Effects of dexmedetomidine on ADAMTS5 mediated matrix degradation in chondrocytes and expressions of related factors / 西安交通大学学报(医学版)

【Objective】 To investigate the effects of dexmedetomidine (DEX) intervention on the expressions of chondrocytes and related factors in vitro and its possible molecular mechanisms. 【Methods】 C28/I2 normal human chondrocyte lines were cultured in vitro, and dexmedetomidine at the concentration of 1 μmol/L was selected to intervene for 24 h and 48 h, respectively. The morphology and cell density of chondrocytes were observed after DEX culture at different time points. Immunofluorescence technique was used to detect the expression levels of disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) in chondrocytes in each group. The expression levels of Adamts5, aggrecan (Acan), versican (Vcan), Furin, proprotein convertase subtilisin/kexin type 6 (Pcsk6), collagen type Ⅰ alpha 1 (Col1a1), collagen type Ⅱ alpha 1 (Col2a1), collagen type X alpha 1 (Col10a1), and SRY2 related high mobility group box gene9 (Sox9) were detected by RT-PCR. Adamts5 gene knockout chondrocytes were constructed by lentivirus transfection technology and treated with DEX; RT-PCR was used to detect the effects of DEX on the expression levels of Acan, Vcan, Furin, Pcsk6 and Sox9 after Adamts5 gene knockout. 【Results】 After 24 and 48 h of intervention with 1 μmol/L DEX, the morphology and size of chondrocytes did not change significantly, but the cell density increased slightly. Immunofluorescence assay showed that the expression of ADAMTS5 increased at first and then decreased after DEX treatment for 24 and 48h, respectively (P=0.032). RT-PCR results showed that with the extension of intervention time, the expression of Adamts5 first increased and then decreased. The expression difference between 48 and 24 h after culture was statistically significant (P=0.032). The change trend of Pcsk6 was the same as that of Adamts5, while the change trend of Acan expression was opposite that of Adamts5. Chondrocytes knocked out Adamts5 gene and intervened with DEX for 24 and 48 h. The results of RT-PCR showed that the expression of Pcsk6 decreased while that of Acan increased and the changes were significant. 【Conclusion】 Dexmedetomidine may activate ADAMTS5 zymogen through Pcsk6, thereby promoting proteoglycan degradation in chondrocytes.

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.

ADAMTS proteoglycanases downregulation with impaired oocyte quality in PCOS

ABSTRACT Objective: A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4) and ADAMTS-5 normal expression levels are essential for ovulation and subsequent fertilization. The objective of the present study was to assess expression pattern of these genes in cumulus cells (CCs) taken from patients with polycystic ovary syndrome (PCOS) and to investigate any possible relationship with the oocyte quality. Subjects and methods: ADAMTS-4 and -5 expression levels within CCs containing oocytes at the metaphase II (MII) and germinal vesicle (GV) stages, taken from 35 patients with PCOS and 35 women with normal ovarian function, were investigated using RT-qPCR. Moreover, possible correlations between ADAMTS-4, ADAMTS-5, and progesterone receptors (PRs) expression as well as oocyte quality were evaluated. Results: ADAMTS-4 and -5 expression levels were dramatically diminished in the CCs of the PCOS patients when compared to the controls. ADAMTS-4 and -5 expression levels were correlated with each other and with the oocyte quality. Furthermore, lower expression levels of ADAMTS-4 and -5 in the PCOS patients were strongly correlated with the diminished PRs expression levels. Conclusions: Downregulation of ADAMTS-4 and -5 in the human CCs of the PCOS patients correlated with the decline in the PRs expression, and impaired oocyte quality may cause lower oocyte recovery, maturation, and fertilization rate.

Changes in Composition of ADAMTS-5 and CD-68 in the Knee Joint Synovial Fluid Cells of Meniscal Tears Patients an Immunohistochemical Study / Cambios en la Composición de ADAMTS-5 y CD-68 en las Células del Líquido Sinovial de la Articulación de la Rodilla en Pacientes con Desgarro de Menisco. Un Estudio Inmunohistoquímico

SUMMARY: Meniscus tear is an important injury affecting the quality of life. This work is aimed to investigate the activity of CD68 and ADAMTS-5 in cells in synovial fluid in male and female patients with meniscal tear. In this study ,18 male and 22 female patients with meniscal tears were included. Local pain sensation during patients' physical examination, swelling, performing daily activities and difficulty in running-walking complaints were determined. 5 cc synovial fluids were aspirated from the lateral suprapatellar pouch part of the knees with meniscal pain. After routine histological follow-up of the samples, they were embedded in paraffin and sectioned with microtome and 5 micrometer thickness. CD68 and ADAMTS-5 primary antibodies were used for immunohistochemical analysis. Sections were taken and evaluated with a stylish microscope. The distribution of blood cells after meniscus tear was higher in female patients than in male patients. CD68 distribution in female patients appeared higher than in male patients. CD68 expression was high in macrophage cell cytoplasm. ADAMTS-5 expression was higher in female patients in degenerative cells and apoptotic cells. ADAMTS-5 is an important metallo-protein involved in the development of apoptotic signal and extracellular matrix synthesis in patients with ADAMTS-5 meniscus tear, and it may be an important criterion for the treatment developed after injury. CD68 and ADAMTS-5 activity was thought to be one of the important signal pathways that can be identified in the treatment of meniscus tear.

RESUMEN: La rotura del menisco es una lesión importante que afecta la calidad de vida. El objetivo fue investigar la actividad de CD68 y ADAMTS-5 en células del líquido sinovial en pacientes masculinos y femeninos con desgarro meniscal. Se incluyeron 18 pacientes masculinos y 22 femeninos con desgarros meniscales. Se determinó la sensación de dolor local durante el examen físico de los pacientes, la hinchazón, la realización de actividades diarias y la dificultad al correr y caminar. Se aspiraron 5 cc de líquido sinoviale de la parte de la bolsa suprapatelar lateral de las rodillas de los pacientes con dolor meniscal. Después del seguimiento histológico de rutina, las muestras se incluyeron en parafina y se seccionaron con un micrótomo de grosor de 5 micrómetros. Para el análisis inmunohistoquímico se usaron los anticuerpos primarios CD68 y ADAMTS-5. La distribución de las células sanguíneas después del desgarro del menisco fue mayor en pacientes femeninos que en pacientes masculinos. La distribución de CD68 en pacientes femeninos fue más alta que en pacientes masculinos. Además la expresión de CD68 fue alta en el citoplasma de los macrófagos. La expresión de ADAMTS-5 fue mayor en pacientes femeninos en las células degenerativas y células apoptóticas. ADAMTS-5 es una metaloproteína importante en el desarrollo de la señal apoptótica y la síntesis de matriz extracelular en pacientes con rotura de menisco ADAMTS-5, y puede ser un criterio importante para el tratamiento después de la lesión. La actividad de CD68 y ADAMTS-5 era una de las vías de señal importantes que se pueden identificar en el tratamiento de la rotura del menisco.

Genetic variation of aggrecanase-2 (ADAMTS5) in susceptibility to osteoarthritis

Aggrecanase-2 (ADAMTS5) gene is responsible for aggrecan degradation that may contribute to cartilage destruction in a mouse osteoarthritis (OA) model. We aimed to investigate the effects of ADAMTS5 gene polymorphisms on OA risk in a Chinese population. A total of 300 OA patients and 300 controls were recruited and their genotypes for ADAMTS5 gene rs226794 and rs2830585 polymorphisms were determined using a custom-by-design 48-Plex single nucleotide polymorphism Scan™ kit. ADAMTS5-associated genes were identified by co-expression analysis and their functions were investigated by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. Bioinformatics analysis showed that ADAMTS5 was significantly related to the components, structural constituent, and organization of the extracellular matrix. The rs2830585 polymorphism, but not rs226794 polymorphism, was significantly associated with an increased risk of knee OA. Stratified analysis further confirmed this significant association in patients at age ≥55 years. In conclusion, the ADAMTS5 rs2830585 polymorphism may be involved in the development of knee OA by destroying the extracellular matrix, but this finding should be further confirmed by larger studies.

LncRNA MALAT1/MiR-145 Adjusts IL-1β-Induced Chondrocytes Viability and Cartilage Matrix Degradation by Regulating ADAMTS5 in Human Osteoarthritis

PURPOSE: Accumulating evidence suggests that microRNA-145 (miR-145) plays an important role in osteoarthritis (OA), which is a chronic progressive joint disease. Long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) promotes metastasis in cancers and functions as a sponge for miR-145. However, the role of MALAT1/miR-145 in OA pathogenesis has not yet been elucidated. MATERIALS AND METHODS: The expression of MALAT1 and miR-145 was examined by quantitative real-time PCR; the interaction between miR-145, MALAT1 and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 5 was verified by luciferase reporter assay. Correlations among MALAT1, miR-145, and ADAMTS5 were analyzed by Spearman rank analysis. Chondrocytes viability and cartilage extracellular matrix (ECM) degradation were investigated with cell viability assay and Western blotting analyzing expression of ADAMTS5, collagen type 2 alpha 1 (COL2A1), aggrecan (ACAN), and cartilage oligomeric matrix protein (COMP). RESULTS: MALAT1 was upregulated, and miR-145 was downregulated in OA samples and IL-1β-induced chondrocytes. Mechanically, miR-145 could directly bind to MALAT1 and ADAMTS5. Moreover, miR-145 expression was negatively correlated with MALAT1 and ADAMTS5 expression in OA patients, whereas MALAT1 and ADAMTS5 expression was positively correlated. Functionally, overexpression of MALAT1 inhibited chondrocyte viability and promoted cartilage ECM degradation in IL-1β-induced chondrocytes. In support thereof, MALAT1 silencing and miR-145 upregulation exerted the opposite effect in IL-1β-induced chondrocytes. Moreover, the effect of MALAT1 was counteracted by miR-145 upregulation, and ADAMTS5 restoration could abate miR-145 effects. CONCLUSION: An MALAT1/miR-145 axis contributes to ECM degradation in IL-1β-induced chondrocytes through targeting ADAMTS5, suggesting that MALAT1/miR-145/ADAMTS5 signaling may underlie human OA pathogenesis.

Expression and clinical significance of ADAMTS5 in human gastric cancer / 实用医学杂志

Objective To investigate the expression status of A Disintegrin and Metalloproteinase with Thrombospondin motifs 5(ADAMTS5 )and its clinical significance in primary human gastric cancer(GC). Meth-ods ADAMTS5 protein expression in 176 GC tissues and 24 adjacent normal tissues was detected by immunohisto-chemistry(IHC),and its correlation to clinicopathological features and prognosis in patients with gastric cancer was analyzed. Results ADAMTS5 expression was lower in GC tissues compared with adjacent normal tissues. Moreover,low expression of ADAMTS5 was associated with gender,histologic type,degree of differentiation,M stage,TNM stage and vascular invasion,which was also an independent indicator for poor prognosis for GC pa-tients. Conclusions ADAMTS5 expression was lower in GC tissues,which was associated with clinicopathological features and prognosis,suggesting a novel potential prognostic indicator and therapeutic target in GC.

The effects of recombinant human interleukin-1 receptor antagonist on the cartilage repair in rat temporomandibular joint with osteoarthritis / 实用口腔医学杂志

Objective:To investigate the effects of recombinant human interluekin-1 receptor antogonist (rhIL-1Ra) on the cartilage repair in rat temporomandibular joint(TMJ) with osteoarthritis(OA).Methods:Collagenase-Ⅱ was injected into bilateral TMJs of 24 adult rats for the induction of bilateral TMJOA,1 week after injection,5μg rhIL-1Ra(diluted in 0.05 ml normal saline) was injected into each right TMJ and the left joint received the same amount of normal saline injection as the control.12 animals were sacrificed at 2and 4 weeks after the first injection respectively.HE staining,immunnohistochemical method and RT-PCR examination were conducted.Mankins scere was used to evaluate the TMJOA degree.1 adult SD rat was used as healthy control,and sacrificed at 2 weeks of the experiment.Results:The TMJs of both sides showed typical OA-related cartilage degradation 2 week after IL-1Ra treatment,the Mankin~ score of the IL-1Ra treated and control joints was 1.33±0.52 and 2.00±6.63 (P＞0.05),4 week after treatment that was 3.00± 0.63 and 6.50 ± 0.84 (P＜0.05),respectively.Lower expression of ADAMTS-4 and ADAMTS-5 was observed in the treated joints than in the controls (P＜0.05).Conclusion:Intra-articular injection of IL-1Ra into TMJ can alleviate the cartilage lesion,the mechanism may lie in the inhibition of the expression of ADAMTS-4 and ADAMTS-5.