced catabolic effects including the decrease in proteoglycan content, suppression of pericellular matrix formation, and loss of proteoglycan through the decreased expression of cartilage-degrading enzymes like matrix metalloproteinase (MMP)-13, MMP-1, and MMP-3 in primary rat chondrocytes. Moreover, catabolic oxidative stress mediators like nitric oxide, inducible nitric oxide synthase, cyclooxygenase-2, and prostaglandin E2 were significantly downregulated by formononetin in primary rat chondrocytes treated with IL-1ß. Sequentially, the upregulation of pro-inflammatory cytokines (like IL-1α, IL-1ß, IL-6, and tumor necrosis factor α), chemokines (like fractalkine, monocyte chemoattractant protein-1, and macrophage inflammatory protein-3α), and vascular endothelial growth factor were significantly downregulated by formononetin in primary rat chondrocytes treated with IL-1ß. These data suggest that formononetin may suppress IL-1ß-induced severe catabolic effects and osteoarthritic condition. Furthermore, formononetin may be a promising candidate for the treatment and prevention of osteoarthritis.

Aqueous extract of Codium fragile alleviates osteoarthritis through the MAPK/NF-κB pathways in IL-1ß-induced rat primary chondrocytes and a rat osteoarthritis model.

BACKGROUND: Codium fragile (Suringar) Hariot has been used as a potential remedy in traditional medicine because of its anti-inflammatory and anti-oxidant effects. Osteoarthritis is a chronic progressive joint disease, characterized by complex mechanisms related to inflammation and degeneration of articular cartilage. In this study, we aimed to evaluate the cartilage protective effect of an aqueous extract of Codium fragile (AECF) using rat primary chondrocytes and the osteoarthritis animal model induced by destabilization of the medial meniscus (DMM). METHODS: In vitro, rat primary cultured chondrocytes were pre-treated with AECF (0.5, 1, and 2mg/mL) for 1h and then incubated with interleukin-1ß (10ng/mL) for 24h. Nitrite production was detected by the Griess reagent. Alteration of the protein levels of iNOS, MMP-13, ADAMTS-4, ADAMTS-5, mitogen-activated protein kinases (MAPKs), and nuclear factor-κB (NF-κB) was detected by western blotting. In vivo, osteoarthritis was induced by DMM of Sprague Dawley (SD) rats. The rats subjected to destabilization of the medial meniscus (DMM) surgery were orally administered with AECF (50, 100, and 200mg/kg bodyweight) or distilled water for 8w. The severity of cartilage lesions was evaluated by safranin O staining and the Osteoarthritis Research Society International (OARSI) score. RESULTS: These results demonstrated that AECF significantly inhibited nitrite production and inhibited the levels of iNOS, MMP-13, ADAMTS-4, and ADAMTS-5 in interleukin-1ß-induced rat primary cultured chondrocytes. Moreover, AECF suppressed interleukin-1ß-induced NF-κB activation in the nucleus and phosphorylation of ERK1/2 and JNK in the cytosol. In vivo, the cartilage lesions in AECF-treated osteoarthritis rats exhibited less proteoglycan loss and lower OARSI scores. CONCLUSIONS: These results suggested that AECF is a potential therapeutic agent for the alleviation of osteoarthritis progression.

Coumestrol Counteracts Interleukin-1ß-Induced Catabolic Effects by Suppressing Inflammation in Primary Rat Chondrocytes.

In the present study, we investigated the anti-catabolic effects of coumestrol, a phytoestrogen derived from herbal plants, against interleukin-1ß-induced cartilage degeneration in primary rat chondrocytes and articular cartilage. Coumestrol did not affect the viability of human normal oral keratinocytes and primary rat chondrocytes treated for 24 h and 21 days, respectively. Although coumestrol did not significantly increase the proteoglycan contents in long-term culture, it abolished the interleukin-1ß-induced loss of proteoglycans in primary rat chondrocytes and knee articular cartilage. Furthermore, coumestrol suppressed the expression of matrix-degrading enzymes such as matrix metalloproteinase-13, -3, and -1 in primary rat chondrocytes stimulated with interleukin-1ß. Moreover, the expression of catabolic factors such as nitric oxide synthase, cyclooxygenase-2, prostaglandin E2, and inflammatory cytokines in interleukin-1ß-stimulated primary rat chondrocytes was suppressed by coumestrol. In summary, these results indicate that coumestrol counteracts the catabolic effects induced by interleukin-1ß through the suppression of inflammation. Therefore, based on its biological activity and safety profile, coumestrol could be used as a potential anti-catabolic biomaterial for osteoarthritis.

Biological Effects of the Herbal Plant-Derived Phytoestrogen Bavachin in Primary Rat Chondrocytes.

The aim of this study was to examine the anabolic and anticatabolic functions of bavachin in primary rat chondrocytes. With bavachin treatment, chondrocytes survived for 21 d without cell proliferation, and the proteoglycan content and extracellular matrix increased. Short-term monolayer culture of chondrocytes showed that gene induction of both aggrecan and collagen type II, major extracellular matrix components, was significantly upregulated by bavachin. The expression and activities of cartilage-degrading enzymes such as matrix metalloproteinases and a disintegrin and metalloproteinase with thrombospondin motifs were inhibited significantly by bavachin, while tissue inhibitors of metalloprotease were significantly upregulated. Bavachin inhibits the expression of inducible nitric oxide synthase, a representative catabolic factor, and downregulated the expression of nitric oxide, cyclooxygenase-2, and prostaglandin E2 in a dose-dependent manner in chondrocytes. Our results suggest that the bavachin has anabolic and potent anticatabolic biological effects on chondrocytes, which may have considerable promise in treating articular cartilage degeneration in the future.

Berberine induces FasL-related apoptosis through p38 activation in KB human oral cancer cells.

In the present study, we examined the anticancer properties of berberine in KB oral cancer cells with a specific focus on its cellular mechanism. Berberine did not affect the cell viability of the primary human normal oral keratinocytes that were used as a control. However, the viability of KB cells was found to decrease significantly in the presence of berberine in a dose-dependent manner. Furthermore, in KB cells, berberine induced the fragmentation of genomic DNA, changes in cell morphology, and nuclear condensation. In addition, caspase-3 and -7 activation, and an increase in apoptosis were observed. Berberine was also found to upregulate significantly the expression of the death receptor ligand, FasL. In turn, this upregulation triggered the activation of pro-apoptotic factors such as caspase-8, -9 and -3 and poly(ADP-ribose) polymerase (PARP). Furthermore, pro-apoptotic factors such as Bax, Bad and Apaf-1 were also significantly upregulated by berberine. Anti-apoptotic factors such as Bcl-2 and Bcl-xL were downregulated. Z-VAD-FMK, a cell-permeable pan-caspase inhibitor, suppressed the activation of caspase-3 and PARP. These results clearly indicate that berberine-induced cell death of KB oral cancer cells was mediated by both extrinsic death receptor-dependent and intrinsic mitochondrial-dependent apoptotic signaling pathways. In addition, berberine-induced upregulation of FasL was shown to be mediated by the p38 MAPK signaling pathway. We also found that berberine-induced migration suppression was mediated by downregulation of MMP-2 and MMP-9 through phosphorylation of p38 MAPK. In summary, berberine has the potential to be used as a chemotherapeutic agent, with limited side-effects, for the management of oral cancer.

Licochalcone-A induces intrinsic and extrinsic apoptosis via ERK1/2 and p38 phosphorylation-mediated TRAIL expression in head and neck squamous carcinoma FaDu cells.

We investigated Licochalcone-A (Lico-A)-induced apoptosis and the pathway underlying its activity in a pharyngeal squamous carcinoma FaDu cell line. Lico-A purified from root of Glycyrrhiza inflata had cytotoxic effects, significantly increasing cell death in FaDu cells. Using a cell viability assay, we determined that the IC50 value of Lico-A in FaDu cells was approximately 100 µM. Chromatin condensation was observed in FaDu cells treated with Lico-A for 24 h. Consistent with this finding, the number of apoptotic cells increased in a time-dependent manner when FaDu cells were treated with Lico-A. TRAIL was significantly up-regulated in Lico-A-treated FaDu cells in a dose-dependent manner. Apoptotic factors such as caspases and PARP were subsequently activated in a caspase-dependent manner. In addition, levels of pro-apoptotic factors increased significantly in response to Lico-A treatment, while levels of anti-apoptotic factors decreased. Lico-A-induced TRAIL expression was mediated in part by a MAPK signaling pathway involving ERK1/2 and p38. In xenograft mouse model, Lico-A treatment effectively suppressed the growth of FaDu cell xenografts by activating caspase-3, without affecting the body weight of mice. Taken together, these data suggest that Lico-A has potential chemopreventive effects and should therefore be developed as a chemotherapeutic agent for pharyngeal squamous carcinoma.

Anticancer activity of Saussurea lappa extract by apoptotic pathway in KB human oral cancer cells.

CONTEXT: Saussurea lappa Dence (Compositae) is used as a traditional herbal medicine to treat abdominal pain and tenesmus in East Asia. Current studies have shown that S. lappa has anticancer activity in divergent of cancer cells. However, the effects of S. lappa on oral cancer and its mechanisms of action have yet to be elucidated. OBJECTIVE: To explore its potential chemotherapeutic effects and mechanism of cell growth inhibition on human oral cancer cells. MATERIALS AND METHODS: The dried roots of S. lappa were used in this study. Cell viability of KB cells was evaluated by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide assay after treatment with 30 µg/ml of methanol extract from the dried roots of S. lappa. To understand whether its effect on cell death is related with apoptosis pathway, we performed DNA fragmentation assay, western blot, caspase activity assay and fluorescence-activated cell sorting (FACS) analysis. RESULTS: Treatment of S. lappa extract onto KB cells reduced cell viability significantly with an IC50 value of 30 µg/ml. The formation of a DNA ladder was observed starting at the 24 h treatment. In western blotting analysis, the S. lappa extract induced the proteolytic processing of caspase-3, -9 and poly (ADP-ribose) polymerase, a significant increase of Bax and marked reduction of Bcl-2. We also confirmed the activation of caspase-3/-7 in living KB cells by fluorescence microscopy. CONCLUSION: These results suggested that S. lappa extract inhibited cell proliferation through the apoptosis pathway in KB human oral cancer cells.

Enhancement of rutin in Fagopyrum esculentum hairy root cultures by the Arabidopsis transcription factor AtMYB12.

Common buckwheat (Fagopyrum esculentum Moench) is rich in phenolic compounds and may be useful for the treatment of metabolic syndrome in humans. To improve the production of rutin in buckwheat, we overexpressed the flavonol-specific transcription factor, AtMYB12 using Agrobacterium rhizogenes into hairy root culture systems. This induced the expression of flavonoid biosynthetic genes encoding phenylalanine ammonia lyase, cinnamate 4-hydroxylase, 4-coumarate:CoA ligase, chalcone synthase, chalcone isomerase, flavone 3-hydroxylase, flavonoid 3'-hydroxylase, and flavonol synthase. This led to the accumulation of rutin in buckwheat hairy roots up to 0.9 mg/g dry wt. PAP1 expression, however, did not correlate with the production of rutin.

Osteogenic activity of the mixture of chitosan and particulate dentin.

To evaluate the healing effect of a mixture of chitosan and particulate dentin, a 8-mm-diameter critical size defect was created in the calvarium of 75 rats. The rats were divided into four experimental groups and a control group (no treatment). The defects in the experimental groups were grafted either with pig particulate dentin (group 1), a mixture of particulate dentin and plaster of Paris (group 2), particulate dentin and chitosan (group 3), or chitosan only (group 4). Rats in each group were sacrificed 2, 4, and 8 weeks after implantation. All experimental groups showed more new bone formation when compared to the control group. Additionally, all groups exhibited more bone growth at 8 weeks than at 4 weeks after implantation. It was concluded from this study that defects treated with particulate dentin powder-chitosan mixture may mediate an excellent effect on the formation of new bones.

Expression of amino acid transporter LAT1 and 4F2hc in the healing process after the implantation of a tooth ash and plaster of Paris mixture.

BACKGROUND: In order to elucidate the expression pattern of L-type amino acid transporter 1 (LAT1) in the bone formation process, the expressions of LAT1 and its subunit 4F2 heavy chain (4F2hc) were investigated in the healing process after the implantation of a tooth ash-plaster of Paris mixture in rats with calvarial osseous defect. MATERIALS AND METHODS: Circular calvarial defects (8 mm in diameter) were made midparietally. The rats were divided into 2 groups, 1 control group and 1 experimental group. In the control group, the defect was only covered with a soft tissue flap (control group); in the experimental group, it was filled with a mixture of tooth ash and plaster of Paris (2:1 by weight; mixture group). The rats were sacrificed at 1, 2, 4 and 8 weeks after operation and RT-PCR and immunohistochemical analyses were performed. RESULTS: In the RT-PCR analysis, the expressions of the LAT1 and 4F2hc mRNAs were slightly stronger in the experimental group than in the control group. In the immunohistochemical analysis, at 1 week after operation, the LAT1 protein and its subunit 4F2hc protein were mainly expressed in the osteoblasts, osteocytes and interstitial tissues of the area around the defect and the inner part of newly forming bone in both groups. The expressions of LAT1 and 4F2hc proteins were decreased at 2 and 4 weeks after operation. The LAT1 and 4F2hc proteins were scarcely expressed at 8 weeks after operation in both groups. The expressions of LAT1 and 4F2hc proteins were slightly stronger in the mixture group than in the control group. CONCLUSION: These results suggest that the LAT1 and its subunit 4F2hc are highly expressed in the early stage of new bone formation and may have an important role in providing cells with neutral amino acids, including several essential amino acids, at that stage.

Effect of low-level laser therapy on osteoarthropathy in rabbit.

The aim of this study was to determine whether low-level laser therapy (LLLT) aided the recovery of damaged articular cartilage in joints with artificially induced osteoarthropathy (OA). OA was induced by injecting hydrogen peroxide (H2O2) into the articular spaces of both knees in rabbits, twice a week for 4 weeks. The induction of OA and the effect of LLLT were evaluated by biochemical, radiological and histopathological analysis. Superoxide dismutase (SOD) activity increased about 40% in the OA group, as compared to the controls. Although SOD activity in the OA group was not significantly different from the 2-week groups, it was significantly different from the 4-week control and treatment groups. There was also a significant difference between the 4-week control and treatment groups. Simple radiographs and three-dimensional computed tomographs (3D CT) did not show detectable arthropathy in the OA group, nor any particular changes in the 2-week groups. In contrast, distinct erosions were seen in the distal articular cartilage of the femur, with irregularity of the articular surface, in the 4-week control group, while the erosions were reduced and arthropathy improved slightly in the 4-week treatment group. Grossly, erosions formed on the articular surface in the OA group. In comparison, severe erosions damaged the articular cartilage in the 4-week control group, but not in the 2-week control and treatment groups. Regeneration of articular cartilage was seen in gross observations in the 4-week treatment group. Histopathologically, there was slight irregularity of the articular surface and necrosis in the OA group, and serious cartilage damage, despite slight chondrocyte regeneration, in the 4-week control group. Conversely, the 4-week treatment group showed chondrocyte replacement, with sometimes close to normal articular cartilage on the articular surface. These results suggest that LLLT was effective in the treatment of chemically-induced OA.

Effect of Ga-as laser on the regeneration of injured sciatic nerves in the rat.

Laser irradiation is one of the therapeutic methods for the recovery of degenerated peripheral nerves. The aim of the present study was to determine if low-power laser treatment stimulates the regeneration process of damaged nerves. A standardized crush to the sciatic nerve was applied to cause extensive axonal degeneration. After this procedure, low-power infrared laser irradiation was administered transcutaneously to the injured sciatic nerve, 3 minutes daily to each of four treatment groups for 1, 3, 5 and 7 weeks, respectively. A nerve conduction study was done, and a morphological assessment was performed using both light and electron microscopy. With trauma of the nerve, both amplitude of compound motor action potential and nerve conduction velocity decreased significantly compared to the pre-trauma state. Morphologically, the numbers of myelinated axons and degenerated axons were decreased and increased, respectively, compared with the control. Typical aspects were of onion skin-type lamellation, fragmentation, edematous swelling and rarefaction in the myelin sheath. All these parameters recovered almost to the level of the pre-trauma state with laser irradiation, in direct proportion to the time spent for treatment. These results suggest that low-power infrared laser irradiation can relieve the mechanical damage of sciatic nerves and stimulate the regeneration of peripheral nerves.

Effects on bone formation in ovariectomized rats after implantation of tooth ash and plaster of Paris mixture.

PURPOSE: Our goal was to report on tooth ash and plaster of Paris mixture in bone defects in an ovariectomized rat osteoporosis model. MATERIALS AND METHODS: Sixty rats were randomly assigned to 4 groups and each group was further divided into 3 subgroups: 4, 8, and 16 weeks after implantation. The defect was filled with different grafting conditions as follows: group 1, ovariectomy and nongraft group; group 2, ovariectomy and tooth ash-plaster graft group; group 3, nonovariectomy and nongraft group; and group 4, nonovariectomy and tooth ash-plaster graft group. Histologic sections and histomorphometric analysis of defects were obtained 4, 8, and 16 weeks after surgery. RESULTS: For the 4-week ovariectomy group, there was significantly greater bone formation in tooth ash-plaster group compared with the nongraft group. In the nonovariectomy group, the tooth ash-plaster group also showed better bone formation than the nongraft group. However, there was no statistical significance. In both the ovariectomy and nonovariectomy groups, a significant increase in bone formation was observed according to the elapse of time. The nonovariectomy group showed increased new bone formation compared with the ovariectomy group, with the tooth ash-plaster group showing statistical significance in each subgroup (P =.048). CONCLUSIONS: Ovariectomy acts as a negative factor in new bone formation. For a critical size bony defect, the tooth ash-plaster treatment of the osseous defect produces more stable, effective, and rapid new bone formation.

Use of particulate dentin-plaster of Paris combination with/without platelet-rich plasma in the treatment of bone defects around implants.

PURPOSE: To evaluate the effect of particulate dentin-plaster of Paris with and without platelet-rich plasma (PRP) on bone healing and new bone formation around titanium dental implants in a canine model. Histologic sections and histomorphometric analysis of the defects were obtained at 6 and 12 weeks after surgery. MATERIALS AND METHODS: Three circular bone defects were surgically prepared in iliac crest sites in each of 10 animals. A total of 30 Avana dental implants were placed in the animals. They were self-tapping, screw-type implants, 10 mm in length and 4 mm in diameter, all made of commercially pure titanium. A titanium implant was placed centrally in each defect. In each dog, the defects were treated with 1 of the following 3 treatment modalities: (1) no treatment (control); (2) grafting with particulate dentin-plaster of Paris; (3) grafting with particulate dentin-plaster of Paris and PRP. RESULTS: Histologic analysis showed that all of the bone defects surrounding the implants that were treated with particulate dentin-plaster of Paris, with and without PRP, were filled with new bone. The defects that were not treated (control) demonstrated new bone formation only in the inferior threaded portion of the implants. DISCUSSION: Histomorphometric results revealed a higher percentage of bone contact with particulate dentin-plaster of Paris and PRP compared to the control and particulate dentin-plaster of Paris. CONCLUSIONS: These results suggested that bone defects around titanium implants can be treated successfully with particulate dentin-plaster of Paris, and that the outcome can be improved if PRP is also used.