RESUMEN
OBJECTIVE: Osteoarthritis (OA) is characterized by progressive matrix destruction of articular cartilage. This study aimed to investigate the potential antioxidative and chondroprotective effects and underlying mechanism of Icariin (ICA) in interleukin-1 beta (IL-1ß)-induced extracellular matrix (ECM) degradation of OA cartilage. METHODS: Human chondrocyte cell line HC-A was treated with different doses of ICA, and then MTT assay and PI staining were used to estimate ICA-induced chondrocyte apoptosis. Intracellular ROS and superoxide dismutase (SOD) and glutathione peroxidase (GPX) were measured after treatment by IL-1ß with or without ICA. The mRNA and protein expression levels of redox transcription factor Nrf2 and the downstream effector SOD-1, SOD-2, NQO-1 and HO-1 were assayed to explore the detailed mechanism by which ICA alleviates ECM degradation. Finally, to expound the role of Nrf2 in ICA-mediated chondroprotection, we specifically depleted Nrf2 in human chondrocytes and then pretreated them with ICA followed by IL-1ß. RESULTS: ICA had no cytotoxic effects on human chondrocytes and 10-9 M was selected as the optimum concentration. ROS induced by IL-1ß could drastically activate matrix-degrading proteases and ICA could significantly rescue the matrix degradation and excess ROS generation caused by IL-1ß. We observed that ICA activated the Nrf2/ARE pathway, consequently upregulating the generation of GPX and SOD. Ablation of Nrf2 abrogated the chondroprotective and antioxidative effects of ICA in IL-1ß-treated chondrocytes. CONCLUSION: ICA alleviates IL-1ß-induced matrix degradation and eliminates ROS by activating the Nrf2/ARE pathway.
Asunto(s)
Elementos de Respuesta Antioxidante/efectos de los fármacos , Condrocitos/efectos de los fármacos , Medicamentos Herbarios Chinos/farmacología , Flavonoides/farmacología , Interleucina-1beta/antagonistas & inhibidores , Factor 2 Relacionado con NF-E2/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Células Cultivadas , Condrocitos/metabolismo , Relación Dosis-Respuesta a Droga , Medicamentos Herbarios Chinos/química , Flavonoides/química , Humanos , Interleucina-1beta/metabolismo , Conformación Molecular , Factor 2 Relacionado con NF-E2/metabolismo , Especies Reactivas de Oxígeno/análisis , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
PURPOSE: Liver fibrosis is a worldwide health issue. Development of effective new drugs for treatment of this disease is of great importance. This study investigated the therapeutic effects of ferulic acid on liver fibrosis in vitro and in vivo. MATERIALS AND METHODS: Human hepatic stellate cell line (HSC) LX-2 was used for in vitro assays. Transforming growth factor ß1 (TGF-ß1) was used to induce hepatic fibrosis in LX-2 cells. Western blot was used to detect protein levels of collagen I, fibronectin, α-smooth muscle actin (SMA), p-Smad2, p-Smad3, p-p38, and p-JNK. Gene expression was measured by RT-qPCR. Fluorescence staining was used to determine localization of Smad4. CCl4-induced hepatic fibrosis in SD rats was used as an in vivo model. Histological features were detected by hematoxylin and eosin staining. Levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), hexadecenoic acid (HA), and hydroxyproline (Hyp) were measured by ELISA. RESULTS: TGF-ß1 treatment significantly increased levels of collagen I, fibronectin, α-SMA, p-Smad2, p-Smad3, and Smad4 in LX-2 cells. Ferulic acid improved TGF-ß1-induced hepatic fibrosis via regulation of the TGF-ß1/Smad pathway. Consistent with in vitro data, CCl4 caused severe hepatic fibrosis in SD rats, as determined by ALT, AST, HA, and Hyp upregulation. Protein levels of p-Smad2 and p-Smad3 in liver tissues were significantly increased following treatment with CCl4. All CCL4-induced changes were markedly attenuated by ferulic acid treatment. CONCLUSION: Ferulic acid potently improved hepatic fibrosis via inhibition of the TGF-ß1/Smad pathway in vitro and in vivo. These findings provided evidence for potential use of ferulic acid to treat or prevent liver fibrosis.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Ácidos Cumáricos/farmacología , Células Estrelladas Hepáticas/efectos de los fármacos , Cirrosis Hepática Experimental/prevención & control , Hígado/efectos de los fármacos , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Actinas/metabolismo , Animales , Tetracloruro de Carbono , Línea Celular , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Colágeno Tipo I/metabolismo , Citoprotección , Fibronectinas/metabolismo , Células Estrelladas Hepáticas/metabolismo , Células Estrelladas Hepáticas/patología , Humanos , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática Experimental/inducido químicamente , Cirrosis Hepática Experimental/metabolismo , Cirrosis Hepática Experimental/patología , Masculino , Fosforilación , Ratas Wistar , Transducción de Señal/efectos de los fármacosRESUMEN
AIM: To observe the effect of Danshao Huaxian capsule (DHC) on the expression of Gremlin and bone morphogenetic protein-7 (BMP-7) in the liver of hepatic fibrosis rats. METHODS: A total of 75 male Wistar rats were randomly divided into a normal control group (A), a CCl4-induced hepatic fibrosis model group (B), a natural recovery group (C), a low-dose DHC-treated group (D), and a high-dose DHC-treated group (E), with 15 rats in each group. Liver fibrosis was induced by subcutaneous injections of carbon tetrachloride (CCl4) and a high-lipid/low-protein diet for 8 wk, except for the rats in group A. Then, the rats in the two DHC-treated groups were administered 0.5 and 1.0 g/kg DHC by gastrogavage once per day for 8 successive weeks, respectively. By the end of the experiment, the level of transforming growth factor ß1 (TGF-ß1) in the liver homogenate was determined by an enzyme-linked immunosorbent assay. The mRNA and protein expression of Gremlin and BMP-7 in the liver tissue was determined by reverse-transcription polymerase chain reaction, an immunohistochemical assay, and Western blot analysis. RESULTS: Compared with group A, the level of TGF-ß1 and the mRNA and protein expression of Gremlin were significantly higher in group B (TGF-ß1: 736.30 ± 24.40 µg/g vs 284.20 ± 18.32 µg/g, P < 0.01; mRNA of Gremlin: 80.40 ± 5.46 vs 49.83 ± 4.20, P < 0.01; positive protein expression rate of Gremlin: 38.46% ± 1.70% vs 3.83% ± 0.88%, P < 0.01; relative protein expression of Gremlin: 2.81 ± 0.24 vs 0.24 ± 0.06, P < 0.01), and the mRNA and protein expression of BMP-7 was significantly lower in group B (mRNA: 54.00 ± 4.34 vs 93.99 ± 7.03, P < 0.01; positive protein expression rate: 28.97% ± 3.14% vs 58.29% ± 6.02, P < 0.01; relative protein expression: 0.48 ± 0.31 vs 1.05 ± 0.12, P < 0.01). Compared with groups B and C, the degree of hepatic fibrosis was significantly improved, and the level of TGF-ß1 and the mRNA and protein expression of Gremlin were significantly lowered in the two DHC-treated groups (TGF-ß1: 523.14 ± 21.29 µg/g, 441.86 ± 23.18 µg/g vs 736.30 ± 24.40 µg/g, 651.13 ± 15.75 µg/g, P < 0.01; mRNA of Gremlin: 64.86 ± 2.83, 55.82 ± 5.39 vs 80.40 ± 5.46, 70.37 ± 4.01, P < 0.01; positive protein expression rate of Gremlin: 20.78% ± 1.60%, 17.43% ± 2.02% vs 38.46% ± 1.70%, 29.50% ± 2.64%, P < 0.01; relative protein expression of Gremlin: 1.95 ± 0.26, 1.65 ± 0.20 vs 2.81 ± 0.24, 2.22 ± 0.63, P < 0.01), and the mRNA and protein expression of BMP-7 was higher in the two DHC-treated groups (mRNA: 73.52 ± 4.56, 81.78 ± 5.38 vs 54.00 ± 4.34, 62.28 ± 4.51, P < 0.01; positive protein expression rate: 41.44% ± 4.77%, 47.49% ± 4.59% vs 28.97% ± 3.14%, 35.85% ± 3.50%, P < 0.01; relative protein expression: 0.71 ± 0.06, 0.81 ± 0.07 vs 0.48 ± 0.31, 0.60 ± 0.37, P < 0.01). CONCLUSION: The therapeutic mechanism of DHC for hepatic fibrosis in rats may be associated with inhibition of the expression of Gremlin and up-regulation of the expression of BMP-7.