Effect of pravastatin on bleomycin‐induced acute lung injury and pulmonary fibrosis

1. Pravastatin is best known for its antilipidemic action. Recent studies have shown that statins have immunomodulatory and anti‐inflammatory effects. The present study aimed to determine whether or not pravastatin can attenuate acute lung injury and fibrosis in a mouse model. 2. Bleomycin was given to C57BL6 mice through intratracheal instillation. Pravastatin was given through intraperitoneal injection. To study the effect of pravastatin on the early inflammatory phase and the late fibrotic phase, mice were killed on days 3, 7, 14 and 21. 3. Pravastatin attenuated the histopathological change of bleomycin‐induced lung injury and fibrosis. The accumulation of neutrophils and increased production of tumor necrosis factor‐α in bronchoalveolar lavage fluid were inhibited in the early inflammatory phase. Pravastatin effectively inhibited the increase of lung hydroxyproline content induced by bleomycin. Furthermore, pravastatin reduced the increased expression of transforming growth factor (TGF)‐β1, connective tissue growth factor (CTGF), RhoA and cyclin D1. The increased levels of TGF‐β1 and CTGF mRNA expression were also significantly inhibited by pravastatin. 4. Pravastatin effectively attenuated bleomycin‐induced lung injury and pulmonary fibrosis in mice. Our results provide evidence for the therapeutic potential of pravastatin in the treatment of acute lung injury and pulmonary fibrosis.     

Icariin attenuates high glucose‐induced type IV collagen and fibronectin accumulation in glomerular mesangial cells by inhibiting transforming growth factor‐β production and signalling through G protein‐coupled oestrogen receptor 1

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Lanatoside C protects mice against bleomycin‐induced pulmonary fibrosis through suppression of fibroblast proliferation and differentiation

It has been established that lanatoside C, a FDA‐approved cardiac glycoside, reduces proliferation of cancer cell lines. The proliferation of fibroblasts is critical to the pathogenesis of pulmonary fibrosis (PF), a progressive and fatal fibrotic lung disease lacking effective treatment. In this study we have investigated the impact of lanatoside C on a bleomycin (BLM)‐induced mouse model of PF and through the evaluation of fibroblast proliferation and activation in vitro. We evaluated explanted lung tissue by histological staining, western blot analysis, qRT‐PCR and survival analysis, demonstrating that lanatoside C was able to protect mice against BLM‐induced pulmonary fibrosis. The proliferation of cultured pulmonary fibroblasts isolated from BLM‐induced PF mice was suppressed by lanatoside C, as hypothesized, through the induction of cell apoptosis and cell cycle arrest at the G2/M phase. The Akt signalling pathway was involved in this process. Interestingly, the production of α‐SMA, fibronectin, and collagen I and III in response to TGF‐β1 in healthy mouse fibroblasts was suppressed following lanatoside C administration by inhibition of TGF‐β1/Smad signalling. In addition, TGF‐β1‐induced migration in lung fibroblasts was also impeded after lanatoside C treatment. Together, our data revealed that lanatoside C alleviated BLM‐induced pulmonary fibrosis in mice via attenuation of growth and differentiation of fibroblasts, suggesting that it has potential as a candidate therapy for PF patients.     

The role of Krüppel‐like factor 4 in transforming growth factor‐β–induced inflammatory and fibrotic responses in human proximal tubule cells

Krüppel‐like factor 4 (KLF4) is known to mitigate inflammation in several cell types. Using human proximal tubule cells, the present study aimed to investigate the role of KLF4 in regulating transforming growth factor (TGF)‐β₁ induced inflammatory and fibrotic responses. Human kidney proximal tubular cells were exposed to high glucose, or TGF‐β₁ and KLF4 expressions were determined. Cells were then transfected with empty vector or KLF4 and exposed to 2‐ng/mL TGF‐β₁ for up to 72 h. Inflammatory proteins (macrophage migration inhibitory factor and monocyte chemoattractant protein‐1) and pro‐fibrotic proteins (fibronectin and collagen IV) were measured after 72 h by enzyme‐linked immunosorbent assay and western blot, respectively. To determine the relevance to in vivo models of chronic kidney disease, KLF4 protein expression in streptozotocin‐induced diabetic mice was determined. Krüppel‐like factor 4 messenger RNA (mRNA) levels were significantly reduced in high glucose‐treated human kidney proximal tubular cells. High glucose increased TGF‐β₁ mRNA expression, which significantly increased migration inhibitory factor and monocyte chemoattractant protein‐1 protein secretion. Transforming growth factor‐β₁ significantly increased fibronectin and collagen IV protein expression. The overexpression of KLF4 significantly reduced TGF‐β–mediated increases in migration inhibitory factor and monocyte chemoattractant protein‐1 but had no effect on TGF‐β–mediated fibronectin and collagen IV mRNA and protein expression. The levels of KLF4 mRNA were significantly reduced in the diabetic kidney, and diabetic animals had a significant reduction in renal tubular expression of KLF4 proteins. This data suggest that KLF4 reduces inflammation induced by TGF‐β₁, suggesting a therapeutic role for KLF4 in diabetic nephropathy.     

Antifibrosis effects of triterpene acids of Eriobotrya japonica (Thunb.) Lindl. leaf in a rat model of bleomycin‐induced pulmonary fibrosis

Objective The aim of this study was to investigate the prophylactic effect and some mechanisms of action of triterpene acids of loquat (TAL) on bleomycin A5‐induced pulmonary fibrosis rats. Methods A model of pulmonary fibrosis was induced by injecting rats with a single dose of bleomycin A5 (5 mg/kg) into the trachea. From the second day, rats in the preventive groups were treated with TAL (50, 150 or 450 mg/kg) or dexamethasone (1.2 mg/kg). On the 28th day after medication, the rats were killed and haematoxylin‐eosin or masson staining was used to evaluate the degree of pulmonary fibrosis. Tumour necrosis factor‐α (TNF‐α) and transforming growth factor‐β1 (TGF‐β1) levels in alveolar macrophage culture supernatant were detected by ELISA. The mRNA expression of TNF‐α and TGF‐β1 in alveolar macrophage was observed by RT‐PCR. Key findings Lung histopathological examination showed TAL could ameliorate the structure of the lung and alleviate fibrogenesis. At the same time, TAL (150 or 450 mg/kg dose group) could reduce the expression of TNF‐α and TGF‐β1 in alveolar macrophage of rats with pulmonary fibrosis at either the protein or mRNA level. Conclusions TAL had a positive prophylactic effect on lung fibrosis, which might have been related to its reduction on TNF‐α or TGF‐β1 expression in the alveolar macrophage of pulmonary fibrosis rats.     

Tracking anti‐fibrotic pathways of nilotinib and imatinib in experimentally induced liver fibrosis: An insight

The tyrosine kinase inhibitors imatinib and nilotinib have been suggested to have promising antifibrotic activity in experimental models of liver fibrosis. The aim of the present study was to investigate new pathways underlying this beneficial effect. Hepatic injury was induced in male Wistar rats by intraperitoneal injection of CCl₄ for 12 weeks. During the last 8 weeks of treatment, rats were also injected daily intraperitoneally with 20 mg/kg imatinib or 20, 10 or 5 mg/kg nilotinib. At the end of treatment, effects on fibrosis were assessed by measuring serum fibrotic markers and profibrogenic cytokines, as well as by histopathological examination. Possible anti‐inflammatory effects were estimated by measuring levels of inflammatory cytokines in liver tissue. Liver expression of α‐smooth muscle actin, transforming growth factor (TGF)‐β1 antibodies and platelet‐derived growth factor receptor β (PDGFRβ) was evaluated by immunohistochemical staining techniques. Nilotinib (5 and 10 mg/kg) significantly (P < 0.05) decreased all serum fibrotic markers measured, but 20 mg/kg of either nilotinib or imatinib had limited effects. At all doses tested, nilotinib significantly (P < 0.05) decreased the CCl₄‐induced increases in tissue inflammatory cytokines. Furthermore, 5 and 10 mg/kg nilotinib significantly decreased TGF‐β1 levels and tissue expression of its antibody, as well expression of PDGFRβ. In conclusion, low doses (5 and 10 but not 20 mg/kg) of nilotinib, rather than imatinib, can control hepatic fibrosis by regulating levels of proinflammatory cytokines, primarily interleukin (IL)‐1 and IL‐6. Nilotinib also controls the signalling pathways of profibrogenic cytokines by lowering TGF‐β1 levels and decreasing expression of PDGFRβ.     

GRK2/β‐arrestin mediates arginine vasopressin‐induced cardiac fibroblast proliferation

Cardiac fibrosis is a pathological feature commonly found in hearts exposed to haemodynamic orneurohormonal stress. Elevated levels of arginine vasopressin (AVP) are closely associated with the progression of heart failure and could be an underlying cause of cardiac fibrosis. The aim of this study is to characterize the effect of AVP on neonatal rat cardiac fibroblasts (NRCFs) and to illustrate its signalling mechanism. The proliferative effect of AVP was assessed by methylthiazolyldiphenyl‐tetrazolium assay and 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation assay, and the amounts of cellular signalling proteins α‐smooth muscle actin (α‐SMA), matrix metalloproteinase (MMP) 2, MMP9, and phosphorylated ERK_1/2 were determined by western blotting. AVP, in a time‐ and concentration‐dependent manner, promoted NRCF proliferation and the expression of MMP2 and MMP9. Inhibition of G protein‐coupled receptor kinase2 (GRK2) by the inhibitory peptide GRK2‐Ct or knock‐down of GRK2 suppressed AVP‐induced BrdU incorporation and the expression of MMP2 and α‐SMA in NRCFs. Moreover, shRNA‐mediated silencing of β‐arrestin1 or β‐arrestin 2 abolished AVP‐induced BrdU incorporation and MMP2 expression. AVP‐induced NRCF proliferation depended on the phosphorylation of ERK_1/2, and inhibition of GRK2 or silencing of β‐arrestins blocked AVP‐induced ERK_1/2 phosphorylation. The effects of AVP on NRCF proliferation and α‐SMA expression were blocked by SR45059, a vasopressin receptor type1A (V_1AR) selective antagonist. In conclusion, AVP promotes NRCF proliferation through V_1AR‐mediated GRK2/β‐arrestin/ERK_1/2 signalling.     

Inhibition of connective tissue growth factor attenuates paraquat‐induced lung fibrosis in a human MRC‐5 cell line

Chronic exposure to Paraquat (PQ) may result in progressive pulmonary fibrosis and subsequent chronic obstructive pulmonary malfunction. Connective tissue growth factor (CTGF) has been proposed as a key determinant in the development of lung fibrosis. We investigated thus whether knock down of CTGF can prevent human lung fibroblasts (MRC‐5) activation and proliferation with the subsequent inhibition of PQ‐induced fibrosis. MRC‐5 was transfected with CTGF‐siRNAs and exposed to different concentrations of PQ. The siRNA‐silencing efficacy was evaluated using western blotting analyses, qRT‐PCR and flow cytometry. Next, the viability and migration of MRC‐5 was determined. MMP‐2, MMP‐9, and TIMP‐1 accumulation were quantified to evaluate the lung fibrosis exposure to PQ. Over expression of CTGF mRNA was observed in human MRC‐5 cell as early as 6 h following PQ stimulation. CTGF gene expression in MRC‐5 cells was substantially reduced by RNAi, which significantly suppressed the expression of the lung fibrosis markers such as tissue inhibitor of metalloproteinase‐2 (TIMP‐2), Matrix metalloproteinase‐2 (MMP‐2) and Matrix metalloproteinase‐9 (MMP‐9) that were stimulated by PQ. Inhibition of CTGF expression suppressed impeded the proliferation and migration ability of MRC‐5 cells and resulted in cell‐extracellular matrix (ECM) protein accumulation in cells. Our results suggest that CTGF promoted the development of PQ‐induced lung fibrosis in collaboration with transforming growth factor β1 (TGFβ1). Furthermore, the observed arresting effects of CTGF knock down during this process suggested that CTGF is the potential target site for preventing PQ‐induced pulmonary fibrosis. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1620–1626, 2016.     

Artemisinin inhibits tumour necrosis factor‐α‐induced vascular smooth muscle cell proliferation in vitro and attenuates balloon injury‐induced neointima formation in rats

The aim of this study was to evaluate the effect of artemisinin (ART) on rat vascular smooth muscle cell (VSMC) proliferation induced by tumour necrosis factor (TNF)‐α, cell cycle arrest, and apoptosis, and its effect on neointima formation after balloon injury of rat carotid artery. Primary rat VSMC were identified by immunofluorescence assay. The proliferation of VSMC induced by TNF‐α was significantly inhibited by ART treatment in a dose‐dependent manner. Treatment with 100‐μM ART significantly reduced the expression of proliferating cell nuclear antigen. In contrast, the same treatment arrested the cell cycle in G0/G1 phase. Western blot analysis showed that the cell cycle‐related proteins cyclin D1, cyclin E, cyclin‐dependent kinase 2, and cyclin‐dependent kinase 4 were downregulated by ART in TNF‐α‐stimulated VSMC. For apoptosis induced by ART, cleaved caspase‐3/‐9 was detected, and the pro‐apoptotic protein Bcl‐2‐associated X protein was upregulated while the anti‐apoptotic protein Bcl‐2 was downregulated. The results suggest that ART can effectively inhibit the proliferation of VSMC induced by TNF‐α through the apoptotic induction pathway and cell cycle arrest. Also, balloon injury indicated that ART significantly inhibited neointima formation in the rat carotid arteries.     

Inflammatory cytokines tumour necrosis factor‐α and interleukin‐8 enhance airway smooth muscle contraction by increasing L‐type Ca2+ channel expression

Inflammation elevates intracellular calcium concentrations ([Ca²⁺]_i) in airway smooth muscle (ASM). The L‐type Ca²⁺ channel (L‐VDCC) plays an important role in regulating Ca²⁺ influx in ASM. However, the role of L‐VDCC in the inflammatory cytokine‐induced pathology of ASM remains unclear. In the present study, we used calcium imaging and isometric tension measurements to assess the role of L‐VDCC in agonist‐induced [Ca²⁺]_i rise and the associated contractions in mouse ASM, and we used immunoblotting to identify L‐VDCC protein expression levels in mouse ASM after exposure to tumour necrosis factor alpha (TNF‐α) or interleukin‐8 (IL‐8). Our results showed that high‐K⁺‐ or carbachol‐induced contractions of mouse ASM were significantly greater after pretreatment with TNF‐α or IL‐8 for 24 hours. Both verapamil and nifedipine, L‐VDCC inhibitors, abolished this increased contraction induced by TNF‐α or IL‐8 pretreatment. Moreover, TNF‐α treatment enhanced carbachol‐induced Ca²⁺ influx in ASM cells, and this effect was abrogated by verapamil. Additionally, immunoblotting results showed that preincubation of mouse ASM with TNF‐α or IL‐8 also enhanced L‐VDCC protein expression. On the basis of these findings, we concluded that proinflammatory cytokines, such as TNF‐α and IL‐8, increase the expression level of L‐VDCC, which in turn contributes to augmented agonist‐induced ASM contractions. This effect of inflammation on L‐VDCC expression in ASM may be associated with airway hyper‐responsiveness and involved in the development of asthma.     

Impairment of α1‐adrenoceptor‐mediated contractile activity in caudal arterial smooth muscle from Type 2 diabetic Goto‐Kakizaki rats

1. In the present study, we compared the responsiveness of de‐endothelialized caudal artery smooth muscle strips, isolated from Type 2 diabetic Goto‐Kakizaki (GK) and normal Wistar rats, to α₁‐adrenoceptor stimulation (cirazoline) and membrane depolarization (K⁺). 2. The contractile and myosin 20 kDa light chain (LC₂₀) phosphorylation responses to 0.3 μmol/L cirazoline of caudal artery strips isolated from 12‐week‐old GK rats were significantly reduced compared with those of age‐matched Wistar rats, whereas the contractile and LC₂₀ phosphorylation responses to 60 mmol/L K⁺ were unaltered. 3. Stimulation of fura 2‐AM‐loaded strips from GK rats with 0.3 μmol/L cirazoline induced a significantly smaller rise in [Ca²⁺]_i (by ～20%) compared with that in strips from Wistar rats, whereas comparable Ca²⁺ transients were evoked by K⁺ in both. 4. Using quantitative polymerase chain reaction, no significant differences were detected in the mRNA expression of α_1A‐, α_1B‐ and α_1D‐adrenoceptor subtypes between GK and Wistar rats. 5. Cirazoline (1 μmol/L)‐ and caffeine (20 mmol/L)‐induced contractions in the absence of extracellular Ca²⁺ were unaltered in GK rats, suggesting that the release of Ca²⁺ from the sarcoplasmic reticulum in response to cirazoline does not differ between GK and Wistar rats. 6. The results of the present study suggest that Ca²⁺ entry from the extracellular space via α₁‐adrenoceptor‐activated, Ca²⁺‐permeable channels, but not via membrane depolarization and voltage‐gated L‐type Ca²⁺ channels, is impaired in caudal artery smooth muscle of GK rats.     

Epigallocatechin gallate attenuates fibroblast proliferation and excessive collagen production by effectively intervening TGF‐β1 signalling

Pulmonary fibrosis (PF) poses a huge burden to the patients and society due to lack of an effective treatment drug. Activation of fibrocyte, fibroblast and myofibroblasts are important steps in the development of PF. Targeting this common pathway with natural chemicals may lead to the development of new drug regimens for PF treatment. In this study, PF was induced in male Wistar rats by intratracheal administration of Bleomycin (BLM). Epigallocatechin gallate (EGCG) was administered to one of the groups of rats to test its efficacy against the development of PF. Bleomycin‐induction resulted in significant elevation of matrix metalloproteinase (MMP)‐2 and ‐9 expression, increased RNA and protein expression of transforming growth factor (TGF)‐β1, Smads and alpha‐smooth muscle actin (α‐SMA). EGCG treatment normalized the BLM induced aberrations in these rats. The protective role of EGCG was also validated in vitro using the WI‐38 fibroblast cell line. TGF‐β1 incubated cells exhibited increased fibroblast proliferation and hydroxyproline levels with a concomitant decrease in the expression of MMPs 2 and 9. An increase in protein expression levels of p‐Smad, α‐SMA and type I collagen (COL1A) was also exhibited by fibroblasts upon TGF‐β1 incubation. Simultaneous treatment of EGCG to WI‐38 cells significantly decreased these protein expressions alongside normalizing the MMPs expression. The study revealed that EGCG inhibited fibroblast activation and collagen accumulation by inhibiting TGF‐β1 signalling and thus can be considered as an effective drug against PF.     

Protective effects of β‐sheet breaker α/β‐hybrid peptide against amyloid β‐induced neuronal apoptosis in vitro

Alzheimer's disease is most common neurodegenerative disorder and is characterized by increased production of soluble amyloid‐β oligomers, the main toxic species predominantly formed from aggregation of monomeric amyloid‐β (Aβ). Increased production of Aβ invokes a cascade of oxidative damages to neurons and eventually leads to neuronal death. This study was aimed to investigate the neuroprotective effects of a β‐sheet breaker α/β‐hybrid peptide (BSBHp) and the underlying mechanisms against Aβ₄₀‐induced neurotoxicity in human neuroblastoma SH‐SY5Y cells. Cells were pretreated with the peptide Aβ₄₀ to induce neurotoxicity. Assays for cell viability, cell membrane damage, cellular apoptosis, generation of reactive oxygen species (ROS), intracellular free Ca²⁺, and key apoptotic protein levels were performed in vitro. Our results showed that pretreatment with BSBHp significantly attenuates Aβ₄₀‐induced toxicity by retaining cell viability, suppressing generation of ROS, Ca²⁺ levels, and effectively protects neuronal apoptosis by suppressing pro‐apoptotic protein Bax and up‐regulating antiapoptotic protein Bcl‐2. These results suggest that α/β‐hybrid peptide has neuroprotective effects against Aβ₄₀‐induced oxidative stress, which might be a potential therapeutic agent for treating or preventing neurodegenerative diseases.