Bleomycin-induced Systemic Sclerosis Research Articles

MicroRNA-21a-5p inhibition alleviates systemic sclerosis by targeting STAT3 signaling

BackgroundMicroRNA (miRNA)-21-5p participates in various biological processes, including cancer and autoimmune diseases. However, its role in the development of fibrosis in the in vivo model of systemic sclerosis (SSc) has not been reported. This study investigated the effects of miRNA-21a-5p overexpression and inhibition on SSc fibrosis using a bleomycin-induced SSc mouse model.MethodsA murine SSc model was induced by subcutaneously injecting 100 μg bleomycin dissolved in 0.9% NaCl into C57BL/6 mice daily for 5 weeks. On days 14, 21, and 28 from the start of bleomycin injection, 100 μg pre-miRNA-21a-5p or anti-miRNA-21a-5p in 1 mL saline was hydrodynamically injected into the mice. Fibrosis analysis was conducted in lung and skin tissues of SSc mice using hematoxylin and eosin as well as Masson’s trichrome staining. Immunohistochemistry was used to examine the expression of inflammatory cytokines, phosphorylated signal transducer and activator of transcription-3 (STAT3) at Y705 or S727, and phosphatase and tensin homologue deleted on chromosome-10 (PTEN) in skin tissues of SSc mice.ResultsMiRNA-21a-5p overexpression promoted lung fibrosis in bleomycin-induced SSc mice, inducing infiltration of cells expressing TNF-α, IL-1β, IL-6, or IL-17, along with STAT3 phosphorylated cells in the lesional skin. Conversely, anti-miRNA-21a-5p injection improved fibrosis in the lung and skin tissues of SSc mice, reducing the infiltration of cells secreting inflammatory cytokines in the skin tissue. In particular, it decreased STAT3-phosphorylated cell infiltration at Y705 and increased the infiltration of PTEN-expressing cells in the skin tissue of SSc mice.ConclusionMiRNA-21a-5p promotes fibrosis in an in vivo murine SSc model, suggesting that its inhibition may be a therapeutic strategy for improving fibrosis in SSc.

Tanshinone IIA ameliorates the development of dermal fibrosis in systemic sclerosis.

We previously revealed the role of tanshinone IIA (TAN IIA) on endothelial cells and the impact of TAN IIA on the endothelial-to-mesenchymal transition in systemic sclerosis (SSc). In this study, we sought to further determine whether TAN IIA can directly act on the skin fibroblasts of scleroderma and look into its underlying anti-fibrotic mechanisms. Bleomycin was used to establish the SSc mouse model. After TAN IIA treatment, dermal thickness, type I collagen and hydroxyproline content were measured. Primary fibroblasts were acquired from SSc patients and cultured in vitro, and the effects of TAN IIA on proliferation, apoptosis and the cell cycle of fibroblasts were detected. In a bleomycin-induced SSc model, we discovered that TAN IIA significantly improved skin thickness and collagen deposition, demonstrating a potent anti-fibrotic action. TAN IIA inhibits the proliferation of skin fibroblasts derived from SSc patients by causing G2/M cell cycle arrest and promoting apoptosis. Additionally, TAN IIA downregulated extracellular matrix gene transcription and collagen protein expression in skin fibroblasts in a dose-gradient-dependent manner. Furthermore, we showed how TAN IIA can reduce the activation of the transforming growth factor-β (TGF-β)/Smad and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways, which are important factors in SSc. In summary, these data suggest that TAN IIA can reduce SSc-related skin fibrosis by modulating the TGF-β/Smad and MAPK/ERK signalling pathways. More importantly, our results imply that TAN IIA can directly act on the skin fibroblasts of SSc, therefore, inhibiting fibrosis.

ACSL4 inhibition prevents macrophage ferroptosis and alleviates fibrosis in bleomycin-induced systemic sclerosis model

BackgroundSystemic sclerosis (SSc), with unclear pathophysiology, is a paradigmatic rheumatic disease of immunity dysfunction-driven multi-organ inflammation and ultimate fibrosis. Pathogenesis breakthroughs are urgently needed for available treatments halting its unremitting stiffness. This study aims to investigate whether ferroptosis can regulate the progressive SSc fibrosis.MethodsIn vivo, bleomycin (BLM)-induced mice model was subjected to ferroptosis detection using western blotting, malondialdehyde (MDA), and glutathione (GSH) assays. Pharmacological inhibitor of the acyl-CoA synthetase long-chain family member 4 (ACSL4) was utilized to explore its potential therapeutic effects for fibrosis, from histological, biochemical, and molecular analyses. In vitro, bone marrow-derived macrophages (BMDM) were activated into inflammatory phenotype and then the relationship was evaluated between activation level and ferroptosis sensitivity in lipopolysaccharide (LPS) incubation with gradient concentrations. The potential calpain/ACSL4 axis was analyzed after calpain knockdown or over-expression in Raw264.7.ResultsBoth skin and lung tissue ferroptosis were present in SSc mice with enhanced ACSL4 expression, while ACSL4 inhibition effectively halted fibrosis progressing and provides protection from inflammatory milieu. Meanwhile, a positive regulation relationship between LPS-induced macrophage activity and ferroptosis sensitivity can be observed. After calpain knockdown, both inflammatory macrophage ferroptosis sensitivity and ACSL4 expression decreased, while its over-expression renders ACSL4-envoking condition. Also, calpain pharmacological inhibition reduced both ferroptosis and fibrosis aptitude in mice.ConclusionsACSL4 induces inflammatory macrophage ferroptosis to aggravate fibrosis progressing. ACSL4 and its upregulators of calpains may be potential therapeutic targets for BLM model of SSc.

Mitigative effect of Naringenin in bleomycin induced systemic sclerosis model; role of Notch signaling pathway

Background An immunological, proinflammatory, and vascular disorder called systemic sclerosis (SSc) frequently leading to progressive tissue fibrosis. Reactive oxidizing species are thought to have a substantial influence on disease initiation and progression, based on a variety of studies. Also, Notch signaling is known to modulate fibroblast homeostasis, angiogenesis, and lymphocyte development. Aim This experimental study was designed to shed light on the possible ameliorating mechanism of naringenin as antioxidant in bleomycin-induced SSc model via focusing on Notch signaling cascade. Materials and methods 50 male albino mice were employed for the test, while being allocated randomly to one of five equal groups as follows: Control group (group I), Naringenin-treated group (group II), Bleomycin-treated group (group III), and group IV (Naringenin/Bleomycin co-treated group) and group V (Bleomycin followed by Naringenin treated group). Plasma hydrogen peroxide level, skin tissue hydrogen peroxide and hydroxyproline levels were measured using colorimetric assay. A Disintegrin and metalloproteinase domain containing protein 17 and neuregulin 1 levels in skin tissue were measured by Enzyme-linked immunosorbent assay. Histological evaluation was also performed. Results Plasma hydrogen peroxide, skin tissue hydrogen peroxide, hydroxyproline, a Disintegrin and metalloproteinase domain containing protein 17 and neuregulin 1 levels in the bleomycin-treated group were significantly increased than other studied groups. Naringenin administration in parallel with the induction mitigated the obtained biochemical changes and protected against the chemical induction of SSc. Conclusion Naringenin could protect against bleomycin-induced SSc through its antioxidant role.

Blockade of IL-11 Trans-Signaling or JAK2/STAT3 Signaling Ameliorates the Profibrotic Effect of IL-11

ABSTRACT Objectives Systemic sclerosis (SSc) is a rare rheumatic disease characterized by vascular damage, dysregulated immune response, and fibrosis. Interleukin−11 (IL−11) is upregulated in SSc. This study aimed to investigate the pathological and therapeutic role of the IL−11 trans-signaling pathway in SSc. Methods Plasma IL−11 level was evaluated in 32 patients with SSc and 15 healthy controls, while the expression levels of ADAM10, ADAM17, IL−11, IL−11 Rα, or IL−11 co-stained with CD3 or CD163 in the skin of SSc patients and healthy controls were analyzed. Fibroblasts were treated with IL−11 and ionomycin to evaluate the profibrotic effect of IL−11 trans-signaling pathway. TJ301 (sgp130Fc) and WP1066 (a JAK2/STAT3 inhibitor) intervention groups were set up to investigate the antifibrotic effect of targeting IL−11. Results Levels of plasma IL−11 were extremely low in most SSc patients and healthy controls. In contrast, levels of IL−11, IL−11 Rα, and ADAM10, but not ADAM17, were significantly elevated in the skin of SSc patients. Moreover, the numbers of IL−11+ CD3+ cells and IL−11+ CD163+ cells were increased in the skin of SSc patients. Besides, IL−11 and ADAM10 were also elevated in the skin and pulmonary of bleomycin-induced SSc mouse. Fibroblasts co-stimulated with IL−11 and ionomycin showed increased expression of COL3 and phosphorylation of STAT3, which could be inhibited by TJ301 or WP1066. TJ301 also ameliorated skin and lung fibrosis in BLM-induced SSc mouse. Conclusions IL−11 induces fibrosis in SSc by regulating the trans-signaling pathway. Blockage of sgp130Fc or inhibition of the JAK2/STAT3 pathway could ameliorate the profibrotic effect of IL−11.

Evaluation of renal damage in a bleomycin-induced murine model of systemic sclerosis.

Systemic sclerosis (SSc) is an autoimmune disease of unknown etiology with a high mortality rate. Renal crisis has been reported as one of the predictors of early mortality in these patients. The present study was performed to evaluate bleomycin-induced SSc using an osmotic minipump as a possible model for the analysis of renal damage in SSc. Male CD1 mice were implanted with osmotic minipumps loaded with saline or bleomycin and sacrificed at 6 and 14 days. Histopathological analysis was performed through hematoxylin and eosin (H&E) and Masson's trichrome staining. The expression of endothelin 1 (ET-1), inducible nitric oxide synthase (iNOS), transforming growth factor β (TGF-β), and 8-hydroxy-2-deoxyguanosine (8-OHdG) was also evaluated by immunohistochemistry. The administration of bleomycin induced a decrease in the length of Bowman's space (3.6 μm, P<0.001); an increase in collagen deposition (14.6%, P<0.0001); and an increase in the expression of ET-1 (7.5%, P<0.0001), iNOS (10.8%, P<0.0001), 8-OHdG (161 nuclei, P<0.0001), and TGF-β (2.4% µm, P<0.0001) on Day 6. On Day 14, a decrease in the length of Bowman's space (2.6 μm, P<0.0001); increased collagen deposition (13.4%, P<0.0001); and increased expression of ET-1 (2.7%, P<0.001), iNOS (10.1%, P<0.0001), 8-OHdG (133 nuclei, P<0.001), and TGF-β (0.6%, P<0.0001) were also observed. Systemic administration of bleomycin via an osmotic minipump produces histopathological changes in the kidneys, similar to kidney damage in SSc. Therefore, this model would allow the study of molecular alterations associated with SSc-related renal damage.

ADAR1 promotes systemic sclerosis via modulating classic macrophage activation.

As a multisystem autoimmune disorder disease, systemic sclerosis (SSc) is characterized by inflammation and fibrosis in the skin and other internal organs. However, mechanisms underlying the inflammatory response that drives the development of SSc remain largely unknown. ADAR1 heterozygous knockout (AD1+/-) mice and myeloid-specific ADAR1 knockout mice were used to determine the function of ADAR1 in SSc. Histopathological analyses and western blot confirmed the role of ADAR1 in bleomycin-induced increased skin and lung fibrosis. In this study, we discover that adenosine deaminase acting on RNA (ADAR1), a deaminase converting adenosine to inosine (i.e., RNA editing) in RNA, is abundantly expressed in macrophages in the early stage of bleomycin-induced SSc. Importantly, ADAR1 is essential for SSc formation and indispensable for classical macrophage activation because ADAR1 deficiency in macrophages significantly ameliorates skin and lung sclerosis and inhibits the expression of inflammation mediator inducible NO synthase (iNOS) and IL-1β in macrophages. Mechanistically, deletion of ADAR1 blocks macrophage activation through diminishing NF-κB signaling. Our studies reveal that ADAR1 promotes macrophage activation in the onset of SSc. Thus, targeting ADAR1 could be a potential novel therapeutic strategy for treating sclerosis formation.

024 Antifibrotic effects of a calpain inhibitor ALLN on bleomycin-induced systemic sclerosis model via antagonizing TGF-β/Smad signaling pathway

Systemic sclerosis (SSc) is a connective tissue disorder representing fibrosis in the skin and internal organs such as lungs. An activated differentiation of local progenitor cells to myofibroblasts is likely a key mechanism underlying overproduction of extracellular matrix and resultant tissue fibrosis in SSc. Calpains are family members of Ca2+-dependent cysteine proteases for which the biological action may contribute to fibrosis in various organs. However, the precise mechanism of calpain-dependent fibrosis and therapeutic utility of their inhibitors in SSc remain unclear.

Targeting Multiple End Organs in Lupus and Other Systemic Rheumatic Diseases by Inhibiting Bruton's Tyrosine Kinase.

Bruton tyrosine kinase (Btk) plays a vital role in activating and differentiating B-cells and regulating signaling in myeloid cells. Indeed, the potential use of Btk inhibitors in preventing lupus has been reported. Here, we extend these observations to 4 additional models of end-organ inflammation: (a) BWF1 lupus nephritis mice, (b) anti-GBM nephritis, (c) bleomycin-induced systemic sclerosis like skin disease, and (d) bleomycin-induced lung disease. In agreement with the previous studies, BTK inhibitor (BTKB66) treatment was effective in treating lupus nephritis in terms of reducing renal damage both functionally and histologically, accompanied by significant decrease in proteinuria. Both low-dose and high-dose BTKB66 profoundly blocked renal disease in the anti-GBM nephritis model, with efficacy that was comparable to that seen with dexamethasone. This study provides the first evidence that BTK inhibition has both therapeutic and preventative effects in bleomycin-induced SSc-like disease, in terms of reducing skin thickness, fibrosis, collagen deposition, and inflammation. Likewise, significantly lower lung inflammatory cell infiltration was observed after treatment with BTKB66. Therapeutic benefit was associated with lower numbers of macrophages, proliferating macrophages and activated T-cells in the respective injured organs. The observation that these immune cells play key roles in driving end organ inflammation in multiple systemic rheumatic diseases have broad implications for the use of BTKB66 in managing patients with systemic rheumatic diseases where multiple end organs are afflicted, including lupus and systemic sclerosis.

Wenyang Huazhuo Tongluo formula alleviates pulmonary vascular injury and downregulates HIF-1α in bleomycin-induced systemic sclerosis mouse model

BackgroundVascular damage, autoimmune abnormalities, and fibrosis are the three pathological features of systemic sclerosis (SSc).However, pulmonary vascular damage is the main factor affecting the progression and prognosis of SSc. The main purpose of this study was to explore the molecular mechanism of Wenyang Huazhuo Tongluo Formula in alleviating pulmonary vascular injury in bleomycin-induced SSc mouse model.MethodsMasson staining and H&E staining were used to analyze the degree of pulmonary vascular fibrosis and the infiltration of leukocyte cells in lung tissue ofbleomycin-induced SSc mouse models treated with saline (BLM group), Wenyang Huazhuo Tongluo Formula (WYHZTL group) and HIF-1α inhibitor KC7F2 (KC7F2 group). Blood vessel exudation was determined by analyzing the cell number and albumin concentration in bronchoalveolar lavage fluid using a cell counter and ELISA assay, respectively. The degree of vascular injury was assessed by measuring the expression levels of vWF, E-selectin, ICAM-1, VCAM-1, VE-cadherin and claudin-5 in serum and pulmonary vascular endothelial cells using ELISA and immunofluorescence staining. Finally, the effect of Wenyang Huazhuo Tongluo Formula on the expression of HIF-1α was detected using immunofluorescence staining.ResultsWenyang Huazhuo Tongluo Formula and KC7F2 significantly inhibited bleomycin-induced pulmonary vascular fibrosis and the level of perivascular inflammatory cell infiltration. The number of cells and the concentration of albumin were significantly reduced in the bronchoalveolar lavage fluid of the WYHZTL group and KC7F2 group compared with the BLM group. In addition, treatment with Wenyang Huazhuo Tongluo Formula and KC7F2 significantly downregulated the expression levels of vWF, E-selectin, ICAM-1, VCAM-1 and HIF-1α, but upregulated the expression of VE-cadherin and claudin-5 in serum and pulmonary vascular endothelial cells, compared with treatment with saline.ConclusionsThis study reveals that Wenyang Huazhuo Tongluo Formula plays a new role in the treatment of SSc by alleviating pulmonary vascular damage. Furthermore, we found that Wenyang Huazhuo Tongluo Formula alleviates pulmonary vascular injury and inhibits HIF-1α expression.

Elevated Alpha 1(I) to Alpha 2(I) Collagen Ratio in Dermal Fibroblasts Possibly Contributes to Fibrosis in Systemic Sclerosis.

Systemic sclerosis (SSc) is characterized by excessive collagen deposition in the skin and internal organs. Activated fibroblasts are the key effector cells for the overproduction of type I collagen, which comprises the α1(I) and α2(I) chains encoded by COL1A1 and COL1A2, respectively. In this study, we examined the expression patterns of α1(I) and α2(I) collagen in SSc fibroblasts, as well as their co-regulation with each other. The relative expression ratio of COL1A1 to COL1A2 in SSc fibroblasts was significantly higher than that in control fibroblasts. The same result was observed for type I collagen protein levels, indicating that α2(I) collagen is more elevated than α2(I) collagen. Inhibition or overexpression of α1(I) collagen in control fibroblasts affected the α2(I) collagen levels, suggesting that α1(I) collagen might act as an upstream regulator of α2(I) collagen. The local injection of COL1A1 small interfering RNA in a bleomycin-induced SSc mouse model was found to attenuate skin fibrosis. Overall, our data indicate that α2(I) collagen is a potent regulator of type I collagen in SSc; further investigations of the overall regulatory mechanisms of type I collagen may help understand the aberrant collagen metabolism in SSc.

Iguratimod inhibits skin fibrosis by regulating TGF-β1/Smad signalling pathway in systemic sclerosis.

Iguratimod (T-614), exerting a powerful anti-inflammatory ability, has therapeutic efficacy in multiple autoimmune diseases. However, the effect of T-614 on systemic sclerosis (SSc) is unclear. Here, we investigate the effect and molecular mechanism of T-614 in experimental SSc models. In vitro, cultured dermal fibroblasts from four SSc patients were subjected to different doses of T-614 in the presence or absence of TGF-β1stimulation. Cell proliferation, apoptosis and migration were determined by CCK-8, flow cytometry and transwell assay, respectively. Fibrosis markers and smad signalling pathway-related proteins were detected by immunoblotting and immunofluorescence. In vivo, a bleomycin-induced SSc mouse model was used to evaluate the effect of T-614 on skin fibrosis. Pathological changes in skin tissues were evaluated by HE, Masson staining and immunohistochemistry. In the study, we found T-614 inhibited TGF-β1-induced cell proliferation, migration and promoted apoptosis in a dose-dependent manner (all p<0.01). T-614 partially reversed TGF-β1-induced upregulation of fibrosis markers and phosphorylation of smad2 and smad3 and blocked p-Smad3 nuclear translocation (all p<0.05), suggesting T-614may inhibit dermal fibroblasts activation by regulating TGF-β1/smad pathway. In vivo experiments, T-614 alleviated skin thickness in bleomycin-induced SSc mice (all p<0.05). The expression of fibrosis markers and the infiltration of macrophages in skin tissue were significantly decreased after T-614 treatment (all p<0.05). Our preliminary data indicated T-614 inhibited dermal fibroblasts activation and skin fibrosis at least partly by regulating TGF-β1/smad pathway in experimental SSc models and may be a promising therapeutic agent for SSc.

Involvement of Multiple Variants of Soluble CD146 in Systemic Sclerosis: Identification of a Novel Profibrotic Factor.

Systemic sclerosis (SSc) is an autoimmune disorder characterized by excessive fibrosis, immune dysfunction, and vascular damage, in which the expression of many growth factors is deregulated. CD146 was recently described as a major actor in SSc. Since CD146 also exists as a circulating soluble form (sCD146) that acts as a growth factor in numerous angiogenic- and inflammation-related pathologies, we sought to identify the mechanisms underlying the generation of sCD146 and to characterize the regulation and functions of the different variants identified in SSc. We performed in vitro experiments, including RNA-Seq and antibody arrays, and in vivo experiments using animal models of bleomycin-induced SSc and hind limb ischemia. Multiple forms of sCD146, generated by both shedding and alternative splicing of the primary transcript, were discovered. The shed form of sCD146 was generated from the cleavage of both long and short membrane isoforms of CD146 through ADAM-10 and TACE metalloproteinases, respectively. In addition, 2 novel sCD146 splice variants, I5-13-sCD146 and I10-sCD146, were identified. Of interest, I5-13-sCD146 was significantly increased in the sera of SSc patients (P < 0.001; n = 117), in particular in patients with pulmonary fibrosis (P < 0.01; n = 112), whereas I10-sCD146 was decreased (P < 0.05; n = 117). Further experiments revealed that shed sCD146 and I10-sCD146 displayed proangiogenic activity through the focal adhesion kinase and protein kinase Cε signaling pathways, respectively, whereas I5-13-sCD146 displayed profibrotic effects through the Wnt-1/β-catenin/WISP-1 pathway. Variants of sCD146, and in particular the novel I5-13-sCD146 splice variant, could constitute novel biomarkers and/or molecular targets for the diagnosis and treatment of SSc and other angiogenesis- or fibrosis-related disorders.

The involvement of leucine-rich α-2 glycoprotein in the progression of skin and lung fibrosis in bleomycin-induced systemic sclerosis model

Objective Systemc sclerosis (SSc) is an autoimmune disorder characterized by fibrosis of the skin and internal organs. Recently, it has been shown that leucine-rich α-2 glycoprotein (LRG) functions as a modulator of transforming growth factor-β (TGF-β) signaling in fibrosis. We aimed to characterize the effect of LRG in SSc model and SSc patients. Methods Histological analysis was performed on LRG knockout (KO) and wild type (WT) mouse in the skin and the lung after bleomycin administration. Serum LRG levels were measured during the injection period. Gene expression analysis of the skin and lung tissue from LRG KO and WT mice was performed. In addition, serum LRG levels were determined in SSc patients and healthy controls. Results LRG KO mice display an inhibition of fibrosis in the skin in association with a decrease of dermal thickness, collagen deposition, and phospho-Smad3 expression after bleomycin. Serum LRG concentration significantly increased in WT mice after bleomycin. There was also a suppression of inflammation and fibrosis in the LRG KO mouse lung indicated by a reduction of lung weight, collagen content, and phospho-Smad3 expression after bleomycin. Gene expressions of TGF-β and Smad2/3 were significantly reduced in LRG KO mice. Serum LRG levels in SSc patients were significantly higher than those in controls. Conclusion LRG promotes fibrotic processes in SSc model through TGF-β-Smad3 signaling, and LRG can be a biomarker for SSc in humans and also a potential therapeutic target for SSc.

MicroRNA-30c attenuates fibrosis progression and vascular dysfunction in systemic sclerosis model mice.

Systemic sclerosis (SSc) is characterized by peripheral circulatory disturbance and fibrosis in skin and visceral organs. We recently demonstrated that α2-antiplasmin (α2AP) is elevated in SSc dermal fibroblasts and SSc model mice, and is associated with fibrosis progression and vascular dysfunction. In the present study, we predicted that α2AP could be a target of microRNA-30c (miR-30c) using TargetScan online database, and investigated the effect of miR-30c on the pathogenesis of SSc using a bleomycin-induced SSc model mice. miR-30c attenuated α2AP expression, and prevented the pro-fibrotic changes (increased dermal thickness, collagen deposition, myofibroblast accmulation) and the vascular dysfunction (the reduction of vascular endothelial cells (ECs) and blood flow) in the skin of SSc model mice. Furthermore, miR-30c suppressed pulmonary fibrosis progression in the SSc model mice. miR-30c exerts the anti-fibrotic and anti-angiopathy effects on SSc model mice, and might provide a basis for clinical strategies for SSc.

A novel bispecific antibody alleviates bleomycin-induced systemic sclerosis injury

Systemic sclerosis (SSc) is induced by variety of factors and eventually causes multiple organ damage. In recent years, biological agents targeting cytokines and cell surface molecules have gradually come to market. Here, the anti-inflammatory and antifibrotic effects of a novel bispecific antibody (FL-BsAb1/17) targeting interleukin-17A (IL-17A) and interleukin-1β (IL-1β) were detected. Bleomycin (BLM) was subcutaneously injected for 21 consecutive days to establish the SSc mouse model, and mice were subsequently treated with etanercept or different doses (1, 5, 10 mg/kg) of FL-BsAb1/17. The results showed that FL-BsAb1/17 treatment (10 mg/kg, 5 mg/kg) significantly attenuated BLM-induced SSc-like inflammation by inhibiting the expression of inflammatory factors (IL-17A, IL-1β, IL-8, IL-22, IL-23, IL-6) and fibrosis, with specific outcomes of dermis thickening and lung fibrosis, by inhibiting the expression of fibrotic factors (TGF-β, α-sma, Col-1, Col-3) in the serum, skin and lungs. In addition, FL-BsAb1/17 (10 mg/kg, 5 mg/kg) downregulated protein levels of TGF-β and phosphorylated Smad2/3 in the skin and lungs and reduced collagen 1 protein levels. This indicated that FL-BsAb1/17 can inhibit the development of fibrosis by inhibiting the TGF-β/Smad2/3 signaling pathway. FL-BsAb1/17 (10 mg/kg, 5 mg/kg) could also effectively reduce the content of MDA, increase the activity of SOD and CAT, and improve the total antioxidant capacity (T-AOC). In conclusion, FL-BsAb1/17 alleviated BLM-induced SSc by downregulating inflammatory cascades, relieving oxidative stress and inhibiting TGF-β/Smad2/3 signaling. These data suggest that FL-BsAb1/17 has potential as a novel therapeutic candidate for SSc.

Amelioration of bleomycin-induced pulmonary fibrosis via TGF-\u03b2-induced Smad and non-Smad signaling pathways in galectin-9-deficient mice and fibroblast cells

BackgroundGalectin-9 is a β-galactoside-binding protein with two carbohydrate recognition domains. Recent studies have revealed that galectin-9 regulates cellular biological reactions and plays a pivotal role in fibrosis. The aim of this study was to determine the role of galectin-9 in the pathogenesis of bleomycin-induced systemic sclerosis (SSc).MethodsHuman galectin-9 levels in the serum of patients with SSc and mouse sera galectin-9 levels were measured by a Bio-Plex immunoassay and enzyme-linked immunosorbent assay. Lung fibrosis was induced using bleomycin in galectin-9 wild-type and knockout mice. The effects of galectin-9 on the fibrosis markers and signaling molecules in the mouse lung tissues and primary lung fibroblast cells were assessed with western blotting and quantitative polymerase chain reaction.ResultsGalectin-9 levels in the serum were significantly higher (9-fold) in patients compared to those of healthy individuals. Galectin-9 deficiency in mice prominently ameliorated epithelial proliferation, collagen I accumulation, and α-smooth muscle actin expression. In addition, the galectin-9 knockout mice showed reduced protein expression levels of fibrosis markers such as Smad2/3, connective tissue growth factor, and endothelin-1. Differences between the wild-type and knockout groups were also observed in the AKT, mitogen-activated protein kinase, and c-Jun N-terminal kinase signaling pathways. Galectin-9 deficiency decreased the signal activation induced by transforming growth factor-beta in mouse primary fibroblasts, which plays a critical role in fibroblast activation and aberrant catabolism of the extracellular matrix.ConclusionsOur findings suggest that lack of galectin-9 protects against bleomycin-induced SSc. Moreover, galectin-9 might be involved in regulating the progression of fibrosis in multiple pathways.

Celastrol is a novel selective agonist of cannabinoid receptor 2 with anti-inflammatory and anti-fibrotic activity in a mouse model of systemic sclerosis

BackgroundIncreasing evidence indicated that the cannabinoid receptors were involved in the pathogenesis of organ fibrogenesis. PurposeThe purpose of this study was to discover novel cannabinoid receptor 2 (CB2) agonist and assess the potential of CB2 activation in treating systemic sclerosis. MethodsA gaussia princeps luciferase–based split luciferase complementation assay (SLCA) was developed for detection of the interaction between CB2 and β-arrestin2. A library of 366 natural products was then screened as potential CB2 agonist using SLCA approach. Several GPCR functional assays, including HTRF-based cAMP assay and calcium mobilization were also utilized to evaluated CB2 activation. Bleomycin-induced experimental systemic sclerosis was used to assess the in vivo anti-fibrotic effects. Dermal thickness and collagen content were evaluated via H&E and sirius red staining. ResultsCelastrol was identified as a new agonist of CB2 by using SLCA. Furthermore, celastrol triggers several CB2-mediated downstream signaling pathways, including calcium mobilization, inhibition of cAMP accumulation, and receptor desensitization in a dose-dependent manner, and it has a moderate selectivity on CB1. In addition, celastrol exhibited the anti-inflammatory properties on lipopolysaccharide (LPS) treated murine Raw 264.7 macrophages and primary macrophages. Finally, we found that celastrol exerts anti-fibrotic effects in the bleomycin-induced systemic sclerosis mouse model accompanied by reduced inflammatory conditions. ConclusionTaken together, celastrol is identified a novel selective CB2 agonist using a new developed arrestin-based SLCA, and CB2 activation by celastrol reduces the inflammatory response, and prevents the development of dermal fibrosis in bleomycin-induced systemic sclerosis mouse model.

Gelatinases Increase in Bleomycin-induced Systemic Sclerosis Mouse Model

Systemic sclerosis is a fibrotic autoimmune disease in which aberrant remodeling of the extracellular matrix in organs disturbs their functionalities. The aim of this study was to investigate the expression of gelatinases on systemic sclerosis. Consequently, a mouse model of systemic sclerosis was employed and the gelatinolytic activity of gelatinases was evaluated on the fibrotic tissues of this model. Two groups of ten mice were considered in this work: a group of systemic sclerosis model and control group. For the generation of systemic sclerosis model, mice received bleomycin, while the control group was subjected to phosphate buffered saline (PBS) reception. Mice were tested for fibrosis by using trichrome staining, hydroxyproline measurement and α-SMA detection in tissue sections. Additionally, the gelatinolytic activity of matrix metalloproteinase 2 and matrix metalloproteinase 9 were measured using gelatin zymography in lungs and skin tissue homogenates. The obtained results indicated that subcutaneous injection of bleomycin-induced fibrosis in skin and lung tissues of mice. Pro and active forms of matrix methaloproteinase 9 were increased in fibrotic lung tissues (p<0.05 and p<0.01, respectively), while, the gelatinolytic activity of MMP2 was unaffected in these tissues. Additionally, in skin tissues of bleomycin-treated animals, both pro and active forms of MMP9 and MMP2 were increased (p<0.05). Pro and active forms of gelatinases increase differently in skin and lung tissues of bleomycin-induced scleroderma.

Antifibrotic effects of 2-carba cyclic phosphatidic acid (2ccPA) in systemic sclerosis: contribution to the novel treatment

BackgroundCyclic phosphatidic acid (cPA) has an inhibitory effect on the autotaxin (ATX)/lysophosphatidic acid (LPA) axis, which has been implicated to play an important role in the progression of fibrosis in systemic sclerosis (SSc). The purpose of this study is to assess the antifibrotic activity of cPA for the treatment of SSc using SSc skin fibroblasts and an animal model of bleomycin-induced skin fibrosis.MethodsWe used a chemically stable derivative of cPA (2ccPA). First, we investigated the effect of 2ccPA on extracellular matrix (ECM) expression in skin fibroblasts. Next, the effect of 2ccPA on the intracellular cAMP levels was determined to investigate the mechanisms of the antifibrotic activity of 2ccPA. Finally, we administered 2ccPA to bleomycin-induced SSc model mice to evaluate whether 2ccPA prevented the progression of skin fibrosis.Results2ccPA decreased ECM expression in SSc skin fibroblasts and TGF-β1-treated healthy skin fibroblasts without LPA stimulation. 2ccPA increased the intracellular cAMP levels in skin fibroblasts, suggesting that the antifibrotic effect of 2ccPA was the consequence of the increase in the intracellular cAMP levels. Administration of 2ccPA also ameliorated the progression of bleomycin-induced skin fibrosis in mice.ConclusionsOur data indicated that 2ccPA had inhibitory effects on the progression of skin fibrosis by abrogating ECM production from activated skin fibroblasts. These cells were repressed, at least in part, by increased intracellular cAMP levels. 2ccPA may be able to be used to treat fibrotic lesions in SSc.