Introduction

Pulmonary fibrosis (PF), also referred to as lung fibrosis, presents a significant global health challenge with a high mortality rate and few effective treatment options. It is a progressive lung disease characterized by the gradual formation of scar tissue within the lung parenchyma, ultimately leading to respiratory failure and death.1 The prevalence of IPF varies globally, with incidence rates ranging from 0.09 to 1.30 per 10,000 individuals and prevalence rates ranging from 0.33 to 4.51 per 10,000 individuals. It predominantly affects males aged over 50 years and shows considerable geographic diversity.2 The pathogenesis of pulmonary fibrosis has redirected the focus from fibroblast-driven mechanisms to those centered on epithelial cells. This evolving perspective highlights that recurrent micro-injuries result in the dysfunction of alveolar type II epithelial cells (ATII). These dysfunctional ATII cells not only fail to sustain normal lung regeneration but also contribute to aberrant epithelial-mesenchymal crosstalk. Consequently, this promotes fibrotic processes over the regeneration of healthy lung tissue.3,4 PF is a significant component of various interstitial lung disorders (ILDs), with idiopathic PF being particularly severe and life-threatening.5 However, the precise origin of myofibroblast involvement in fibrosis remains unclear. Numerous studies have identified the Epithelial-Mesenchymal Transition (EMT) as a plausible contributor to fibrotic development.6 EMT is a biological process that involves the conversion of epithelial cells to mesenchymal cells, resulting in enhanced migratory and invasive characteristics.7 EMT encompasses several key features, such as disruption of cell-cell interactions, reconfiguration of the actin cytoskeleton, and cell separation, leading to the transformation of epithelial cell layers into fibroblast-like cells, marked by increased motility and specific genetic marker expression.8 Despite the availability of anti-inflammatory and immunosuppressive treatments, a complete cure for lung fibrosis remains a challenge. The most frequently used animal model to study PF is intratracheal instillation of the anticancer drug bleomycin (BLM).9 BLM can produce reactive oxygen metabolites such as superoxide and hydroxyl radicals, which can initiate DNA damage by inducing strand cleavage.10 This, in turn, may trigger lipid peroxidation, carbohydrate oxidation, alterations in prostaglandin synthesis and degradation in the lungs, and increased collagen synthesis in the pulmonary tissues. BLM-induced lung injury is characterized by a complex inflammatory process involving various immune cell types including macrophages, neutrophils, and lymphocytes.11 Further, a considerable alternation in the expression of oxidative stress markers has been associated with BLM-induced lung injury including superoxide dismutase (SOD), catalase, and glutathione (GSH), as well as measures of lipid peroxidation such as malondialdehyde (MDA).12

Long non-coding RNAs (lncRNAs) are RNA sequences comprising over 200 nucleotides that play crucial roles in controlling transcription and regulating gene expression through epigenetic mechanisms and offer distinct advantages in the realm of epigenetic regulation.13,14 The lncRNA CBR3-AS1, also known as plncRNA-1, is a lncRNA encoded on chromosome 21q22.12 with a transcriptional length of 749 nt and was initially found to be highly expressed in prostate cancer cells.15 A study conducted by Liu et al shed light on the molecular mechanisms of lncRNA CBR3-AS1 in non-small cell lung cancer development.16 The authors observed that lncRNA CBR3-AS1 was overexpressed in NSCLC tissues compared to adjacent normal tissues. Downregulation of lncRNA CBR3-AS1 levels led to a decrease in cell proliferation, migration, and invasiveness; hindered cell cycle progression; and promoted apoptosis in NSCLC cells. The lncRNA CBR3-AS1 has also emerged as a key player in Notch/Jagged1 and TGF-B1 components, which are pivotal in fibrotic processes.17,18 Remarkably, a recent study showed that the lncRNA CBR3-AS1 is associated with the inhibition of miRNA-29-mediated cell invasion and migration in colorectal cancer cells.19 This interconnectedness suggests that lncRNA CBR3-AS1 may act as a central node that coordinates communication between different targets of the lung fibrosis pathway and, therefore, may be a promising candidate for drug intervention in lung fibrosis. MicroRNAs (miRNAs) are a category of non-coding RNAs responsible for the regulation of gene networks. Previous studies have shown that systemic gene transfer of miRNA-29 results in the reduction of lung fibrosis in animal models.20 Furthermore, in two distinct groups of patients with IPF, lower levels of miRNA-29 were associated with higher mortality rates when observed in peripheral blood. This finding suggests that individuals with decreased miRNA-29 concentration in their blood could potentially be identified as a target population for treatment strategies.21 Previous studies have established that downregulation of miRNA-29 is associated with an increase in the expression of Smad and TGF-β.20 This molecular cascade contributes significantly to pathological processes and cellular responses that underlie the development and progression of lung fibrosis.20,22 This finding provides support for the exploration of miRNA-29-based treatments as potential approaches for managing IPF.

The induction of myofibroblast differentiation has been attributed to the presence of Inflammatory Zone 1 (FIZZ1), which is believed to trigger the Notch signaling pathway and contribute to the development of fibrosis.23 The efficacy of FIZZ1 in upregulating the expression of the activated intracellular domain of Notch1 and its ligand Jagged1 has been established. The observed upregulation has been associated with increased levels of α-smooth muscle actin expression (α-SMA), which serves as an indicator of myofibroblast differentiation in the context of fibrosis.24 The Jagged ligand/Notch receptor signaling pathway has been implicated in the regulation of epithelial function and the production of EMT during embryogenesis and cancer.25 Fibrogenesis is primarily initiated by transforming growth factor-β (TGF-β), which is regulated by Smad-dependent or non-Smad pathways and is influenced by co-receptors and interaction networks.26 The TGF signaling pathway is initiated in fibroblasts through the activation of TGF type I receptor kinase (ALK5).27 The stimulatory effect of TGF-on fibrogenesis has been well-documented in both in vivo and in vitro studies. However, owing to the multifaceted nature of TGF activity, the development of antifibrotic medicines that specifically target the TGF axis poses significant challenges.28 In recent years, hydroxyproline has garnered growing interest as a significant biomarker for PF, owing to its specific interaction with collagen.29 Nuclear factor kappa B (NFκB) represents a group of dimeric transcription factors with the capacity to bind to DNA, thereby assuming a central role in regulating a wide array of biological reactions that are triggered in response to cellular stress.30 In lung fibrosis, NF-κB serves as a crucial metric for the initiation and progression of fibrotic changes within pulmonary tissue.31

Among the key regulatory factors coordinating immune cell migration are chemokines, a specialized category of cytokines known for their role in guiding immune cell trafficking via specific receptor interactions.32 Interleukin (IL)-18, a member of the IL-1 gene family, functions as a proinflammatory cytokine and plays a role in both acquired and innate immunity.33 The activation of T helper type 1 cells and induction of IL-2 production were observed upon stimulation with IL-18. Furthermore, the protein known as Intercellular Adhesion Molecule-1 (ICAM-1) belongs to the immunoglobulin superfamily and acts as a binding agent for lymphocyte function-associated antigen I alpha. It has been observed that ICAM-1 is significantly involved in inflammatory pulmonary conditions such as bronchial asthma and damage caused by hyperoxia-induced damage.34 In the intricate network of molecules contributing to PF, the MMP7 gene takes center stage. This gene encodes an ECM-degrading enzyme, a member of the matrix metalloproteinase (MMP) family, which plays a role in various biological processes including tissue remodeling and atherosclerosis progression. Interestingly, mice lacking MMP-7 exhibited non-functional crypts, emphasizing the importance of this gene in maintaining tissue integrity. Moreover, studies have demonstrated that MMP-7 plays a pivotal role in the induction of PF, as evidenced by the protection of MMP-7-deficient mice from lung injury caused by BLM exposure.35,36

Current therapies for PF aim to manage symptoms and slow disease progression. However, a definitive cure remains elusive. Lung transplantation is the only feasible treatment for individuals with advanced stages of lung fibrosis.37 Trimetazidine (TMZ) is a commonly used anti-ischemic medication for the treatment of coronary artery disease, which inhibits the long-chain 3-ketoacyl coenzyme in the mitochondria, particularly thiolase.38 Recent studies have shown that TMZ administration in the early stages can reduce the incidence and progression of cardiac fibrosis in rat models of diabetic cardiomyopathy, indicating that its benefits extend beyond cardiomyocytes.39 Accordingly, TMZ may possess considerable pharmacological potential for the attenuation of PF by improving cellular metabolic processes by enhancing glucose oxidation and blocking the β-oxidation of fatty acids. Furthermore, TMZ exhibits anti-apoptotic properties by enhancing cellular autophagic capability and mitigating induced apoptosis.40 This may help to maintain energy metabolism during ischemia, preserving cellular homeostasis and ionic pump function.41 Remarkably, TMZ has been shown to substantially inhibit the TGF-1β/Smad/α-SMA signaling pathway in CCl4-induced hepatic fibrosis, leading to the attenuation of fibrosis and cell proliferation.42 Taken together, these findings suggest that TMZ may possess a potential pharmacological effect against lung fibrosis by targeting several molecular cascades.

Based on the above-mentioned facts, there is a possible correlation between miRNA-29 and lncRNA CBR3-AS1 expression in lung fibrosis and FIZZ1-mediated Notch/Jagged1 pathway (Figure S1). In our continuous efforts to decipher novel therapeutic targets for human diseases,43–51 we aimed to explore a novel molecular interplay of the lncRNA CBR3-AS1/ miRNA-29/ FIZZ1 axis in modulating the Notch/Jagged1/Smad3/TGF-ß1 pathway and unveil how this molecular cascade jointly regulates the development and progression of lung fibrosis. Furthermore, we explored the pharmacological potential of TMZ to ameliorate lung fibrosis by targeting the proposed lncRNA CBR3-AS1/ miRNA-29/ FIZZ1 axis.

Material and Methods Chemicals and Drugs

The study utilized BLM, trimetazidine, and thiopental sodium obtained from the Sigma Chemical Company (Cairo, Egypt).

Animals

All animal procedures were approved by the Institutional Animal Ethics Committee of Ain Shams, University Faculty of Medicine (FMASU R212/2023). Twenty-eight male Wistar rats weighing 150 g and 200 g were acquired from the National Research Institute in Cairo, Egypt. The animals were housed in a temperature-controlled animal facility with a 12-hour light/dark cycle. Before beginning the experimental protocol, the rats were given a one-week adaptation period to familiarize themselves with their new surroundings. This was done to ensure that ethical guidelines were followed and that the animals were in the best possible condition for the duration of the study.

Experimental Design

To create a rat model of PF, 5 mg/kg BLM sulfate salt was dissolved in 0.1 mL of normal saline and instilled into the trachea. The rats rotated vertically to ensure proper dispersion within the lung tissues. Anesthesia was achieved by intraperitoneal injection of 20 mg/kg of thiopental sodium. A midline incision was made in the neck to expose the trachea for BLM administration. The incision was then surgically sutured and a topical application of 2% sodium fusidate was applied to the wound.52 Twenty-eight male Wistar rats were randomly assigned to one of four groups (n=7).

1) Control group: Rats were treated with 0.1 mL of normal saline by intratracheal instillation.

2) BLM-treated group: Rats were given BLM (5 mg/kg, diluted in 0.1 mL of normal saline) via intratracheal instillation.53–55

3) TMZ-treated group: Rats were orally treated with TMZ at a dosage of 15 mg/kg/day for four weeks.

4) TMZ + BLM group: Rats were treated with BLM (5 mg/kg) via intratracheal instillation, followed by the oral administration of TMZ at a dose of 15 mg/kg/day for four weeks.56

The health and activity of rats were monitored throughout the experiment. After the experiment, blood and lung tissue samples were collected from all the rats for biochemical, histopathological, and immunohistochemical analyses. The purpose of this analysis was to evaluate changes in lung tissue at the molecular and cellular levels due to experimental interventions (Figure 1). Blood samples were obtained from the retro orbital region and collected into plain tubes. Subsequently, the samples were centrifuged at 3000 rpm for 15 min to separate the serum, which was then stored at −80°C for subsequent biochemical analysis. The lungs of all rats were excised through an incision in the chest area, followed by two washes with a cold saline solution. Right lung tissues were preserved in 10% formalin solution for microscopic examination. Tissue homogenization was performed to analyze the chemical properties of the left lung tissues tissue.

Figure 1 The designed workflow aims to elucidate the molecular interplay of the LncRNA CBR3-AS1/miRNA-29/FIZZ1 axis in modulating lung fibrosis, and the potential protective effects of TMZ. The workflow details the following key steps: identification of target interactions through experimental validation, functional assays to assess the impact on cellular processes relevant to fibrosis, and comprehensive analysis of gene expression changes in lung tissue samples to understand the regulatory mechanisms involved. Finally, histological and immunochemical assessments of lung tissues to examine the architectural structure of lung tissues.

RNA Isolation from Lung Sample

We retrieved lncRNAs that regulate miRNAs with many target genes involved in lung fibrosis, using the InCeDB database (http://gyanxetbeta.com/lncedb/index.php). We selected LncRNA-CBR-AS1 related to be involved in cell proliferation and lung fibrosis. Total RNA was extracted from lung tissue samples using the RNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The concentration of RNA in each sample was measured using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, USA). A ratio of absorbance at A260/A280 of 1.8–2.1 was generally accepted as“pure”for RNA. Reverse transcription was performed using MiScript II RT PCR kits (Qiagen catalog no. 218161, Hilden, Germany) according to the manufacturer’s protocol. The reverse transcription reactions were stored at −20°C prior to real-time PCR.

Lnc-CBR-AS1 Expression Analysis

Lnc-CBR-AS1 was quantified by adding 10μL 2x RT2 SYBR Green ROX qPCR Mastermix, followed by RT2 lncRNAq PCR Assay for Rat Hoxb1 (XM_001081344) (cat no: 330001, ID PPR45880A). 2μL of template ecDNA and RNase-free water were added to a final volume of 20μL. The ACTB_1_SG Quanti Tect Primer Assay (NM_001101) was used as a housekeeping gene for the normalization of our raw data and compared with a reference sample. The PCR program for the relative quantification of lncRNAs was as follows: denaturation at 95°C for 10 min, followed by 45 cycles of denaturation 15sat 95°C; then annealing 30sat 55°C and extension for 30s. Relative quantification of serum RNAs was calculated using Schmittgen and Livak (2008) for each examined sample. The threshold cycle (Ct) value of each sample was calculated using the Rotor Gene real-time PCR detection system (Qiagen, Hilden, Germany). The melting curve was analyzed to confirm the specificity of the amplicons for qPCR.

Assessment of mi-RNA 29 by Real-Time PCR

Tissue Homogenization: Tissue samples were suspended in a 2.0 mL screw cap tube containing 200 µL of phosphate buffer saline (PBS), a single 5 mm stainless steel bead (Qiagen) was added to each tube, and the samples were homogenized at maximum speed (30 Hz) for 2 min using the Qiagen Tissue Lyser system. Following homogenization, the samples were spun for 1 min at maximum speed to reduce foaming, and the homogenate was applied to a filter column for RNA extraction. miRNA and total mRNA extraction and purification: miRNA and total mRNA were extracted using a miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Reverse transcription cDNA was synthesized by reverse transcription reaction using the miScript II RT Kit (Qiagen, Hilden, Germany). miRNA expression analysis. The quantification of miR-29 levels was performed using the SYBR-Green fluorescent-based primer assay (hsa-miR-29: cat no: MS00003528), and Hs_RUN6-2_11 (cat no: MS00033740) was used as a housekeeping gene for normalization. qPCR was performed using a 5-plex Rotor Gene PCR System (Qiagen). The reaction mixture (20 µL) consisted of 2×QuantiTect SYBR Green PCR master mix, 10× Miscript universal primers, 2 µL primer assay, and 50 pg–3 ng cDNA. Both targets were amplified in duplicates for each sample. The thermal protocol consisted of 15 min of hot start Taq DNA polymerase activation at 95◦C followed by 40 cycles of denaturation at 94 ◦C for 15 min, primer annealing for 30s at 55 ◦C and extension at 70◦C for 30s). The 2∆∆Ct method was used to analyze miR-145 expression levels, using RUN6 as an endogenous reference control for normalization purposes.

Assessment of Inflammatory Biomarkers in Lung Tissue by Real-Time PCR

To investigate the impact of TMZ on potential markers (FIZZ1, Jagged1, Notch, TGFβ, Smad3, and NFKB) in lung tissue, lung samples from the left lung were stored at −80°C. Total RNA was extracted using an RNeasy Mini Kit (catalog no. 74104) and reverse-transcribed using an iScript One-Step RT-PCR Kit with SYBR® Green (catalog no. D045-1 and D045-2) was purchased from Qiagen (Hilden, Germany). PCR primers were designed according to standard guidelines, with a length ranging from 18 to 25 nucleotides and a GC percentage ranging between 40% and 65% (Table 1). The primers and primer pairs were designed to avoid internal secondary structures and complementarity at the 3’ end with careful consideration. To optimize reaction efficiency and specificity, the primer concentration varied from 100 to 500 nM, and a final concentration of 300 nM per primer was generally effective for most reactions. To achieve the best results, it is recommended to use equal concentrations of each primer, while restricting the amplicon size to a range of 50–200 base pairs. For the template, the suggested input quantities range from 1 pg to 100 ng of total RNA or 10 fg to 100 ng of polyA(+) RNA. First-strand synthesis was performed between 40°C and 52°C, with the best outcomes observed at 50°C for 10 min. Higher temperatures may lead to difficulties in detecting non-specific amplification artifacts. Prior to use, all components except iScript reverse transcriptase were thawed at room temperature. Mix gently and centrifuge at 4°C to ensure proper collection of contents at the bottom of the tube, and then chill on ice. To minimize pipetting errors and enhance assay accuracy in quantitative PCR applications, it is essential to prepare a reaction cocktail by combining all necessary constituents, except for the sample template (total RNA), and then equally distribute aliquots into individual reaction tubes. Finally, the target sample was added in volumes ranging from 5 to 10 µL to improve the assay precision. For the replicated samples, a master mix was assembled by incorporating a single addition of the sample template.

Table 1 The List of Primer Pairs Used in qRT-PCR Analysis for the Selected Genes of Their miRNA Expression

Biochemical Measurements Assessment of Serum Immunological Markers

Blood samples (1 mL) were collected from the rat hearts and placed in centrifuge tubes (1.5 mL). The serum was extracted by centrifuging the blood for 10 min at 3000 rpm at 4 °C. The serum IL18 and ICAM1 levels were detected using an ELISA kit (Jianglai Biological Technology, Shanghai, China) according to the manufacturer’s instructions.

Assessment of Serum Oxidative Stress Markers

The serum malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) levels were determined using advanced laboratory techniques. Enzyme-linked immunosorbent assay (ELISA) was employed for the assessment, utilizing specific assay kits for each biomarker. The MDA levels were quantified using the OxiSelect “TBARS; Thiobarbituric Acid Reactive Substances” assay kit from CELL BIOLABS, USA. GPx activity was measured using a GPx assay kit (Cayman Chemical, Ann Arbor, MI, USA). SOD levels were quantified using an assay kit (catalog no. MBS266897; BioSource).

Assessment of Serum and Tissue Fibrogenic Markers

Fibrogenic markers, including Matrix Metalloproteinase-7 (MMP-7), Tissue Inhibitor of Metalloproteinase-1 (TIMP-1), and Connective Tissue Growth Factor (CTGF), were assessed using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis,),)SAin according to the manufacturer’s specifications. The evaluation protocol strictly adhered to the guidelines. Additionally, the fibrogenic impact in the lung tissue was quantified by measuring the hydroxyproline content, following the method outlined in the hydroxyproline assay kit sourced from Sigma-Aldrich Co. (Germany). The absorbance was recorded at 560 nm using a microplate reader. To determine lung collagen content, hydroxyproline assay results were used in the calculation:

Lung collagen content = Hydroxyproline content (13.517) The results were expressed as μg of collagen per milligram of wet lung tissue.57 ELISA kits from R&D Systems were used for the measurements following the manufacturer’s protocol.

Western Blot Study

The immunoblotting assay was employed for protein quantification and analysis. Homogenized lung tissue samples were processed using the ReadyPrep™ Protein Extraction Kit (Bio-Rad, Cat. #163-2086), For each sample, 20 µg of protein was combined with an equivalent volume of 2x Laemmli sample buffer. This buffer comprised 4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue, and 0.125 M Tris-HCl (pH 6.8) (Bio Basic Inc., Cat. #SK3041). To denature the proteins, the mixture was heated at 95°C for 5 minutes before being loaded onto a polyacrylamide gel (TGX Stain-Free™ Cat # 161–0181) for electrophoresis. Post-electrophoresis, the proteins were transferred to a PVDF membrane using the BioRad Trans-Blot Turbo system. The transfer sandwich, comprising filter paper, the PVDF membrane, the gel, and another filter paper, was assembled and placed in the transfer tank with 1x transfer buffer (25 mM Tris, 190 mM glycine, and 20% methanol). The transfer was executed at 25 V for 7 minutes To block non-specific binding, the membrane was incubated in TBST buffer at room temperature for 1 hour. The membrane was probed with targeted primary antibodies, including CTGF/CCN2 Antibody (NB100-724, Novus Biologicals, USA), IL-18 (11E5) (Santa Cruz Biotech., Cat. #sc-52012), SOD-2 (A-2) (Santa Cruz Biotech., Cat. #sc-133134), MDA-7 (Y14) (Santa Cruz Biotechnology, Cat. #sc-80184), Catalase (H-9) (Santa Cruz Biotech., Cat. #sc-27180), Jagged1 (E-12) (Santa Cruz Biotech., Cat. #sc-390177), Glutathione Reductase (B-12) (Santa Cruz Biotech, Cat. #sc-133159), FIZZ1/RELM Alpha Antibody (NBP2-29355, Novus Biologicals, USA), ICAM-1 (G-5) (Santa Cruz Biotech., Cat. #sc-8439), TGFβ1 (3C11) (Santa Cruz Biotech., Cat. #sc-130348), NFκB (Santa Cruz Biotech., Cat. # sc-8008), Smad3 (38-Q) (Santa Cruz Biotech., Cat. #sc-101154), MMP7 Polyclonal Antibody (Protein tech, Cat. #10374-2-AP), NOTCH1 Polyclonal Antibody (Elabscience, Cat. #E-AB-12815, USA), α-Smooth Muscle Actin Antibody (Cell Signaling Tech., Cat. #14968, USA), and TIMP1 (D10E6) mAb (Cell Signaling Tech., Cat. #8946, USA). For protein detection, primary antibodies were diluted in TBST and incubated overnight at 4°C. Chemiluminescence was developed using Clarity™ Western ECL Substrate (Bio-Rad, Cat. #170-5060) and captured with a CCD camera. Protein band intensities were analyzed and normalized against beta-actin using the ChemiDoc MP system.

Histological Studies Light Microscopic Study

After obtaining the right lung from all rats, it was preserved in a solution of neutral-buffered formaldehyde (10%) and placed in paraffin blocks. We cut 5-μm serial sections and stained them with hematoxylin and eosin (H&E) and Masson’s trichrome. The sections were analyzed using an Olympus CKX41 light microscope (Olympus Corporation, Tokyo, Japan) to detect microscopic changes or tissue fibrosis. We evaluated histological changes in five randomly chosen fields in all sections, but not consecutively.58

Immunohistochemical Analysis

To detect lung fibrosis, we used an immunohistochemical method with an avidin-biotin-peroxidase technique (Lab Vision, CA, USA) to test for the α-SMA antibody. We diluted it at 1:800 and left it for one and a half hours. Next, the secondary antibody (DAKO, Denmark) was added to the sections for 30 min. The reaction was developed for 10 min using DAB solution (purchased from DAKO, Denmark), and the slides were counterstained with Mayer’s hematoxylin. We prepared a positive control using smooth muscles with the same protocol and a negative control with the same protocol, but without using the primary antibody.59

Morphometric Study

The Histology Department at Ain Shams University utilized a Leica Qwin 500 computer system for image analysis to measure the following parameters.

Average thickness of the interalveolar septum in H&E-stained sections was measured in micrometers (µm) using a ×40 power lens. Average percentage of collagen fibers in Masson’s trichrome-stained sections using a ×40 power lens. Average percentage of α-SMA positive immunostained sections using a ×20 power lens.Statistical Analysis

We used two software programs for statistical analysis: GraphPad Prism version 5.0 (2007) (Inc., CA, USA) and SPSS 21 program (IBM Inc., Chicago, IL, USA). To compare statistical differences between groups, a one-way analysis of variance (ANOVA) followed by a post-hoc Tukey’s test was conducted. SPSS 21 was used to calculate the mean values and standard deviations (SD) of the histological parameters, and another ANOVA test was conducted to compare variations between different groups. The significance level was determined based on the p-value, where a p-value less than 0.05 was considered statistically significant and a p-value less than 0.0001 was highly significant. GraphPad StatMate software version 1.01i Jan was used to determine the optimal sample size. To perform the morphometric analysis, we measured various parameters in five distinct fields from two consecutive sections of each animal within each group. The mean and standard deviation (SD) of these measurements were computed using SPSS 21 software (IBM Inc., Chicago, IL, USA). To compare the results, we applied a one-way Analysis of Variance (ANOVA) with a subsequent Least Significant Difference (LSD) post-hoc test. The significance of the data was assessed based on the calculated P-value, where a value of P < 0.05, was considered indicative of statistical significance.

Results TMZ Upregulated the Expression of miRNA-29 Gene by Targeting the Epigenetic Regulator Lnc CBR3-AS1 in BLM-Induced Lung Fibrosis

First, we explored the effect of epigenetic regulators on lung fibrosis by assessing the expression of lnc CBR3-AS1 in a BLM-induced lung fibrosis model. The long noncoding RNA CBR3-AS1 plays a vital role in facilitating the epithelial-mesenchymal transition and regulating the signaling pathways of both TGF-β1 and Notch/jagged1. As shown in Figure 2, BLM treatment significantly (P<0.0001) increased the expression of lnc CBR3-AS1 as compared to that in the control group. These results indicate that lnc CBR3-AS1 plays a molecular epigenetic regulatory role not only in lung cancer, but also in lung fibrosis. We next assessed the effect of TMZ administration by evaluating the expression levels of lnc CBR3-AS1 in the TMZ-treated control and TMZ-treated model groups. The results indicated that TMZ had no significant effect on lnc CBR3-AS1 expression levels as compared to the control group, while it demonstrated a significant attenuation (P<0.0001) in the expression of lnc CBR3-AS1 gene as compared to the BLM-treated group. These results further emphasize the role of molecular epigenetic regulation of lnc CBR3-AS1 gene expression in lung fibrosis and suggest that TMZ is an antifibrotic drug for the treatment of lung fibrosis by modulating the epigenetic regulation of lnc CBR3-AS1. To identify the target miRNA of the master regulator lnc CBR3-AS1, we assessed the expression levels of miRNA-29 and explored its role in lung fibrosis. Despite the expression of both miRNA-29 and lnc CBR3-AS1 in lung tissues, the interplay between these genes in lung fibrosis remains poorly understood. In this regard, we evaluated the expression of miRNA-29 in lung tissue after BLM treatment and compared it to that in the control group. As indicated in Figure 2, our findings revealed the BLM administration significantly (P<0.0001) reduced miRNA-29 expression compared to that in the control group. These findings are in agreement with previous reports of reduced expression levels of miRNA-29 in individuals with IPF.21 These results further suggest the exploration of miRNA-29-based treatments as a potential therapeutic approach for lung fibrosis. In contrast, TMZ treatment showed no considerable effect on the expression of miRNA-29 in the TMZ-treated control group, as compared to the control group. However, there was a substantial reduction (P<0.0001) in the expression levels of miRNA-29 compared with those in the BLM-induced lung fibrosis group. These results suggest that lnc CBR3-AS1 is an epigenetic regulator of miRNA-29, and that both genes play crucial roles in modulating the molecular cascade of lung fibrosis. Furthermore, our findings highlighted the possible therapeutic potential of TMZ in lung fibrosis by targeting epigenetic regulators.

Figure 2 Effect of trimetazidine treatment (15 mg/kg/day; for four weeks) on the expression levels of epigenetic regulator Lnc CBR3-AS1 (A), and miRNA-29 (B) in BLM-induced lung fibrosis. The data is expressed as mean±SD, with a sample size of n=7. Multiple comparisons were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test.

Notes: The differences between groups were considered significant when toward (*P<0.05; **P<0.01; ***P<0.0001).

TMZ Significantly Attenuated the Expression of Inflammatory Biomarkers Jagged1 and Notch by Targeting FIZZ-1 in BLM-Induced Lung Fibrosis

FIZZ1 promotes the transformation of lung fibroblasts into myofibroblasts through the activation of the Notch signaling pathway, which is a significant characteristic of lung fibrosis. FIZZ1 also elevated the levels of the activated intracellular domain of Notch1 and its ligand Jagged1, which is linked to increased expression of α-smooth muscle actin (α-SMA) in lung fibrosis. Toward this end, we next focused on assessing the expression of FIZZ1 in lung tissues and explored its interplay with the inflammatory signaling cascade. As shown in Figure 3, the administration of BLM resulted in a notable increase (P<0.0001) in the expression of FIZZ1 compared to the control group. In agreement with previous studies, these findings suggest that BLM administration leads to FIZZ1 upregulation, which subsequently activates inflammatory pathways in lung tissues, thereby contributing to the development of lung fibrosis. Next, we assessed the therapeutic potential of TMZ in lung fibrosis by assessing the expression of FIZZ1 in the TMZ-treated control and TMZ-treated lung fibrosis model groups. Our results revealed that TMZ administration showed no statistical difference in the expression of FIZZ1 in the TMZ-treated control group compared to that in the control group. Conversely, the TMZ-treated model group exhibited a significant reduction (P<0.0001) in FIZZ1 expression compared to the BLM-induced lung fibrosis group. These findings indicated that the antifibrogenic potential of TMZ may be associated with its ability to modulate inflammatory cascades in lung tissues by targeting FIZZ1.

Figure 3 Effect of trimetazidine treatment (15 mg/kg/day; for four weeks) on FIZZ1 (A), Jagged1 (B) and Notch (C) genes and proteins (D) expression in BLM-induced lung fibrosis. The correlation study analysis between the expression of tissue FIZZ1 and Lnc CBR3-AS1 (E), miRNA-29 (F) in BLM-induced lung fibrosis model. The data is expressed as mean±SD, with a sample size of n=7. Multiple comparisons were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test.

Notes: The differences between groups were considered significant when P<0.05 (*P<0.05; **P<0.01; ***P<0.001, ****P<0.0001).

To examine the possible correlation between lnc CBR3-AS1/miRNA-29 and FIZZ1, we performed correlation analysis using Pearson’s correlation coefficient between the expression of lnc CBR3-AS1/miRNA-29 and FIZZ1. According to the findings (Figure 3), statistical analysis conducted using Pearson’s correlation coefficient indicated a significant (p<0.05) positive association between tissue FIZZ 1 and lnc CBR3-AS1 expression (R= 0.7723). Furthermore, our analysis showed a significant negative association between miRNA-29 and FIZZ1 expression (R=−0.7535). Taken together, these results indicate that lnc CBR3-AS1 could be an epigenetic regulator of FIZZ1, and further suggest a possible correlation between miRNA-29 and lncRNA CBR3-AS1 gene expression and FIZZ1-mediated inflammatory pathways in lung fibrosis.

To explore the inflammatory markers associated with FIZZ1, we conducted computational networking analysis using the STRING database to assess the protein-protein association interaction network between FIZZ1 (Retnla) and the inflammatory markers. As shown in Figure S2, the results indicated significant (PPI enrichment with a p-value of 9.41e−07) interactions between FIZZ1, and the inflammatory markers NOTCH, Jagged (Jag1), Smad3, TGFB, and RelA (NF-kB) (Table S1). The Jagged1/Notch pathway plays a significant role in regulating epithelial-to-mesenchymal transition and myofibroblast formation, a process initiated by FIZZ-1 in alveolar epithelial cells, as part of the progression of chronic lung fibrosis. To gain more insight into the association between FIZZ1 and the Jagged1/Notch inflammatory pathways in BLM-induced lung fibrosis, we assessed the expression levels of Jagged1 and Notch genes. As depicted in Figure 3, our analysis revealed that the administration of BLM significantly upregulated (P<0.0001) the expression levels of Jagged1 and Notch genes compared to the control group. These results are in agreement with the previously observed increase in FIZZ1 expression in the BLM-treated model group and indicate the successful induction of fibrosis in lung tissues. Assessment of the therapeutic potential of TMZ revealed that administration of TMZ did not significantly affect the expression of Jagged1 and Notch genes in the TMZ-treated control group compared to the control group. Furthermore, treatment of the BLM-induced lung fibrosis model with TMZ demonstrated a substantial reduction in the expression levels of Jagged1 (P<0.0001) and Notch (P<0.01) genes, as compared to the BLM-treated group. Notch/ Jagged1 genes have been previously recognized as target downstream genes for lncRNA CBR3-AS1. To further underscore our results, we assessed the protein expression of FIZZ1, Jagged1, and Notch proteins in lung tissue across the various treatment groups by conducting Western blot analysis. As shown in Figure S3, the Western blot analysis corroborated our PCR findings across the different groups. The BLM-treated group exhibited, compared to the control group, a significant augmentation in the expression of FIZZ1, Jagged1, and Notch proteins (Figure S3). Conversely, TMZ treatment notably attenuated the expression of examined proteins in the BLM-induced model. Together, our findings suggest a molecular interplay of the lnc CBR3-AS1/miRNA-29/FIZZ1 axis in regulating the Notch/Jagged1 inflammatory pathway and provide novel molecular insights into how these cascades jointly regulate the progression of lung fibrosis. These results further implied that the observed antifibrogenic potency of TMZ could be attributed to its ability to regulate the lncRNA CBR3-AS1/miRNA-29/FIZZ1 axis in lung tissues.

TMZ Significantly Downregulated the Expression of Inflammatory Biomarkers Smad3 and NFKB by Targeting TGF-β in BLM-Induced Lung Fibrosis

As previously stated, our computational network analysis revealed a significant interaction between FIZZ1 and the inflammatory Smad3, TGFB, and RelA (NF-kB) markers (Figure S2 and Table S1). Accordingly, the expression of TGF-β genes was analyzed to investigate the proinflammatory pathway in lung fibrosis. The TGF-β signaling pathway is associated with fibrosis and inflammation in lung tissues. As presented in Figure 4, BLM administration resulted in a significant increase (P<0.0001) in the expression levels of TGF-β compared to the control group. These results indicate that BLM treatment successfully triggered the expression of genes responsible for inflammation and fibrosis in lung tissues. In contrast, treatment of the control group with TMZ showed no considerable difference in TGF-β gene expression, as compared to the control group, while in the BLM-induced lung fibrosis model, there was a significant attenuation (P<0.0001) in the expression of the TGF-β gene, as compared to the BLM-treated group. These findings further affirm the therapeutic potential of TMZ in modulating the deteriorating effects of BLM-induced lung fibrosis by targeting the proinflammatory TGF-β pathway. We further explored the inflammatory pathway associated with TGF-β signaling in lung fibrosis by assessing the expression levels of Smad3 and NF-κB. Aligned with TGF-β expression, our findings revealed a significant upregulation (P<0.0001) in the expression levels of Smad3 and NF-κB genes in the BLM-treated group compared to the control group (Figure 4). Crosstalk between the Smad3 signaling pathway, driven by the presence of TGF-β, and the activation of NFкB due to lung tissue damage plays a pivotal role in promoting lung fibrosis by orchestrating the production of inflammatory cytokines and fibroblasts within the lung parenchymal tissue. Our results further indicated that BLM treatment induces the inflammatory signaling pathway in lung tissues, resulting in the upregulation of inflammatory cytokines and fibroblasts. Next, we explored the antifibrotic activity of TMZ in lung fibrosis by assessing its ability to target the proinflammatory Smad3 and NF-κB signaling pathways in BLM-induced PF. As depicted in Figure 4, treatment of the BLM-induced PF model with TMZ significantly reduced (P<0.0001) the expression levels of Smad3 and NF-κB genes, as compared to the BLM-treated model. Remarkably, no significant change was observed in the TMZ-treated control group compared with that in the control group. These findings were further validated by conducting Western blot analysis to measure the expression levels of Smad3, NF-κB, and TGF-β proteins in lung tissue across different treatment groups. As illustrated in Figure S4, the Western blot analysis reinforced the PCR results, demonstrating consistency across the different groups. In comparison to the control group, the lung fibrosis model group (BLM-treated group) exhibited a significant increase in the expression of Smad3, NF-κB, and TGF-β proteins, whereas the expression levels were significantly reduced in the TMZ-treated group (Figure S4). The interaction between TGF-β and Smad3/ NFkB triggers the expression of genes responsible for matrix expression, fibroblast differentiation, and proliferation. Furthermore, TGF-β and Smad3 are known as target genes for miRNA-29 and lnc CBR3-AS1, suggesting that the lnc CBR3-AS1/miRNA-29/FIZZ1 axis could be a molecular cascade that modulates inflammatory and fibrotic progression in lung fibrosis. Our findings further indicate that TMZ possesses considerable antifibrotic and anti-inflammatory activities, which are associated with its ability to target the TGF-β, NFкB, and Smad3 signaling pathways in lung fibrosis.

Figure 4 Effect of trimetazidine treatment (15 mg/kg/day; for four weeks) on inflammatory biomarkers TGFB (A), Smad3 (B), and NFKB (C) genes and proteins (D) expression in BLM-induced lung fibrosis. The data is expressed as mean±SD, with a sample size of n=7. Multiple comparisons were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test.

Notes: The differences between groups were considered significant when P<0.05 (*P<0.05; **P<0.01; ****P<0.0001).

TMZ Modulated the Expression of Oxidative Stress MDA, CAT, SOD and GSH in BML-Induced Lung Fibrosis

An increase in fibroblast growth and myoblast differentiation has been observed under excessive oxidative stress. Previous reports showed that the miRNA-29 and Fizz1 play critical roles in regulating the oxidative stress process in type 2 diabetes Mellitus and pulmonary fibrosis, respectively.60–64 Accordingly, certain markers of oxidative stress have been identified as valuable in predicting lung fibrosis. To investigate the effect of oxidative stress on the development of lung fibrosis, we assessed the expression of various oxidative stress biomarkers, including malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), and glutathione (GSH). As depicted in Figure 5, the BLM-induced lung fibrosis model exhibited a significant (P<0.0001) upregulation in the expression of MDA, coupled with a significant (P<0.0001) reduction in serum SOD, CAT, and GSH levels compared to the control group. MDA is a marker of lipid peroxidation, while SOD inhibits oxidative stress. The upregulation of MDA levels following BLM treatment suggested a higher level of oxidative damage in the lungs. SOD and CAT are enzymes that help neutralize harmful reactive oxygen species, whereas GSH is an essential antioxidant. The observed downregulation of serum SOD, CAT, and GSH levels implies a reduction in antioxidant defense mechanisms to counteract oxidative stress. Together, these findings reveal that BLM exacerbates oxidative stress and weakens the capacity of the lungs to defend against it, which may be relevant to the development and progression of lung fibrosis. We further assessed the antifibrotic potential of TMZ by assessing its ability to modulate oxidative stress in BLM-induced lung fibrosis. As shown in Figure 5, in the TMZ-treated control group, TMZ treatment demonstrated no significant effect on the serum expression of MDA, CAT, SOD, and GSH levels, as compared to the control group. Nevertheless, TMZ administration showed the ability to significantly attenuate (P<0.0001) the expression levels of MDA, as well as significantly upregulate the expression levels of SOD (P<0.0001), CAT (P<0.0001) and GSH (P<0.01) in BLM-treated model group. Additionally, Western blot analysis was utilized to quantify the expression of oxidative stress proteins in lung tissue across different treatment groups, as depicted in Figure 5. The lung fibrosis model group exhibited, compared to the control group, a significant elevation in the MDA protein expression, while the expression levels of SOD, CAT, and GSH proteins were substantially attenuated. On the other hand, following the TMZ administration, these alterations were reversed. As compared to the BLM-induced PF model, the expression of SOD, CAT, and GSH proteins was significantly augmented, along with significant mitigation in the expression of MAD protein (Figure S5). These findings further affirm the therapeutic potential of TMZ as an antifibrotic drug by reducing the expression of markers that oppose oxidative stress while increasing the expression of markers that activate antioxidant defense mechanisms, ultimately countering oxidative stress.

Figure 5 Effect of trimetazidine treatment (15 mg/kg/day; for four weeks) on serum oxidative stress markers MDA (A), CAT (B), SOD (C), and GSH (D), and their protein expression by Western blot analysis (E) in BLM-induced lung fibrosis. The data is expressed as mean±SD, with a sample size of n=7. Multiple comparisons were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test.

Notes: The differences between groups were considered significant when P<0.05 (*P<0.05; **P<0.01; ***P<0.001, ****P<0.0001).

TMZ Significantly Reduced the Expression Levels of Serum Immunological Biomarkers ICAM-I and IL-18 in BLM-Induced Lung Fibrosis

Next, we employed computational networking analysis to explore the protein-protein association interaction network between FIZZ1 (Retnla), and immunomodulatory markers. Our findings revealed a significant PPI enrichment (p-value of 2.87e−07) with interacting nodes linked to the functional protein partners, including ICAM1, IL18, and FIZZ1 (RetLna) (Figure S2 and Table S2). ICAM-1 and IL-18 are well-known immunological chemokines associated with pulmonary inflammation and have garnered attention as promising diagnostic and/or prognostic biomarkers for IPF. The overexpression of ICAM-1 on the surface of endothelial cells plays a significant role in inducing the inflammatory signaling pathway in lung fibrosis. To gain further insight into the immune response in lung fibrosis, we assessed the expression levels of various immunological markers including ICAM-1 and IL-18. As presented in Figure 6, the BLM-treated group demonstrated a significant elevation (P<0.0001) in the expression levels of the immunological markers ICAM-1 and IL-18 compared with the control group. These markers are associated with inflammation and immune responses in the lungs, and their elevated expression suggests a heightened immune and inflammatory response. Consistent with previous findings, our results suggest that these markers may play a role in the pathophysiology of lung fibrosis, making them potential targets for further research into the development of potential therapies. To further explore the immunomodulatory function of TMZ, we assessed the expression of ICAM-1 and IL-18 markers in TMZ-treated control and BLM-treated model groups after treatment with TMZ. Our investigations revealed that TMZ treatment had no significant effect on the expression of immunological markers in the TMZ-treated control group compared with the control group. However, there was a significant attenuation (P<0.0001) in the expression levels of ICAM-1 and IL-18 compared to those in the BLM-induced lung fibrosis group (Figure 6). These findings were further affirmed by Western blot analysis to evaluate the expression of ICAM-1 and IL-18 proteins in lung tissue across different treatment groups. Our results, aligned with PCR analysis, revealed that the lung fibrosis model group displayed a significant elevation in ICAM-1 and IL-18 protein expression compared to the control group (Figure S6). In contrast, the TMZ-treated BLM-induced PF model showed a significant mitigation in the expression of these proteins. These findings provide compelling evidence for the immunomodulatory role of TMZ in lung fibrosis, indicating its potential as a therapeutic agent to mitigate the immune and inflammatory aspects of this condition.

Figure 6 Effect of trimetazidine treatment (15 mg/kg/day; for four weeks) on immunological biomarker ICAM-1 (A), IL18 (B) and their protein expression by Western blot analysis (C) in BLM-induced lung fibrosis. The data is expressed as mean±SD, with a sample size of n=7. Multiple comparisons were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test.

Notes: The differences between groups were considered significant when P<0.05 (*P<0.05; **P<0.01; ****P<0.0001).

TMZ Modulated the Expression of Various Serum and Tissue Fibrogenic Markers (MMP7, TIMP1, CTGF, Hydroxyproline, and Collagen Content) in BLM-Induced Lung Fibrosis

Finally, we assessed the protein-protein association interaction network between FIZZ1 (Retnla), and fibrotic markers utilizing computational networking analysis. As shown in Figure S2, a significant PPI enrichment (p-value < 1.0e−16) has been observed between MMP7, TIMP1, CTGF (CNN2), collagen (COL1A1), and FIZZ1 (RetLna) (Table S3). Accordingly, the expression of fibrogenic biomarkers CTGF, MMP7, and TIMP1 associated with the initiation and progression of lung fibrosis have been examined among the experimental groups. As shown in Figure 7, BLM administration resulted in a significant increase (P<0.0001) in the expression levels of serum MMP7, TIMP1, and CTGF compared with the control group. The observed upregulation of these fibrogenic markers in the BLM-treated group indicated that BLM treatment successfully induced fibrosis and inflammation in lung tissue. Next, we explored the therapeutic potential of TMZ for modulating fibrogenic markers. Our assessments revealed that the administration of TMZ to the control group had no significant effect on the expression levels of these serum fibrogenic markers compared to the control group (Figure 7A–7C). Conversely, TMZ treatment substantially attenuated (P<0.0001) the expression levels of serum MMP7, TIMP1, and CTGF markers compared to those in the BLM-induced lung fibrosis group. To further affirm these findings, we examined the expression of CTGF, MMP7, and TIMP1 proteins in lung tissue across different treatment groups using Western blot analysis. As shown in Figure 7, the results revealed a significant elevation in the protein expression of CTGF, MMP7, and TIMP1 in the BLM-treated group, as compared to the control model. While the TMZ-administrated BLM-induced PF model displayed a substantial attenuation in the expression of these proteins (Figure S7). Together, our findings further affirmed the antifibrotic activity of TMZ and its ability to mitigate the detrimental effects of BLM treatment on lung tissues.

Figure 7 Effect of trimetazidine treatment (15 mg/kg/day; for four weeks) on fibrogenic markers MMP7 (A), TIMP1 (B), CTGF (C), hydroxyproline (D), and collagen (E), and their protein expression by Western blot analysis (F) in BLM-induced lung fibrosis. The data is expressed as mean±SD, with a sample size of n=7. Multiple comparisons were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test.

Notes: The differences between groups were considered significant when P<0.05 (*P<0.05; **P<0.01; ****P<0.0001).

We further extended our investigations to assess the expression levels of lung hydroxyproline and collagen. As shown in Figure 7, administration of BLM resulted in a significant elevation (p<0.0001) in the expression levels of hydroxyproline and collagen in lung tissue compared to the control group. Elevated expression of hydroxyproline and collagen has been linked to the development of fibrous tissues in the lungs. Next, we explored the therapeutic potential of TMZ in modulating these fibrogenic markers. Our assessments revealed that the administration of TMZ to the control group had no significant effect on the expression levels of lung tissue hydroxyproline and collagen content, as compared to the control group (Figure 7D). Alternatively, TMZ treatment displayed significant mitigation (p<0.0001) in the expression levels of hydroxyproline and collagen content in lung tissue compared to the BLM-induced lung fibrosis group. These findings further affirm the antifibrotic potency of TMZ and its ability to reduce lung hydroxyproline and collagen contents, suggesting an effect of TMZ in attenuating the development of fibrosis tissues in the lung.

TMZ Attenuated the Inflammatory Cell Infiltrates and Interalveolar Septum Thickness in BLM-Induced Lung Fibrosis

Analysis of lung sections stained with hematoxylin and eosin (H&E) in the control group revealed the presence of the typical normal architecture of the lung, with thin-walled- alveoli and unobstructed lumina. The alveolar wall comprises of cells with flattened nuclei (pneumocytes Type I), and cubical cells with rounded, bulging nuclei (pneumocytes Type II). A thin interalveolar septum is observed between adjacent alveoli. The average thickness of the interalveolar septa was 7.758 ± 1.256 µm, as indicated by mean values and standard deviations (Figure 8A).

Figure 8 (A–D) Photomicrograph of the H&E-stained lung section of the control group (A); BLM-Model group (B); and TMZ+BLM treated group (C). (A) The control group shows the normal architecture of the lung, with thin-walled- alveoli and patent lumina. The alveolar wall comprises flat cells with flattened nuclei (pneumocytes Type (I), and cubical cells with rounded, bulging nuclei (pneumocytes Type II). Notice the thin interalveolar septa. (B): The BLM-model group shows marked distortion of the lung architecture with the replacement of the lung tissue with collagen fibers (arrowheads), and most of the alveoli collapsed (A). Notice markedly thickened interalveolar septa (blue arrows) and numerous congested blood vessels were observed (black arrows). (C) The TMZ+BLM treated group shows restoration of the normal lung architectures with numerous alveoli having thin walls and patent lumina. However, numerous blood vessels were congested with erythrocytes (arrows). (D): The mean thickness of interalveolar septum ±SD (in µm) in different groups (n=3).

Examination of lung sections from rat