First study on the effect of transforming growth factor beta 1 and insulin-like growth factor 1 on the chondrogenesis of elephant articular chondrocytes in a scaffold-based 3D culture model.

Background and Aim: Osteoarthritis (OA) is recognized as a degenerative joint disease that leads to chronic pain and low quality of life in animals. Captive elephants, the largest land mammals with a long lifespan, are more prone to develop OA due to restricted spaces and insufficient physical activity. This study aimed to investigate the effect of transforming growth factor-ß1 (TGF-ß1) and insulin-like growth factor 1 (IGF-1) on elephant chondrogenesis in a scaffold culture of articular chondrocytes. Materials and Methods: Elephant chondrocytes-seeded gelatin scaffolds were cultured in chondrogenic media with or without 10 ng/mL of TGF-ß1 or IGF-1 alone or 5-10 ng/mL of their combination for up to 21 days. The mRNA expression of cartilage-specific anabolic genes, ACAN and COL2A1, was analyzed using a real-time reverse transcription-polymerase chain reaction. The amounts of sulfated glycosaminoglycans (sGAGs) in conditioned media and contents in cultured scaffolds were determined through dimethylmethylene blue assay. Cell morphology, accumulation of proteoglycans, and details of the cultured scaffolds were determined using hematoxylin-eosin staining, safranin O staining, and scanning electron microscopy (SEM), respectively. Results: TGF-ß1 alone significantly upregulated ACAN gene expression but not COL2A1, while IGF-1 alone did not enhance both ACAN and COL2A1 genes. The combination significantly upregulated both mRNA expression levels of ACAN and COL2A1 gene at day 14. The sGAGs accumulation and contents in the treatment groups, except IGF-1 tended to be higher than the controls, concomitantly with the production of the extracellular matrix, showed the formation of a cartilage-like tissue through histological and SEM analyses. Conclusion: Together, our results suggest that the single treatment of TGF-ß1 has a selective effect on ACAN gene, while the combined growth factors seem to be an advantage on elephant chondrogenesis. This three-dimensional culture model is probably helpful for developing cartilage regeneration in vitro and is further applied in tissue engineering for OA treatment in vivo.

Relationship among Serum Progestagens, Cortisol, and Prolactin in Pregnant and Cycling Asian Elephants in Thailand.

The aim of this study was to examine relationships among serum progestagens, cortisol, and prolactin in pregnant and normal cycling Asian elephants living in tourist camps in northern Thailand. Samples were collected twice a month for 22 months from nine elephants. Of those, four were pregnant (24.3 ± 2.9 years of age; range 21−28 years) and five (20.2 ± 9.6 years; range 8−34 years) exhibited normal ovarian cycles based on serum progestagen analyses. Gestation was divided into three periods: 1st (week 1−31), 2nd (week 32−62), and 3rd (week 63 to parturition), while the estrous cycle was divided into the follicular and luteal phases. Serum progestagens were higher during the luteal phase of the cycle (p < 0.003), whereas cortisol and prolactin were similar. In pregnant elephants, there were no differences in serum progestagens or cortisol concentrations across the three gestational periods, whereas prolactin concentrations increased significantly during the 2nd and 3rd periods (p < 0.0001). By contrast, prolactin concentrations in nonpregnant elephants were consistently low throughout the ovarian cycle. In one cycling female, prolactin concentrations were similar to pregnant elephants, perhaps because she was an allomother to two calves. Another cycling female exhibited consistently elevated cortisol concentrations, 5 to 10 times higher than the other elephants. There were no correlations between serum progestagens, cortisol, and prolactin throughout gestation; however, serum progestagens and cortisol were positively related in cycling elephants (r = 0.386, p < 0.001). From our results, there were a number of individual differences in reproductive hormonal patterns, so it is important to develop personalized monitoring programs for each elephant to enhance breeding success and create sustaining captive populations of elephants in Asia.

Kaempferia parviflora Extract Alleviated Rat Arthritis, Exerted Chondroprotective Properties In Vitro, and Reduced Expression of Genes Associated with Inflammatory Arthritis.

Kaempferia parviflora Wall. ex Baker (KP) has been reported to attenuate cartilage destruction in rat model of osteoarthritis. Previously, we demonstrated that KP rhizome extract and its active components effectively suppressed mechanisms associated with RA in SW982 cells. Here, we further evaluated the anti-arthritis potential of KP extract by using multi-level models, including a complete Freund's adjuvant-induced arthritis and a cartilage explant culture model, and to investigate the effects of KP extract and its major components on related gene expressions and underlying mechanisms within cells. In arthritis rats, the KP extract reduced arthritis indexes, with no significant changes in biological parameters. In the cartilage explant model, the KP extract exerted chondroprotective potential by suppressing sulfated glycosaminoglycans release while preserving high accumulation of proteoglycans. In human chondrocyte cell line, a mixture of the major components equal to their amounts in KP extract showed strong suppression the expression of genes-associated inflammatory joint disease similar to that of the extract. Additionally, KP extract significantly suppressed NF-κB and MAPK signaling pathways. The suppressing expression of necroptosis genes and promoted anti-apoptosis were also found. Collectively, these results provided supportive evidence of the anti-arthritis properties of KP extract, which are associated with its three major components.

Proinflammatory cytokines and lipopolysaccharides up regulate MMP-3 and MMP-13 production in Asian elephant (Elephas maximus) chondrocytes: attenuation by anti-arthritic agents.

BACKGROUND: Osteoarthritis (OA), the most common form of arthritic disease, results from destruction of joint cartilage and underlying bone. It affects animals, including Asian elephants (Elephas maximus) in captivity, leading to joint pain and lameness. However, publications regarding OA pathogenesis in this animal are still limited. Therefore, this study aimed to investigate the effect of proinflammatory cytokines, including interleukin-1 beta (IL-1ß), IL-17A, tumor necrosis factor-alpha (TNF-α), and oncostatin M (OSM), known mediators of OA pathogenesis, and lipopolysaccharides on the expression of cartilaginous degrading enzymes, matrix metalloproteinase (MMP)-3 and MMP-13, in elephant articular chondrocytes (ELACs) cultures. Anti-arthritic drugs and the active compounds of herbal plants were tested for their potential attenuation against overproduction of these enzymes. RESULTS: Among the used cytokines, OSM showed the highest activation of MMP3 and MMP13 expression, especially when combined with IL-1ß. The combination of IL-1ß and OSM was found to activate phosphorylation of the mitogen-activated protein kinase (MAPK) pathway in ELACs. Lipopolysaccharides or cytokine-induced expressions were suppressed by pharmacologic agents used to treat OA, including dexamethasone, indomethacin, etoricoxib, and diacerein, and by three natural compounds, sesamin, andrographolide, and vanillylacetone. CONCLUSIONS: Our results revealed the cellular mechanisms underlying OA in elephant chondrocytes, which is triggered by proinflammatory cytokines or lipopolysaccharides and suppressed by common pharmacological or natural medications used to treat human OA. These results provide a more basic understanding of the pathogenesis of elephant OA, which could be useful for adequate medical treatment of OA in this animal.

Proinflammatory Effects of IL-1ß Combined with IL-17A Promoted Cartilage Degradation and Suppressed Genes Associated with Cartilage Matrix Synthesis In Vitro.

Combinations of IL-1ß and other proinflammatory cytokines reportedly promote the severity of arthritis. We aimed to investigate the effects of IL-1ß combined with IL-17A on cartilage degradation and synthesis in in vitro models. Cartilage explant degradation was determined using sulfated glycosaminoglycans (S-GAGs) levels, matrix metalloproteinase (MMP13) gene expression, uronic acid, and collagen contents. Cell morphology and accumulation of proteoglycans were evaluated using hematoxylin-eosin and safranin O staining, respectively. In the pellet culture model, expressions of cartilage-specific anabolic and catabolic genes were evaluated using real-time qRT-PCR. Early induction of MMP13 gene expression was found concomitantly with significant S-GAGs release. During the prolonged period, S-GAGs release was significantly elevated, while MMP-13 enzyme levels were persistently increased together with the reduction of the cartilaginous matrix molecules. The pellet culture showed anabolic gene downregulation, while expression of the proinflammatory cytokines, mediators, and MMP13 genes were elevated. After cytokine removal, these effects were restored to nearly basal levels. This study provides evidence that IL-1ß combined with IL-17A promoted chronic inflammatory arthritis by activating the catabolic processes accompanied with the suppression of cartilage anabolism. These suggest that further applications, which suppress inflammatory enhancers, especially IL-17A, should be considered as a target for arthritis research and therapy.

TGF-ß1 upregulates the expression of hyaluronan synthase 2 and hyaluronan synthesis in culture models of equine articular chondrocytes.

We investigated the effect of transforming growth factor beta 1 (TGF-ß1) on equine hyaluronan synthase 2 (HAS2) gene expression and hyaluronan (HA) synthesis in culture models of articular chondrocytes. Equine chondrocytes were treated with TGF-ß1 at different concentrations and times in monolayer cultures. In three-dimensional cultures, chondrocyte-seeded gelatin scaffolds were cultured in chondrogenic media containing 10 ng/mL of TGF-ß1. The amounts of HA in conditioned media and in scaffolds were determined by enzyme-linked immunosorbent assays. HAS2 mRNA expression was analyzed by semi-quantitative reverse transcription polymerase chain reaction. The uronic acid content and DNA content of the scaffolds were measured by using colorimetric and Hoechst 33258 assays, respectively. Cell proliferation was evaluated by using the alamarBlue assay. Scanning electron microscopy (SEM), histology, and immunohistochemistry were used for microscopic analysis of the samples. The upregulation of HAS2 mRNA levels by TGF-ß1 stimulation was dose and time dependent. TGF-ß1 was shown to enhance HA and uronic acid content in the scaffolds. Cell proliferation and DNA content were significantly lower in TGF-ß1 treatments. SEM and histological results revealed the formation of a cartilaginous-like extracellular matrix in the TGF-ß1-treated scaffolds. Together, our results suggest that TGF-ß1 has a stimulatory effect on equine chondrocytes, enhancing HA synthesis and promoting cartilage matrix generation.

Comparison of physical body growth and metabolic and reproductive endocrine functions between north and south climates of Japan in trained Thoroughbred yearling horses.

This study aimed to compare body growth, metabolic, and reproductive hormonal changes in trained Thoroughbred yearling horses under different climate conditions with and without light supplementation (LS). Thoroughbred yearlings raised at research centers of the Japan Racing Association in Hokkaido (north) or Miyazaki (south) were divided into control and LS groups. In the LS groups, 44 colts and 47 fillies from Hokkaido and 11 colts and 11 fillies from Miyazaki were exposed to LS with an extended photoperiod of 14.5 hr of daylight and 9.5 hr of darkness. One week before and once a month after LS, circulating total thyroxine (T4), insulin-like growth factor-I (IGF-1), prolactin (PRL), cortisol, and progesterone (P4) concentrations were measured by radioimmunoassay and fluoroimmunoassay, respectively. Growth parameters, including body weight, height, girth, and cannon bone circumferences, were measured monthly. Hair coat (HC) condition was scored. Under natural conditions, the T4 concentrations of Hokkaido yearlings tended to be higher, whereas the IGF-1 (colt) and PRL levels were significantly lower than those of yearlings in Miyazaki. Growth parameters and HC scores were lower in Hokkaido yearlings. With LS, the PRL and P4 concentrations in Hokkaido and Miyazaki were higher, and the first ovarian activity tended to be earlier than in the controls. Only LS Hokkaido yearlings showed significantly higher HC scores than the controls. Comparing the different climates among the LS yearlings, the levels of PRL and P4 and the HC scores in Hokkaido yearlings increased and reached levels similar to those in Miyazaki yearlings. The body weight and girth increment percentages of Hokkaido yearlings in January dramatically decreased and then eventually increased to levels similar to those of Miyazaki yearlings. This suggested that yearlings in naturally colder Hokkaido exhibit higher basal metabolism to maintain homeostasis. However, providing LS may help to improve growth and early development of reproductive function in Hokkaido yearlings to levels equal to those of Miyazaki horses.

Sensitive radioimmunoassay of total thyroxine (T4) in horses using a simple extraction method.

Most thyroid hormone determinations in animals are based on immunoassays adapted from those used to test human samples, which may not reflect the actual values of thyroid hormone in horses because of the presence of binding proteins. The aims of the present study were i) to establish a novel radioimmunoassay (RIA) using a more simple and convenient method to separate binding proteins for the measurement of total thyroxine (T4) in horses and ii) to validate the assay by comparing total T4 concentrations in yearling horses raised in different climates. Blood samples were collected from trained yearlings in Hokkaido (temperate climate) and Miyazaki (subtropical climate) in Japan and from adult horses in estrus and diestrus. T4 was extracted from both serum and plasma using modified acid ethanol cryo-precipitation and sodium acetate ethanol methods. Circulating total T4 concentrations were determined by RIA. T4 concentration by sodium acetate ethanol was appropriately detectable rather than sodium salicylate method and was the same as for acid ethanol method. Furthermore, this sodium acetate ethanol method required fewer extraction steps than the other methods. Circulating T4 concentrations in yearlings were 225.98 ± 20.89 ng/ml, which was higher than the previous reference values. With respect to climate, T4 levels in Hokkaido yearlings tended to be higher than those in Miyazaki yearlings throughout the study period. These results indicated that this RIA protocol using a modified sodium acetate ethanol separation technique might be an appropriate tool for specific measurement of total T4 in horses.

Andrographolide Exerts Chondroprotective Activity in Equine Cartilage Explant and Suppresses Interleukin-1 ß -Induced MMP-2 Expression in Equine Chondrocyte Culture.

Cartilage erosion in degenerative joint diseases leads to lameness in affected horses. It has been reported that andrographolide from Andrographis paniculata inhibited cartilage matrix-degrading enzymes. This study aimed to explore whether this compound protects equine cartilage degradation in the explant culture model and to determine its effect on matrix metalloproteinase-2 (MMP-2) expression, a matrix-degrading enzyme, in equine chondrocyte culture. Equine articular cartilage explant culture was induced by 25 ng/mL interleukin-1ß, a key inducer of cartilage degeneration, in cultures with or without andrographolide ranging from 10 to 50 µM. After 3-21 days, they were analyzed for the markers of cartilage degradation. It was found that interleukin-1ß increased the release of sulfated glycosaminoglycans and hyaluronan from the explants into the culture media consistently with the decrease in uronic acid and collagen content in the cartilage explants. These catabolic effects were inhibited when cotreated with interleukin-1ß and andrographolide. In primary equine chondrocytes, andrographolide suppressed interleukin-1ß-induced MMP-2 mRNA expression and MMP-2 activity in the culture medium. These results confirmed the in vitro potent chondroprotective activities of this compound which were performed in cartilage explants and on a cellular level. These may indicate the application of andrographolide for therapeutic use in equine degenerative joint diseases.