Melatonin inhibits the formation of chemically induced experimental encapsulating peritoneal sclerosis through modulation of T cell differentiation by suppressing of NF-κB activation in dendritic cells.

Encapsulating peritoneal sclerosis (EPS) is a severe complication of peritoneal dialysis (PD). Surgery is a therapeutic strategy for the treatment of complete intestinal obstruction. However, complete intestinal obstruction in long-term PD results in high mortality and morbidity rates after surgery. Immunopathogenesis participates in EPS formation: CD8, Th1, and Th17 cell numbers increased during the formation of EPS. The anti-inflammatory and immunomodulatory effects of melatonin may have beneficial effects on this EPS. In the present study, we determined that melatonin treatment significantly decreases the Th1 and Th17 cell populations in mice with EPS, decreases the production of IL-1ß, TNF-α, IL-6, and IFN-γ, and increases the production of IL-10. The suppression of Th1 and Th17 cell differentiation by melatonin occurs through the inhibition of dendritic cell (DC) activation by affecting the initiation of the NF-κB signaling pathway in DCs. Our study suggests that melatonin has preventive potential against the formation of EPS in patients with PD.

Enhancing anatomy education through cooperative learning: harnessing virtual reality for effective gross anatomy learning.

The advent of virtual reality (VR) in education offers unique possibilities for facilitating cooperative learning strategies, particularly in fields demanding intricate spatial understanding, such as gross anatomy. This study investigates the impact of integrating cooperative learning strategies within a VR-based gross anatomy curriculum, focusing on enhancing students' anatomy knowledge and skills. We analyzed the performance of two cohorts of first-year nursing students across five semesters (2016-2020), where traditional learning methods were used in the first three semesters (2016-2018), and a VR-based cooperative learning approach was adopted in the last two semesters (2019-2020). Our findings suggest that the VR-based cooperative learning group achieved significantly higher scores in their gross anatomy laboratory courses compared to their counterparts learning through traditional methods. This research provides valuable insights into how the integration of VR technology and cooperative learning strategies can not only enhance learning outcomes but also improve the VR learning experience by reducing motion sickness. It accentuates the potential of VR-based cooperative learning as an impactful educational tool in anatomy education. Future research should further explore the optimal integration of VR and cooperative learning strategies in diverse course types and their potential to enhance educational outcomes and the learning experience.

Interleukin 26 Induces Macrophage IL-9 Expression in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease with chronic inflammation, bone erosion, and joint deformation. Synovial tissue in RA patients is full of proinflammatory cytokines and infiltrated immune cells, such as T help (Th) 9, Th17, macrophages, and osteoclasts. Recent reports emphasized a new member of the interleukin (IL)-10 family, IL-26, an inducer of IL-17A that is overexpressed in RA patients. Our previous works found that IL-26 inhibits osteoclastogenesis and conducts monocyte differentiation toward M1 macrophages. In this study, we aimed to clarify the effect of IL-26 on macrophages linking to Th9 and Th17 in IL-9 and IL-17 expression and downstream signal transduction. Murine and human macrophage cell lines and primary culture cells were used and stimulated by IL26. Cytokines expressions were evaluated by flow cytometry. Signal transduction and transcription factors expression were detected by Western blot and real time-PCR. Our results show that IL-26 and IL-9 colocalized in macrophage in RA synovium. IL-26 directly induces macrophage inflammatory cytokines IL-9 and IL-17A expression. IL-26 increases the IL-9 and IL-17A upstream mechanisms IRF4 and RelB expression. Moreover, the AKT-FoxO1 pathway is also activated by IL-26 in IL-9 and IL-17A expressing macrophage. Blockage of AKT phosphorylation enhances IL-26 stimulating IL-9-producing macrophage cells. In conclusion, our results support that IL-26 promotes IL-9- and IL-17-expressing macrophage and might initiate IL-9- and IL-17-related adaptive immunity in rheumatoid arthritis. Targeting IL-26 may a potential therapeutic strategy for rheumatoid arthritis or other IL-9 plus IL-17 dominant diseases.

Intermittent Exposure of Hypercapnia Suppresses Allograft Rejection via Induction of Treg Differentiation and Inhibition of Neutrophil Accumulation.

BACKGROUND: In the management of major burn wounds, allogeneic skin transplantation is a critical procedure to improve wound repair. Our previous works found that intermittent exposure to carbon dioxide leads to permissive hypercapnia (HCA) and prolongs skin allograft survival. However, the modulatory effects of HCA exposure on the immune system are not well understood. OBJECTIVES: Our purpose was to investigate how intermittent exposure to HCA can effectively reduce the immune reaction to allogeneic skin graft rejection. METHODS: A fully major histocompatibility complex-incompatible skin transplant from BALB/c to C57BL/6 mice model was utilized. Immune cells from splenic and draining lymph nodes were analyzed by flow cytometry. Serum proinflammatory cytokines were analyzed by ELISA. RESULTS: Serum levels of IFN-γ, IL-2, IL-6, and TNF-α were significantly decreased in the HCA group. Additionally, the percentage of CD8+ cells in draining lymph nodes was significantly lower in HCA than in the control group. Moreover, the generation rate of FoxP3+ regulatory T cells (Tregs) from spleen naïve CD4+ T cells was increased by intermittent exposure to carbon dioxide. The infiltrated neutrophils were also eliminated by HCA. Taken together, we concluded that intermittent hypercapnia exposure could effectively suppress skin rejection by stimulating Treg cell generation and suppressing immune reactions.

Interleukin-26 Has Synergistic Catabolic Effects with Palmitate in Human Articular Chondrocytes via the TLR4-ERK1/2-c-Jun Signaling Pathway.

The inflammatory cytokine interleukin-26 (IL-26) is highly expressed in the serum and synovial fluid of patients with inflammatory arthritis. The effect of IL-26 on human articular chondrocytes (HACs) remains unclear. Obesity is associated with disability of patients with rheumatoid arthritis and disease activity in those with ankylosing spondylitis. The saturated free fatty acid palmitate with IL-1ß can synergistically induce catabolic effects in HACs. The aim of this study was to evaluate the effects of IL-26 and palmitate in HACs. In this study, palmitate markedly synergizes the IL-26-induced proinflammatory effects and matrix protease, including COX-2, IL-6, and MMP-1, in HACs via the toll-like receptor 4 (TLR4)-ERK1/2-c-Jun signal transduction pathway. The synergistic catabolic effects of palmitate and IL-26 were attenuated by inhibitors of TLR4 (TAK242), ERK1/2 (U0126), or c-Jun (SP600125) in HACs and cartilage matrix. In addition, metformin, a potential inhibitor of TLR4, also decreased expression of COX-2 and IL-6 induced by co-incubation with IL-26 and palmitate. IL-26 and palmitate synergistically induced expression of inflammatory and catabolic mediators, resulting in articular cartilage matrix breakdown. The present study also revealed a possible mechanism and therapeutic targets against articular cartilage degradation by increased saturated fatty acids in patients with inflammatory arthritis.

Valproic Acid Suppresses Autoimmune Recurrence and Allograft Rejection in Islet Transplantation through Induction of the Differentiation of Regulatory T Cells and Can Be Used in Cell Therapy for Type 1 Diabetes.

Type 1 diabetes mellitus (T1D) results from the destruction of insulin-producing ß cells in the islet of the pancreas by lymphocytes. Non-obese diabetic (NOD) mouse is an animal model frequently used for this disease. It has been considered that T1D is a T cell-mediated autoimmune disease. Both CD4+ and CD8+ T cells are highly responsible for the destruction of ß cells within the pancreatic islets of Langerhans. Previous studies have revealed that regulatory T (Treg) cells play a critical role in the homeostasis of the immune system as well as immune tolerance to autoantigens, thereby preventing autoimmunity. Valproic acid (VPA), a branched short-chain fatty acid, is widely used as an antiepileptic drug and a mood stabilizer. Previous reports have demonstrated that VPA treatment decreases the incidence and severity of collagen-induced arthritis and experimental autoimmune neuritis by increasing the population of Treg cells in these mouse disease models. Given the effect of VPA in the induction of Treg cells' population, we evaluated the therapeutic potential and the protective mechanism of VPA treatment in the suppression of graft autoimmune rejection and immune recurrence in syngeneic or allogenic islet transplantation mouse models. In our study, we found that the treatment of VPA increased the expression of forkhead box P3 (FOXP3), which is a critical transcription factor that controls Treg cells' development and function. Our data revealed that 400 mg/kg VPA treatment in recipients effectively prolonged the survival of syngeneic and allogenic islet grafts. The percentage of Treg cells in splenocytes increased in VPA-treated recipients. We also proved that adoptive transfer of VPA-induced Tregs to the transplanted recipients effectively prolonged the survival of islet grafts. The results of this study provide evidence of the therapeutic potential and the underlying mechanism of VPA treatment in syngeneic islet transplantation for T1D. It also provides experimental evidence for cell therapy by adoptive transferring of in vitro VPA-induced Tregs for the suppression of autoimmune recurrence.

Expression of heme oxygenase-1 in type II pneumocytes protects against heatstroke-induced lung damage.

Heatstroke (HS) is an acute clinical disease characterized by abnormal hyperthermia and multi-organ dysfunction. Heme oxygenase (HO)-1, also called heat shock protein (HSP)32, is induced by hyperthermia and also plays protective roles in many lung disease models. Based on this phenomenon, we investigated the protective role of endogenous HO-1 in heat-induced lung damage in rats. Male Sprague-Dawley (SD) rats were separated into three groups: (a) normothermic sham, (b) HS, and (c) SnPP (inhibitor of HO-1) pretreatment rats. In the HS group, rats were killed at various time points (1, 3, 6, and 12 h after heat exposure) in order to analyze messenger ribonucleic acid (mRNA) and protein levels. Lung sections were examined for tissue damage and localization of HO-1 using immunofluorescence double labeling. We found that HS induced lung pathology (congested and thickened lung septa). The level of HO-1 mRNA was increased at 1 h, and the protein level peaked at 6 h after heat exposure. Pretreatment with SnPP (tin-protoporphyrin IX, 30 mg/kg, intraperitoneal injection for 1 h before heat exposure) aggravated the lung damage. Furthermore, we demonstrated HO-1 expression in lung type II pneumocytes. Our results suggest that endogenous HO-1 is protective against HS-induced lung damage. Induction of HO-1 may be a potential therapeutic strategy for treating heat-related diseases.

Decoy Receptor 3 Promotes Preosteoclast Cell Death via Reactive Oxygen Species-Induced Fas Ligand Expression and the IL-1α/IL-1 Receptor Antagonist Pathway.

PURPOSE: Interleukin-1α (IL-1α) is a potent cytokine that plays a role in inflammatory arthritis and bone loss. Decoy receptor 3 (DCR3) is an immune modulator of monocytes and macrophages. The aim of this study was to investigate the mechanism of DCR3 in IL-1α-induced osteoclastogenesis. METHODS: We treated murine macrophages with DCR3 during receptor activator of nuclear factor kappa Β ligand- (RANKL-) plus IL-1α-induced osteoclastogenesis to monitor osteoclast formation by tartrate-resistant acid phosphatase (TRAP) staining. Osteoclast activity was assessed using a pit formation assay. The mechanisms of inhibition were studied by biochemical analyses, including RT-PCR, immunofluorescent staining, flow cytometry, an apoptosis assay, immunoblotting, and ELISA. RESULTS: DCR3 suppresses IL-1α-induced osteoclastogenesis in both primary murine bone marrow-derived macrophages (BMM) and RAW264.7 cells as it inhibits bone resorption. DCR3 induces RANKL-treated osteoclast precursor cells to express IL-1α, secretory IL-1ra (sIL-1ra), intracellular IL-1ra (icIL-1ra), reactive oxygen species (ROS), and Fas ligand and to activate IL-1α-induced interleukin-1 receptor-associated kinase 4 (IRAK4). The suppression of DCR3 during RANKL- or IL-1α-induced osteoclastogenesis may be due to the abundant secretion of IL-1ra, accumulation of ROS, and expression of Fas ligand in apoptotic osteoclast precursor cells. CONCLUSIONS: We concluded that there is an inhibitory effect of DCR3 on osteoclastogenesis via ROS accumulation and ROS-induced Fas ligand, IL-1α, and IL-1ra expression. Our results suggested that the upregulation of DCR3 in preosteoclasts might be a therapeutic target in inflammatory IL-1α-induced bone resorption.

Interleukin 26 Skews Macrophage Polarization Towards M1 Phenotype by Activating cJUN and the NF-κB Pathway.

Interleukin 26 (IL-26) is a new member of the IL-10 family that is highly expressed in rheumatoid arthritis (RA). However, the functions of IL-26 produced by macrophages in RA have not been elucidated. In the present work, we evaluated the effects and the mechanisms of IL-26 on M1 and M2 macrophage differentiation. Human or mouse macrophage cells were treated with lipopolysaccharides (LPS), interferon gamma (IFNγ), or IL-4 alone or concurrently treated with IL-26 to monitor M1 or M2 macrophage subtypes. The expression level of M1 or M2 macrophage genes was evaluated by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). The molecular mechanisms of downstream signaling activation during differentiation were investigated by immunoblotting assay. Our results found that IL-26 promoted macrophage cells from CD80+ M1 macrophage differentiation, not from the CD206+ M2 phenotype. The messenger RNA of M1-type macrophage markers tumor necrosis factor alpha (TNFα) and inducible nitric oxide synthase (iNOS) was up-regulated in the IL-26-treated group. Also, the M1-related proinflammatory cytokines TNFα and IL-6 were induced after IL-26 stimulation. Interestingly, IL-10, a cytokine marker of M2 macrophage, was also elevated after IL-26 stimulation. Moreover, the M1-like macrophage stimulated by IL-26 underwent cJUN, nuclear factor kappa B (NF-κB), and signal transducer and activator of transcription 1 (STAT1) activation. Our findings suggested the role of IL-26 in synovial macrophages of active rheumatoid arthritis and provided a new insight into IL-26 as a candidate therapeutic target in rheumatoid arthritis.

Adoptive transfer of DMSO-induced regulatory T cells exhibits a similar preventive effect compared to an in vivo DMSO treatment for chemical-induced experimental encapsulating peritoneal sclerosis in mice.

Encapsulating peritoneal sclerosis (EPS) is a severe complication of peritoneal dialysis (PD). This disease leads to intestinal obstruction with or without peritonitis. The imbalance between the populations of Th17 and regulatory T (Treg) cells (higher Th17 cells and lower Treg cells) is part of the pathogenesis of EPS formation. We demonstrated that dimethyl sulfoxide (DMSO) effectively inhibited autoimmune diabetes recurrence in the islet transplantation of NOD mice via the induction of the differentiation of Treg cells. In this study, we investigated the therapeutic potential of DMSO in the inhibition of EPS formation by a mouse model. Under DMSO treatment, the thickening of the parietal and visceral peritoneum was significantly reduced. The populations of CD4, CD8, and IFN-γ-producing CD4 and CD8 T cells were decreased. The populations of IL-4-producing CD4 T lymphocytes, IL-10-producing CD4 T lymphocytes, CD4 CD69 T lymphocytes and Treg lymphocytes were increased. The expression levels of the cytokines IFN-γ, IL-17a, TNF-α and IL-23, in ascites, were significantly decreased following the DMSO treatment. Furthermore, the differentiation of Treg cells was induced by DMSO from naïve CD4 T cells in vitro, and these cells were adoptively transferred into the EPS mice and significantly prevented EPS formation, exhibiting a comparable effect to the in vivo DMSO treatment. We also demonstrated that the differentiation of Treg cells by DMSO occurred via the activation of STAT5 by its epigenetic effect, without altering the PI3K-AKT-mTOR or Raf-ERK pathways. Our results demonstrated, for the first time, that in vivo DMSO treatment suppresses EPS formation in a mouse model. Furthermore, the adoptive transfer of Treg cells that were differentiated from naïve CD4 T cells by an in vitro DMSO treatment exhibited a similar effect to the in vivo DMSO treatment for the prevention of EPS formation.

Interleukin 26 suppresses receptor activator of nuclear factor κB ligand induced osteoclastogenesis via down-regulation of nuclear factor of activated T-cells, cytoplasmic 1 and nuclear factor κB activity.

OBJECTIVE: IL-26 has been shown to have high expression in RA. However, the effects of IL-26 on bone destruction in RA have not been evaluated. The aim of this study was to investigate the effects and mechanisms of IL-26 on RANK ligand (RANKL)-induced osteoclastogenesis. METHODS: We treated cells with IL-26 in RANKL-induced oseteoclastogenesis to monitor osteoclast formation by tartrate-resistant acid phosphatase (TRAP) staining. Osteoclast activity was assessed by pit formation assay and F-actin ring formation. The mechanism of the inhibition was studied by biochemical analyses such as RT-PCR, immunofluorescence staining and immunoblotting. In addition, cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. RESULTS: IL-26 inhibited RANKL-induced TRAP-positive multinucleated cells and inhibited RANKL-induced nuclear factor κB (NF-κB) activation and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) nuclear translocation in RAW264.7 cells. Also, IL-26 significantly inhibited the bone-resorbing activity and F-actin ring formation ability of mature osteoclasts. Moreover, IL-26 suppressed RANKL-induced mitogen-activated protein kinase activation and NFATc1 downstream gene expression. CONCLUSION: We suggest that the inhibitory activity of IL-26 on osteoclastogenesis is via down-regulation of RANKL-induced NF-κB and NFATc1 expression. Our results suggest IL-26 as a possible new remedy against osteolytic bone destruction.

Hypercapnic acidosis prolongs survival of skin allografts.

BACKGROUND: Evidence reveals that hypercapnic acidosis (HCA) modulates immune responses. However, the effect of HCA on allogenic skin graft rejection is unknown. We examined whether HCA might improve skin graft survival in a mouse model of skin transplantation. METHODS: A major histocompatibility-complex-incompatible BALB/c to C57BL/6 mouse skin transplantation model was used. Animals were divided into sham control, air, and HCA groups. Mice in the HCA group were exposed daily to 5% CO2 in air for 1 h. Skin grafts were harvested for histologic analyses. Nuclear factor (NF)-κB activation was determined in harvested draining lymph nodes. Spleen weights and serum levels of tumor necrosis factor-α and chemokine (C-X-C motif) ligand 2 were serially assessed after skin transplantation. RESULTS: Skin allografts survived significantly longer in the HCA group of mice than those in the air group. Allografted mice in the air group underwent a 2.1-fold increase in spleen weight compared with a 1.1-fold increase in the mice with HCA on day 3. There were increased inflammatory cell infiltration, folliculitis, focal dermal-epidermal separation, and areas of epidermal necrosis in the air group that were reduced with HCA treatment. In the HCA group, CD8(+) T cell infiltration at day 7 decreased significantly but not CD4(+) T cell infiltration. In addition, HCA significantly suppressed serum tumor necrosis factor-α on days 1 and 3 and chemokine (C-X-C motif) ligand 2 on days 1 and 10. Furthermore, the HCA group had remarkably suppressed NF-κB activity in draining lymph nodes. CONCLUSIONS: HCA significantly prolonged the survival of incompatible skin allografts in mice by reducing proinflammatory cytokine production, immune cell infiltration, and NF-κB activation.

Decoy receptor 3 suppresses RANKL-induced osteoclastogenesis via down-regulating NFATc1 and enhancing cell apoptosis.

OBJECTIVE: Decoy receptor 3 (DCR3) has been known to modulate immune functions of monocyte or macrophage. In the present study, we investigated the mechanism and the effect of DCR3 on RANK ligand (RANKL)-induced osteoclastogenesis. METHODS: We treated cells with DCR3 in RANKL-induced osteoclastogenesis to monitor osteoclast formation by tartrate-resistant acid phosphatase (TRAP) staining. Osteoclast activity was assessed by pit formation assay. The mechanism of inhibition was studied by biochemical analysis such as RT-PCR and immunoblotting. In addition, cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell apoptosis and apoptosis signalling were evaluated by immunoblotting and using flow cytometry. RESULTS: DCR3 inhibited RANKL-induced TRAP(+) multinucleated cells and inhibited RANKL-induced nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation and nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) nuclear translocation in RAW264.7 cells. Also, DCR3 significantly inhibited the bone-resorbing activity of mature osteoclasts. Moreover, DCR3 enhanced RANKL-induced cell apoptosis and enhanced RANKL-induced Fas ligand expression. The mechanisms were mediated via the intrinsic cytochrome c and activated caspase 9 apoptosis pathway. CONCLUSION: We postulated that the inhibitory activity of DCR3 on osteoclastogenesis occurs via down-regulation of RANKL-induced NFATc1 expression and induction of cell apoptosis. Our results postulated DCR3 as a possible new remedy against inflammatory bone destruction.

A benzamide-linked small molecule NDMC101 inhibits NFATc1 and NF-κB activity: a potential osteoclastogenesis inhibitor for experimental arthritis.

PURPOSE: Using receptor activator of NF-κB ligand (RANKL) induced osteoclast differentiation on RAW264.7 as a screening tool; we synthesize and identify small-molecule inhibitors preserving immunomodulatory effects as therapeutics for rheumatoid arthritis. METHODS: Differentiation into osteoclast-like cells was examined by tartrate-resistant acid phosphatase (TRAP) staining and expression of osteoclast differentiation markers. Collagen-induced arthritis (CIA) mice were administered test articles by gavages to assess its efficacy. Then clinical, histological, and biochemical parameters were assessed to determine the effects of N-(4-chloro-2-fluorophenyl)-2-hydroxybenzamide (NDMC101) on synovial inflammation and bone erosion by hematoxlin and eosin staining and Enzyme-linked immunosorbent assay (ELISA). RESULTS: NDMC101 markedly inhibited RANKL-induced formation of TRAP+ multinucleated cells in RAW264.7 and bone marrow macrophage cells (BMMs). Moreover, pit formation assay showed that NDMC101 significantly reduced the bone-resorbing activity of mature osteoclasts. In CIA mice, oral administration of NDMC101 reduced arthritic index and mitigated bone erosion. Serum TNF-α and IL-1ß concentrations in these mice were decreased significantly at the higher dose of 62.5 mg/kg. CONCLUSIONS: Screening of our chemical library, our findings suggest that NDMC101 inhibits osteoclastogenesis which also ameliorates paw swelling and inflammatory bone destruction. Its efficacy is associated with the inhibition of such transcription factors as NF-κB and NFATc1 as well as multiple protein kinases, including p38, ERK, and JNK. There results guarantee further clinical tests of NDMC101 for its therapeutic potential in the treatment of inflammation-induced bone diseases.

Decoy receptor 3 attenuates collagen-induced arthritis by modulating T cell activation and B cell expansion.

OBJECTIVE: To investigate the immune-modulated effects of decoy receptor 3 (DCR3) in an experimental model of rheumatoid arthritis (RA). METHODS: We delivered DCR3 plasmid into collagen-induced arthritis (CIA) mice using the hydrodynamic method and evaluated the serum level of DCR3 protein by ELISA. After immunization, we assessed disease severity of arthritis incidence, arthritis scores, paw thickness, and means of arthritic limbs, and used hematoxylin and eosin staining to observe synovial hyperplasia. We analyzed numbers of murine splenocytes and inguinal lymphocyte cells, cell populations, and serum proinflammatory cytokines by flow cytometry. We investigated B cell proliferation by carboxyfluorescein succinimidyl ester assay. We evaluated serum levels of total IgG2a and type II collagen-specific IgG and IgG2a using ELISA. RESULTS: DCR3 expression in sera significantly attenuated disease severity in CIA mice. We found that DCR3 inhibited the volume of inguinal lymph nodes, numbers of CD19+ B cells, and populations of interferon-γ, interleukin 4 (IL-4), IL-17A, and Foxp3-producing CD4+ T cell in vivo. We found that DCR3 inhibited Pam3CSK4 (Toll-like receptor 1/2 ligand)-induced B220+ B cell proliferation in vitro. DCR3 treatment reduced the serum level of IL-6, total IgG2a, and CII-specific IgG2a antibody. CONCLUSION: We postulated that the protective effects of DCR3 in CIA resulted from modulation of the immune system by maintaining the B/T cell balance and decreasing lymphocyte expansion. We suggest DCR3 as a prophylactic and potential therapeutic agent in the treatment of RA.

Plectranthus amboinicus attenuates inflammatory bone erosion in mice with collagen-induced arthritis by downregulation of RANKL-induced NFATc1 expression.

OBJECTIVE: Plectranthus amboinicus has been known to treat inflammatory diseases or swelling symptoms. We investigated whether P. amboinicus exhibited an inhibitory effect on osteoclastogenesis in vitro and inflammatory bone erosion in collagen-induced arthritis (CIA) mice, an animal model of rheumatoid arthritis. We attempted to identify the active component of P. amboinicus involved in regulation of osteoclastogenesis. METHODS: We treated M-CSF- and RANKL-stimulated murine bone marrow-derived macrophages (BMM) and RANKL-induced RAW264.7 cells with different concentrations of P. amboinicus or rosmarinic acid, a phytopolyphenol purified from P. amboinicus, to monitor osteoclast formation by TRAP staining. The mechanism of the inhibition was studied by biochemical analysis such as RT-PCR and immunoblotting. CIA mice were administered gavages of P. amboinicus (375 mg/kg) or placebo. Then clinical, histological, and biochemical measures were assessed to determine the effects of P. amboinicus on synovial inflammation and bone erosion by H&E staining of the inflamed joints and ELISA. RESULTS: Rosmarinic acid strongly inhibited RANKL-induced NF-κB activation and nuclear factor of activated T cells c1 (NFATc1) nuclear translocation in BMM, and also inhibited RANKL-induced formation of TRAP-positive multinucleated cells. A pit formation assay and the CIA animal model showed that P. amboinicus significantly inhibited the bone-resorbing activity of mature osteoclasts. CONCLUSION: We postulated that rosmarinic acid conferred the inhibitory activity on P. amboinicus for inhibition of osteoclastogenesis via downregulation of RANKL-induced NFATc1 expression. Our results indicated the possibility of P. amboinicus as a new remedy against inflammatory bone destruction.