RESUMO
The study aimed to investigate the preservative effects of genistein on articular cartilage in an experimental model of knee osteoarthritis in rats. Thirty male Wistar rats were assigned to 3 equal groups: sham group, osteoarthritis control group (OAG), and genistein-treated osteoarthritis group (GTG). Intra-articular injections of monosodium iodoacetate were used for osteoarthritis induction. After 2 weeks of rest for the induction of the inflammatory process, genistein (30 mg/kg/day) vs. saline gavage was administered for 8 weeks. The expression of matrix metalloproteinase (MMP)-8 and MMP-13, Sox5/Sox6, Indian hedgehog (IHH), and Col2 were evaluated in medial femoral condyle sections by immunohistochemical staining. The number of chondrocytes and cartilage thicknesses were also measured and compared among the groups. No significant change in cartilage thickness was observed in GTG compared with OAG (p = 0.188). Chondrocyte count was significantly higher in the articular cartilage of GTG compared with OAG (p = 0.006). Induction of osteoarthritis significantly increased the expression of MMP-8, MMP-13, and IHH, but decreased Col2, Sox5, and Sox6 expression (p < 0.001); these were partially prevented in the GTG. Our findings support the effectiveness of genistein treatment in the prevention of articular cartilage damage in the experimental model of knee osteoarthritis. The proposed mechanism of action is through the suppression of the MMP, IHH, and Col2 pathways, besides the induction of Sox5 and Sox6 expression. Novelty: Genistein prevents articular cartilage damage in the experimental model of knee osteoarthritis. The osteoprotective effect is manifested by the modulation of expression of MMP, Sox, IHH, and Col2 proteins.
Assuntos
Anti-Inflamatórios/farmacologia , Cartilagem Articular/efeitos dos fármacos , Genisteína/farmacologia , Articulação do Joelho , Osteoartrite do Joelho/prevenção & controle , Fitoestrógenos/farmacologia , Animais , Anti-Inflamatórios/uso terapêutico , Cartilagem Articular/citologia , Contagem de Células , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Genisteína/uso terapêutico , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Imuno-Histoquímica , Masculino , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 8 da Matriz/genética , Metaloproteinase 8 da Matriz/metabolismo , Osteoartrite do Joelho/metabolismo , Fitoestrógenos/uso terapêutico , Ratos Wistar , Fatores de Transcrição SOXD/genética , Fatores de Transcrição SOXD/metabolismoRESUMO
Here we describe a single-molecule quantitative assay of mRNA levels based on mRNA mediate-ligation and BEAMing (beads, emulsion, amplification, and magnetics) technique, which allows accurate and parallel measurement of multiple genes from a small amount of cells. In this method, a pair of oligos complementary target mRNA was used to probe transcripts for each gene of interest. The ligated products of oligos pair were clonally amplified on beads in millions of parallel compartmentalized droplets in a water-in-oil emulsion. The levels of each transcript within a sample were measured by counting the number of the correspondingly amplified beads which were immobilized on a glass surface. To demonstrate its utility, this method has been applied to the quantitation of the mRNA levels for two transcription factors, Klf4 and Sox5, and a housekeeping gene, Gapdh, in human leukemia K562 cells before and after induction with phorbol 12-myristate 13-acetate. Interestingly, we found a significant downregulation of the mRNA level of Sox5 after phorbol 12-myristate 13-acetate treatment. The mRNA mediate-ligation and BEAMing technique provides an accurate and sensitive way to quantify the amount of multiple specific mRNA in a very small number of cells, which may be valuable in the studies requiring precise and parallel quantization of multiple mRNA in the defined cell populations.