Immunofluorescence studies: Representative photographs showing excessive P-LKB1/LC3-II (+) expression and markedly diminished CTFβ(+)-cells under treatment with cilostazol (10 μM) in the activated N2aSwe cells, as compared with vehicle-treated group.

<p>Immunofluorescence studies: Representative photographs showing excessive P-LKB1/LC3-II (+) expression and markedly diminished CTFβ(+)-cells under treatment with cilostazol (10 μM) in the activated N2aSwe cells, as compared with vehicle-treated group.</p

Comparison of cilostazol- and resveratrol-stimulated P-LKB1 and P-AMPKα expressions in N2a cells that LKB1 is detectable, and in HeLa cells that lack LKB1.

<p><b>A</b>. Immunoblot of P-LKB significantly increased after pretreating N2a cells with cilostazol (CSZ, 10 μM) or resveratrol (RES, 20 μM), whereas P-LKB expression was not appeared in HeLa cells. <b>B</b>. The significant increases in P-AMPK expression by cilostazol (CSZ, 10 μM) or resveratrol (Res, 20 μM) were not observed in HeLa cells, whereas they were obviously identified in N2a cells. Results are expressed as the means ± SDs of 4 experiments. ***<i>P</i> < 0.001 vs. vehicle (Veh). <b>C</b>. Proposed signal pathways for the neuroprotective effect of cilostazol against Aβ-induced neurotoxicity: Cilostazol upregulates autophagy through activating SIRT1/LKB1/AMPK1α signal pathways and depletes intracellular Aβ and APP-CTFβ accumulation, and thereby results in decreased neurotoxicity.</p

Pueraria montana Merr.

原著和名: タイワンクズ科名: マメ科 = Leguminosae採集地: 沖縄県 石垣島 (琉球 石垣島)採集日: 1977/9/23採集者: 萩庭丈壽整理番号: JH017540国立科学博物館整理番号: TNS-VS-96754

Suppression of endothelial cell tube formation by cilostazol (CSZ).

<p>Photographs of tube formation induced by HMGB1 (100 ng/ml), which was applied directly to the HMVECs in the absence and the presence of 10 µM of cilostazol (A) and by conditioned medium (CM) derived from cultures of HMGB1 (100 ng/ml for 48 hr)-treated RA synovial fibroblasts (C). Diluted CM was added to the culture medium after adding HMVECs to Matrigel. Cultures were photographed after 9 hr (×100). (B and D) Quantitative analyses: Results are mean numbers ±SEM of tubes per well for 4–5 experiments. **<i>P</i><0.01, ***<i>P</i><0.001 vs. PBS (phosphate-buffered saline); ^##<i>P</i><0.01, ^###<i>P</i><0.001 vs. HMGB1 alone; ^†††<i>P</i><0.001 vs. 10 µM cilostazol.</p

Hypothetical model.

<p>Scheme illustrating the dual effects of cilostazol, namely, (1) its inhibition of HMGB1-activated NF-κB nuclear translocation and subsequent HIF-1α expression, and (2) its inactivation of HIF-1α by increasing SIRT1 deacetylase activity in RA SFs. These effects lead to the down-regulation of VEGF and to the inhibition of synovial angiogenesis.</p

Suppression of HMGB1-induced elevations of VEGF mRNA and protein expressions by cilostazol.

<p>(A, B, C and D) Time- and concentration-dependent increases in VEGF mRNA and protein expressions stimulated by HMGB1. (E) Concentration-dependent suppression of HMGB1-stimulated VEGF mRNA expression by cilostazol (1–100 µM). (F) HMGB1-induced increase in VEGF releases and the attenuation of VEGF release by 10 µM cilostazol, by 30 µM YC-1 (HIF-1α inhibitor), and by 10 nM chetomin. The effect of cilostazol was blocked by KT5720 (KT, 1 µM) pretreatment. (G) Representative immunofluorescence photographs showing greater VEGF (+)-staining in HMGB1-treated synovial fibroblasts (24 hr), compared with control synovial fibroblasts. After treatment with cilostazol (10 µM), VEGF (+)-staining was obviously reduced. Results are expressed as means ±SEM of 4–5 experiments. **<i>P</i><0.01, ***<i>P</i><0.001 vs. no treatment; ^##<i>P</i><0.01, ^###<i>P</i><0.001 vs. 100 ng/ml HMGB1 alone; ^†††<i>P</i><0.001 vs. 10 µM cilostazol.</p

Elevations of HIF-1α mRNA and protein expressions by HMGB1 and their inhibition by cilostazol.

<p>(A and B) Concentration-dependent HMGB1 (10–500 ng/ml)-induced increases in the mRNA and protein expressions of HIF-1α. (C) Time (3, 6, 12, and 24 hr)-dependent increases in HIF-1α protein levels in the presence of 100 ng/ml of HMGB1. (D) Concentration-dependent decrease of HMGB1 (100 ng/ml)-induced HIF-1α protein by cilostazol (CSZ; 1, 10 and 30 µM) and resveratrol (RES, 20 µM). (E and F) Reverse of cilostazol-induced decrease in HIF-1α mRNA (E) and protein (F) by KT5720 (1 µM; a cAMP-dependent protein kinase inhibitor), and significant suppression of HMGB1-induced HIF-1α mRNA by chetomin (10 nM, an inhibitor of hypoxia-inducible transcription) and by Bay11–7082 (1 µM, an inhibitor of I<i>κ</i>Bα phosphorylation). Results are the means ±SEM of 4–5 experiments. **<i>P</i><0.01, ***<i>P</i><0.001 vs. no treatment; ^#<i>P</i><0.05, ^##<i>P</i><0.01, ^###<i>P</i><0.001 vs. HMGB1 alone; ^†††<i>P</i><0.001 vs. 10 µM cilostazol.</p

Increased SIRT1 expression and deacetylase activity by cilostazol.

<p>(A) Time (3–24 hr)-dependent decrease in SIRT1 protein expression after exposure to HMGB1 (100 ng/ml), and (B) recovery of reduced SIRT1 expression by cilostazol (1–30 µM) and by resveratrol (RES, 20 µM). (C) HMGB1-induced increase in H4 acetyl-k16 (H4 Ac-K16) immunoreactivity was decreased by cilostazol (10 and 30 µM) and resveratrol (RES, 20 µM). Cilostazol-induced decrease in H4 Ac-K16 was prevented by sirtinol (20 µM). (D) Analyses of SIRT1 knockout RA SFs compared with negative control cells. Cilostazol (10 µM) failed to induce expression of SIRT1 in RA SF cells transfected with SIRT1 siRNA oligonucleotide (100 nM) as contrasted to negative control. (E and F) Loss of the effect of cilostazol on the elevated expressions of p65 and acetyl (AC)-p65 induced by HMGB1 (100 ng/ml) in SIRT1 gene silenced fibroblasts, which contrasted to the effects of cilostazol in the negative control cells. Results are the means ±SEM of 4 experiments. **<i>P</i><0.01, ***<i>P</i><0.001 vs. no treatment or vehicle (Veh); ^##<i>P</i><0.01, ^###<i>P</i><0.001 vs. HMGB1; ^††<i>P</i><0.01 vs. 10 µM cilostazol.</p

Inhibition of NF-κB p65 expression and activity by cilostazol.

<p>(A, B and C) HMGB1-induced increase in phosphorylated IκBα (A) and increase in the nuclear translocation of NF-κB (B) and DNA binding activity of NF-κB in synovial fibroblasts (C). These effects were inhibited by glycyrrhizin (50 µM, an inhibitor of HMGB1 binding to DNA) and by Bay11–7082 (1 µM). (D, E and F) Cilostazol (CSZ; 1, 10 and 30 µM) and resveratrol (RES; 20 µM) decreased phosphorylated IκBα (D), nuclear NF-κB (E) levels and DNA binding activity of NF-κB (F). (G, H) Suppression of HMGB1-stimulated production of proinflammatory cytokines, IL-6 and MCP-1, by cilostazol (10 µM) and the reversal of this suppression by KT5720 (1 µM) in cultured medium. Results are the means ±SEM of 3–4 experiments. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 vs. no treatment; ^#<i>P</i><0.05, ^##<i>P</i><0.01, ^###<i>P</i><0.001 vs. HMGB1 alone; ^††<i>P</i><0.01 vs. 10 µM cilostazol.</p

Immunohistochemistry (H & E and CD31) and immunofluorescence (SIRT1, HIF-1α and VEGF) assay.

<p>(A) Photographs showing SIRT1(+)-, HIF-1α (+)- and VEGF (+)-spots in joint sections of vehicle control and cilostazol (CSZ)-treated CIA mice. (B) Tibiotarsal joint tissue sections were stained with anti-CD31 antibody, and rabbit anti-human vWF (a marker of endothelial cells). Numbers of blood vessels/field and percentages of vWF stained area were analyzed in joint sections of CIA mice. Results are the means ±SEM of 4 experiments. *<i>P</i><0.05, **<i>P</i><0.01 vs. control; ^#<i>P</i><0.05 vs. vehicle (Veh)-treated CIA mice. The pictures shown are representative of 4 experiments that produced similar results.</p