Transcriptome Analysis Reveals Novel Genes Associated with Cartilage Degeneration in Posttraumatic Osteoarthritis Progression.

OBJECTIVE: The current therapeutic strategy for posttraumatic osteoarthritis (PTOA) focuses on early intervention to attenuate disease progression, preserve joint function, and defer joint replacement timing. Sequential transcriptomic changes of articular cartilage in a rat model were investigated to explore the molecular mechanism in early PTOA progression. DESIGN: Anterior cruciate ligament transection and medial meniscectomy (ACLT + MMx)-induced PTOA model was applied on male Wistar rats. Articular cartilages were harvested at time 0 (naïve), 2 week, and 4 weeks after surgery. Affymetrix Rat genome 230 2.0 array was utilized to analyze the gene expression changes of articular cartilages. RESULTS: We identified 849 differentially expressed genes (DEGs) at 2 weeks and 223 DEGs at 4 weeks post-ACLT + MMx surgery compared with time 0 (naïve group). Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to gain further insights from these DEGs. 22 novel genes and 1 novel KEGG pathway (axon guidance) in cartilage degeneration of osteoarthritis were identified. Axon guidance molecules-Gnai1, Sema4d, Plxnb1, and Srgap2 commonly dysregulated in PTOA progression. Gnai1 gene showed a concordant change in protein expression by immunohistochemistry staining. CONCLUSIONS: Our study identified 22 novel dysregulated genes and axon guidance pathway associated with articular cartilage degeneration in PTOA progression. These findings provide the potential candidates of biomarkers and therapeutic targets for further investigation.

Dextromethorphan upregulates osteoblast and osteoclast activity but does not attenuate ovariectomy-induced osteoporosis.

AIMS: Study on the in vivo regulatory role of glutamate in osteoblast (OB) and osteoclast (OC) differentiation is less advanced. The present study investigated the effect of dextromethorphan (DXM), an N-methyl-d-aspartate receptors (NMDARs) antagonist, on osteoporosis development. MAIN METHODS: In order to examine the role of glutamate in bone metabolism, ovariectomized (Ovx) female Wistar rats were injected three times per week for 8weeks with either saline, or 15µg/kg of ß-estrodiol, or DXM (40mg/kg) intraperitoneally. Serum samples were collected every two weeks for measuring osteocalcin and C-terminal telopeptide of type I collagen (CTX-1) level. Rats were then sacrificed at week 8 and the femurs harvested for micro-CT scanning and mechanical strength. KEY FINDINGS: In saline-treated group, osteocalcin level significantly lower than that of sham-operated rats at 8weeks after operation, while CTX-1 levels were not affected. Estrogen treatment, as a positive control, partially inhibited the Ovx-induced reduction of osteocalcin serum level. DXM injection prevented the Ovx-induced reduction of osteocalcin expression and significantly upregulated CTX-1 expression. The micro-CT scan showed that the bone volume density decreased significantly in DXM treated rats compared to the sham-operated rats. In the mechanical strength assay, the maximum failure load for DXM treatment was significantly lower than the other groups. SIGNIFICANCE: Treatment with DXM upregulated OB and OC markers in Ovx rats, however with a greater effect on the OC marker, and had no significant benefit on bone volume density or bone strength.

Sulfasalazine attenuates ACL transection and medial menisectomy-induced cartilage destruction by inhibition of cystine/glutamate antiporter.

We had previously demonstrated that excitatory amino acid glutamate plays a role in the progression and severity of knee osteoarthritis (OA), and early hyaluronic acid injection attenuates the OA progression by attenuation of knee joint glutamate level, which was also related to the cystine/glutamate antiporter system X (system XC-) expression. System XC- uptakes cystine into chondrocytes for glutathione (GSH) synthesis, but the role of system XC- in OA is rarely addressed. Sulfasalazine (SSZ) is a system XC- inhibitor; SSZ was applied intra-articularly to study the function of system XC- in the development of OA in rats subjected to anterior cruciate ligament transection and medial meniscectomy (ACLT + MMx). Moerover, the system XC- activator N-acetylcysteine (NAC) was also applied to verify the role of system XC-. The intra-articular injection of SSZ significantly attenuated knee swelling and cartilage destruction in the knees of ACLT + MMx rats and this effect was blocked by NAC. The results showed that inhibition of system XC- function can attenuate ACLT + MMx-induced cartilage destruction. In the present study, system XC- inhibitor SSZ was shown to reduce glutamate content in synovial fluid and GSH in chondrocytes. It was also showed SSZ could attenuate ACLT + MMx-induced cartilage destruction, and treatment of NAC reversed the protective effect of SSZ.

Hylan G-F 20 attenuates posttraumatic osteoarthritis progression: Association with upregulated expression of the circardian gene NPAS2.

AIMS: The study was to examine the effect of Hylan G-F 20 on the progression of posttraumatic osteoarthritis (PTOA) and the expression of the circadian genes neuronal PAS domain protein 2 (NPAS2) and period 2 (Per2). MAIN METHODS: We used the anterior cruciate ligament transaction and medial menisectomy (ACLT+MMx) model in Wistar rats. The rats were divided into three groups, the sham-operated group, the Hylan G-F 20-treated group, and the saline-treated group. Rats which underwent ACLT + MMx surgery were injected intraarticularly with, respectively, Hylan G-F 20 or saline once a week for 3 consecutive weeks, starting 7days after surgery. The gross morphology and histopathology of the experimental knee joints were evaluated at the end of week 6. Expression of the NPAS2 and Per2 genes was measured by real-time PCR. KEY FINDINGS: Hylan G-F 20 suppressed the articular cartilage destruction and synovitis compared to the saline-treated group. Compared to the sham-operated group, the Hylan G-F 20-treated group showed significantly upregulated expression of NPAS2 in cartilage (2.53±0.08-fold higher; p<0.05) and a non-significant increase in Per2 expression (2.35±1.26-fold higher p=0.28), while the saline-treated group showed significant downregulation of NPAS2 expression and a non-significant decrease in Per2 expression. SIGNIFICANCE: Our data suggested that early intraarticular injection of Hylan G-F 20 attenuates the progression of PTOA and significantly upregulates NPAS2 expression. These findings provide a new direction for studying associations between the use of a pharmacological agent, the degenerative process, and circadian gene expression.

Ultra-low dose (+)-naloxone restores the thermal threshold of morphine tolerant rats.

BACKGROUND/PURPOSE: As known, long-term morphine infusion leads to tolerance. We previously demonstrated that both co-infusion and post-administration of ultra-low dose (±)-naloxone restores the antinociceptive effect of morphine in morphine-tolerant rats. However, whether the mechanism of the action of ultra-low dose (±)-naloxone is through opioid receptors or not. Therefore, in the present study, we further investigated the effect of ultra-low dose (+)-naloxone, it does not bind to opioid receptors, on the antinociceptive effect of morphine. METHODS: Male Wistar rats were implanted with one or two intrathecal (i.t.) catheters; one catheter was connected to a mini-osmotic pump, used for morphine (15 µg/h), ultra-low dose (+)-naloxone (15 pg/h), morphine plus ultra-low dose (+)-naloxone (15 pg/h) or saline (1 µl/h) infusion for 5 days. On day 5, either ultra-low dose (+)-naloxone (15 pg) or saline (5 µl) was injected via the other catheter immediately after discontinued morphine or saline infusion. Three hours later, morphine (15 µg in 5 µl saline) or saline were given intrathecally. All rats received nociceptive tail-flick test every 30 minutes for 120 minutes after morphine challenge at different temperature (45-52°C, respective). RESULTS: Our results showed that, both co-infusion and post-treatment of ultra-low dose (+)-naloxone with morphine preserves the antinociceptive effect of morphine. Moreover, in the post administration rats, ultra-low dose (+)-naloxone further enhances the antinociceptive effect of morphine. CONCLUSION: This study provides an evidence for ultra-low dose (+)-naloxone as a therapeutic adjuvant for patients who need long-term opioid administration for pain management.

Early intraarticular injection of hyaluronic acid attenuates osteoarthritis progression in anterior cruciate ligament-transected rats.

SUBJECT: Hyaluronic acid (HA) is widely used to relieve the symptoms of osteoarthritis (OA). An association of reduction of glutamate content with the synovial fluid of OA rats was reported previously. DESIGN: Anterior cruciate ligament transaction (ACLT) was performed on one knee in male Wistar rats, the other knee was assigned to sham control and HA or saline was injected intraarticularly into the ACLT knee from week 3 to week 7. Knee dialysate was collected for amino acid measurement at week 20. Morphology and histopathology of the femoral medial condyles and synovium were examined and evaluated using Mankin and synovitis scores. RESULTS: HA injection provided better cartilage (3.38 ± 0.03 vs. 5.45 ± 0.0.02) and synovial condition (3 ± 0.02 vs. 6.03 ± 0.02) than saline controls. Moreover, HA injection reduced the concentration of glutamates in knee dialysates compared to saline controls (1.11 ± 0.14-folds and 2.21 ± 0.19-folds of the sham-operated knee, respectively). Cystine/glutamate antiporter system [Formula: see text] expression was significantly downregulated in the saline group, but not in the HA group (0.32 ± 0.08-folds and 0.71 ± 0.10-folds of the sham-operated knee, respectively). CONCLUSION: Early intraarticular injection of HA attenuates the progression of cartilage destruction in the ACLT knee, and the downregulation of the cystine/glutamate antiporter system [Formula: see text] was accompanied by the progression of OA.

Ultra-low-dose naloxone enhances the antinociceptive effect of morphine in PTX-treated rats: regulation on global histone methylation.

OBJECTIVE: Epigenetic reprogramming may have a possible role in neuropathic pain development; the present study examined the global patterns of lysine histone modification. In this serial study we analyzed the levels of histone 3 lysine 4 monomethylation, histone 3 lysine 4 dimethylation, and histone 3 lysine 9 trimethylation in pertussis toxin (PTX)-induced thermal hyperalgesic rat spinal cords. METHODS: Male Wistar rats implanted with an intrathecal catheter received a single intrathecal PTX (1 µg in 5 µl saline) injection. Four days later, they were randomly assigned to receive either a single injection of saline, or ultra-low-dose naloxone (15 ng in 5 µl saline), followed by morphine (10 µg in 5 µl saline) injection 30 minutes later. RESULTS: The results showed that PTX injection induced thermal hyperalgesia and significant increase of global histone methylation in the spinal cords. Intrathecal morphine alone did not affect the thermal hyperalgesia and global histone methylation. In contrast, intrathecal administration of ultra-low-dose naloxone plus morphine significantly attenuated the PTX-induced thermal hyperalgesia and down-regulated the global histone methylation. CONCLUSION: The results suggest that ultra-low-dose naloxone might be clinical valuable for neuropathic pain management via regulating global histone modification.

Ultra-low dose naloxone restores the antinocicepitve effect of morphine in PTX-treated rats: association of IL-10 upregulation in the spinal cord.

AIMS: Ultra-low dose naloxone has been shown to restore the antinociceptive effect of morphine in pertussis toxin (PTX)-treated rats by suppressing spinal microglia activation and inhibiting inflammatory cytokine expression. This study was further investigated the mechanism by which ultra-low dose naloxone promotes analgesia in pertussis toxin-treated rats. MAIN METHODS: Male Wistar rats were implanted with an intrathecal (i.t.) catheter and injected either saline or PTX (1 µg). Four days later, rats randomly received either saline, or ultra-low dose naloxone, or recombinant rat interleukin-10 (rrIL-10) (1 µg) injection followed by saline or morphine (10 µg) 30 min later. In some experiments, mouse anti-rat IL-10 antibody (10 µg) was injected intrathecally into PTX injected rats daily on days 4, 5, 6, and 7. On day 7, ultra-low dose naloxone was given 1h after antibody injection with or without subsequent morphine injection. KEY FINDINGS: PTX injection induced notable thermal hyperalgesia and mechanical allodynia. Injection of ultra-low dose naloxone preserved the antinociceptive effect of morphine in PTX-treated rats and associated an increasing of IL-10 protein expression. Intrathecal injection rrIL-10 alone or in combination with morphine, not only reversed mechanical allodynia but also partially restored the antinociceptive effect of morphine; injection of anti-rat IL-10 antibody attenuated the effect of morphine plus ultra-low dose naloxone on mechanical allodynia and completely inhibited the antinociceptive effect of morphine. SIGNIFICANCE: These results indicate that intrathecal ultra-low dose naloxone induces IL-10 expression in spinal neuron and microglia, which suppresses PTX-induced neuroinflammation and restores the antinociceptive effect of morphine.

Resveratrol regulates N-methyl-D-aspartate receptor expression and suppresses neuroinflammation in morphine-tolerant rats.

BACKGROUND: In the present study, we examined the effects and mechanisms of the Chinese herb resveratrol on attenuation of morphine tolerance in rats. METHODS: Male Wistar rats were implanted with 2 intrathecal catheters; one catheter was connected to a mini-osmotic pump, used for either morphine (15 µg/h) or saline (1 µL/h) infusion for 5 days. On day 5, resveratrol (7.5, 15, 30, or 60 µg), dimethyl sulfoxide (5 µL), or saline (5 µL) was injected via the other catheter immediately after the discontinued morphine infusion. Three hours later, intrathecal morphine (15 µg in 5 µL saline) was given. All rats received the nociceptive tail-flick test every 30 minutes for 120 minutes after the morphine challenge. RESULTS: Long-term morphine infusion induced antinociceptive tolerance and up-regulated N-methyl-D-aspartate receptor (NMDAR) subunit NR1 and NR2B expression in the synaptosome fraction of the tolerant spinal cord dorsal horn. Resveratrol pretreatment provided a significant antinociceptive effect of morphine in morphine-tolerant rats, and it was associated with reversal of the up-regulated NR1 and NR2B subunits in the synaptosome fraction of morphine-tolerant rat spinal cords. NR1/NR2B-specific antagonist ifenprodil treatment produced a similar effect as that of resveratrol. Furthermore, an increase of postsynaptic density-95/NR1/NR2B complex immunoprecipitation in morphine-tolerant rat spinal cord was also inhibited by resveratrol pretreatment. Moreover, chronic morphine infusion activated glial cells with an increase of proinflammatory cytokine tumor necrosis factor-α, interleukin-1ß, and interleukin-6 mRNA expression in morphine-tolerant rat spinal cords and these effects were suppressed by resveratrol pretreatment before the morphine challenge. CONCLUSIONS: Resveratrol attenuates morphine tolerance by inhibiting neuroinflammation and down-regulating NMDAR NR1 and NR2B subunit expression. Resveratrol regulates the NMDAR expression, which might be involved in a loss of scaffolding postsynaptic density-95 protein.

Conditional antisense-knockdown of zebrafish cardiac troponin C as a new animal model for dilated cardiomyopathy.

BACKGROUND: Mutations of cardiac troponin C (cTnC) can cause dilated cardiomyopathy in humans. METHODS AND RESULTS: Plasmids were constructed such that the reverse tetracycline-controlled transactivator (rtTA) was driven by the cardiac myosin light chain 2 promoter. This heart-specific rtTA bound another bidirectional promoter to express the green fluorescence protein reporter gene and the antisense RNA of cTnC in the presence of doxycycline. A transgenic line of zebrafish (CA17) with cTnC dysfunction was also generated. The heart rates of the embryos in the CA17 line were significantly slower than those of embryos in the control T03 transgenic line at 6 and 12 days post fertilization (dpf). In the CA17 line, cardiac chambers in the F2 embryos were significantly greater and the ventricular ejection fraction was lower than those in the T03 at both 6 and 12 dpf. The mortality rate of F2 adult fish of the CA17 line was also significantly higher (P<0.001). CONCLUSIONS: Using conditional expression of antisense RNA of zebrafish cTnC, a new animal model with phenotypes simulating dilated cardiomyopathy has been created.

Glycogen synthase kinase 3 alpha and 3 beta have distinct functions during cardiogenesis of zebrafish embryo.

BACKGROUND: Glycogen synthase kinase 3 (GSK3) encodes a serine/threonine protein kinase, is known to play roles in many biological processes. Two closely related GSK3 isoforms encoded by distinct genes: GSK3alpha (51 kDa) and GSK3beta (47 kDa). In previously studies, most GSK3 inhibitors are not only inhibiting GSK3, but are also affecting many other kinases. In addition, because of highly similarity in amino acid sequence between GSK3alpha and GSK3beta, making it difficult to identify an inhibitor that can be selective against GSK3alpha or GSK3beta. Thus, it is relatively difficult to address the functions of GSK3 isoforms during embryogenesis. At this study, we attempt to specifically inhibit either GSK3alpha or GSK3beta and uncover the isoform-specific roles that GSK3 plays during cardiogenesis. RESULTS: We blocked gsk3alpha and gsk3beta translations by injection of morpholino antisense oligonucleotides (MO). Both gsk3alpha- and gsk3beta-MO-injected embryos displayed similar morphological defects, with a thin, string-like shaped heart and pericardial edema at 72 hours post-fertilization. However, when detailed analysis of the gsk3alpha- and gsk3beta-MO-induced heart defects, we found that the reduced number of cardiomyocytes in gsk3alpha morphants during the heart-ring stage was due to apoptosis. On the contrary, gsk3beta morphants did not exhibit significant apoptosis in the cardiomyocytes, and the heart developed normally during the heart-ring stage. Later, however, the heart positioning was severely disrupted in gsk3beta morphants. bmp4 expression in gsk3beta morphants was up-regulated and disrupted the asymmetry pattern in the heart. The cardiac valve defects in gsk3beta morphants were similar to those observed in axin1 and apcmcr mutants, suggesting that GSK3beta might play a role in cardiac valve development through the Wnt/beta-catenin pathway. Finally, the phenotypes of gsk3alpha mutant embryos cannot be rescued by gsk3beta mRNA, and vice versa, demonstrating that GSK3alpha and GSK3beta are not functionally redundant. CONCLUSION: We conclude that (1) GSK3alpha, but not GSK3beta, is necessary in cardiomyocyte survival; (2) the GSK3beta plays important roles in modulating the left-right asymmetry and affecting heart positioning; and (3) GSK3alpha and GSK3beta play distinct roles during zebrafish cardiogenesis.

Molecular structure and developmental expression of three muscle-type troponin T genes in zebrafish.

Troponin T (Tnnt), a troponin component, interacts with tropomyosin and is crucial to the regulation of striated muscle contraction. To gain insight into the molecular evolution and developmental regulation of Tnnt gene (Tnnt) in lower vertebrates, zebrafish Tnnt1 (slow Tnnt), Tnnt2 (cardiac Tnnt), and Tnnt3b (fast Tnnt isoform b) were characterized. The polypeptides of zebrafish Tnnt1, Tnnt2, and Tnnt3b were conserved in the central tropomyosin- and C-terminal troponin I-binding domains. However, the N-terminal hypervariable regions were highly extended and rich in glutamic acid in polypeptides of Tnnt1 and Tnnt2, but not Tnnt3b. The Tnnt2 and Tnnt3b contain introns, whereas Tnnt1 is intron-free. During development, large to small, alternatively spliced variants were detected in Tnnt2, but not in Tnnt1 or Tnnt3. Whole-mount in situ hybridization showed zebrafish Tnnt1 and Tnnt2 are activated during early somitogenesis (10 hr postfertilization, hpf) and cardiogenesis (14 hpf), respectively, but Tnnt3b is not activated until middle somitogenesis (18 hpf). Tnnt2 and Tnnt3b expression was cardiac- and fast-muscle specific, but Tnnt1 was expressed in both slow and fast muscles. We propose that three, distinct, muscle-type Tnnt evolved after the divergence of fish and deuterostome invertebrates. In zebrafish, the developmental regulation of Tnnt during somitogenesis and cardiogenesis is more restricted and simpler than in tetrapods. These new findings may provide insight into the developmental regulation and molecular evolution of vertebrate Tnnt.

Isolation and expression of two zebrafish homologues of parvalbumin genes related to chicken CPV3 and mammalian oncomodulin.

Full-length cDNA clones coded for two beta-type homologues of parvalbumin genes, pvalb3a and pvalb3b, were isolated from zebrafish. The homology and phylogenetic analyses, based on the deduced amino acid sequences, revealed that PVALB3A and PVALB3B are co-orthologues to chicken CPV3 and mammalian oncomodulin (OCM) but are divergent from alpha-type PVALB of tetrapods and muscle-type PVALB of bony fish. Whole-mount in situ hybridization revealed that the spatio-temporal expression of pvalb3a and pvalb3b were distinct and highly development-regulated during early embryogenesis. Unlike their counterparts of CPV3 in chicken and OCM in mammals, zebrafish pvalb3a transcripts were widely expressed in mucous cells, the olfactory epithelium, anterior pituitary, pharyngeal teeth germ, macrophages, inner ear and lateral line neuromasts, whereas, pvalb3b transcripts were more restrictedly expressed in the yolk syncytial layer, inner ear and pronephric ducts.

Isolation and expression of two zebrafish homologues of parvalbumin genes related to chicken CPV3 and mammalian oncomodulin.

Full-length cDNA clones coded for two beta-type homologues of parvalbumin genes, pvalb3a and pvalb3b, were isolated from zebrafish. The homology and phylogenetic analyses, based on the deduced amino acid sequences, revealed that PVALB3A and PVALB3B are co-orthologues to chicken CPV3 and mammalian oncomodulin (OCM) but are divergent from alpha-type PVALB of tetrapods and muscle-type PVALB of bony fish. Whole-mount in situ hybridization revealed that the spatio-temporal expression of pvalb3a and pvalb3b were distinct and highly development-regulated during early embryogenesis. Unlike their counterparts of CPV3 in chicken and OCM in mammals, zebrafish pvalb3a transcripts were widely expressed in mucous cells, the olfactory epithelium, anterior pituitary, pharyngeal teeth germ, macrophages, inner ear and lateral line neuromasts, whereas, pvalb3b transcripts were more restrictedly expressed in the yolk syncytial layer, inner ear and pronephric ducts.