Masataka Sunagawa

Interleukin-17A promotes rheumatoid arthritis synoviocytes migration and invasion under hypoxia by increasing MMP2 and MMP9 expression through NF-κB/HIF-1α pathway.

Both hypoxia and interleukin-17A (IL-17A) promote the migration and invasion of fibroblast-like synoviocytes (FLSs), which are critical for the pathogenesis of rheumatoid arthritis (RA). However, the biochemical pathways regulating IL-17A combined with hypoxia are not well defined. In this study, we found that co-stimulating RA-FLSs with IL-17A and hypoxia did not appear to promote the epithelial-mesenchymal transition (EMT), but did increase cell motility. We further showed that a proinvasive effect of IL-17A on FLSs under hypoxia might be through upregulation of matrix metalloproteinase 2 (MMP2) and MMP9. Moreover, IL-17A-induced expression of MMP2 and MMP9 under hypoxia was accompanied by increased activation of nuclear factor-κB (NF-κB)/hypoxia-inducible factor-1α (HIF-1α). Knockdown or inhibition of HIF-1α and NF-κB by small interfering RNA or specific small molecule inhibitors blocked IL-17A-mediated and hypoxia-mediated MMP2 and MMP9 expression, cell migration, and invasion. In addition, the inhibition of NF-κB led to a marked decrease in the expression of HIF-1α, which indicated that IL-17A activated HIF-1α via the NF-κB pathway in hypoxia. Taken together, our observations suggest a synergetic effect of IL-17A and hypoxia that might contribute to the migration and invasion of RA-FLSs by upregulating the expression of MMP2 and MMP9 by activation of the NF-κB/HIF-1α pathway.

Celastrol Inhibits Lipopolysaccharide-Stimulated Rheumatoid Fibroblast-Like Synoviocyte Invasion through Suppression of TLR4/NF-κB-Mediated Matrix Metalloproteinase-9 Expression

Invasion of fibroblast-like synoviocytes (FLSs) is critical in the pathogenesis of rheumatoid arthritis (RA). The metalloproteinases (MMPs) and activator of Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) pathway play a critical role in RA-FLS invasion induced by lipopolysaccharide (LPS). The present study aimed to explore the anti-invasive activity of celastrol on LPS-stimulated human RA-FLSs, and to elucidate the mechanism involved. We investigated the effect of celastrol on LPS-induced FLS migration and invasion as well as MMP expression and explored the upstream signal transduction. Results showed that celastrol suppressed LPS-stimulated FLS migration and invasion by inhibiting MMP-9 expression and activity. Furthermore, our results revealed that celastrol inhibited the transcriptional activity of MMP-9 by suppressing the binding activity of NF-κB in the MMP-9 promoter, and suppressed the TLR4/MyD88/NF-κB pathway. Administration of celastrol (0.5 mg/kg and 1 mg/kg, intraperitoneally) daily for 3 weeks in a collagen-induced arthritis rat model markedly alleviated the clinical signs, synovial hyperplasia and inflammatory cell infiltration of joints. In conclusion, celastrol might inhibit FLS migration and invasion induced by LPS by suppressing TLR4/NF-κB-mediated MMP-9 expression, providing a theoretical foundation for the clinical treatment of RA with celastrol.

Celastrol inhibits interleukin-17A-stimulated rheumatoid fibroblast-like synoviocyte migration and invasion through suppression of NF-κB-mediated matrix metalloproteinase-9 expression.

Interleukin-17A (IL-17A)-induced migration and invasion of fibroblast-like synoviocytes (FLSs) is critical for the pathogenesis of rheumatoid arthritis (RA). More than 30% of RA patients are resistant to available therapies, despite the introduction of novel biologic agents. Therefore, it is necessary to develop new anti-arthritic agents. Recent studies have demonstrated that celastrol has anti-arthritic activity in an adjuvant-induced arthritis (AIA) model. However, the effect and molecular mechanisms of celastrol on the migration and invasion of RA-FLSs are not yet understood. Results showed that treatment of RA-FLSs with celastrol suppressed the IL-17A-induced migration and invasion abilities of the cells. In addition, celastrol inhibited IL-17A-induced matrix metalloproteinase (MMP)-9 mRNA and protein expression, and the proteolytic activity of MMP-9 in RA-FLSs. Furthermore, our results revealed that celastrol inhibited the transcriptional activity of MMP-9 by suppression of the binding activity of nuclear factor-κB (NF-κB) in the MMP-9 promoter, and inhibited IκBα phosphorylation and nuclear translocation of NF-κB. In conclusion, celastrol can inhibit IL-17A-induced migration and invasion by suppressing NF-κB-mediated MMP-9 expression in RA-FLSs. These results provide a strong rationale for further testing and validation of celastrol as an adjunct with conventional drugs for the treatment of RA in humans.

Anti-invasive effects of celastrol in hypoxia-induced fibroblast-like synoviocyte through suppressing of HIF-1α/CXCR4 signaling pathway

Rheumatoid arthritis (RA) joints are in a hypoxic condition. Hypoxia-induced migration and invasion of fibroblast-like synoviocytes (FLSs) are considered to play a critical role in the pathogenesis of RA. Among the key genes upregulated by hypoxia-inducible factor-1α (HIF-1α), CXC chemokine receptor 4 (CXCR4) plays an important role in FLS migration and invasion. Our previous studies have shown that celastrol exerts anti-arthritic effects by inhibiting FLS migration and invasion under normoxic conditions. However, the effect and molecular mechanisms underlying the effect of celastrol on hypoxia-induced FLS migration and invasion are poorly understood. In the present study, we assessed the effect of celastrol on hypoxia-induced FLS migration and invasion. Results showed that celastrol suppressed hypoxia-induced FLS migration and invasion. In addition, we also found that celastrol inhibited hypoxia-induced CXCR4 expression at both the mRNA and the protein levels in RA-FLSs. Meanwhile, it is revealed that celastrol inhibited the transcriptional activity of CXCR4 under hypoxic conditions by suppressing the binding activity of HIF-1α in the CXCR4 promoter, and blocked hypoxia-induced accumulation of nuclear HIF-1α. Furthermore, treatment with HIF-1α inhibitor reduced the hypoxia-induced expression and transcriptional activity of CXCR4. In conclusion, our results indicate that celastrol inhibits hypoxia-induced migration and invasion via suppression of HIF-1α mediated CXCR4 expression in FLSs under hypoxic conditions. These results provide a strong rationale for further testing and validation of the use of celastrol as a new alternative for using in the treatment of RA.

Blood Fluidity Enhancement by Electrical Acupuncture Stimulation is Related to an Adrenergic Mechanism

We have reported that electrical acupuncture stimulation (ACU) increases blood fluidity by decreasing platelet aggregation. In this study, we investigated the mechanism causing the increase of blood fluidity. The effects of ACU on blood fluidity and platelet adhesion were examined using a Micro Channel Array Flow Analyzer (MC-FAN) and a laser scattering platelet aggregometer (PA-20). Male Wistar rats (7-8 weeks old) were used in the study. ACU (1 or 100 Hz, 3-5 V), which causes slight muscle twitching, was applied to the ZuSanli (ST-36) acupoint for 15 or 60 minutes once/day. Blood samples were collected from the inferior vena cava. ACU applied to ST-36 revealed significant increases in blood fluidity, while platelet adhesion activity decreased, regardless of the difference of stimulus time. The acupuncture had an immediate effect. Even if naloxone was administered during acupuncture stimulus, the blood flow time shortened in a similar way, as in the only acupuncture stimulus group. In addition, the effect of acupuncture on blood fluidity was inhibited by a β-antagonist. The results indicate that ACU affects blood fluidity depending on the acupoints, and that the effect of ACU might involve an endogenous adrenergic mechanism.

Moxibustion at mingmen reduces inflammation and decreases IL-6 in a collagen-induced arthritis mouse model.

The purpose of this study was to compare the effectiveness of moxibustion (MOX) treatment at the GV4 and CV12 acupoints, and to determine the correlations between MOX treatment and interleukin (IL)-6 and corticosterone levels in a collagen-induced arthritis (CIA) mouse model. CIA mice were immunized twice intradermally over a 3-week interval with bovine type II collagen. After the second immunization (day 21), MOX was applied to the mouse equivalent of the GV4 and CV12 acupoints with a 1mg moxa cone five times/day. Clinical symptoms of CIA were observed three times/week until day 35. The concentrations of IL-6 and corticosterone in the blood samples were measured by immunoassay kits. At day 35, the incidence of CIA was significantly decreased in mice treated with MOX at the GV4 acupoint (78%, n=23, p<0.05), compared to untreated CIA mice (100%) and mice treated with MOX at the CV12 acupoint (100%). IL-6 and corticosterone levels were significantly increased by immunization. IL-6 levels significantly decreased in mice treated with MOX at the GV4 acupoint. These results suggest that MOX treatment suppressed CIA at the GV4 acupoint, not at the CV12 acupoint, possibly through inhibition of IL-6 production.

Suppressive Effect of Juzentaihoto on Vascularization Induced by B16 Melanoma Cells In Vitro and In Vivo

Juzentaihoto (JTT) is well known to be one of Japanese herbal medicines, and used for the supplemental therapy of cancer patients with remarkable success. The present study, therefore, was undertaken to examine the possible therapeutic mechanisms of JTT on cancer using B16 melanoma cell (B16 cell)/experimental mouse system. JTT was well mixed with rodent chow at 3.0% concentrations, and was administered orally ad libitum. Administration of JTT was started one week before tumor cell injection and continued throughout the experiment. Administration of JTT into mice significantly inhibited tumor metastasis in lungs after intravenous injection of 2 × 105 B16 cells in a volume of 50 μL. JTT also significantly suppressed enlargement of tumor size in hind footpad after the subcutaneous injection of 2 × 105 (50 μL) B16 cells. In the second part of experiments, the chamber that containing B16 cells was buried in the murine back. In JTT administrated group, vascular endothelial growth factor (VEGF) of chamber internal fluid significantly decreased, and vascularization of chamber circumference was also inhibited. These results strongly suggest that oral administration of JTT caused decrease in the generation of VEGF, which is responsible for vascularization, and results in inhibition of B16 cell metastasis.

Promotion of blood fluidity by inhibition of platelet adhesion using electroacupuncture stimulation.

Stress applied to rats is known to result in a quick decrease in blood fluidity. Although electrical acupuncture stimulation (ACU) attenuates stress responses, the influence of ACU on blood fluidity has not been well examined. In the present study, the effect of ACU on blood fluidity and platelet adhesion was examined using a Micro Channel Array Flow Analyzer and a laser scattering platelet aggregometer (PA-20), respectively. Male Wistar rats (7-8 weeks old) were used. ACU (1 Hz, 3-5 V), which causes slight muscle twitching, was applied to acupoints for 60 minutes/day once or on 2 consecutive days. Stimulated acupoints were as follows: ZuSanli (ST-36), Sanyinjiao (SP-6), Hegu (L-I4), Neiguan (P-6), and Shenshu (BL-23). ACU applied to ST-36, SP-6, and L-14 revealed significant increases in blood fluidity while platelet adhesion activity decreased. No significant changes were observed when ACU was applied to P-6 and BL-23. Results indicate that ACU affects blood fluidity depending on the acupoints. Blood fluidity changed with ACU within 1 day. In other words, the effect of acupuncture has an immediate effect. In addition, platelet aggregation decreased with ACU, suggesting that an increase in blood fluidity is associated with platelet aggregation ability.

Inductive Effect of Palmatine on Apoptosis in RAW 264.7 Cells

Osteoporosis is a serious public health problem characterized by low bone density and deterioration of the bone microarchitecture. Current treatment options target either osteoclast resorption or osteoblast formation. It has been reported that berberine, a close structural analog of palmatine, inhibited bone loss in an osteoporosis model. In this study, osseous metabolism was observed in vitro with osteoclast bone resorbing cells. We proved that mouse preosteoclastic cell line (RAW 264.7) has a higher sensitivity to palmatine than mouse osteoblastic cell line (MC3T3-E1); the cell survival rates significantly decreased at 40 μM palmatine. The NO2 − level, a metabolic product of nitric monoxide (NO), and iNOS mRNA expression, an osteoclast with NO induced enzyme, also increased with higher dosage of palmatine. Furthermore, it was recognized that the cell viability decrease from palmatine was caused by apoptosis rather than necrosis. Additionally, osteoclast apoptosis from palmatine did not occur when iNOS was inhibited with NG-nitro-L-arginine methyl ester hydrochloride (pan NOS inhibitor). These results indicate that palmatine plays an important role in osteoclast apoptosis via the NOS system. Hence, palmatine could be considered as a viable pharmaceutical candidate for osteoporosis bone resorption inhibitor.

Anti-invasive effects of Celastrus Orbiculatus extract on interleukin-1 beta and tumour necrosis factor-alpha combination-stimulated fibroblast-like synoviocytes.

BackgroundInvasion of fibroblast-like synoviocytes (FLSs) is critical in the pathogenesis of rheumatoid arthritis (RA). The metalloproteinases (MMPs) and activator of nuclear factor-kappa B (NF-κB) pathway play a critical role in RA-FLS invasion induced by interleukin-1 beta (IL-1β) and tumour necrosis factor-alpha (TNF-α). The present study aimed to explore the anti-invasive activity and mechanism of Celastrus orbiculatus extract (COE) on IL-1β and TNF-α combination-stimulated human RA-FLSs.MethodsWe investigated the effect of COE on IL-1β and TNF-α combination-induced FLS invasion as well as MMP expression and explored upstream signal transduction.ResultsCOE suppressed IL-1β and TNF-α combination-stimulated FLSs invasion by inhibiting MMP-9 expression and activity. Furthermore, our results revealed that COE inhibited the transcriptional activity of MMP-9 by suppression of the binding activity of NF-κB in the MMP-9 promoter, and inhibited IκBα phosphorylation and nuclear translocation of NF-κB.ConclusionsCOE inhibits IL-1β and TNF-α combination-induced FLSs invasion by suppressing NF-κB-mediated MMP-9 expression.