Prophylactic effect of Potentilla fulgens on renal ischemia-reperfusion injury in rats / Efecto profiláctico de Potentilla fulgens sobre la lesión por reperfusión por isquemia renal en rata

SUMMARY: We aimed to investigate the possible protective effects of Potentilla fulgens on kidney tissue with ischemia- reperfusion using immunohistochemical methods. Wistar rats were grouped as sham, ischemia, ischemia-reperfusion (I/R) and I/R treated with Potentilla fulgens. Renal vessels of the left rat kidney were clamped for 60 minutes for ischemia, IR group had 6 h of reperfusion. 400 mg/kg Potentilla fulgens were given intraperitoneally 5 days before ischemia+reperfusion procedure. Biochemical analysis (MDA, GSH and MPO) of samples were performed. Kidney tissues were fixed with 10 % neutral formalin and routine paraffin tissue follow-up protocol was applied, stained with routine Hematoxylin and Eosin. ADAMTS-5 and Caspase-3 immunostaining was applied for immunohistochemistry and examined under a light microscope. In the ischemia group, inflammation and congestion in the vessels and increased ADAMTS-5 expression in glomerular cells and tubule cells were observed. In reperfusion, an increase in degenerative glomerular cells, tubule cells and intertubular connective tissue and inflammatory cells ADAMTS-5 expression was observed. In the P. fulgens group, degeneration and inflammation decreased and positive ADAMTS-5 expression was observed. In the ischemia and ischemia reperfusion group, increased apoptotic appearance and Caspase-3 positive expression in glomerular and tubular cells, and negative expression in most cells in the P. fulgens group. Potentilla fulgens are thought to stop apoptotic cell development at a certain stage, which affects the cytokine mechanism and plays an important role in the reduction of inflammatory cells and angiogenic regulation.

RESUMEN: El objetivo de este estudio fue investigar los posibles efectos protectores de Potentilla fulgens en el tejido renal con isquemia-reperfusión utilizando métodos inmunohistoquímicos. Se agruparon ratas Wistar como simulación, isquemia, isquemia-reperfusión (I / R) e I / R tratadas con Potentilla fulgens. Los vasos renales del riñón iz- quierdo de las ratas se fijaron durante 60 min por isquemia, el grupo de IR tuvo 6 h de reperfusión. Se administraron 400 mg / kg de Potentilla fulgens por vía intraperitoneal 5 días antes del procedimiento de isquemia + reperfusión. Se realizaron análisis bioquímicos (MDA, GSH y MPO) de muestras. Los tejidos renales se fijaron con formalina neutra al 10 % y se aplicó el protocolo de seguimiento de tejido de parafina de rutina y teñido con hematoxilina y eosina. Se aplicó inmunotinción de ADAMTS-5 y Caspasa-3 para inmunohistoquímica y se examinó con un microscopio óptico. En el grupo de isquemia, se observó inflamación y congestión en los vasos y el aumento de la expresión de ADAMTS-5 en células glomerulares y células tubulares. En la reperfusión, se observó un aumento en la expresión de ADAMTS-5 de células glomerulares degenerativas, células tubulares y tejido conjuntivo intertubular y células inflamatorias. En el grupo de Potentilla fulgens, la degeneración y la inflamación disminuyeron y se observó expresión positiva de ADAMTS-5. En el grupo de isquemia y reperfusión de isquemia, aumentó la apariencia apoptótica y expresión positiva de Caspasa-3 en células glomerulares y tubulares, y expresión negativa en la mayoría de las células del grupo de Potentilla fulgens. Se cree que Potentilla fulgens detiene el desarrollo de las células apoptóticas en una determinada etapa, lo que afecta el mecanismo de las citocinas y juega un papel importante en la reducción de las células inflamatorias y la regulación angiogénica.

Matrix-degrading protease ADAMTS-5 cleaves inter-α-inhibitor and releases active heavy chain 2 in synovial fluids from arthritic patients.

Destruction of the cartilage matrix in joints is an important feature of arthritis. Proteolytic degradation of cartilage glycoproteins can contribute to the loss of matrix integrity. Human inter-α-inhibitor (IαI), which stabilizes the extracellular matrix, is composed of the light-chain serine proteinase inhibitor bikunin and two homologous heavy chains (HC1 and HC2) covalently linked through chondroitin 4-sulfate. Inflammation promotes the transfer of HCs from chondroitin 4-sulfate to hyaluronan by tumor necrosis factor-stimulated gene-6 protein (TSG-6). This reaction generates a covalent complex between the heavy chains and hyaluronan that can promote leukocyte invasion. This study demonstrates that both IαI and the HC-hyaluronan complex are substrates for the extracellular matrix proteases ADAMTS-5 and matrix metalloprotease (MMP) -3, -7, and -13. The major cleavage sites for all four proteases are found in the C terminus of HC2. ADAMTS-5 and MMP-7 displayed the highest activity toward HC2. ADAMTS-5 degradation products were identified in mass spectrometric analysis of 29 of 33 arthropathic patients, indicating that ADAMTS-5 cleavage occurs in synovial fluid in arthritis. After cleavage, free HC2, together with TSG-6, is able to catalyze the transfer of heavy chains to hyaluronan. The release of extracellular matrix bound HC2 is likely to increase the mobility of the HC2/TSG-6 catalytic unit and consequently increase the rate of the HC transfer reaction. Ultimately, ADAMTS-5 cleavage of HC2 could alter the physiological and mechanical properties of the extracellular matrix and contribute to the progression of arthritis.

Velvet antler polypeptide partially rescue facet joint osteoarthritis-like phenotype in adult ß-catenin conditional activation mice.

BACKGROUND: Wnt/ß-catenin signaling pathway is closely related to osteoarthritis. In our preliminary study, ß-catenin conditional activation (cAct) mice that specifically over-express ß-catenin gene in cartilage chondrocyte exhibits osteoarthritis-like phenotype in the lumbar disc and knee joint. Therefore, we used the mice to model FJ-OA and test the potential curative effect of Velvet Antler Polypeptide (VAP) on this mice model. METHODS: We tested the effect of VAP on ß-catenin conditional activation mice, and used Cre negative littermates as controls. Micro-CT, histology and histomorphometry analysis were performed to evaluate the curative effect of VAP on mice facet joint-like phenotype. Expression of ß-catenin and collagen II was detected by immunohistochemistry (IHC) and western-blot., MMP13, ADAMTS4 and ADAMTS5 was detected by immunofluorescence (IF). RT-PCR analysis was preformed to detect mRNA expression of cartilage degrading enzymes, such as MMP13, ADAMTS4 and ADAMTS5. RESULTS: Results of micro-CT (µCT) analysis showed that VAP could partially reverse lumbar disc osteophyte formation observed in ß-catenin(ex3)Col2ER mice. Histology data revealed VAP partially improved facet joint cartilage tissue invades. Histomorphometry analysis showed an increase in total cartilage area after VAP treatment. IHC show that VAP reduced ß-catenin protein levels and moderately up-regulated collagen II protein levels. RT-PCR and IF data showed that VAP down-regulated the expression of extracellular matrix synthesis (ECM) degradation enzymes MMP13, ADAMTS4 and ADAMTS5. CONCLUSION: Taken together, VAP may modulate ECM by inhibits MMP13, ADAMTS4 and ADAMTS5 via Wnt /ß-catenin signaling pathway. Velvet Antler Polypeptide may be a potential medicine for FJ-OA.

ADAMTS-9 in Mouse Cartilage Has Aggrecanase Activity That Is Distinct from ADAMTS-4 and ADAMTS-5.

A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 are the principal aggrecanases in mice and humans; however, mice lacking the catalytic domain of both enzymes (TS-4/5∆cat) have no skeletal phenotype, suggesting there is an alternative aggrecanase for modulating normal growth and development in these mice. We previously identified aggrecanase activity that (a) cleaved at E↓G rather than E↓A bonds in the aggrecan core protein, and (b) was upregulated by retinoic acid but not IL-1α. The present study aimed to identify the alternative aggrecanase. Femoral head cartilage explants from TS-4/5∆cat mice were stimulated with IL-1α or retinoic acid and total RNA was analysed by microarray. In addition to ADAMTS-5 and matrix metalloproteinase (MMP)-13, which are not candidates for the novel aggrecanase, the microarray analyses identified MMP-11, calpain-5 and ADAMTS-9 as candidate aggrecanases upregulated by retinoic acid. When calpain-5 and MMP-11 failed to meet subsequent criteria, ADAMTS-9 emerged as the most likely candidate for the novel aggrecanase. Immunohistochemistry revealed ADAMTS-9 expression throughout the mouse growth plate and strong expression, particularly in the proliferative zone of the TS-4/5-∆cat mice. In conclusion, ADAMTS-9 has a novel specificity for aggrecan, cleaving primarily at E↓G rather than E↓A bonds in mouse cartilage. ADAMTS-9 might have more important roles in normal skeletal development compared with ADAMTS-4 and ADAMTS-5, which have key roles in joint pathology.

Curcumin downregulates expression of opioid-related nociceptin receptor gene (OPRL1) in isolated neuroglia cells.

BACKGROUND: Curcumin (CC) exerts polyvalent pharmacological actions and multi-target effects, including pain relief and anti-nociceptive activity. In combination with Boswellia serrata extract (BS), curcumin shows greater efficacy in knee osteoarthritis management, presumably due to synergistic interaction of the ingredients. AIM: To elucidate the molecular mechanisms underlying the analgesic activity of curcumin and its synergistic interaction with BS. METHODS: We performed gene expression profiling by transcriptome-wide mRNA sequencing in human T98G neuroglia cells treated with CC (Curamed), BS, and the combination of CC and BS (CC-BS; Curamin), followed by interactive pathways analysis of the regulated genes. RESULTS: Treatment with CC and with CC-BS selectively downregulated opioid-related nociceptin receptor 1 gene (OPRL1) expression by 5.9-fold and 7.2-fold, respectively. No changes were detected in the other canonical opioid receptor genes: OPRK1, OPRD1, and OPRM1. Nociceptin reportedly increases the sensation of pain in supra-spinal pain transduction pathways. Thus, CC and CC-BS may downregulate OPRL1, consequently inhibiting production of the nociception receptor NOP, leading to pain relief. In neuroglia cells, CC and CC-BS inhibited signaling pathways related to opioids, neuropathic pain, neuroinflammation, osteoarthritis, and rheumatoid diseases. CC and CC-BS also downregulated ADAM metallopeptidase gene ADAMTS5 expression by 11.2-fold and 13.5-fold, respectively. ADAMTS5 encodes a peptidase that plays a crucial role in osteoarthritis development via inhibition of a corresponding signaling pathway. CONCLUSION: Here, we report for the first time that CC and CC-BS act as nociceptin receptor antagonists, selectively downregulating opioid-related nociceptin receptor 1 gene (OPRL1) expression, which is associated with pain relief. BS alone did not affect OPRL1 expression, but rather appears to potentiate the effects of CC via multiple mechanisms, including synergistic interactions of molecular networks.

Epigallocatechin-3-O-gallate modulates global microRNA expression in interleukin-1ß-stimulated human osteoarthritis chondrocytes: potential role of EGCG on negative co-regulation of microRNA-140-3p and ADAMTS5.

PURPOSE: MicroRNAs (miRNAs) are short, non-coding RNAs involved in almost all cellular processes. Epigallocatechin-3-O-gallate (EGCG) is a green tea polyphenol and is known to exert anti-arthritic effects by inhibiting genes associated with osteoarthritis (OA). This study was undertaken to investigate the global effect of EGCG on interleukin-1ß (IL-1ß)-induced expression of miRNAs in human chondrocytes. METHODS: Human chondrocytes were derived from OA cartilage and then treated with EGCG and IL-1ß. Human miRNA microarray technology was used to determine the expression profile of 1347 miRNAs. Microarray results were verified by taqman assays and transfection of chondrocytes with miRNA inhibitors. RESULTS: Out of 1347 miRNAs, EGCG up-regulated expression of 19 miRNAs and down-regulated expression of 17 miRNAs, whereas expression of 1311 miRNAs remains unchanged in IL-1ß-stimulated human OA chondrocytes. Bioinformatics approach showed that 3`UTR of ADAMTS5 mRNA contains the 'seed-matched-sequence' for hsa-miR-140-3p. IL-1ß-induced expression of ADAMTS5 correlated with down-regulation of hsa-miR-140-3p. Importantly, EGCG inhibited IL-1ß-induced ADAMTS5 expression and up-regulated the expression of hsa-miR-140-3p. This EGCG-induced co-regulation between ADAMTS5 and hsa-miR-140-3p becomes reversed in OA chondrocytes transfected with anti-miR-140-3p. CONCLUSIONS: This study provides an important insight into the molecular basis of the reported anti-arthritic effects of EGCG. Our data indicate that the potential of EGCG in OA chondrocytes may be related to its ability to globally inhibit inflammatory response via modulation of miRNAs expressions.

Expression of ADAMTS2 and ADAMTS5 in the salivary gland of rats after radioiodine therapy.

OBJECTIVE: The aim of this study was to investigate the presence of ADAMTS2 and ADAMTS5 in the salivary gland (SG) of rats after high-dose radioiodine therapy. METHODS: A total of 36 male Wistar albino rats were used for this study. Thirty-six male rats were divided randomly into six groups: control and five radioactive iodine (RAI) treatment groups of six rats each. All animals were killed. The evaluation of biodistribution and histopathological studies were carried out on the SGs removed. Real-time PCR and immunohistochemical analysis were carried out to determine mRNA and protein expression levels of ADAMTS genes. Differences between the groups were evaluated statistically. RESULTS: In RAI-treated groups, ADAMTS2 and ADAMTS5 gene expression was observed to increase, whereas there was no mRNA or protein expression in the control group. There were statistically significant increases in the mRNA expression of ADAMTS2 (all RAI-administered groups in parathyroid gland and at 4, 24, and 48 h in submandibular gland) and ADAMTS5 (all RAI-administered groups, except on the 30th day in the parathyroid gland and all RAI groups in submandibular gland). Through immunohistochemical analysis, the staining pattern in the extracellular source was also observed in the overexpressed ADAMTS2 and ADAMTS5 groups. Nuclear coarsening and partial focal subnuclei vacuolization were determined in all RAI-administered groups with histopathological examinations. CONCLUSION: An increase in the mRNA expression levels of ADAMTS2 and ADAMTS5 genes was detected in the RAI-administered groups. These results suggested that ADAMTS2 and ADAMTS5 genes might play a role in radiation exposure and radioiodine-induced SG changes.

Hedgehog signal expression in articular cartilage of rat temporomandibular joint and association with adjuvant-induced osteoarthritis.

OBJECTIVE: The aim of this study was to investigate the changes in hedgehog (Hh) expression and its possible effects on cartilage degeneration in adjuvant-induced temporomandibular joint osteoarthritis (TMJOA) of rats. METHODS: Forty-eight male Sprague-Dawley rats were randomly divided into experimental osteoarthritis (OA) and sham control groups. The bilateral TMJs of six rats from each group were harvested at three, seven, 14, and 28 days. Histological changes in condylar cartilage were assessed by hematoxylin and eosin, toluidine blue, and safranin O staining. The expression of Hh signal-related proteins including Indian hedgehog (Ihh), patched-1 (Ptch1), smoothened (Smo), glioma-associated oncogene homologue1 (Gli1) in cartilage was assessed by immunohistochemistry and Western blot. The protein expression of matrix metalloproteinase-13 (MMP-13), type X collagen, and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5) in cartilage was evaluated by Western blot. RESULTS: The histological analysis showed marked cartilage degeneration in adjuvant-induced OA groups, including reduced cartilage cellular density, thinner and degraded cartilage, and decreased proteoglycan content in the extracellular matrix. Compared with matched control groups, the expression of Ihh, Ptch1, Smo, and Gli1 in the OA groups was higher in a time-dependent manner. The protein levels of MMP-13, type X collagen, and ADAMTS-5 were substantially increased in OA cartilage compared with those in matched control rats. CONCLUSION: These results indicate that the activation of Ihh signaling may be correlated with pathological changes of condylar cartilage in adjuvant-induced TMJOA.

Anti-arthritic actions of ß-cryptoxanthin against the degradation of articular cartilage in vivo and in vitro.

An inverse correlation between the morbidity of rheumatoid arthritis and daily intake of ß-cryptoxanthin has been epidemiologically shown. In this study, we investigated the effects of ß-cryptoxanthin on the metabolism of cartilage extracellular matrix in vivo and in vitro. Oral administration of ß-cryptoxanthin (0.1-1 mg/kg) to antigen-induced arthritic rats suppressed the loss of glycosaminoglycans in articular cartilage, which is accompanied by the interference of aggrecanase-mediated degradation of aggrecan. Inhibition of the interleukin 1α (IL-1α)-induced aggrecan degradation by ß-cryptoxanthin was also observed with porcine articular cartilage explants in culture. ß-Cryptoxanthin (1-10 µM) dose-dependently down-regulated the IL-1α-induced gene expression of aggrecanase 1 (ADAMTS-4) and aggrecanase 2 (ADAMTS-5) in cultured human chondrocytes. Moreover, ß-cryptoxanthin was found to augment the gene expression of aggrecan core protein in chondrocytes. These results provide novel evidence that ß-cryptoxanthin exerts anti-arthritic actions and suggest that ß-cryptoxanthin may be useful in blocking the progression of rheumatoid arthritis and osteoarthritis.