ABSTRACT
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.
Subject(s)
Animals , Male , Rats , Plant Extracts/administration & dosage , Reperfusion Injury/drug therapy , Potentilla/chemistry , Kidney Diseases/drug therapy , Immunohistochemistry , Plant Extracts/pharmacology , Rats, Wistar , Protective Agents , Disease Models, Animal , Caspase 3/metabolism , ADAMTS5 Protein/metabolismABSTRACT
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.
Subject(s)
Female , Humans , Oocytes , Polycystic Ovary Syndrome , Polycystic Ovary Syndrome/genetics , ADAMTS4 Protein/genetics , ADAMTS5 Protein/genetics , Down-RegulationABSTRACT
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.
Subject(s)
Humans , Male , Female , Tibial Meniscus Injuries/metabolism , Tibial Meniscus Injuries/pathology , Knee Joint/metabolism , Knee Joint/pathology , Synovial Fluid/chemistry , Immunohistochemistry , Antigens, CD/analysis , Synoviocytes/metabolism , ADAMTS5 Protein/analysis , Knee Joint/cytologyABSTRACT
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.
Subject(s)
Humans , Male , Female , Aged , Polymorphism, Genetic/genetics , Genetic Variation/genetics , Osteoarthritis, Knee/genetics , Genetic Predisposition to Disease/genetics , ADAMTS5 Protein/genetics , Enzyme-Linked Immunosorbent Assay , Case-Control Studies , GenotypeABSTRACT
<p><b>OBJECTIVE</b>To establish a model of chondrocyte degeneration in vitro.</p><p><b>METHODS</b>Chondrocytes were isolated from articular cartilages of newly born SD rats by digestion with typeⅡ collagenase. The chondrocytes were cultured with H-DMEM medium containing 10%FBS, 50 ng/mL IL-1β+10%FBS, 2.5% rat serum and 5% rat serum, respectively; and the chondrocytes at passage one were used in the experiments. The morphology changes were investigated under phase contrast microscope after chondrocytes were treated with rat serum and IL-1β. Proliferation of chondrocytes was detected by MTT method. The protein expression levels of PCNA, typeⅡ collagen and MMP-13 were examined by Western blotting. The levels of ADAMTS5, MMP-9, Aggrecan and SOX-9 mRNA were detected by real-time PCR.</p><p><b>RESULTS</b>The cell morphology was changed from polygon to spindle in both rat serum groups and IL-1β group, and the proliferation of chondrocytes in these groups was much higher than that in control group. The results showed that the expression levels of typeⅡ collagen, Aggrecan and SOX-9 decreased while the expression levels of MMP-13, MMP-9 and ADMATS5 were up-regulated in rat serum and IL-1β-treated groups compared with control group.</p><p><b>CONCLUSION</b>The results indicate that rat serum can induce chondrocyte degeneration and may be used for osteoarthritis model in vitro.</p>
Subject(s)
Animals , Rats , ADAM Proteins , Metabolism , ADAMTS5 Protein , Aggrecans , Metabolism , Cartilage, Articular , Cell Biology , Cells, Cultured , Chondrocytes , Pathology , Collagen Type II , Metabolism , Disease Models, Animal , Interleukin-1beta , Pharmacology , Matrix Metalloproteinase 13 , Metabolism , Matrix Metalloproteinase 9 , Metabolism , Osteoarthritis , Pathology , Proliferating Cell Nuclear Antigen , Metabolism , RNA, Messenger , Rats, Sprague-Dawley , SOX9 Transcription Factor , Metabolism , Serum , Up-RegulationABSTRACT
<p><b>BACKGROUND</b>There is a difficulty in evaluating the in vivo functionality of individual chondrocytes, and there is much heterogeneity among cartilage affected by osteoarthritis (OA). In this study, in vitro cultured chondrocytes harvested from varying stages of degeneration were studied as a projective model to further understand the pathogenesis of osteoarthritis.</p><p><b>METHODS</b>Cartilage of varying degeneration of end-stage OA was harvested, while cell yield and matrix glycosaminoglycan (GAG) content were measured. Cell morphology, proliferation, and gene expression of collagen type I, II, and X, aggrecan, matrix metalloproteinase 13 (MMP-13), and ADAMTS5 of the acquired chondrocytes were measured during subsequent in vitro culture.</p><p><b>RESULTS</b>Both the number of cells and the GAG content increased with increasing severity of OA. Cell spreading area increased and gradually showed spindle-like morphology during in vitro culture. Gene expression of collagen type II, collagen type X as well as GAG decreased with severity of cartilage degeneration, while expression of collagen type I increased. Expression of MMP-13 increased with severity of cartilage degeneration, while expression of ADAMTS-5 remained stable. Expression of collagen type II, X, GAG, and MMP-13 substantially decreased with in vitro culture. Expression of collagen type I increased with in vitro cultures, while expression of ADAMTS 5 remained stable.</p><p><b>CONCLUSIONS</b>Expression of functional genes such as collagen type II and GAG decreased during severe degeneration of OA cartilage and in vitro dedifferentiation. Gene expression of collagen I and MMP-13 increased with severity of cartilage degeneration.</p>