The Effects Of Quercetin On The Apoptosis Of Human Rheumatoid Arthritis Fibroblast-like Synoviocytes And Associated Molecular Mechanisms

Part One: The effects of quercetin on the apoptosis of human rheumatoid arthritis fibroblast-like synoviocytesObject:Quercetin is a natural flavonoid, possessing antitumor and anti-inflammatory properties. In the present study, we sought to investigate the effects of quercetin on the apoptosis of human rheumatoid arthritis fibroblast-like synoviocytes(RAFLSs).Methods:Fibroblasts were isolated from 39 patients with RA during joint replacement surgery in the First Affiliated Hospital of Zhengzhou University. Fresh synovial tissues were chopped into fragments, washed in sterile phosphate-buffered saline(PBS), and digested with collagenase 1 to obtain synovial fibroblasts. Human RAFLS morphology was observed under light microscope. Flow cytometric analysis was used to measure the expression of cluster of differentiation 3(CD3), CD14, CD19 and CD90 on the cell surface of RAFLSs. Human RAFLSs were treated with different concentrations of quercetin(0, 100, 200, and 300 μm) for 24 h. DNA fragmentation assay was performed to analyze the effects of quercetin on the apoptosis of human RAFLSs. Western blot analysis was used to detect the expression of apoptosis-associated protein cleaved caspase-3 and cleaved caspase-9. The expression of both proteins were normalized to the internal control of β-actin in human RAFLSs with different treatments. Caspase-3 and-9 activities were determined by a colorimetric assay using kits from R&D System according to the manufacturer’s protocol. Loss of mitochondrial membrane potential was assessed by flow cytometry, using the fluorescent dye rhodamine 123(Rh123). Western blot analysis was performed to measure the expression of cytosolic cytochrome C(cyt C), anti-apoptotic protein B-cell lymphoma 2(Bcl-2) and pro-apoptotic protein Bcl-2 associated X protein(Bax).Results:The cells were morphologically homogeneous and exhibited the appearance of synovial fibroblasts, with typical bipolar configuration under light microscopy. Flow cytometric analysis confirmed the isolated RAFLSs as a homogeneous population(>95% CD90, <2% CD14, <1% CD19, and <1% CD3 positive). DNA fragmentation assay showed that quercetin induced apoptosis in a dose-dependent manner in human RAFLSs. Western blot assay showed that quercetin significantly enhanced the accumulation of cleaved caspase-3 and-9. Caspase activity assays indicated that there was a concentration-dependent elevation of caspase-3 and-9 activities in quercetin-treated RAFLSs. To explore whether mitochondrial damage is involved in quercetin-induced apoptosis, we used Rh123, accumulating within mitochondria in a potential-dependent manner, to stain human RAFLSs. Notably, quercetin caused a concentration-dependent loss of mitochondrial membrane potential. The release of cyto c was also measured by Western blot analysis. In line with the results of mitochondrial damage, the release of cyto c from mitochondria into cytosol was also dose-dependently enhanced by quercetin treatment. Furthermore, Western blot analysis revealed that Bcl-2 expression was diminished and Bax expression was elevated by quercetin treatment and the Bcl-2/Bax ratio was markedly reduced in quercetin-treated human RAFLSs.Conclusion:Quercetin induces human RAFLS apoptosis through enhancing the accumulation of cleaved caspase-3 and caspase-9 and promoting their activities, coupled with a concentration-dependent loss of mitochondrial membrane potential, release of cyto c into the cytoplasm and increase of Bax/Bcl-2 ratio.Part Two: The involvement of p53 in quercetin-induced apoptosis of rheumatoid arthritis fibroblast-like synoviocytesObject:P53, a tumor suppressor, plays an important role in cell apoptosis. In this study, we sought to investigate the effects of p53 in quercetin-induced apoptosis.Methods:Different concentrations of quercetin(0, 100, 200, and 300 μm) were used to treat human RAFLSs for 24 h. Western blot analysis was conducted to analyze the phosphorylation levels of p53 in quercetin-treated human RAFLSs. Different concentrations of p53 inhibitor, pifithrin-alpha(PFT-α)(0, 10, 20, and 30 μm) were employed to pretreat human RAFLSs for 30 min prior to the treatment of RAFLSs with 200 μm of quercetin. The phosphorylation levels of p53 were measured by Western blot analysis to investigate the effects of PFT-α on quercetin-induced phosphorylation of p53. Transfection of human RAFLSs with scrambled RNA or p53-specific si RNA was performed using cationic lipopolyamines. After incubation for 4 h, fresh growth medium containing 10% FCS was added and cells were treated as described above. The silencing of p53 by specific si RNAs was analyzed by Western blot analysis. The effects of p53 knockdown on quercetin-induced human RAFLS apoptosis were investigated by DNA fragmentation.Results:Western blot analysis showed that quercetin treatment promoted the phosphorylation levels of p53 in a dose-dependent manner.PFT-a pretreatment significantly diminished the phosphorylation of p53 at the concentration of 30 mM and dose dependently reduced quercetin-induced apoptosis of human RAFLSs. To further investigate the functional effects of p53 in quercetin-induced apoptosis of RAFLSs, we used si RNAs to target p53. Western blot analysis showed that the residual expression of total p53 protein was 13.2±2.5% and 3.1±0.4% for 2 and 5 mg of si RNA, respectively. Moreover, p53 knockdown significantly decreased quercetin-induced apoptosis(P < 0.01).Conclusion:Quercetin promotes the phosphorylation level of p53 and apoptosis of human RAFLSs, which is suppressed by PFT-a pretreatment. Targeting p53 inhibits quercetin-mediated RAFLS apoptosis. Our results indicate that quercetin-induced RAFLS apoptosis is associated with the phosphorylation of p53.