A new species of the Cyrtodactylusbrevipalmatus group (Squamata, Gekkonidae) from Tak Province, northwestern Thailand.

An integrative taxonomic analysis was used to delimit and diagnose a new species of the Cyrtodactylusbrevipalmatus group from Tak Province in western Thailand. Although Bayesian phylogenetic analyses place C.denticulatussp. nov. within the brevipalmatus group, the new species is neither nested within nor is it the sister species of any other species in the brevipalmatus group. Furthermore, based on the mitochondrial NADH dehydrogenase subunit 2 gene (ND2) and adjacent tRNAs, it bears an uncorrected pairwise sequence divergence of 7.87-21.94% from all other species in the brevipalmatus group. Cyrtodactylusdenticulatussp. nov. is differetiated from all other species in the brevipalmatus group by having a number of unique charateristics such as denticulate ventrolateral body folds and ventrolateral subcaudal ridges, characters not seen in any other species of the group (n = 51 individuals). Additionally, based on a multiple factor anlaysis, C.denticulatussp. nov. does not overlap with any other species in multivariate space. The discovery of C.denticulatussp. nov. underscores the unrealized diversity of upland ecosystems across Thailand and the urgent need for increased exploration and conservation of these unique imperiled montane refugia, especially in this era of climate change.

RPA/CRISPR-cas12a as a specific, sensitive and rapid method for diagnosing Ehrlichia canis and Anaplasma platys in dogs in Thailand.

Rickettsial pathogens including Ehrlichia canis and Anaplasma platys are bacteria that cause parasitic infections in dogs such as canine monocytic ehrlichiosis (CME) and canine cyclic thrombocytopenia (CCT), respectively affecting mortality and morbidity worldwide. An accurate, sensitive, and rapid method to diagnose these agents is essential for effective treatment. In this study, a recombinase polymerase amplification (RPA) coupled with CRISPR-Cas12a methods was established to detect E. canis and A. platys infection in dogs based on the 16S rRNA. The optimal condition for DNA amplification by RPA was 37 °C for 20 min, followed by CRISPR-Cas12a digestion at 37 °C for one hour. A combination of RPA and the cas12a detection method did not react with other pathogens and demonstrated strong sensitivity, detecting as low as 100 copies of both E. canis and A. platys. This simultaneous detection method was significantly more sensitive than conventional PCR. The RPA-assisted cas12a assay provides specific, sensitive, rapid, simple and appropriate detection of rickettsial agents in canine blood at the point-of-care for diagnostics, disease prevention and surveillance.

Herbal species authentication by melting fingerprint coupled with high resolution melting analysis (MF-HRM).

BACKGROUND: Herbal medicines have recently attracted increasing attention for use as food supplements with health benefits; however, species authentication can be difficult due to incomplete morphological characters. Here, a molecular tool was developed for the identification of species in the National List of Essential Medicinal Plants in Thailand. METHODS: The identification process used DNA fingerprints including start codon targeted (SCoT) and inter simple sequence repeat (ISSR) polymorphisms, coupled with high resolution melting (HRM), to produce melting fingerprint (MF)-HRM. RESULTS: Results indicated that MF-HRM, SCoT-HRM and ISSR-HRM could be used for DNA fingerprints as S34, S36, S9 and S8 of SCoT and UBC873, S25 and UBC841 of ISSR. The melting fingerprints obtained from S34 of SCoT exhibited the best primers for identification of herbal species with 87.5% accuracy and relatively high repeatability. The presence of intraspecific variation in a few species affected the shift of melting fingerprints within species. MF-HRM using S34 showed improved species prediction compared to DNA fingerprints. The concentration of DNA with 10 ng/µl was recommended to perform MF-HRM. MF-HRM enabled species authentication of herbal commercialized products at only 20% resulting from the low quality of DNA isolated, while admixture of multiple product species interfered with the MF process. CONCLUSION: Findings suggested that MF-HRM showed promise as a molecular tool for the authentication of species in commercial herbal products with high specificity, moderate repeatability and rapidity without prior sequence information. This information will greatly improve quality control and traceability during the manufacturing process.

Sensitive and rapid detection of Babesia species in dogs by recombinase polymerase amplification with lateral flow dipstick (RPA-LFD).

Canine babesiosis is a tick-borne disease caused by Babesia spp., which infects and destroys healthy erythrocytes, leading to mortality and morbidity in dogs. The diagnosis of babesiosis is tedious and time-consuming, especially in latent and chronic infections. Here, a recombinase polymerase amplification combined with a lateral flow dipstick (RPA-LFD) assay was developed for rapid and accurate detection of Babesia spp. in canine blood specimens based on the 18S rRNA region. The RPA-LFD assay using rpaBab264 gave specificity to Babesia spp. in dogs (B. vogeli and B. gibsoni) without cross-amplification to other parasites (apicomplexans and non-apicomplexans), with detection limit of at least 22.5 copies/µl (0.1 fg/µl) at 40 °C for at least 10 min. The whole process of DNA amplification by RPA and readout by LFD did not exceed 30 min. To determine the performance of the RPA-LFD assay, a total of 30 clinical samples was examined and compared with conventional PCR (cPCR) and multiplex HRM (mHRM). Eight dogs (26.67%) were detected as positive by RPA-LFD, while seven and six were found positive by cPCR and mHRM, respectively. RPA-LFD and cPCR showed high agreement with Babesia spp. detection with kappa > 0.9. We confirmed that the dogs were infected by B. vogeli from sequences of positive PCR results. Our findings suggested that RPA-LFD using the rpaBab264 assay offered a rapid, accurate, cost-effective and simple method for Babesia spp. detection that is feasibly applicable to be rapid kit at a pet hospital or point-of-care testing.

Bar-cas12a, a novel and rapid method for plant species authentication in case of Phyllanthus amarus Schumach. & Thonn.

Rapid and accurate species diagnosis accelerates performance in numerous biological fields and associated areas. However, morphology-based species taxonomy/identification might hinder study and lead to ambiguous results. DNA barcodes (Bar) has been employed extensively for plant species identification. Recently, CRISPR-cas system can be applied for diagnostic tool to detect pathogen's DNA based on the collateral activity of cas12a or cas13. Here, we developed barcode-coupled with cas12a assay, "Bar-cas12a" for species authentication using Phyllanthus amarus as a model. The gRNAs were designed from trnL region, namely gRNA-A and gRNA-B. As a result, gRNA-A was highly specific to P. amarus amplified by RPA in contrast to gRNA-B even in contaminated condition. Apart from the large variation of gRNA-A binding in DNA target, cas12a- specific PAM's gRNA-A as TTTN can be found only in P. amarus. PAM site may be recognized one of the potential regions for increasing specificity to authenticate species. In addition, the sensitivity of Bar-cas12a using both gRNAs gave the same detection limit at 0.8 fg and it was 1,000 times more sensitive compared to agarose gel electrophoresis. This approach displayed the accuracy degree of 90% for species authentication. Overall, Bar-cas12a using trnL-designed gRNA offer a highly specific, sensitive, speed, and simple approach for plant species authentication. Therefore, the current method serves as a promising tool for species determination which is likely to be implemented for onsite testing.

Phylogenetic analyses of distantly related clades of bent-toed geckos (genus Cyrtodactylus) reveal an unprecedented amount of cryptic diversity in northern and western Thailand.

Cyrtodactylus species are the most diverse of the geckos and are widely distributed in Southeast Asia, including Thailand. However, their patterns of distribution, especially in northern and western parts of Thailand, remain unknown because few Cyrtodactylus species in these regions have been described. Thus, a data set of mitochondrial NADH dehydrogenase 2 (ND2) gene and flanking tRNAs from Cyrtodactylus found in northern and western Thailand, including contiguous areas, was assembled to elucidate phylogenetic relationships and identify the distribution patterns of these geckos. The results showed four well-supported clades, a northwestern clade (A), a northern clade (B), a western clade (C), and a special clade characterized by specific morphological features (D). Clades A-C were grouped with strong support by the geography of their localities from northern Thailand (Mae Hong Son and Chiang Mai Provinces) along the Tenasserim mountain ranges to Phang-Nga Province, Thailand. Clade D is a distinct clade of Cyrtodactylus species characterized by a tuberculate and prehensile tail and distributed widely in mainland Southeast Asia. Overall, the results suggest a pattern of geographic separation and distribution of Cyrtodactylus in northern and western Thailand. Additionally, this study provides evidence of a hidden biodiversity of Cyrtodactylus in these regions.

A new species Cyrtodactytlus Gray (Squamata: Gekkonidae) from western Thailand and the phylogenetic placement of C. inthanon and C. doisuthep.

A new species of Cyrtodactylus from Tak Province, Thailand, Cyrtodactylus amphipetraeus sp. nov., is described using an integrative taxonomic analysis based on morphology, color pattern, and the mitochondrial gene NADH dehydrogenase subunit 2 (ND2). The phylogenetic analyses place the new species within the C. sinyineensis group which was previously thought to be endemic to the Salween Basin in southern Myanmar. The phylogeny also places C. inthanon in the C. sinyneensis group which is expanded herein to also include the group's sister species C. doisuthep. Along with C. amphipetraeus sp. nov., these are the first three species of the C. sinyineensis group to be found outside of Myanmar east of the Tenasserim Mountains. The Tenasserim Mountain region is discussed as an area of cladogeneic turnover.

Simultaneous differential detection of canine blood parasites: Multiplex high-resolution melting analysis (mHRM).

Recently, the incidence of canine infection by the tick-borne parasites Babesia spp., Hepatozoon canis, Ehrlichia canis and Anaplasma platys has been increasing globally. We have developed a multiplex high-resolution melting analysis (mHRM) technique to reduce the time demands and costs associated with detecting haemoparasites in canine blood, while increasing the degree of reliability of this method of analysis. We have designed primers that are specific for protozoans (B. vogeli and H. canis) and Rickettsia-like bacteria (E. canis and A. platys) based on the 18S or 16S rDNA sequences, respectively. Two primer pairs (Protz18S-C and Bact16S-A) were found to be suitable for detecting these agents since their melting temperatures (Tm) exhibited discernible differences among the four haemoparasites, A. platys, B. vogeli, E. canis and H. canis (83.10 °C, 82.41 °C, 80.37 °C and 78.56 °C, respectively). The sequences acquired from these PCR products were >94 % identical to those of A. platys, B. vogeli, E. canis and H. canis in GenBank. The limit of detection (LOD) for B. vogeli, E. canis and A. platys was 103 copies/µl, while the LOD for H. canis was 104 copies/µl. Of the 68 dogs tested, 28 (41 %) were infected with these agents. The most commonly occurring infection involved E. canis, followed by B. vogeli, A. platys and H. canis, with infection percentages of 26 %, 13 %, 7 % and 6 %, respectively. These results demonstrate that mHRM can serve as a rapid, economical and reliable tool for the detection of parasitic diseases in canine blood for diagnosis and epidemiology.

Genetic assessment of three Fagaceae species in forest restoration trials.

Restoring isolated patches of forest ecosystems in degraded landscapes could potentially lead to genetic loss and inbreeding. Therefore, this study determined the occurrence of genetic diversity among the tree species Castanopsis tribuloides, C. calathiformis, and Lithocarpus polystachyus all of which were proven previously to be effective native tree species in the restoration of upland evergreen forests in northern Thailand when using the seed sample collection method. We tested our hypothesis as to whether the genetic diversity of a plant population that had been planted from the seeds of 4-6 adult trees would be lower and whether incidences of fixation index (Fis) would be higher among the second generation seedlings of these three Fagaceae species in isolated forest restoration trial plots. Microsatellite primers were selected from the entire genome sequence of C. tribuloides and the genetic sequences of C. tribuloides, L. polystachyus, and C. calathiformis were analyzed. Our results indicated a high degree of genetic diversity (He) in C. tribuloides (0.736) and C. calathiformis (0.481); however, a low level of genetic diversity was observed in L. polystachyus (0.281) within the restored forest. The fixation index for the second generation of L. polystachyus and C. calathiformis in the restored forest showed evidence of inbreeding. These results imply the efficiency of the seed sample collection method and verify that it does not reduce the level of genetic diversity in C. tribuloides and C. calathiformis. However, it may result in incidences of an inbreeding phenomena, suggesting the need to increase the number of adult trees used at the seed collection stage.

Ethanolic extract of Kaempferia parviflora interrupts the mechanisms-associated rheumatoid arthritis in SW982 culture model via p38/STAT1 and STAT3 pathways.

BACKGROUND: Kaempferia parviflora Wall. ex Baker (KP) has long been used in traditional medicine to treat various diseases because active compounds in rhizome extracts are important anti-inflammatory agents. PURPOSE: This study aims to investigate the effects of an ethanolic extract of KP on the molecular mechanisms associated with rheumatoid arthritis (RA), which was induced by a combination of proinflammatory cytokines (IL-1ß or TNF-α with IL-17A) in a human synovial sarcoma cell line (SW982) culture model. METHODS: SW982 cells pretreated with cytokines were incubated with KP extract at 3-30 µg/ml, or three major compounds of KP (5,7-dimethoxyflavone, 5,7,4'-trimethoxyflavone, and 3,5,7,3',4'-pentamethoxyflavone) for up to 72 h. Dexamethasone was used as positive control. RA-associated genes and inflammatory products were measured in parallel with cell death genes. Apoptosis by flow cytometry and migration assay were also analyzed. Western blotting was used to examine the effects on intracellular signaling mechanisms. RESULTS: KP extract markedly reduced the expression of genes and levels of proinflammatory cytokines, inflammatory mediators, and matrix-degraded enzymes, but neither induced apoptosis nor altered the cell cycle. Its major constituents differently exerted suppressive effects on inflammatory genes. The KP extract downregulated the expression of genes associated with autophagosome and necroptosome formations. The extract also inhibited cell migration, reduced the mRNA expression of cadherin-11, and selectively reduced the phosphorylation of p38 MAPK, STAT1, and STAT3 signaling molecules, but did not interfere with the NF-κB pathway. CONCLUSION: These results suggest that the anti-arthritic potential of KP extract results from anti-inflammation and anti-migration via the suppression of the cytokines-induced p38/STAT1 and STAT3 pathways.

Determination of two fluoroquinolones and their combinations with hyaluronan effect in in vitro canine cartilage explants.

BACKGROUND: Previous studies reported the effect of enrofloxacin (Enro) and marbofloxacin (Mar) on cell death and alteration of the key genes involved in catabolic and anabolic processes and demonstrated the beneficial effects of hyaluronan (HA) combined with fluoroquinolones (FQs) on primary canine chondrocytes. This study further determines the effects of these treatments on canine cartilage explants in both normal and interleukin-1 beta (IL-1ß)-stimulated conditions. METHODS: We examined sulfate glycosaminoglycan (s-GAG) release, uronic acid (UA) content, and safranin-O staining, as well as the expression patterns of inflammatory, extracellular matrix (ECM) component and enzymes. RESULTS: Enro treatment alone effectively stimulated proteoglycan anabolism by increasing UA content and glycosaminoglycans (GAGs) in normal and pre-IL-1ß-stimulated explant, whereas Mar showed opposite results. The combination of HA and FQs increased s-GAG release and UA content in normal explants in addition to effective down-regulated expression of MMP3. HA reduced the adverse effects of Mar by enhancing UA and GAG contents in both normal and pre-IL-1ß-explants. Moreover, HA effectively induced HAS1and ACANup-regulation and reduced MMP9, TNF, PTGS2,and NFKB1 expression for a long term. DISCUSSION: Our results suggest the direct effects of Enro and Mar may selectively stimulate the conditioned explants to express MMP-codinggenes and promote gene expression involved in matrix production, pro-inflammatory cytokines, and cell degradation in different directions. HA successfully reduced the adverse effects of FQs by enhancing s-GAG and UA contents and down-regulated expression of MMPs.

Dystroglycan 1: A new candidate gene for patellar luxation in Chihuahua dogs.

AIM: The objective of this study was to uncover new candidate genes related to patellar luxation (PL) in dogs to select for those with low susceptibility for breeding purposes. MATERIALS AND METHODS: The inter simple sequence repeat (ISSR) technique was performed to construct DNA fingerprints of 61 Chihuahua dogs with PL and 30 healthy Chihuahua dogs. DNA polymorphisms were detected by comparing the sequences between the affected and unaffected dogs, using the pairwise alignments in MultAlin. Genotyping was performed using allele-specific polymerase chain reaction (AS-PCR). The association analysis of ISSR DNA fingerprints and genotypes or phenotypes was performed using the Chi-square (χ 2) model and generalized linear model (GLM), respectively. RESULTS: Two single nucleotide polymorphisms (SNPs), namely SNP1UBC811 (g.91175C>G) and SNP2UBC811 (g.92259T>C), were found in the intron of the Dystroglycan 1 (DAG1) gene, which was obtained using the PL-related marker UBC811 primer (p=0.02), and genotyped by AS-PCR. When investigated using the GLM, g.91175C>G had a significant association with PL (p=0.0424), whereas g.92259T>C did not have such an association (p=0.0959). CONCLUSION: DAG1 might be one of the genes related to PL in Chihuahuas and could aid the process of marker-assisted selection in genetic breeding for Chihuahua dogs without PL.

In Vitro Chondroprotective Potential of Extracts Obtained from Various Phyllantus Species.

Phyllanthus amarus has been proven to exhibit chondroprotection. Regarding the morphological similarities among Phyllanthus species, we were attracted to evaluate the chondroprotective potential of Phyllanthus species including P. amarus obtained from Chiang Mai and Phuket, Phyllanthus urinaria L., Phyllanthus urinaria subsp. chamaepeuce, Phyllanthus debilis, and Phyllanthus airy-shawii using interleukin-1ß-induced degradation of cartilage explants. The ethanolic extracts of the plants were evaluated for major lignans, phyllanthin, and hypophyllanthin by HPLC and further measurements of the total contents of flavonoids and phenolic compounds along with the assays for antioxidant and anti-collagenase activities. The interleukin-1ß-induced cartilage explant degradation was performed with/without the extracts at concentrations of 50-250 µg/mL. After 4-14 days of incubation, the medium was assayed for the level of sulfated glycosaminoglycans while the explants were measured for the remaining content of uronic acid. Proteoglycan intensity in the explants was determined by safranin O staining. Diacerein, the antiarthritic agent, was used as the positive control. Although the two major lignans were found in P. amarus from Chiang Mai, P. amarus from Phuket, and P. urinaria L. extracts, similar chondroprotective activities were observed in all Phyllanthus extracts. Total phenolic content and total flavonoid content of the extracts showed a correlation with antioxidation, whereas the total phenolic content correlated with anti-collagenase activity. Among the six extracts, P. airy-shawii showed the greatest antioxidant and collagenase inhibitory activities. The results revealed that chondroprotective activities of all of the extracts of Phyllanthus species might result from an additive or synergistic influence of some constituents of these plants, which could be considered for antiarthritic purposes.

Differences in osteon structure histomorphometry between puppyhood and adult stages in the Golden Retriever.

Osteon structure has been widely studied in mammals, but osteon structure in dogs has received relatively little attention, especially in terms of whether aging has any effect on osteon structure. The aim of this study was to compare the osteon structure of both flat (scapula and os coxae) and long bones (humerus, radius, ulna, metacarpus, femur and tibia) of male puppy and adult Golden Retrievers. We examined five parameters: Haversian canal diameter, Haversian canal area, osteon diameter, osteon area, and number of lacunae per osteon. Our results show that the values for Haversian canal diameter were significantly higher in the os coxae and tibia, but significantly lower in the femur of adult dogs as compared to those of puppies. The Haversian canal diameter of the other bones investigated did not show any significant differences between puppies and adult dogs. The Haversian canal area was significantly greater in the os coxae, radius and femur of adult dogs than in those of puppies. The osteon diameter and area of every bone examined were significantly smaller in puppies than in adult dogs. Lastly, the number of lacunae per osteon showed the same trend as osteon diameter and area. Plexiform bone could be found in three bones in puppies, i.e. the femur, humerus and tibia. Overall, the results of this study should provide basic knowledge on the microanatomy of cortical bone in dogs and on the possible influence age.

In vitro effects of triamcinolone acetonide and in combination with hyaluronan on canine normal and spontaneous osteoarthritis articular cartilage.

The purposes of this study were to examine the cartilage degradation effects of triamcinolone acetonide (TA) on normal and osteoarthritic (OA) primary canine chondrocytes and cartilage explants and to examine the cartilage degradation effects of TA in combination with low-molecular-weight hyaluronan (LMWHA). To assess the effects of these drugs on cell culture, 3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and real-time PCR were used to measure chondrotoxicity and determine gene expression, respectively. Uronic acid and hydroxyproline remaining in cartilage and histopathology were used to estimate the effects of these drugs on cartilage explants. In chondrocyte cultures, TA reduced chondrocyte viability in a concentration-dependent manner. LMWHA 2.5 mg/ml combined with TA at IC20 (0.09 mg/ml) could increase the viability of normal chondrocytes when compared with TA-treated alone. TA at IC20 induced down-regulation of ACAN and induced up-regulation of ADAMTS5 in canine normal chondrocytes. TA at IC20 (0.11 mg/ml) up-regulated ADAMTS5, MMP2, MMP3, MMP13, and ACAN expression in canine OA chondrocytes. In explant culture, TA at 1.25, 2.5, and 5 mg/ml increased the severity of structural damage, chondrocyte loss and cluster formation, and proteoglycan loss in OA cartilage. LMWHA could decrease the chondrotoxicity of TA at IC20 only in normal chondrocytes, as observed by chondrocyte viability. The combination of LMWHA and TA did not show clearly beneficial effects in all other normal and OA samples. Consequently, using TA alone or in combination with LMWHA in OA cartilage should be of concern because it may lead to cartilage destruction.

Effects of corticosteroids and their combinations with hyaluronanon on the biochemical properties of porcine cartilage explants.

BACKGROUND: Intra-articular injection of corticosteroids is used to treat the inflammatory pain of arthritis and osteoarthritis (OA), but our previous study found a deleterious effect of these steroids on chondrocyte cells. Hyaluronic acid (HA) injection has been suggested as a means to counteract negative side effects through replenishment of synovial fluid that can decrease pain in affected joints. To better understand the effects of corticosteroids on these processes, dexamethasone (Dex) and prednisolone (Pred) were administered to porcine cartilage explants at several concentrations with and without HA. We examined corticoid effects by determining sulfate-glycosaminoglycan (s-GAG) and uronic acid (UA) content of the explant media, and safranin-O staining of the cells. Analysis of lactate dehydrogenase (LDH) activity was conducted to assess cell cytotoxicity. RESULTS: Dex treatment significantly reduced cellular cytotoxicity compared to the other treatment groups, especially with regards to the release of s-GAG, and protects against superficial proteoglycan damage. However, there was no difference between Pred and Dex, with and without HA, in the UA content remaining in porcine cartilage explants. CONCLUSIONS: The data suggest that combinations of Dex and Pred with HA did not have a significant effect on protection or enhancement of the articular cartilage matrix under the current conditions.

Effects of low molecular weight hyaluronan combined with carprofen on canine osteoarthritis articular chondrocytes and cartilage explants in vitro.

Intra-articular injection with non-steroidal anti-inflammatory drugs (NSAIDs) is used to treat inflammatory joint disease, but the side effects of NSAIDs include chondrotoxicity. Hyaluronan has shown positive effects on chondrocytes by reducing apoptosis and increasing proteoglycan synthesis. The purposes of this study were to evaluate the effects of low molecular weight hyaluronan (low MW HA), carprofen 25 mg/ml, carprofen 12.5 mg/ml, and a combination of HA and carprofen on canine osteoarthritis (OA) articular chondrocytes and a cartilage explant model in terms of cell viability, extracellular matrix remaining, and gene expression after exposure. In chondrocyte culture, MTT assay was used to evaluate the chondrotoxicity of IC50 and IC80 of carprofen with HA. In cartilage explant culture, two kinds of extracellular matrix (uronic acid and collagen) remaining in cartilage were used to evaluate cartilage damage for 14 d after treatment. Expression of COL2A1, AGG, and MMP3 was used to evaluate the synthesis and degradation of the matrix for 7 d after treatment. In chondrocyte culture, low MW HA could preserve OA chondrocyte viability but could not reduce the chondrotoxicity level of carprofen (P < 0.05). In explant culture, low MW HA combined with 12.5 mg/ml carprofen caused less destruction of uronic acid and collagen structure when compared with the control (P < 0.05). Low MW HA caused high expression levels of COL2A1 and AGG in OA cartilage (P < 0.05); HA combined with carprofen resulted in higher COL2A1 and AGG expression levels than carprofen alone.

Chondroprotective potential of Phyllanthus amarus Schum. & Thonn. in experimentally induced cartilage degradation in the explants culture model.

Phyllanthus amarus Schum. & Thonn. (P. amarus) has been reported to exhibit anti-inflammation and antiarthritis properties leading to our interest to examine its beneficial effect in osteoarthritis. Thus, this study aimed to explore the chondroprotective potential of P. amarus extract (PAE) and its major compounds, phyllanthin and hypophyllanthin, in a cartilage explant model. Various concentrations of P. amarus extract, phyllanthin and hypophyllanthin, were treated on porcine articular cartilage explants induced with 25 ng/ml of interleukin-1 beta (IL-1ß). After 4 days of incubation, the culture medium was measured for the release of sulfate glycosaminoglycans (s-GAGs) and matrix metalloproteinase-2 (MMP-2) activity by DMMB binding assay and zymography, respectively. The explant tissues were analyzed for the remaining of uronic acid content by colorimetric assay and stained with safranin-O for investigation of proteoglycan content. Cell viability of this model was evaluated by lactate dehydrogenase (LDH) assay. Chondroprotective potential of PAE and the major components against IL-1ß-induced cartilage explant degradation were revealed by the decreased s-GAGs level and MMP-2 activity in culture medium consistent with an increase in uronic acid and proteoglycan contents in the explants when compared to the IL-1ß treatment. These results agreed with those of diacerein and sesamin which used as positive controls. In addition, better chondroprotective activities of P. amarus crude extracts than those of the purified components were disclosed in this study. Hence, this is a pioneering study presenting the chondroprotective potential of PAE which may augment its application for therapeutic use as an antiarthritic agent.

Articular cartilage gene expression after coxofemoral joint luxation in the dog.

This study examined the relationship between days of hip luxation and the expression of various mRNA. Twenty-six articular cartilages were used in the experiment: 3 samples were from normal dogs and 23 samples were collected from the femoral heads of hips that had been luxated for different lengths of time. Ten mRNA, including nonapoptotic genes (AGG, COL2A1, MMP-3, HAS-1, HAS-2, and TIMP-1) and apoptotic genes (BAX, BCL-2, CAS-3, and CAS-9), were studied for their expression using real-time PCR. We found very high correlation between expression level and luxation days (r (2) > 0.9) in COL2A1, MMP-3, HAS-1, HAS-2, TIMP-1, BAX, and CAS-9, while the others (AGG, BCL-2, and CAS-3) also showed high correlation (r (2) = 7-9). And we found a significant difference (P < 0.05) in the expression of transcripts depending on the number of luxation days. In conclusion, a delay in joint reduction may increase the chances of development of osteoarthritis.