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Advanced Science Volume 6 ,Issue 7 ,2019-02-15
Au Clusters Treat Rheumatoid Arthritis with Uniquely Reversing Cartilage/Bone Destruction
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Fuping Gao 1 Qing Yuan 1 , 2 Pengju Cai 1 Liang Gao 1 , 2 Lina Zhao 1 Meiqing Liu 1 Yawen Yao 1 Zhifang Chai 1 , 3 Xueyun Gao 1 , 2
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DOI:10.1002/advs.201801671
Received 2018-09-26,
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摘要

Abstract Super‐small nanoclusters may intrinsically trigger specific molecular pathway for disease treatment in vitro/vivo. To prove the hypothesis the super‐small nanoclusters, e.g., Au clusters, are directly used to treat rheumatoid arthritis (RA) in vitro/vivo. RA is a chronic autoimmune disease that is characterized by the inflammation of joints and the unreversible destruction of the cartilage/bone. Au clusters significantly suppress lipopolysaccharide (LPS)‐induced proinflammatory mediator production in the murine macrophage cell line by inhibiting the signaling pathways that regulate the major proinflammatory mediator genes. In preclinical rat RA studies, Au clusters strongly prevent type II collagen‐induced rat RA without systemic side effects. Compared with the clinical first‐line anchored anti‐RA drug, methotrexate, Au clusters equally inhibit inflammation in vivo. Type II collagen‐induced rat RA is characterized with the destruction of cartilage/bone; treatment with Au clusters reverses the destruction of cartilage/bone to its normal state. This is because Au clusters directly inhibit receptor activator of nuclear factor‐κB ligand (RANKL)‐induced osteoclast differentiation and function through the downregulation of osteoclast‐specific genetic marker expression. However the methotrexate almost has no positive effect for this key issue in rat RA therapy. These data prove that the super‐small nanoclusters, e.g., Au clusters, could be a novel candidate nanodrug for RA treatment.

关键词

rheumatoid arthritis;inflammation;bone destruction;Au clusters

授权许可

© 2019 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim

图表

Characterizations of the Au clusters, Au29SG27, synthesized using glutathione (GSH = γ‐Glu‐Cys‐Gly) as the template. a) High‐resolution transmission electron microscope (HRTEM) image of the Au clusters. b) UV–vis absorption (blue lines), photoemission (red lines, λex = 365 nm), and photoexcitation (black lines, λem = 610 nm) spectra of the Au clusters at room temperature. c) Digital photos of the Au clusters in deionized water under visible (left) and UV (right) light. d) Electrospray ionization mass spectrometry (ESI‐MS) of the Au clusters. ESI mass spectra of the clusters display a series of multiple charged cations originating from the addition of formic acid during testing. The molecular weight can be determined from deconvoluted spectra by combinations of Au atoms and GS ligands. The 4+ (m/z 3484.7610), 5+ (m/z 2788.0263), 6+ (m/z 2323.5251), and 7+ (m/z 1991.6510) peaks can be deconvoluted into the corresponding mass of an uncharged cluster (m/z 13 935). Such a cluster could be assigned as Au29SG27 with a molecular weight of 13 983 Da, which may lose a carboxyl group during the ionization process. e) Schematic diagram of the Au29SG27 molecular structure by density functional theory. The Au atoms and S atoms are in orange and yellow, respectively. G indicates the glutathione skeleton without S atoms and H atoms.

Au clusters synthesized with glutathione as the template (Au29SG27, designated GA) inhibit the inflammatory response of lipopolysaccharide (LPS )‐stimulated RAW 264.7 cells. a–e) Concentration‐dependent inhibition of inflammatory mediators NO a), TNF‐α b), IL‐1β c), IL‐6 d), and PGE2 e) in LPS‐stimulated RAW 264.7 cells treated with different concentrations of Au clusters. NO released in the cell supernatants was measured using Griess reagent and an ELISA plate reader. TNF‐α, IL‐1β, IL‐6, and PGE2 were measured by ELISA analysis. Data (n = 6, mean ± SD) are representative of at least two independent experiments. f) Concentration‐dependent inhibition of the i‐NOS, COX‐2, TNF‐α, IL‐1β, and IL‐6 protein levels in LPS‐stimulated RAW 264.7 cells measured by western blot analysis. β‐actin was used as an internal control. Images are representative of two experiments. g) Concentration‐dependent inhibition of the i‐NOS, COX‐2, TNF‐α, IL‐1β, and IL‐6 mRNA levels in LPS‐stimulated RAW 264.7 cells analyzed by real‐time RT‐PCR using specific primers. β‐actin was used as an internal control. Images are representative of two experiments. h) Inhibitory effect of different concentration of Au clusters on IKK, IκBα, and p65 phosphorylation in LPS‐stimulated RAW 264.7 cells analyzed by western blotting. β‐actin was used as an internal control. Images are representative of two experiments. i) Inhibitory effect of different concentrations of Au clusters on MAPK signaling pathways in LPS‐stimulated RAW 264.7 cells analyzed by western blotting. β‐actin was used as an internal control. Images are representative of two experiments. Data are presented as the mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001.

Indirect immunofluorescence assay and confocal fluorescence images of IκBα degradation and p65 nuclear localization. a,b) Concentration‐dependent inhibition of phosphor‐p65 (p‐p65) and p65 nuclear localization by Au clusters in LPS‐stimulated RAW 264.7 cells visualized with confocal microscopy after immunofluorescence staining with p‐p65 antibody a) or p65 antibody b) (green). Cells were stained with DAPI to visualize the nuclei (column labeled nuclei). c,d) Concentration‐dependent inhibition of phosphorylation and degradation of IκBα by Au clusters in LPS‐stimulated RAW 264.7 cells visualized with confocal microscopy after immunofluorescence staining with p‐IκBα antibody c) or IκBα antibody d) (green). Cells were stained with DAPI to visualize the nuclei (column labeled nuclei). The images are representative of two experiments.

Au clusters inhibit inflammatory responses in rat CIA. a–c) Progression of ankle circumference a), clinical arthritis score b), and body weight c) in CIA rats (n = 10) over 6 weeks after starting treatment with vehicle (buffer), MTX, and Au clusters. Nonimmunized normal rats treated with vehicle were used as a control group. d) Representative photographs of CIA rats treated with vehicle, MTX, and Au clusters and nonimmunized normal rats at the 1) initial, 2) middle, and 3) end timepoints of drug administration. e,f) The levels of proinflammatory cytokines (TNF‐a, IL‐1β, and IL‐6) and PGE2 at the end of the study (day 42) in the serum e) and articular tissue f) of CIA rats treated with vehicle (buffer), MTX, and Au clusters. The cytokine and PGE2 levels of nonimmunized rats in serum and articular tissue were used as controls. Data are presented as the mean ± SD; #p < 0.05 and ##p < 0.01 versus nonimmunized control rats for ankle circumference, clinical arthritis score, and body weight; *p < 0.05, **p < 0.01, and ***p < 0.001 versus CIA rats treated with vehicle. For proinflammatory cytokines and PGE2 at the end of the study in serum (e) and articular tissue (f), *p < 0.05, **p < 0.01, ***p < 0.001, and n.s., not significant.

The therapeutic effects of Au clusters in the rat CIA model. a) Representative histopathological images (n = 6 per group) of joint sections from nonimmunized rats and CIA rats treated with vehicle, MTX, and Au clusters on day 42. (Hematoxylin and Eosin staining; Scale bar = 100 µm). b) Histological scores of inflammation and cartilage and bone erosion in CIA rats treated with vehicle, MTX, and Au clusters on day 42. The joint sections of nonimmunized rats were used as controls. c) Representative microCT images (n = 6 per group) of metatarsal bone and ankle articulations in CIA rats treated with vehicle, MTX, and Au clusters on day 42. MicroCT images of metatarsal bone and ankle articulations of nonimmunized rats were used as controls. d–i) Representative images of immunohistochemistry (IHC) of phosphor‐IKK, phosphor‐IκBα, and phosphor‐p65 antigenicity (d,f,h) (3 mice per group) and percentages of p‐IKK+, p‐IκBα+, and p‐ p65+ cells (e,g,i) in joint tissues from nonimmunized rats and CIA rats treated with vehicle, MTX, and Au clusters on day 42 (Scale bar = 100 µm). Synovial cells and inflammatory cells in the articular cavity were stained brown to indicate phosphor‐IKK and phosphor‐p65 antigenicity. Data are presented as the mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001; n.s., not significant.

Au clusters directly inhibit RANKL‐induced OC formation and differentiation. a) Representative TRAP staining images (n = 9) (left) and the number of TRAP‐positive multinucleated cells after 4 days of differentiation (right), demonstrating the effect of different concentrations of Au clusters on OC formation. Scale bar = 100 µm. b) Representative phalloidin staining images (n = 9) from the actin ring formation assays; cell nuclei were stained blue using DAPI (left). Scale bar = 100 µm. The number of intact F‐actin rings (right). c) Representative images (n = 9) of bone resorption pits generated by mature OCs after 6 days and the percentage resorption area (right). Scale bar = 100 µm. M‐CSF (30 ng mL−1) and RANKL (50 ng mL−1) were present in all of the OC formation and differentiation assays. Control cells cultured without RANKL and Au clusters were used as negative controls. d) The relative mRNA expression levels of c‐fos, NFATc1, TRAP, and OSCAR in the presence of RANKL with or without Au clusters, relative to GAPDH. e) western blot analysis of protein expression levels of phosphorylated IKK, IκBα, p65, p38, ERK, and JNK in BMMs pretreated with or without Au clusters for 4 h prior to RANKL stimulation (50 ng mL−1) for the indicated times. β‐actin was used as an internal control. Images are representative of two experiments. Data are presented as the mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001.

通讯作者

Xueyun Gao.CAS Key Laboratory for the Biological Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, 100049, China;Department of Chemistry and Chemical Engineering, Beijing University of Technology, Beijing, 100124, China.gaoxy@ihep.ac.cn

推荐引用方式

Fuping Gao,Qing Yuan,Pengju Cai,Liang Gao,Lina Zhao,Meiqing Liu,Yawen Yao,Zhifang Chai,Xueyun Gao. Au Clusters Treat Rheumatoid Arthritis with Uniquely Reversing Cartilage/Bone Destruction. Advanced Science ,Vol.6, Issue 7(2019)

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