Transcriptional Levels of Intercellular Junction Proteins in an Alveolar Epithelial Cell Line Exposed to Irradiation or Bleomycin.

We performed a comparative study of the effects of X-ray irradiation and bleomycin on the mRNA levels of E-cadherin and tight junction proteins (claudin-3, claudin-4, claudin-18, ZO-2, and occludin) in an alveolar epithelial cell line L2. Irradiation decreased claudin-4 levels and increased occludin levels, while the levels of other mRNAs remained unchanged. Bleomycin increased the expression levels of all proteins examined except claudin-3. Irradiation and bleomycin have different effects on the expression level of intercellular junction proteins, indicating different reactions triggered in alveolar epithelial cells and a great prospects of further comparative studies.

Attenuation of fibroblast activation and fibrosis by adropin in systemic sclerosis.

Fibrotic diseases impose a major socioeconomic challenge on modern societies and have limited treatment options. Adropin, a peptide hormone encoded by the energy homeostasis-associated (ENHO) gene, is implicated in metabolism and vascular homeostasis, but its role in the pathogenesis of fibrosis remains enigmatic. Here, we used machine learning approaches in combination with functional in vitro and in vivo experiments to characterize adropin as a potential regulator involved in fibroblast activation and tissue fibrosis in systemic sclerosis (SSc). We demonstrated consistent down-regulation of adropin/ENHO in skin across multiple cohorts of patients with SSc. The prototypical profibrotic cytokine TGFß reduced adropin/ENHO expression in a JNK-dependent manner. Restoration of adropin signaling by therapeutic application of bioactive adropin34-76 peptides in turn inhibited TGFß-induced fibroblast activation and fibrotic tissue remodeling in primary human dermal fibroblasts, three-dimensional full-thickness skin equivalents, mouse models of bleomycin-induced pulmonary fibrosis and sclerodermatous chronic graft-versus-host-disease (sclGvHD), and precision-cut human skin slices. Knockdown of GPR19, an adropin receptor, abrogated the antifibrotic effects of adropin in fibroblasts. RNA-seq demonstrated that the antifibrotic effects of adropin34-76 were functionally linked to deactivation of GLI1-dependent profibrotic transcriptional networks, which was experimentally confirmed in vitro, in vivo, and ex vivo using cultured human dermal fibroblasts, a sclGvHD mouse model, and precision-cut human skin slices. ChIP-seq confirmed adropin34-76-induced changes in TGFß/GLI1 signaling. Our study characterizes the TGFß-induced down-regulation of adropin/ENHO expression as a potential pathomechanism of SSc as a prototypical systemic fibrotic disease that unleashes uncontrolled activation of profibrotic GLI1 signaling.

High S100A9 level predicts poor survival, and the S100A9 inhibitor paquinimod is a candidate for treating idiopathic pulmonary fibrosis.

BACKGROUND: S100A9 is a damage-associated molecular pattern protein that may play an important role in the inflammatory response and fibrotic processes. Paquinimod is an immunomodulatory compound that prevents S100A9 activity. Its safety and pharmacokinetics have been confirmed in human clinical trials. In this study, we investigated the effects of paquinimod in preventing the development of lung fibrosis in vivo and examined the prognostic values of circulatory and lung S100A9 levels in patients with idiopathic pulmonary fibrosis (IPF). METHODS: The expression and localisation of S100A9 and the preventive effect of S100A9 inhibition on fibrosis development were investigated in a mouse model of bleomycin-induced pulmonary fibrosis. In this retrospective cohort study, the S100A9 levels in the serum and bronchoalveolar lavage fluid (BALF) samples from 76 and 55 patients with IPF, respectively, were examined for associations with patient survival. RESULTS: S100A9 expression was increased in the mouse lungs, especially in the inflammatory cells and fibrotic interstitium, after bleomycin administration. Treatment with paquinimod ameliorated fibrotic pathological changes and significantly reduced hydroxyproline content in the lung tissues of mice with bleomycin-induced pulmonary fibrosis. Additionally, we found that paquinimod reduced the number of lymphocytes and neutrophils in BALF and suppressed endothelial-mesenchymal transition in vivo. Kaplan-Meier curve analysis and univariate and multivariate Cox hazard proportion analyses revealed that high levels of S100A9 in the serum and BALF were significantly associated with poor prognoses in patients with IPF (Kaplan-Meier curve analysis: p=0.037 (serum) and 0.019 (BALF); multivariate Cox hazard proportion analysis: HR=3.88, 95% CI=1.06 to 14.21, p=0.041 (serum); HR=2.73, 95% CI=1.05 to 7.10, p=0.039 (BALF)). CONCLUSIONS: The present results indicate that increased S100A9 expression is associated with IPF progression and that the S100A9 inhibitor paquinimod is a potential treatment for IPF.

Prevention of bleomycin-induced pulmonary fibrosis by vaccination with the Tocilizumab mimotope.

Mimotope, a kind of peptide vaccine, is developed to bind natural receptor and inhibit the downstream signaling. We have demonstrated that the vaccination of Tocilizumab mimotopes could alleviate the renal fibrosis by interfering with both IL-6 and ferroptosis signaling. However, the effect of the vaccination of Tocilizumab mimotopes on the fibroblast was not investigated in previous study. Thus, we sought to explore the changes in the fibroblast induced by the Tocilizumab mimotopes vaccination. Bleomycin instillation was performed to construct the pulmonary fibrosis model after the immunization of Tocilizumab mimotopes. Lung histological analysis showed that the Tocilizumab mimotopes could significantly reduce the maladaptive repairment and abnormal remodeling. Immunoblotting assay and fluorescence staining showed that Immunization with the Tocilizumab mimotopes reduces the accumulation of fibrosis-related proteins. High level of lipid peroxidation product was observed in the animal model, while the Tocilizumab mimotopes vaccination could reduce the generation of lipid peroxidation product. Mechanism analysis further showed that Nrf-2 signaling, but not GPX-4 and FSP-1 signaling, was upregulated, and reduced the lipid peroxidation. Our results revealed that in the BLM-induced pulmonary fibrosis, high level of lipid peroxidation product was significantly accumulation in the lung tissues, which might lead to the occurrence of ferroptosis. The IL-6 pathway block therapy could inhibit lipid peroxidation product generation in the lung tissues by upregulating the Nrf-2 signaling, and further alleviate the pulmonary fibrosis.

Calcium transferring from ER to mitochondria via miR-129/ITPR2 axis controls cellular senescence in vitro and in vivo.

Senescent cells are known to be accumulated in aged organisms. Although the two main characteristics, cell cycle arrest (for dividing cells) and secretion of senescence-associated secretory phenotype (SASP) factors, have been well described, the lack of sufficient senescent markers and incomplete understanding of mechanisms have limited the progress of the anti-senescence field. Calcium transferred from the endoplasmic reticulum (ER) via inositol 1, 4, 5-trisphosphate receptor type 2 (ITPR2) to mitochondria has emerged as a key player during cellular senescence and aging. However, the internal regulatory mechanisms, particularly those of endogenous molecules, remain only partially understood. Here we identified miRNA-129 (miR-129) as a direct repressor of ITPR2. Interestingly, miR-129 controlled a cascade of intracellular calcium signaling, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), DNA damage, and consequently cellular senescence through ITPR2 and mitochondrial calcium uniporter (MCU). In addition, miR-129 was repressed in different senescence models and delayed bleomycin-induced cellular senescence. Importantly, intraperitoneal injection of miR-129 partly postponed bleomycin-accelerated lung aging and natural aging markers as well as reduced immunosenescence markers in mice. Altogether, these findings demonstrated that miR-129 regulated cellular senescence and aging markers via intracellular calcium signaling by directly targeting ITPR2.

Macrophage expressed tartrate-resistant acid phosphatase 5 promotes pulmonary fibrosis progression.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disorder involving scarring of pulmonary tissue and a subsequent decrease in respiratory capacity, ultimately resulting in death. Tartrate resistant acid phosphatase 5 (ACP5) plays a role in IPF but the exact mechanisms are yet to be elucidated. In this study, we have utilized various perturbations of the bleomycin mouse model of IPF including genetic knockout, RANKL inhibition, and macrophage adoptive transfer to further understand ACP5's role in pulmonary fibrosis. Genetic ablation of Acp5 decreased immune cell recruitment to the lungs and reduced the levels of hydroxyproline (reflecting extracellular matrix-production) as well as histological damage. Additionally, gene expression profiling of murine lung tissue revealed downregulation of genes including Ccl13, Mmp13, and Il-1α that encodes proteins specifically related to immune cell recruitment and macrophage/fibroblast interactions. Furthermore, antibody-based neutralization of RANKL, an important inducer of Acp5 expression, reduced immune cell recruitment but did not decrease fibrotic lung development. Adoptive transfer of Acp5-/- bone marrow-derived monocyte (BMDM) macrophages 7 or 14 days after bleomycin administration resulted in reductions of cytokine production and decreased levels of lung damage, compared to adoptive transfer of WT control macrophages. Taken together, the data presented in this study suggest that macrophage derived ACP5 plays an important role in development of pulmonary fibrosis and could present a tractable target for therapeutic intervention in IPF.

Therapeutic effect of sodium alginate on bleomycin, etoposide and cisplatin (BEP)-induced reproductive toxicity by inhibiting nitro-oxidative stress, inflammation and apoptosis.

Impaired spermatogenesis and male infertility are common consequences of chemotherapy drugs used in patients with testicular cancer. The present study investigated the effects of sodium alginate (NaAL) on testicular toxicity caused by bleomycin, etoposide, and cisplatin (BEP). Rats in group 1 received normal saline, while groups 2 and 3 were treated with 25 and 50 mg/kg of NaAL, respectively. Group 4 was treated with a 21-day cycle of BEP (0.5 mg/kg bleomycin, 5 mg/kg etoposide, and 1 mg/kg cisplatin), and groups 5 and 6 received BEP regimen plus 25 and 50 mg/kg of NaAL, respectively. Then, sperm parameters, testosterone levels, testicular histopathology and stereological parameters, testicular levels of malondialdehyde (MDA), nitric oxide (NO), and total antioxidant capacity (TAC), and the expression of apoptosis-associated genes including Bcl2, Bax, Caspase3, p53, and TNF-α were evaluated. Our findings revealed that NaAL improved sperm parameters, testosterone levels, histopathology, and stereology parameters in BEP-administrated rats. NaAL also improved testis antioxidant status by enhancing TAC and ameliorating MDA and NO. Further, modifications to the expression of Bcl2, Bax, Caspase3, p53, and TNF-α suggested that NaAL alleviated BEP-induced apoptosis and inflammation. Collectively, NaAL protects rats' testes against BEP-evoked toxicity damage through the modulation of nitro-oxidative stress, apoptosis, and inflammation.

Investigation of miR-26b and miR-27b expressions and the effect of quercetin on fibrosis in experimental pulmonary fibrosis.

In this study, investigation of the effects of Quercetin on Bleomycin-induced pulmonary fibrosis and fibrosis-associated molecules miR-26b and miR-27b was aimed. Control group was given 10% saline on the 0th day, and saline was administered for 21 days starting from the 8th day. Group 2 was given 50 mg/kg Quercetin for 21 days starting from the 8th day. Group 3 was given 10 mg/kg Bleomycin Sulfate on day 0, and sacrificed on the 22nd and 29th day. Group 4 was given 10 mg/kg Bleomycin Sulfate on the 0th day, and was given 50 mg/kg Quercetin for 14 days, and 21 days starting from day 8. Lung tissues were examined using light and electron microscopic, immunohistochemical and molecular biological methods. Injury groups revealed impaired alveolar structure, collagen accumulation and increased inflammatory cells in interalveolar septum. Fibrotic response was decreased and the alveolar structure was improved with Quercetin treatment. α-SMA expressions were higher in the injury groups, but lower in the treatment groups compared to the injury groups. E-cadherin expressions were decreased in the injury groups and showed stronger immunoreactivity in the treatment groups compared to the injury groups. miR-26b and miR-27b expressions were lower in the injury groups than the control groups, and higher in the treatment groups than the injury groups. Quercetin can be considered as a new treatment agent in the idiopathic pulmonary fibrosis, since it increases the expression levels of miR-26b and miR-27b which decrease in fibrosis, and has therapeutic effects on the histopathological changes.

Hydrogen sulfide inhibits alveolar type II cell senescence and limits pulmonary fibrosis via promoting MDM2-mediated p53 degradation.

AIM: Senescence of alveolar type II (AT2) cells is an important driver of pulmonary fibrosis. This study aimed to investigate whether and how dysregulation of hydrogen sulfide (H2 S) production affected AT2 cell senescence, and then explored the effect of H2 S on the communication between AT2 and fibroblasts. METHODS: ICR mice were intratracheally administered with bleomycin (3 mg/kg). Sodium hydrosulfide (NaHS, 28 µmol/kg/d) was intraperitoneally injected for 2 weeks. The H2 S-generating enzyme cystathionine-ß-synthase (CBS) knockout heterozygous (CBS+/- ) mice were used as a low H2 S production model. RESULTS: Analysis of microarray datasets revealed downregulation of H2 S-generating enzymes in lung tissues of patients with pulmonary fibrosis. Decreased H2 S production was correlated with higher levels of cell senescence markers p53 and p21 in bleomycin-induced lung fibrosis. CBS+/- mice exhibited increased levels of p53 and p21. The numbers of AT2 cells positive for p53 and p21 were increased in CBS+/- mice as compared to control mice. H2 S donor NaHS attenuated bleomycin-induced AT2 cell senescence both in vivo and in vitro. H2 S donor suppressed bleomycin-induced senescence-associated secretory phenotype (SASP) of AT2 cells via inhibiting p53/p21 pathway, consequently suppressing proliferation and myofibroblast transdifferentiation of fibroblasts. Mechanically, H2 S suppressed p53 expression by enhancing the mouse double-minute 2 homologue (MDM2)-mediated ubiquitination and degradation of p53. CONCLUSION: H2 S inactivated p53-p21 pathway, consequently suppressing AT2 cell senescence as well as cell communication between senescent AT2 cells and fibroblasts. Aberrant H2 S synthesis may contribute to the development of pulmonary fibrosis through promoting the activation loop involving senescent AT2 cells and activated fibroblasts.

Time course of histopathology of bleomycin-induced pulmonary fibrosis using an intratracheal sprayer in mice.

Idiopathic pulmonary fibrosis (IPF) is a poor prognosis disease that affects approximately 5 million people worldwide, and the detailed mechanisms underlying the pathogenesis of IPF remain unclear. Bleomycin-induced pulmonary fibrosis has been widely used as a representative animal model of IPF that induces fibrosis in lung tissue. The lungs of rodent consist of five lobes and each bronchus enters each lobe of the lung at a different bifurcation angle, path length, and diameter. The method of administration of bleomycin is considered as important thing to establish appropriate animal models. We conducted a time-dependent histopathological study to examine how pulmonary fibrosis develops in each lung lobe when bleomycin was intratracheally sprayed in ICR mice. And we then explored the suitable points for evaluation of anti-fibrotic agents in this model. As a result, we found that homogeneous fibrosis was induced in the 5 lobes of the lungs following initial inflammation. The expression of transforming growth factor (TGF)-ß1 and phospho-Smad2 (pSmad2) was observed from Day 1, and their positivity increased until Day 21. In conclusion, we have observed a detailed time course of histological changes in bleomycin-induced pulmonary fibrosis in ICR mice using the aerosolization technique. We found that our protocol can induce a highly homogeneous lesion in the lung and that the most suitable time point to assess anti-fibrotic agents is 14 days after treatment in this model.

Bleomycin-treated myoblasts undergo p21-associated cellular senescence and have severely impaired differentiation.

As we age, the ability to regenerate and repair skeletal muscle damage declines, partially due to increasing dysfunction of muscle resident stem cells-satellite cells (SC). Recent evidence implicates cellular senescence, which is the irreversible arrest of proliferation, as a potentiator of SC impairment during aging. However, little is known about the role of senescence in SC, and there is a large discrepancy in senescence classification within skeletal muscle. The purpose of this study was to develop a model of senescence in skeletal muscle myoblasts and identify how common senescence-associated biomarkers respond. Low-passage C2C12 myoblasts were treated with bleomycin or vehicle and then evaluated for cytological and molecular senescence markers, proliferation status, cell cycle kinetics, and differentiation potential. Bleomycin treatment caused double-stranded DNA breaks, which upregulated p21 mRNA and protein, potentially through NF-κB and senescence-associated super enhancer (SASE) signaling (p < 0.01). Consequently, cell proliferation was abruptly halted due to G2/M-phase arrest (p < 0.01). Bleomycin-treated myoblasts displayed greater senescence-associated ß-galactosidase staining (p < 0.01), which increased over several days. These myoblasts remained senescent following 6 days of differentiation and had significant impairments in myotube formation (p < 0.01). Furthermore, our results show that senescence can be maintained despite the lack of p16 gene expression in C2C12 myoblasts. In conclusion, bleomycin treatment provides a valid model of damage-induced senescence that was associated with elevated p21, reduced myoblast proliferation, and aberrant cell cycle kinetics, while confirming that a multi-marker approach is needed for the accurate classification of senescence within skeletal muscle.

miR-302d Targeting of CDKN1A Regulates DNA Damage and Steroid Hormone Secretion in Bovine Cumulus Cells.

(1) Background: DNA damage in cumulus cells hinders oocyte maturation and affects steroid hormone secretion. It is crucial to identify the key factors that regulate cellular DNA damage and steroid hormone secretion. (2) Methods: Treatment of bovine cumulus cells with bleomycin to induce DNA damage. The effects of DNA damage on cell biology were determined by detecting changes in DNA damage degree, cell cycle, viability, apoptosis, and steroid hormones. It was verified that mir-302d targeted regulation of CDKN1A expression, and then affected DNA damage and steroid hormone secretion in cumulus cells. (3) Results: Bleomycin induced increased DNA damage, decreased G1-phase cells, increased S-phase cells, inhibited proliferation, promoted apoptosis, affected E2 and P4 secretion, increased CDKN1A expression, and decreased miR-302d expression. Knockdown of CDKN1A reduced DNA damage, increased G1-phase cells, decreased G2-phase cells, promoted proliferation, inhibited apoptosis, increased E2 and P4 secretion, and increased the expression of BRCA1, MRE11, ATM, CDK1, CDK2, CCNE2, STAR, CYP11A1, and HSD3B1. The expression of RAD51, CCND1, p53, and FAS was decreased. Overexpression of CDKN1A resulted in the opposite results. miR-302d targets CDKN1A expression to regulate DNA damage and then affects the cell cycle, proliferation, apoptosis, steroid hormone secretion, and the expression of related genes. (4) Conclusions: miR-302d and CDKN1A were candidate molecular markers for the diagnosis of DNA damage in bovine cumulus cells.

Histone deacetylase 3 deletion in alveolar type 2 epithelial cells prevents bleomycin-induced pulmonary fibrosis.

BACKGROUND: Epithelial mesenchymal transformation (EMT) in alveolar type 2 epithelial cells (AT2) is closely associated with pulmonary fibrosis (PF). Histone deacetylase 3 (HDAC3) is an important enzyme that regulates protein stability by modulating the acetylation level of non-histones. Here, we aimed to explore the potential role and regulatory mechanisms associated with HDAC3 in PF. METHODS: We quantified HDAC3 expression both in lung tissues from patients with PF and from bleomycin (BLM)-treated mice. HDAC3 was also detected in TGF-ß1-treated AT2. The mechanistic activity of HDAC3 in pulmonary fibrosis and EMT was also explored. RESULTS: HDAC3 was highly expressed in lung tissues from patients with PF and bleomycin (BLM)-treated mice, especially in AT2. Lung tissues from AT2-specific HDAC3-deficient mice stimulated with BLM showed alleviative fibrosis and EMT. Upstream of HDAC3, TGF-ß1/SMAD3 directly promoted HDAC3 transcription. Downstream of HDAC3, we also found that genetic or pharmacologic inhibition of HDAC3 inhibited GATA3 expression at the protein level rather than mRNA. Finally, we found that intraperitoneal administration of RGFP966, a selective inhibitor of HDAC3, could prevent mice from BLM-induced pulmonary fibrosis and EMT. CONCLUSION: TGF-ß1/SMAD3 directly promoted the transcription of HDAC3, which aggravated EMT in AT2 and pulmonary fibrosis in mice via deacetylation of GATA3 and inhibition of its degradation. Our results suggest that targeting HDAC3 in AT2 may provide a new therapeutic target for the prevention of PF.

Superiority of systemic bleomycin to intradermal HOCl for the study of interstitial lung disease.

Systemic sclerosis (SSc) is an autoimmune disease characterized by vasculopathy, immune dysregulation, and multi-organ fibrosis. Interstitial lung disease (ILD) is a complication of SSc and a leading cause of SSc-death. The administration of hypochlorous acid (HOCl) intradermally in the mouse (HOCl-SSc) purportedly shows several features typical of SSc. We studied the model by injecting BALB/c mice daily intradermally with HOCl for 6-weeks, an exposure reported to induce lung fibrosis. On day 42, the skinfold thickness and the dermal thickness were two and three times larger respectively in the HOCl group compared to controls. HOCl treatment did not result in histological features of pulmonary fibrosis nor significant changes in lung compliance. Automated image analysis of HOCl mice lungs stained with picrosirius red did not show increased collagen deposition. HOCl injections did not increase pulmonary mRNA expression of pro-fibrotic genes nor induced the production of serum advanced oxidation protein products and anti-topoisomerase 1 antibodies. Immune cells in bronchoalveolar lavage fluid (BALF) and whole lung digests were not increased in HOCl-treated animals. Since lung fibrosis is proposed to be triggered by oxidative stress, we injected HOCl to Nrf2-/- mice, a mouse deficient in many antioxidant proteins. Lung compliance, histology, and BALF leukocyte numbers were comparable between Nrf2-/- mice and wild-type controls. We conclude that the HOCl-SSc model does not manifest SSc-lung disease.

Saikosaponin-d regulates angiogenesis in idiopathic pulmonary fibrosis through angiopoietin/Tie-2 pathway.

OBJECTIVE: Idiopathic pulmonary fibrosis (IPF) is considered as a chronic interstitial lung disease with underlying mechanism of IPF remaining unclear, while there are no definitive treatment options. In recent years, scientists have gradually paid attention to the influence of angiogenesis on IPF. Because IPF is a progressive with microvascular remodeling disorder, scientists have postulated that angiogenesis may also be one of the initiating and contributing factors of the disease. Bupleurum is a common natural Chinese herbal medicine with antibacterial, anti-inflammatory, anti-tumor and other pharmacological effects. As the most important active monomer of Bupleurum, Saikosaponin-d (SSd) is a new discovery with anti-pulmonary fibrosis (PF) activity. This study attempts to investigate the role of SSd in the interference of PF through regulation of angiogenesis in IPF through Angiopoietin (Angpt) /Tie receptor 2 (Tie2) pathway. METHODS: Randomly, we allocated C57BL/6 mice into four groups (n = 20 in each group). Afterwards, establishment of IPF model was accomplished via intratracheal administration of bleomycin (BLM, 5 mg/kg), while corresponding drug intervention was given accordingly. On 3rd, 7th, 14th and 28th days after modeling, we performed histopathological examination through staining. Meanwhile, immunohistochemistry (IHC) of PF and the expression of related factors were observed, while Ang/Tie2 pathway was assessed by ELISA with the effect of SSd on angiogenesis related proteins in IPF being explored with IHC and Western Blot technique. RESULTS: Our results showed that SSd could reduce inflammation and PF levels in lung tissue of experimental mice, while levels of angiogenesis-related factors, namely Tie-2, Ang-1 and ANGPT2 (Ang-2), fibrosis- associated factors like Alpha-smooth muscle actin (α-SMA), collagen-I and hydroxyproline in SSd and dexamethasone (DXM) mice were significantly reduced at each time point compared to BLM (p < 0.01). Additionally, we discovered substantial decreased expressions of Ang-1, Ang-2, Tie-2, α-SMA and collagen-I at protein level in SSd and DXM mice at each time point compared to BLM (p < 0.05). Besides, insignificant differences were observed between SSd and DXM groups (p > 0.05). CONCLUSION: This study has demonstrated that SSd could down-regulate the expression of ANG-1, Ang-2 and Tie2 in the Ang/Tie2 pathway, and may reduce lung inflammation and PF in BLM-induced mice via inhibition of angiogenesis.

Boningmycin induces AMPK-mediated endoplasmic reticulum-associated degradation of PD-L1 protein in human cancer cells.

Anti-PD-1/PD-L1 monoclonal antibodies have displayed remarkable clinical benefits and revolutionized the treatment of multiple tumor types, but the low response rates and immune-related adverse events limit their application, which promoting the development of small molecule agents to improve the efficacy of PD-1/PD-L1 blockade therapy. Boningmycin (BON), a new small molecule belonging to bleomycin (BLM) family, exhibits potent anticancer activity in vitro and in vivo, as well as negligible lung toxicity, thereby can be an alternative of BLM. However, understandings about the anticancer mechanism of BLM-related compounds are extremely rare, it remains unclear if they affect PD-L1 level in a manner similar to that of other antitumor drugs. In this study, we discover that BON significantly reduces PD-L1 protein level in NCI-H460 and HT-1080 cells. Meanwhile, BON decreases the protein level of PD-L1 in a tumor xenograft model of NCI-H460 cells. Nevertheless, the mRNA level is not influenced after BON exposure. Furthermore, BON-induced PD-L1 reduction is proteasome- dependent. By using specific inhibitors and RNA interference technology, we confirm that the decline of PD-L1 protein by BON is mediated by AMPK-activated endoplasmic reticulum-associated degradation pathway, which is like to the action of metformin. Last but not the least, BON has synergism on gefitinib in vitro and in vivo. In conclusion, it is the first report demonstrating that BON decreases PD-L1 protein level through AMPK-mediated endoplasmic reticulum-associated degradation pathway. These findings will benefit the clinical transformation of BON and aid in the elucidation of molecular mechanism of BLM-related compounds.

AT2 receptor agonist LP2 restores respiratory function in a rat model of bleomycin-induced lung remodelling.

This study aimed to evaluate the prophylactic and therapeutic potential of angiotensin II type 2 receptor peptide agonist LP2 in bleomycin-induced airway and cardiac remodeling in rats. Male Wistar rats were intratracheally instillated with bleomycin. Animals of a prophylactic arm received LP2 from day 0 at intraperitoneal doses of 1, 3 or 10 µg/kg/d, whereas animals from a therapeutic arm received this LP2 treatment from day 7. On day 28 direct lung mechanics were determined and cardiac and lung tissues were collected and (histo)morphologically assessed. Prophylactic LP2 at 1 µg/kg/d with bleomycin, versus bleomycin alone, significantly improved the airway pressure responses at fixed inflation of 4 ml (p < 0.05) and 7 ml volume (p < 0.05), static compliance (p < 0.01), inspiratory capacity (p < 0.05), lung tolerance of increased volume (p < 0.0001), right to left ventricular hypertrophy (p < 0.05). Therapeutic regime showed a similar trend as the prophylactic arm but was less effective, mostly lacking significance. However, and importantly, therapeutic LP2 at 1 µg/kg/d significantly decreased mRNA expression of collagen 1A1 (p < 0.01), of Connective Tissue Growth Factor 1 (p < 0.05) and of Tissue MetalloPeptidase inhibitor 1 (p < 0.05). In conclusion, a very low dose of 1 µg/kg/d LP2 has capacity to counter bleomycin-induced impairment of lung functioning and consequent cardiac remodeling.

A novel BRD4 degrader, ARV-825, attenuates lung fibrosis through senolysis and antifibrotic effect.

BACKGROUND: Recent studies suggest that cellular senescence is related to the pathogenesis of idiopathic pulmonary fibrosis. However, cellular senescence has yet to be targeted therapeutically in clinical practice. ARV825, a recently developed BRD4 degrader, has been reported as a novel senolytic drug. Conversely, it has also been reported that BRD4 regulates the pro-fibrotic gene expression of fibroblasts. Therefore, this study focuses on the senolytic and anti-fibrotic effects of ARV825 and evaluated these effects on lung fibrosis. METHODS: Lung fibroblasts were induced to senescence through serial passage. The expression of senescence markers and pro-fibrotic markers were determined through quantitative PCR or immunoblot analysis. Lung fibrosis was induced in mice through intratracheal administration of bleomycin. Mice treated with ARV825 underwent histological analysis of lung fibrosis using the Ashcroft score. Total lung collagen was quantified through a hydroxyproline assay. Respiratory mechanics analysis was performed using the flexiVent system. RESULTS: For senescent cells, ARV825 induced the expression of an apoptosis marker while reducing the expression of BRD4 and senescence markers. On the other hand, for early passage pre-senescent cells, ARV825 reduced the expression of collagen type 1 and α-smooth muscle actin. In an experimental mouse model of lung fibrosis, ARV825 attenuated lung fibrosis and improved lung function. Immunohistochemical staining revealed a significant decrease in the number of senescent alveolar type 2 cells in lung tissue due to ARV825 treatment. CONCLUSIONS: These results suggest that ARV825 may impact the progressive and irreversible course of fibrotic lung diseases.

[Protective effect of intervention with cannabinoid type-2 receptor agonist JWH133 on pulmonary fibrosis in mice].

Objective: JWH133, a cannabinoid type 2 receptor agonist, was tested for its ability to protect mice from bleomycin-induced pulmonary fibrosis. Methods: By using a random number generator, 24 C57BL/6J male mice were randomly divided into the control group, model group, JWH133 intervention group, and JWH133+a cannabinoid type-2 receptor antagonist (AM630) inhibitor group, with 6 mice in each group. A mouse pulmonary fibrosis model was established by tracheal instillation of bleomycin (5 mg/kg). Starting from the first day after modeling, the control group mice were intraperitoneally injected with 0.1 ml of 0.9% sodium chloride solution, and the model group mice were intraperitoneally injected with 0.1 ml of 0.9% sodium chloride solution. The JWH133 intervention group mice were intraperitoneally injected with 0.1 ml of JWH133 (2.5 mg/kg, dissolved in physiological saline), and the JWH133+AM630 antagonistic group mice were intraperitoneally injected with 0.1 ml of JWH133 (2.5 mg/kg) and AM630 (2.5 mg/kg). After 28 days, all mice were killed; the lung tissue was obtained, pathological changes were observed, and alveolar inflammation scores and Ashcroft scores were calculated. The content of type Ⅰ collagen in the lung tissue of the four groups of mice was measured using immunohistochemistry. The levels of interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) in the serum of the four groups of mice were measured using enzyme-linked immunosorbent assay (ELISA), and the content of hydroxyproline (HYP) in the lung tissue of the four groups of mice was measured. Western blotting was used to measure the protein expression levels of type Ⅲ collagen, α-smooth muscle actin (α-SMA), extracellular signal regulated kinase (ERK1/2), phosphorylated P-ERK1/2 (P-ERK1/2), and phosphorylated ribosome S6 kinase type 1 (P-p90RSK) in the lung tissue of mice in the four groups. Real-time quantitative polymerase chain reaction was used to measure the expression levels of collagen Ⅰ, collagen Ⅲ, and α-SMA mRNA in the lung tissue of the four groups of mice. Results: Compared with the control group, the pathological changes in the lung tissue of the model group mice worsened, with an increase in alveolar inflammation score (3.833±0.408 vs. 0.833±0.408, P<0.05), an increase in Ashcroft score (7.333±0.516 vs. 2.000±0.633, P<0.05), an increase in type Ⅰ collagen absorbance value (0.065±0.008 vs. 0.018±0.006, P<0.05), an increase in inflammatory cell infiltration, and an increase in hydroxyproline levels [(1.551±0.051) µg/mg vs. (0.974±0.060) µg/mg, P<0.05]. Compared with the model group, the JWH133 intervention group showed reduced pathological changes in lung tissue, decreased alveolar inflammation score (1.833±0.408, P<0.05), decreased Ashcroft score (4.167±0.753, P<0.05), decreased type Ⅰ collagen absorbance value (0.032±0.004, P<0.05), reduced inflammatory cell infiltration, and decreased hydroxyproline levels [(1.148±0.055) µg/mg, P<0.05]. Compared with the JWH133 intervention group, the JWH133+AM630 antagonistic group showed more severe pathological changes in the lung tissue of mice, increased alveolar inflammation score and Ashcroft score, increased type Ⅰ collagen absorbance value, increased inflammatory cell infiltration, and increased hydroxyproline levels. Compared with the control group, the expression of α-SMA, type Ⅲ collagen, P-ERK1/2, and P-p90RSK proteins in the lung tissue of the model group mice increased, while the expression of type Ⅰ collagen, type Ⅲ collagen, and α-SMA mRNA increased. Compared with the model group, the protein expression of α-SMA (relative expression 0.60±0.17 vs. 1.34±0.19, P<0.05), type Ⅲ collagen (relative expression 0.52±0.09 vs. 1.35±0.14, P<0.05), P-ERK1/2 (relative expression 0.32±0.11 vs. 1.14±0.14, P<0.05), and P-p90RSK (relative expression 0.43±0.14 vs. 1.15±0.07, P<0.05) decreased in the JWH133 intervention group. The type Ⅰ collagen mRNA (2.190±0.362 vs. 5.078±0.792, P<0.05), type Ⅲ collagen mRNA (1.750±0.290 vs. 4.935±0.456, P<0.05), and α-SMA mRNA (1.588±0.060 vs. 5.192±0.506, P<0.05) decreased. Compared with the JWH133 intervention group, the JWH133+AM630 antagonistic group increased the expression of α-SMA, type Ⅲ collagen, P-ERK1/2, and P-p90RSK protein in the lung tissue of mice, and increased the expression of type Ⅲ collagen and α-SMA mRNA. Conclusion: In mice with bleomycin-induced pulmonary fibrosis, the cannabinoid type-2 receptor agonist JWH133 inhibited inflammation and improved extracellular matrix deposition, which alleviated lung fibrosis. The underlying mechanism of action may be related to the activation of the ERK1/2-RSK1 signaling pathway.

NMDA receptor activation induces damage of alveolar type II cells and lung fibrogenesis through ferroptosis.

Ferroptosis, a newly discovered type of regulated cell death, has been implicated in numerous human diseases. Idiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal interstitial lung disease with poor prognosis and limited treatment options. Emerging evidence has linked ferroptosis and glutamate-determined cell fate which is considered a new light on the etiology of pulmonary fibrosis. Here, we observed that N-methyl d-aspartate receptor (NMDAR) activation promoted cell damage and iron deposition in MLE-12 cells in a dose-, time-, and receptor-dependent manner. This mediated substantial Ca2+ influx, upregulated the expression levels of nNOS and IRP1, and affected intracellular iron homeostasis by regulating the expression of iron transport-related proteins (i.e., TFR1, DMT1, and FPN). Excessive iron load promoted the continuous accumulation of total intracellular and mitochondrial reactive oxygen species, which ultimately led to ferroptosis. NMDAR inhibition reduced lung injury and pulmonary fibrosis in bleomycin-induced mice. Bleomycin stimulation upregulated the expression of NMDAR1, nNOS, and IRP1 in mouse lung tissues, which ultimately led to iron deposition via regulation of the expression of various iron metabolism-related genes. NMDAR activation initiated the pulmonary fibrosis process by inducing iron deposition in lung tissues and ferroptosis of alveolar type II cells. Our data suggest that NMDAR activation regulates the expression of iron metabolism-related genes by promoting calcium influx, increasing nNOS and IRP1 expression, and increasing iron deposition by affecting cellular iron homeostasis, ultimately leading to mitochondrial damage, mitochondrial dysfunction, and ferroptosis. NMDAR activation-induced ferroptosis of alveolar type II cells might be a key event to the initiation of pulmonary fibrosis.