Inhibition of α-SMA by the Ectodomain of FGFR2c Attenuates Lung Fibrosis

The soluble ectodomain of fibroblast growth factor receptor-IIIc (sFGFR2c) is able to bind to fibroblast growth factor (FGF) ligands and block the activation of the FGF-signaling pathway. In this study, sFGFR2c inhibited lung fibrosis dramatically in vitro and in vivo. The upregulation of α-smooth muscle actin (α-SMA) in fibroblasts by transforming growth factor-β1 (TGF-β1) is an important step in the process of lung fibrosis, in which FGF-2, released by TGF-β1, is involved. sFGFR2c inhibited α-SMA induction by TGF-β1 via both the extracellular signal-regulated kinase 1/2 (ERK1/2) and Smad3 pathways in primary mouse lung fibroblasts and the proliferation of mouse lung fibroblasts. In a mouse model of bleomycin (BLM)-induced lung fibrosis, mice were treated with sFGFR2c from d 3 or d 10 to 31 after BLM administration. Then we used hematoxylin and eosin staining, Masson staining and immunohistochemical staining to evaluate the inhibitory effects of sFGFR2c on lung fibrosis. The treatment with sFGFR2c resulted in significant attenuation of the lung fibrosis score and collagen deposition. The expression levels of α-SMA, p-FGFRs, p-ERK1/2 and p-Smad3 in the lungs of sFGFR2c-treated mice were markedly lower. sFGFR2c may have potential for the treatment of lung fibrosis as an FGF-2 antagonist.     

MicroRNA-155 Deficiency Attenuates Liver Steatosis and Fibrosis without Reducing Inflammation in a Mouse Model of Steatohepatitis

Background & Aim

MicroRNAs (miRs) regulate hepatic steatosis, inflammation and fibrosis. Fibrosis is the consequence of chronic tissue damage and inflammation. We hypothesized that deficiency of miR-155, a master regulator of inflammation, attenuates steatohepatitis and fibrosis.

Methods

Wild type (WT) and miR-155-deficient (KO) mice were fed methionine-choline-deficient (MCD) or -supplemented (MCS) control diet for 5 weeks. Liver injury, inflammation, steatosis and fibrosis were assessed.

Results

MCD diet resulted in steatohepatitis and increased miR-155 expression in total liver, hepatocytes and Kupffer cells. Steatosis and expression of genes involved in fatty acid metabolism were attenuated in miR-155 KO mice after MCD feeding. In contrast, miR-155 deficiency failed to attenuate inflammatory cell infiltration, nuclear factor κ beta (NF-κB) activation and enhanced the expression of the pro-inflammatory cytokines tumor necrosis factor alpha (TNFα) and monocyte chemoattractant protein-1 (MCP1) in MCD diet-fed mice. We found a significant attenuation of apoptosis (cleaved caspase-3) and reduction in collagen and α smooth muscle actin (αSMA) levels in miR-155 KO mice compared to WTs on MCD diet. In addition, we found attenuation of platelet derived growth factor (PDGF), a pro-fibrotic cytokine; SMAD family member 3 (Smad3), a protein involved in transforming growth factor-β (TGFβ) signal transduction and vimentin, a mesenchymal marker and indirect indicator of epithelial-to-mesenchymal transition (EMT) in miR-155 KO mice. Nuclear binding of CCAAT enhancer binding protein β (C/EBPβ) a miR-155 target involved in EMT was significantly increased in miR-155 KO compared to WT mice.

Conclusions

Our novel data demonstrate that miR-155 deficiency can reduce steatosis and fibrosis without decreasing inflammation in steatohepatitis.     

Uncoupling between Inflammatory and Fibrotic Responses to Silica: Evidence from MyD88 Knockout Mice

The exact implication of innate immunity in granuloma formation and irreversible lung fibrosis remains to be determined. In this study, we examined the lung inflammatory and fibrotic responses to silica in MyD88-knockout (KO) mice. In comparison to wild-type (WT) mice, we found that MyD88-KO animals developed attenuated lung inflammation, neutrophil accumulation and IL-1β release in response to silica. Granuloma formation was also less pronounced in MyD88-KO mice after silica. This limited inflammatory response was not accompanied by a concomitant attenuation of lung collagen accumulation after silica. Histological analyses revealed that while pulmonary fibrosis was localized in granulomas in WT animals, it was diffusely distributed throughout the parenchyma in MyD88-KO mice. Robust collagen accumulation was also observed in mice KO for several other components of innate immunity (IL-1R, IL-1, ASC, NALP3, IL-18R, IL-33R, TRIF, and TLR2-3-4,). We additionally show that pulmonary fibrosis in MyD88-KO mice was associated with the accumulation of pro-fibrotic regulatory T lymphocytes (T regs) and pro-fibrotic cytokine expression (TGF-β, IL-10 and PDGF-B), not with T helper (Th) 17 cell influx. Our findings indicate that the activation of MyD88-related innate immunity is central in the establishment of particle-induced lung inflammatory and granuloma responses. The development of lung fibrosis appears uncoupled from inflammation and may be orchestrated by a T reg-associated pathway.     

Induction of MiR-21 by Stereotactic Body Radiotherapy Contributes to the Pulmonary Fibrotic Response

Radiation-induced lung fibrosis, the most serious effect of lung cancer radiotherapy on normal tissue, remains a major technical obstacle to the broader application of radiotherapy to patients with lung cancer. This study describes the use of an image-guided irradiation system in mice mimicking stereotactic body radiotherapy (SBRT) to examine the molecular features of chronic fibrotic response after radiation injury. MicroRNA (miR) array analysis of injured pulmonary tissue identified a set of miRs whose expression was significantly increased in damaged lung tissue. In particular, miR-21 expression was increased at the radiation injury site, concurrent with collagen deposition. Although the inhibition of miR-21 by its specific inhibitor anti-miR-21 only marginally affected endothelial-mesenchymal transition (EndMT) in lung endothelial cells, this inhibition significantly reduced collagen synthesis in lung fibroblasts. Furthermore, ectopic expression of miR-21 was sufficient to promote a fibrotic response in lung fibroblasts, enhancing Smad2 phosphorylation concurrent with Smad7 downregulation. These findings indicate that the induction of miR-21 expression is responsible for fibrotic responses observed in mesenchymal cells at the injury site through the potentiation of TGF-β signaling. Local targeting of miR-21 at the injured area could have potential therapeutic utility in mitigating radiation-induced lung fibrosis.     

Deletion of Smad3 protects against C-reactive protein-induced renal fibrosis and inflammation in obstructive nephropathy

Introduction and Aims: Elevated plasma levels of C-reactive protein (CRP) are closely associated with progressive renal injury in patients with chronic kidney disease (CKD). Here, we tested a hypothesis that CRP may promote renal fibrosis and inflammation via a TGF-β/Smad3-dependent mechanism.Methods: Role and mechanisms of TGF-β/Smad3 in CRP-induced renal fibrosis and inflammation were examined in a mouse model of unilateral ureteral obstruction (UUO) induced in CRP Tg/Smad3 KO mice and in a rat tubular epithelial cell line in which Smad3 gene is stably knocked down (S3KD-NRK52E).Results: We found that mice overexpressing the human CRP gene were largely promoted renal inflammation and fibrosis as evidenced by increasing IL-1β, TNF-α, MCP-1 expression, F4/80⁺ macrophages infiltration, and marked accumulation of α-smooth muscle actin (α-SMA), collagen I and fibronectin in the UUO kidney, which were blunted when Smad3 gene was deleted in CRPtg-Smad3KO. Mechanistically, we found that the protection of renal inflammation and fibrosis in the UUO kidney of CRPtg-Smad3KO mice was associated with the inactivation of CD32-NF-κB and TGF-β/Smad3 signaling.Conclusion: In conclusion, Smad3 deficiency protects against CRP-mediated renal inflammation and fibrosis in the UUO kidney by inactivating CD32-NF-κB and TGF-β/Smad3 signaling.     

Cathepsin S deficiency results in abnormal accumulation of autophagosomes in macrophages and enhances Ang II-induced cardiac inflammation

Background

Cathepsin S (Cat S) is overexpressed in human atherosclerotic and aneurysmal tissues and may contributes to degradation of extracellular matrix, especially elastin, in inflammatory diseases. We aimed to define the role of Cat S in cardiac inflammation and fibrosis induced by angiotensin II (Ang II) in mice.

Methods and Results

Cat S-knockout (Cat S^−/−) and littermate wild-type (WT) C57BL/6J mice were infused continuously with Ang II (750 ng/kg/min) or saline for 7 days. Cat S^−/− mice showed severe cardiac fibrosis, including elevated expression of collagen I and α-smooth muscle actin (α-SMA), as compared with WT mice. Moreover, macrophage infiltration and expression of inflammatory cytokines (tumor necrosis factor α, transforming growth factor β and interleukin 1β) were significantly greater in Cat S^−/− than WT hearts. These Ang II-induced effects in Cat S^−/− mouse hearts was associated with abnormal accumulation of autophagosomes and reduced clearance of damaged mitochondria, which led to increased levels of reactive oxygen species (ROS) and activation of nuclear factor-kappa B (NF-κB) in macrophages.

Conclusion

Cat S in lysosomes is essential for mitophagy processing in macrophages, deficiency in Cat S can increase damaged mitochondria and elevate ROS levels and NF-κB activity in hypertensive mice, so it regulates cardiac inflammation and fibrosis.     

Epigallocatechin 3-Gallate Ameliorates Bile Duct Ligation Induced Liver Injury in Mice by Modulation of Mitochondrial Oxidative Stress and Inflammation

Cholestatic liver fibrosis was achieved by bile duct ligation (BDL) in mice. Liver injury associated with BDL for 15 days included significant reactive oxygen/nitrogen species generation, liver inflammation, cell death and fibrosis. Administration of Epigallocatechin 3-Gallate (EGCG) in animals reduced liver fibrosis involving parenchymal cells in BDL model. EGCG attenuated BDL-induced gene expression of pro-fibrotic markers (Collagen, Fibronectin, alpha 2 smooth muscle actin or SMA and connective tissue growth factor or CTGF), mitochondrial oxidative stress, cell death marker (DNA fragmentation and PARP activity), NFκB activity and pro-inflammatory cytokines (TNFα, MIP1α, IL1β, and MIP2). EGCG also improved BDL induced damages of mitochondrial electron transport chain complexes and antioxidant defense enzymes such as glutathione peroxidase and manganese superoxide dismutase. EGCG also attenuated hydrogen peroxide induced cell death in hepatocytes in vitro and alleviate stellate cells mediated fibrosis through TIMP1, SMA, Collagen 1 and Fibronectin in vitro. In conclusion, the reactive oxygen/nitrogen species generated from mitochondria plays critical pathogenetic role in the progression of liver inflammation and fibrosis and this study indicate that EGCG might be beneficial for reducing liver inflammation and fibrosis.     

β-Cryptoxanthin Alleviates Diet-Induced Nonalcoholic Steatohepatitis by Suppressing Inflammatory Gene Expression in Mice

Recent nutritional epidemiological surveys showed that serum β-cryptoxanthin inversely associates with the risks for insulin resistance and liver dysfunction. Consumption of β-cryptoxanthin possibly prevents nonalcoholic steatohepatitis (NASH), which is suggested to be caused by insulin resistance and oxidative stress from nonalcoholic fatty liver disease. To evaluate the effect of β-cryptoxanthin on diet-induced NASH, we fed a high-cholesterol and high-fat diet (CL diet) with or without 0.003% β-cryptoxanthin to C56BL/6J mice for 12 weeks. After feeding, β-cryptoxanthin attenuated fat accumulation, increases in Kupffer and activated stellate cells, and fibrosis in CL diet-induced NASH in the mice. Comprehensive gene expression analysis showed that although β-cryptoxanthin histochemically reduced steatosis, it was more effective in inhibiting inflammatory gene expression change in NASH. β-Cryptoxanthin reduced the alteration of expression of genes associated with cell death, inflammatory responses, infiltration and activation of macrophages and other leukocytes, quantity of T cells, and free radical scavenging. However, it showed little effect on the expression of genes related to cholesterol and other lipid metabolism. The expression of markers of M1 and M2 macrophages, T helper cells, and cytotoxic T cells was significantly induced in NASH and reduced by β-cryptoxanthin. β-Cryptoxanthin suppressed the expression of lipopolysaccharide (LPS)-inducible and/or TNFα-inducible genes in NASH. Increased levels of the oxidative stress marker thiobarbituric acid reactive substances (TBARS) were reduced by β-cryptoxanthin in NASH. Thus, β-cryptoxanthin suppresses inflammation and the resulting fibrosis probably by primarily suppressing the increase and activation of macrophages and other immune cells. Reducing oxidative stress is likely to be a major mechanism of inflammation and injury suppression in the livers of mice with NASH.     

蛹虫草菌粉胶囊抑制肺部炎症及缓解肺纤维化的研究

传统中药可治疗肺炎.新型急性呼吸综合征冠状病毒2型(SARS-CoV-2)外部囊膜上的S蛋白是决定病毒毒力的关键因素及主要抗原.本研究利用SARS-CoV-2病毒的S蛋白和细菌脂多糖(LPS)诱导的肺炎模型,初步探索了蛹虫草菌粉对肺炎模型的促炎性因子、单核/巨噬细胞和髓源抑制性细胞(MDSCs)以及纤维化的调节作用.研...     

Down-regulation of miR-let-7e attenuates LPS-induced acute lung injury in mice via inhibiting pulmonary inflammation by targeting SCOS1/NF-κB pathway

Excessive pulmonary inflammatory response is critical in the development of acute lung injury (ALI). Previously, microRNAs (miRNAs) have been recognized as an important regulator of inflammation in various diseases. However, the effects and mechanisms of miRNAs on inflammatory response in ALI remain unclear. Herein, we tried to screen miRNAs in the processes of ALI and elucidate the potential mechanism. Using a microarray assay, microRNA let-7e (let-7e) was chose as our target for its reported suppressive roles in several inflammatory diseases. Down-regulation of let-7e by antagomiR-let-7e injection attenuated LPS-induced acute lung injury. We also found that antagomiR-let-7e could obviously improve the survival rate in ALI mice. Moreover, antagomiR-let-7e treatment reduced the production of proinflammatory cytokines (i.e., TNF-α, IL-1β and IL-6) in bronchoalveolar lavage fluid (BALF) of LPS-induced ALI mice. Luciferase reporter assays confirmed that suppressor of cytokine signaling 1 (SOCS1), a powerful attenuator of nuclear factor kappa B (NF-κB) signaling pathway, was directly targeted and suppressed by let-7e in RAW264.7 cells. In addition, it was further observed that SOCS1 was down-regulated, and inversely correlated with let-7e expression levels in lung tissues of ALI mice. Finally, down-regulation of let-7e suppressed the activation of NF-κB pathway, as evidenced by the reduction of p-IκBα, and nuclear p-p65 expressions in ALI mice. Collectively, our findings indicate that let-7e antagomir protects mice against LPS-induced lung injury via repressing the pulmonary inflammation though regulation of SOCS1/NF-κB pathway, and let-7e may act as a potential therapeutic target for ALI.     

Dasatinib Reduces Lung Inflammation and Fibrosis in Acute Experimental Silicosis

Silicosis is an occupational lung disease with no effective treatment. We hypothesized that dasatinib, a tyrosine kinase inhibitor, might exhibit therapeutic efficacy in silica-induced pulmonary fibrosis. Silicosis was induced in C57BL/6 mice by a single intratracheal administration of silica particles, whereas the control group received saline. After 14 days, when the disease was already established, animals were randomly assigned to receive DMSO or dasatinib (1 mg/kg) by oral gavage, twice daily, for 14 days. On day 28, lung morphofunction, inflammation, and remodeling were investigated. RAW 264.7 cells (a macrophage cell line) were incubated with silica particles, followed by treatment or not with dasatinib, and evaluated for macrophage polarization. On day 28, dasatinib improved lung mechanics, increased M2 macrophage counts in lung parenchyma and granuloma, and was associated with reduction of fraction area of granuloma, fraction area of collapsed alveoli, protein levels of tumor necrosis factor-α, interleukin-1β, transforming growth factor-β, and reduced neutrophils, M1 macrophages, and collagen fiber content in lung tissue and granuloma in silicotic animals. Additionally, dasatinib reduced expression of iNOS and increased expression of arginase and metalloproteinase-9 in silicotic macrophages. Dasatinib was effective at inducing macrophage polarization toward the M2 phenotype and reducing lung inflammation and fibrosis, thus improving lung mechanics in a murine model of acute silicosis.     

Knockout of Endothelial Cell-Derived Endothelin-1 Attenuates Skin Fibrosis but Accelerates Cutaneous Wound Healing

Endothelin (ET)-1 is known for the most potent vasoconstrictive peptide that is released mainly from endothelial cells. Several studies have reported ET-1 signaling is involved in the process of wound healing or fibrosis as well as vasodilation. However, little is known about the role of ET-1 in these processes. To clarify its mechanism, we compared skin fibrogenesis and wound repair between vascular endothelial cell-specific ET-1 knockout mice and their wild-type littermates. Bleomycin-injected fibrotic skin of the knockout mice showed significantly decreased skin thickness and collagen content compared to that of wild-type mice, indicating that bleomycin-induced skin fibrosis is attenuated in the knockout mice. The mRNA levels of transforming growth factor (TGF)-β were decreased in the bleomycin-treated skin of ET-1 knockout mice. On the other hand, skin wound healing was accelerated in ET-1 knockout mice, which was indicated by earlier granulation tissue reduction and re-epithelialization in these mice. The mRNA levels of TGF-β, tumor necrosis factor (TNF)-α and connective tissue growth factor (CTGF) were reduced in the wound of ET-1 knockout mice. In endothelial ET-1 knockout mouse, the expression of TNF-α, CTGF and TGF-β was down-regulated. Bosentan, an antagonist of dual ET receptors, is known to attenuate skin fibrosis and accelerate wound healing in systemic sclerosis, and such contradictory effect may be mediated by above molecules. The endothelial cell-derived ET-1 is the potent therapeutic target in fibrosis or wound healing, and investigations of the overall regulatory mechanisms of these pathological conditions by ET-1 may lead to a new therapeutic approach.     

Tumor Necrosis Factor - Alpha Is Essential for Angiotensin II-Induced Ventricular Remodeling: Role for Oxidative Stress

The functional crosstalk between angiotensin II (Ang II) and tumor necrosis factor (TNF)-α has been shown to cause adverse left ventricular remodeling and hypertrophy in hypertension. Previous studies from our lab showed that mice lacking TNF-α (TNF-α^-/-) have attenuated hypertensive response to Ang II; however, the signaling mechanisms involved are not known. In this study, we investigated the signaling pathways involved in the Ang II and TNF-α interaction. Chronic Ang II infusion (1μg/kg/min, 14 days) significantly increased cardiac collagen I, collagen III, CTGF and TGF-β mRNA and protein expression in wild-type (WT) mice, whereas these changes were decreased in TNF-α^-/- mice. TNF-α^-/- mice with Ang II infusion showed reduced myocardial perivascular and interstitial fibrosis compared to WT mice with Ang II infusion. In WT mice, Ang II infusion increased reactive oxygen species formation and the expression of NADPH oxidase subunits, indicating increased oxidative stress, but not in TNF-α^-/- mice. In addition, treatment with etanercept (8 mg/kg, every 3 days) for two weeks blunted the Ang II-induced hypertension (133±4 vs 154±3 mmHg, p<0.05) and cardiac hypertrophy (heart weight to body weight ratio, 4.8±0.2 vs 5.6±0.3, p<0.05) in WT mice. Furthermore, Ang II-induced activation of NF-κB, p38 MAPK, and JNK were reduced in both TNF-α^-/- mice and mice treated with etanercept. Together, these findings indicate that TNF-α contributes to Ang II-induced hypertension and adverse cardiac remodeling, and that these effects are associated with changes in the oxidative stress dependent MAPK/TGF-β/NF-κB pathway. These results may provide new insight into the mechanisms of Ang II and TNF-α interaction.     

Dual Targeting of MEK and PI3K Pathways Attenuates Established and Progressive Pulmonary Fibrosis

Pulmonary fibrosis is often triggered by an epithelial injury resulting in the formation of fibrotic lesions in the lung, which progress to impair gas exchange and ultimately cause death. Recent clinical trials using drugs that target either inflammation or a specific molecule have failed, suggesting that multiple pathways and cellular processes need to be attenuated for effective reversal of established and progressive fibrosis. Although activation of MAPK and PI3K pathways have been detected in human fibrotic lung samples, the therapeutic benefits of in vivo modulation of the MAPK and PI3K pathways in combination are unknown. Overexpression of TGFα in the lung epithelium of transgenic mice results in the formation of fibrotic lesions similar to those found in human pulmonary fibrosis, and previous work from our group shows that inhibitors of either the MAPK or PI3K pathway can alter the progression of fibrosis. In this study, we sought to determine whether simultaneous inhibition of the MAPK and PI3K signaling pathways is a more effective therapeutic strategy for established and progressive pulmonary fibrosis. Our results showed that inhibiting both pathways had additive effects compared to inhibiting either pathway alone in reducing fibrotic burden, including reducing lung weight, pleural thickness, and total collagen in the lungs of TGFα mice. This study demonstrates that inhibiting MEK and PI3K in combination abolishes proliferative changes associated with fibrosis and myfibroblast accumulation and thus may serve as a therapeutic option in the treatment of human fibrotic lung disease where these pathways play a role.     

Adiponectin Attenuates Lung Fibroblasts Activation and Pulmonary Fibrosis Induced by Paraquat

Pulmonary fibrosis is one of the most common complications of paraquat (PQ) poisoning, which demands for more effective therapies. Accumulating evidence suggests adiponectin (APN) may be a promising therapy against fibrotic diseases. In the current study, we determine whether the exogenous globular APN isoform protects against pulmonary fibrosis in PQ-treated mice and human lung fibroblasts, and dissect the responsible underlying mechanisms. BALB/C mice were divided into control group, PQ group, PQ + low-dose APN group, and PQ + high-dose APN group. Mice were sacrificed 3, 7, 14, and 21 days after PQ treatment. We compared pulmonary histopathological changes among different groups on the basis of fibrosis scores, TGF-β₁, CTGF and α-SMA pulmonary content via Western blot and real-time quantitative fluorescence-PCR (RT-PCR). Blood levels of MMP-9 and TIMP-1 were determined by ELISA. Human lung fibroblasts WI-38 were divided into control group, PQ group, APN group, and APN receptor (AdipoR) 1 small-interfering RNA (siRNA) group. Fibroblasts were collected 24, 48, and 72 hours after PQ exposure for assay. Cell viability and apoptosis were determined via Kit-8 (CCK-8) and fluorescein Annexin V-FITC/PI double labeling. The protein and mRNA expression level of collagen type III, AdipoR1, and AdipoR2 were measured by Western blot and RT-PCR. APN treatment significantly decreased the lung fibrosis scores, protein and mRNA expression of pulmonary TGF-β₁, CTGF and α-SMA content, and blood MMP-9 and TIMP-1 in a dose-dependent manner (p<0.05). Pretreatment with APN significantly attenuated the reduced cell viability and up-regulated collagen type III expression induced by PQ in lung fibroblasts, (p<0.05). APN pretreatment up-regulated AdipoR1, but not AdipoR2, expression in WI-38 fibroblasts. AdipoR1 siRNA abrogated APN-mediated protective effects in PQ-exposed fibroblasts. Taken together, our data suggests APN protects against PQ-induced pulmonary fibrosis in a dose-dependent manner, via suppression of lung fibroblast activation. Functional AdipoR1 are expressed by human WI-38 lung fibroblasts, suggesting potential future clinical applicability of APN against pulmonary fibrosis.     

Attenuation of CCl4-Induced Hepatic Fibrosis in Mice by Vaccinating against TGF-β1

Transforming growth factor β1 (TGF-β1) is the pivotal pro-fibrogenic cytokine in hepatic fibrosis. Reducing the over-produced expression of TGF-β1 or blocking its signaling pathways is considered to be a promising therapeutic strategy for hepatic fibrosis. In this study, we evaluated the feasibility of attenuating hepatic fibrosis by vaccination against TGF-β1 with TGF-β1 kinoids. Two TGF-β1 kinoid vaccines were prepared by cross-linking TGF-β1-derived polypeptides (TGF-β1²⁵–[41-65] and TGF-β1³⁰–[83-112]) to keyhole limpet hemocyanin (KLH). Immunization with the two TGF-β1 kinoids efficiently elicited the production of high-levels of TGF-β1-specific antibodies against in BALB/c mice as tested by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The antisera neutralized TGF-β1-induced growth-inhibition on mink lung epithelial cells (Mv1Lu) and attenuated TGF-β1-induced Smad2/3 phosphorylation, α-SMA, collagen type 1 alpha 2 (COL1A2), plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) expression in the rat hepatic stellate cell (HSC) line, HSC-T6. Vaccination against TGF-β1 with the kinoids significantly suppressed CCl₄-induced collagen deposition and the expression of α-SMA and desmin, attenuated hepatocyte apoptosis and accelerated hepatocyte proliferation in BALB/c mice. These results demonstrated that immunization with the TGF-β1 kinoids efficiently attenuated CCl₄-induced hepatic fibrosis and liver injury. Our study suggests that vaccination against TGF-β1 might be developed into a feasible therapeutic approach for the treatment of chronic fibrotic liver diseases.     

Innate immune activation by inhaled lipopolysaccharide, independent of oxidative stress, exacerbates silica-induced pulmonary fibrosis in mice

Acute exacerbations of pulmonary fibrosis are characterized by rapid decrements in lung function. Environmental factors that may contribute to acute exacerbations remain poorly understood. We have previously demonstrated that exposure to inhaled lipopolysaccharide (LPS) induces expression of genes associated with fibrosis. To address whether exposure to LPS could exacerbate fibrosis, we exposed male C57BL/6 mice to crystalline silica, or vehicle, followed 28 days later by LPS or saline inhalation. We observed that mice receiving both silica and LPS had significantly more total inflammatory cells, more whole lung lavage MCP-1, MIP-2, KC and IL-1β, more evidence of oxidative stress and more total lung hydroxyproline than mice receiving either LPS alone, or silica alone. Blocking oxidative stress with N-acetylcysteine attenuated whole lung inflammation but had no effect on total lung hydroxyproline. These observations suggest that exposure to innate immune stimuli, such as LPS in the environment, may exacerbate stable pulmonary fibrosis via mechanisms that are independent of inflammation and oxidative stress.     

P2X7 Receptor Modulates Inflammatory and Functional Pulmonary Changes Induced by Silica

Silicosis is an occupational lung disease, characterized by irreversible and progressive fibrosis. Silica exposure leads to intense lung inflammation, reactive oxygen production, and extracellular ATP (eATP) release by macrophages. The P2X7 purinergic receptor is thought to be an important immunomodulator that responds to eATP in sites of inflammation and tissue damage. The present study investigates the role of P2X7 receptor in a murine model of silicosis. To that end wild-type (C57BL/6) and P2X7 receptor knockout mice received intratracheal injection of saline or silica particles. After 14 days, changes in lung mechanics were determined by the end-inflation occlusion method. Bronchoalveolar lavage and flow cytometry analyzes were performed. Lungs were harvested for histological and immunochemistry analysis of fibers content, inflammatory infiltration, apoptosis, as well as cytokine and oxidative stress expression. Silica particle effects on lung alveolar macrophages and fibroblasts were also evaluated in cell line cultures. Phagocytosis assay was performed in peritoneal macrophages. Silica exposure increased lung mechanical parameters in wild-type but not in P2X7 knockout mice. Inflammatory cell infiltration and collagen deposition in lung parenchyma, apoptosis, TGF-β and NF-κB activation, as well as nitric oxide, reactive oxygen species (ROS) and IL-1β secretion were higher in wild-type than knockout silica-exposed mice. In vitro studies suggested that P2X7 receptor participates in silica particle phagocytosis, IL-1β secretion, as well as reactive oxygen species and nitric oxide production. In conclusion, our data showed a significant role for P2X7 receptor in silica-induced lung changes, modulating lung inflammatory, fibrotic, and functional changes.     

Polydatin alleviated radiation‐induced lung injury through activation of Sirt3 and inhibition of epithelial–mesenchymal transition

Radiation‐induced lung injury (RILI) is one of the most common and fatal complications of thoracic radiotherapy. It is characterized with two main features including early radiation pneumonitis and fibrosis in later phase. This study was to investigate the potential radioprotective effects of polydatin (PD), which was shown to exert anti‐inflammation and anti‐oxidative capacities in other diseases. In this study, we demonstrated that PD‐mitigated acute inflammation and late fibrosis caused by irradiation. PD treatment inhibited TGF‐β1‐Smad3 signalling pathway and epithelial–mesenchymal transition. Moreover, radiation‐induced imbalance of Th1/Th2 was also alleviated by PD treatment. Besides its free radical scavenging capacity, PD induced a huge increase of Sirt3 in culture cells and lung tissues. The level of Nrf2 and PGC1α in lung tissues was also elevated. In conclusion, our data showed that PD attenuated radiation‐induced lung injury through inhibiting epithelial–mesenchymal transition and increased the expression of Sirt3, suggesting PD as a novel potential radioprotector for RILI.