Effects of different omega-3 sources, fish oil, krill oil, and green-lipped mussel against cytokine-mediated canine cartilage degradation.

Our purpose was to evaluate the protective effect of three marine omega-3 sources, fish oil (FO), krill oil (KO), and green-lipped mussel (GLM) against cartilage degradation. Canine cartilage explants were stimulated with either 10 ng/mL interleukin-1ß (IL-1ß) or IL-1ß/oncostatin M (10 ng/mL each) and then treated with various concentrations of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA; 3 and 30 µg/mL), FO, KO, or GLM (250, 500, and 1000 µg/mL) for 28 days. Gene expression was then investigated in primary canine chondrocytes. Our results showed that DHA and EPA as well as omega-3 sources could suppress matrix degradation in cytokine-induced cartilage explants by significantly reducing the increase of sulfated glycosaminoglycans (s-GAGs) and preserving uronic acid and hydroxyproline content (except GLM). These agents were not able to reduce IL-1ß-induced IL1B and TNFA expression but were able to down-regulate the expression of the catabolic genes MMP1, MMP3, and MMP13 and up-regulate the anabolic genes AGG and COL2A1; FO and KO were especially effective. Our findings indicated that FO and KO were superior to GLM for their protective effect against proteoglycan and collagen degradation. Hence, FO and KO could serve as promising sources of chondroprotective agents.

Suppression of Cartilage Degradation by Zingerone Involving the p38 and JNK MAPK Signaling Pathway.

Zingerone, an active compound that is present in cooked ginger, has been claimed to be a bioactive ingredient that holds the potential of preventing and/or treating diseases involving inflammation. In this study, zingerone was used to discover its properties against joint inflammation using interleukin-1ß-induced osteoarthritis in cartilage explant and cell culture models. Zingerone was supplemented into the cartilage explant and cell culture media at different concentrations along with the presence of interleukin-1ß, an inducer of osteoarthritis. Markers indicating cartilage degradation, inflammation, and the signaling molecules involved in the inflammatory induction were investigated. Diacerien, an anti-osteoarthritic drug, was used as a positive control. Zingerone at a concentration of 40 µM reduced the level of matrix metalloproteinase-13 to about 31.95 ± 4.33 % compared with the interleukin-1ß-treated group and halted cartilage explant degradation as indicated by reducing the accumulative release of sulfated glycosaminoglycans by falling to the control concomitantly with an elevation of the remaining contents of uronic acid and collagen in the explant tissues when zingerone was added. In the SW1353 cell line model, zingerone efficiently suppressed the expression of TNF-α, interleukin-6, and interleukin-8 mRNA levels and tended to reduce the levels of both p38 and c-Jun N-terminal kinase phosphorylation. From the results of this study, it can be concluded that zingerone potentially reduced cartilage degradation, which is partially involved in p38 and c-Jun N-terminal kinases of the mitogen activator protein kinase signaling pathway leading to the reduction of proinflammatory cytokine amplification effects and cartilage-degrading enzyme syntheses. This finding supports the contention that ginger holds positive pharmaceutical effects against osteoarthritis.

Elemental Analysis of Bone, Teeth, Horn and Antler in Different Animal Species Using Non-Invasive Handheld X-Ray Fluorescence.

Mineralized tissues accumulate elements that play crucial roles in animal health. Although elemental content of bone, blood and teeth of human and some animal species have been characterized, data for many others are lacking, as well as species comparisons. Here we describe the distribution of elements in horn (Bovidae), antler (Cervidae), teeth and bone (humerus) across a number of species determined by handheld X-ray fluorescence (XRF) to better understand differences and potential biological relevance. A difference in elemental profiles between horns and antlers was observed, possibly due to the outer layer of horns being comprised of keratin, whereas antlers are true bone. Species differences in tissue elemental content may be intrinsic, but also related to feeding habits that contribute to mineral accumulation, particularly for toxic heavy metals. One significant finding was a higher level of iron (Fe) in the humerus bone of elephants compared to other species. This may be an adaptation of the hematopoietic system by distributing Fe throughout the bone rather than the marrow, as elephant humerus lacks a marrow cavity. We also conducted discriminant analysis and found XRF was capable of distinguishing samples from different species, with humerus bone being the best source for species discrimination. For example, we found a 79.2% correct prediction and success rate of 80% for classification between human and non-human humerus bone. These findings show that handheld XRF can serve as an effective tool for the biological study of elemental composition in mineralized tissue samples and may have a forensic application.

In vitro cytotoxic screening of 31 crude extracts of Thai herbs on a chondrosarcoma cell line and primary chondrocytes and apoptotic effects of selected extracts.

Thirty-one dichloromethane and methanol crude extracts of 16 herb species used in Thai traditional folk medicine were studied for their cytotoxic activities on the SW 1353 chondrosarcoma cell line and primary chondrocytes. Methyl thiazolyl tetrazolium (MTT) cell viability assay and flow cytometric method were used as screening tools for cytotoxicity testing. The half maximal inhibitory concentration (IC50) was measured and reported for each crude extract. Apoptosis, necrosis, and cell viability were measured by flow cytometry at IC50. Two out of 31 herbal extracts, methanol extracts of Paris polyphylla var. chinensis and Ficus thailandica C.C. Berg & S. Gardner, showed potent anticancer activity. They demonstrated high apoptosis induction activity in SW 1353 cells but had less effect on percentage of viability and necrosis of normal chondrocyte cells. Cytotoxic screening and apoptosis assays suggest the potential anticancer activity of some plants used in Thai traditional medicine and provide information concerning their direct effects.

Effects of one-time and two-time intra-articular injection of hyaluronic acid sodium salt after joint surgery in dogs.

Thirty-one dogs with patellar luxation (grades 2 and 3) were categorized into three groups. Group 1 (G.1; n = 12) had sodium hyaluronate (SHA) intra-articularly injected into the stifle joint that received surgery. Group 2 (G.2; n = 10) received SHA twice: first after surgery and then 1 week later. Group 3 (G.3; n = 9) served as a control, without injection. Blood was collected before injection and then once a week for 4 weeks after injection for evaluation of chondroitin sulfate (CS-WF6) and hyaluronan (HA). The results revealed significantly (p < 0.05) improved clinical scores by the end of week 4 in G.1 and G.2 relative to G.3; however, there was no significant difference between G.1 and G.2. There was a significant decrease (p < 0.05) in serum CS-WF6 levels beginning at week 2 in G.1 and G.2. At weeks 3 and 4, serum HA in G.1 and G.2 differed from that in G.3 (p < 0.05). No significant difference (p > 0.05) was observed in serum biomarkers between G.1 and G.2. In conclusion, intra-articular injection with SHA after joint surgery may improve homeostasis of the joint, retarding the process of OA.

In vitro suppression of the MMP-3 gene in normal and cytokine-treated human chondrosarcoma using small interfering RNA.

BACKGROUND: Matrix metalloproteinase (MMPs) synthesized and secreted from connective tissue cells have been thought to participate in degradation of the extracellular matrix. Increased MMPs activities that degrade proteoglycans have been measured in osteoarthritis cartilage. This study aims to suppress the expression of the MMP-3 gene in in vitro human chondrosarcoma using siRNA. METHODS: CELLS WERE CATEGORIZED INTO FOUR GROUPS: control (G.1); transfection solution treated (G.2); negative control siRNA treated (G.3); and MMP-3 siRNA treated (G.4). All four groups were further subdivided into two groups - treated and non-treated with IL-1beta- following culture for 48 and 72 h. We observed the effects of gene suppression according to cell morphology, glycosaminoglycan (GAG) and hyaluronan (HA) production, and gene expression by using real-time polymerase chain reaction (PCR). RESULTS: In IL-1beta treated cells the apoptosis rate in G.4 was found to be lower than in all other groups, while viability and mitotic rate were higher than in all other groups (p < 0.05). The production of GAG and HA in G.4 was significantly higher than the control group (p < 0.05). MMP-3 gene expression was downregulated significantly (p < 0.05). CONCLUSION: MMP-3 specific siRNA can inhibit the expression of MMP-3 in chondrosarcoma. This suggests that MMP-3 siRNA has the potential to be a useful preventive and therapeutic agent for osteoarthritis.

Evaluation of serum chondroitin sulfate and hyaluronan: biomarkers for osteoarthritis in canine hip dysplasia.

Hip dysplasia (HD) is one of the most important bone and joint diseases in dogs. Making the radiographic diagnosis is sometime possible when the disease has markedly progressed. Chondroitin sulfate (CS) and hyaluronan (HA) are the most important cartilage biomolecules that are elevated in the serum taken from dogs with osteoarthritis. The serum CS and HA can be detected by an ELISA technique, with using monoclonal antibodies against CS epitope 3B3 and WF6 and the HA chain as the primary antibodies. The aim of this study was to compare the levels of serum CS (both epitopes) and HA in non-HD and HD dogs. All 123 dogs were categorized into 2 groups. The non-HD group was composed of 98 healthy dogs, while the HD group was comprised of 25 HD dogs. Blood samples were collected for analyzing the serum CS and HA levels with using the ELISA technique. The results showed that the average serum level of the CS epitope WF6 in the HD group (2,594 +/- 3,036.10 ng/ml) was significantly higher than that in the non-HD group (465 +/- 208.97 ng/ml) (p < 0.01) while the epitope 3B3 in the HD group (105 +/- 100.05 ng/ml) was significantly lower than that in the non-HD group (136 +/- 142.03 ng/ml) (p < 0.05). The amount of serum HA in the HD group (134.74 +/- 59.71 ng/ml) was lower than that in the non HD group (245.45 +/- 97.84 ng/ml) (p < 0.05). The results indicate that the serum CS and HA levels might be used as biomarkers for osteoarthritis in HD dogs.

Selective degradation of maternal and embryonic transcripts in in vitro produced bovine oocytes and embryos using sequence specific double-stranded RNA.

RNA interference (RNAi) has been used for selective degradation of an mRNA transcript or inhibiting its translation to a functional protein in various species. Here, we applied the RNAi approach to suppress the expression of the maternal transcript C-mos and embryonic transcripts Oct-4 in bovine oocytes and embryos respectively, using microinjection of sequence-specific double-stranded RNA (dsRNA). For this, 435 bp C-mos and 341 bp Oct-4 dsRNA were synthesized and microinjected into the cytoplasm of immature oocytes and zygotes respectively. In experiment 1, immature oocytes were categorized into three groups: those injected with C-mos dsRNA, RNase-free water and uninjected controls. In experiment 2, in vitro produced zygotes were categorized into three groups: those injected with Oct-4 dsRNA, RNase-free water and uninjected controls. The developmental phenotypes, the level of mRNA and protein expression were investigated after treatment in both experiments. Microinjection of C-mos dsRNA has resulted in 70% reduction of C-mos transcript after maturation compared to the water-injected and uninjected controls (P<0.01). Microinjection of zygotes with Oct-4 dsRNA has resulted in 72% reduction in transcript abundance at the blastocyst stage compared to the uninjected control zygotes (P<0.01). Moreover, a significant reduction in the number of inner cell mass (ICM) cells was observed in Oct-4 dsRNA-injected embryos compared to the other groups. From oocytes injected with C-mos dsRNA, 60% showed the extrusion of the first polar body compared to 50% in water-injected and 44% in uninjected controls. Moreover, only oocytes injected with C-mos dsRNA showed spontaneous activation. In conclusion, our results demonstrated that sequence-specific dsRNA can be used to knockdown maternal or embryonic transcripts in bovine embryogenesis.