Inhibitory effect of l-mimosine on bleomycin-induced pulmonary fibrosis in rats: Role of eIF3a and p27
Abstract
Previous scientific investigations have elucidated the profound impact of eukaryotic translation initiation factor 3a, commonly abbreviated as eIF3a, on cellular processes, specifically noting that a reduction in its expression levels, often induced by compounds like L-mimosine, can lead to a significant arrest in the cell cycle. This established link between eIF3a and cell cycle regulation provides an important foundation for understanding its broader biological roles. Building upon this, our own prior research has already identified a crucial involvement of eIF3a in the pathogenesis and progression of bleomycin-induced pulmonary fibrosis, a severe and debilitating lung condition characterized by excessive scar tissue formation. However, despite these insights, a significant gap in our understanding persisted regarding the precise mechanistic pathway through which L-mimosine exerts its inhibitory effects on bleomycin-induced pulmonary fibrosis. Specifically, it remained unclear whether the interplay between eIF3a and the cyclin-dependent kinase inhibitor p27, a key regulator of cell proliferation, constitutes a critical signaling axis mediating these beneficial actions. The current study was meticulously designed to address this fundamental question, aiming to unravel the potential involvement of the eIF3a/p27 signal pathway in the therapeutic efficacy of L-mimosine against this challenging fibrotic disease.
To comprehensively investigate this hypothesis, a multi-faceted experimental approach was employed, encompassing both *in vivo* animal models and *in vitro* cell culture systems. For the *in vivo* component, pulmonary fibrosis was experimentally induced in a rat model, a widely accepted and highly relevant system for studying this human disease, through a single intratracheal instillation of bleomycin at a precisely controlled dose of 5 mg per kilogram of body weight. Concurrently, for the *in vitro* investigations, primary pulmonary fibroblasts were carefully isolated and cultured. These cells, being the primary effector cells in fibrotic processes, were then utilized to assess crucial cellular activities, particularly their proliferative capacity. This was rigorously quantified using established methodologies such as the BrdU incorporation method, which measures DNA synthesis in actively dividing cells, and flow cytometry, providing a detailed analysis of cell cycle phases and proliferation rates. Furthermore, to delineate the molecular mechanisms underpinning these cellular and physiological changes, the expression levels of several key target molecules were meticulously analyzed. These included eIF3a and p27, central to our proposed pathway, as well as alpha-smooth muscle actin (α-SMA), a canonical marker for myofibroblast differentiation, and the extracellular matrix components collagen I and collagen III, which are hallmarks of fibrotic tissue accumulation. The quantitative analysis of these molecular markers was performed at both the messenger RNA (mRNA) level, utilizing quantitative polymerase chain reaction (qPCR) to assess transcriptional activity, and the protein level, employing Western blot analysis to determine the functional protein abundance, thereby providing a holistic view of gene expression regulation.
The results obtained from the *in vivo* experiments provided compelling evidence of L-mimosine’s therapeutic potential. Treatment with L-mimosine remarkably and significantly ameliorated the severe histological alterations characteristic of bleomycin-mediated pulmonary fibrosis. This amelioration was evident in reduced scarring, improved lung architecture, and a marked decrease in the overall fibrotic burden within the lung tissue. Crucially, L-mimosine treatment effectively blocked the excessive deposition of collagen, a defining feature of fibrotic progression. These macroscopic and histological improvements were consistently accompanied by a precise reversal of the molecular dysregulations induced by bleomycin. Specifically, L-mimosine treatment counteracted the bleomycin-induced up-regulation of eIF3a, α-SMA, collagen I, and collagen III expression, observed at both the mRNA and protein levels, bringing them closer to physiological norms. Simultaneously, L-mimosine successfully reversed the bleomycin-induced down-regulation of p27 expression, restoring its levels. Parallel investigations conducted *in vitro* on cultured primary pulmonary fibroblasts further corroborated these *in vivo* findings and provided deeper mechanistic insights.
L-mimosine remarkably attenuated the excessive proliferation of these fibroblasts, a critical event in fibrosis, and also significantly suppressed the aberrant expression of α-SMA, collagen I, and collagen III, all of which had been potently induced by transforming growth factor-beta 1 (TGF-β1), a well-established and powerful profibrotic cytokine. This robust inhibitory effect of L-mimosine on fibroblast activation and extracellular matrix production was consistently accompanied by a corresponding inhibition of eIF3a expression and a notable increase in p27 expression, mirroring the molecular changes observed in the animal model. To definitively establish the causal role of eIF3a within this pathway, a targeted genetic intervention was performed. Specifically, the knockdown of eIF3a gene expression, achieved through specific molecular tools, was found to independently reverse the detrimental effects of TGF-β1 on fibroblasts. This included the reversal of TGF-β1-induced fibroblast proliferation, the restoration of p27 expression which had been down-regulated by TGF-β1, and the suppression of the TGF-β1-induced up-regulation of α-SMA, collagen I, and collagen III expression. This direct manipulation of eIF3a expression provided strong evidence that eIF3a is a pivotal upstream mediator in the profibrotic pathway and that its inhibition directly contributes to the beneficial effects observed.
Taken together, the collective and compelling body of evidence generated from these comprehensive *in vivo* and *in vitro* studies strongly suggests that L-mimosine exerts its significant inhibitory effects on the progression of bleomycin-induced pulmonary fibrosis in rats through a precise and orchestrated modulation of the eIF3a/p27 signaling pathway. This research not only identifies a critical mechanism of action for L-mimosine’s antifibrotic properties but also highlights the eIF3a/p27 axis as a promising therapeutic target for the development of novel strategies to combat pulmonary fibrosis.
Keywords: Eukaryotic translation initiation factor 3a; Pulmonary fibroblasts; Pulmonary fibrosis; L-Mimosine; p27.
Introduction
Pulmonary fibrosis represents a profoundly severe and ultimately fatal disease, characterized by its progressive nature and a diverse range of underlying causes. This debilitating condition is histopathologically defined by a relentless and uncontrolled proliferation of fibroblasts, which are cells responsible for synthesizing connective tissue, alongside the excessive and pathological deposition of extracellular matrix components within the delicate lung parenchyma. This culminates in profound tissue remodeling, leading to the destruction of normal lung architecture and impaired respiratory function. Despite ongoing efforts in therapeutic development, the current treatment regimens available for patients suffering from pulmonary fibrosis offer only limited efficacy, resulting in a markedly poor long-term survival rate. Tragically, the average survival period for individuals diagnosed with this devastating disease is a mere two to three years. This dire prognosis underscores an urgent and critical need for the discovery and development of novel pharmacological agents that not only possess superior therapeutic efficacy but also exhibit an improved safety and tolerability profile, thereby offering a more hopeful outlook for affected patients.
A fundamental aspect of cellular biology, translational control, plays an extraordinarily pivotal role in the precise regulation of gene expression within living organisms. This intricate process primarily occurs at the initiation step of protein synthesis, a complex cascade involving a multitude of specialized proteins collectively known as eukaryotic translation initiation factors, or eIFs. Among these critical regulatory proteins, eIF3a, also identified as p170, stands out as the largest and arguably one of the most significant subunits of the multiprotein eIF3 complex. Emerging research has increasingly highlighted the diverse and essential functions of eIF3a, proposing its involvement not only in the selective translation of a specific subset of messenger RNAs, thereby fine-tuning protein production, but also in the meticulous regulation of fundamental cellular processes such as cell cycle progression and overall cell proliferation.
Furthermore, compelling evidence derived from oncological research has demonstrated that a strategic suppression of endogenous eIF3a expression can effectively impede the acquisition of malignant phenotypes in various human cancer cell lines. Conversely, the deliberate overexpression of ectopic eIF3a has been shown to actively promote the malignant transformation of mammalian cells, firmly establishing its pro-oncogenic role. Our own independent and prior investigations have significantly contributed to this understanding by revealing a crucial and previously unrecognized involvement of eIF3a in the pathological development of bleomycin-induced pulmonary fibrosis. Beyond its role in *in vivo* disease models, we also uncovered its critical participation in the transforming growth factor-beta 1 (TGF-β1)-induced proliferation and differentiation of pulmonary fibroblasts, which are key cellular drivers of fibrogenesis. These collective findings robustly suggest that eIF3a’s regulatory influence extends beyond tumorigenesis, encompassing the dynamic process of fibroblast proliferation within the context of pulmonary fibrosis.
In parallel, another compelling compound, L-mimosine, a naturally occurring plant amino acid with the chemical designation (S)-α-amino-β-[1-(3-hydroxy-4-oxopyridine)] propionic acid and the chemical formula C8H10N2O4, has garnered considerable scientific interest. This remarkable compound functions primarily as an iron chelator and has been meticulously characterized for its ability to reversibly impede mammalian cell proliferation. Its mechanism of action involves effectively blocking the cells at the late G1 phase of the cell cycle, a critical checkpoint before DNA replication. This blockage is achieved by preventing DNA synthesis itself and by interfering with the intricate synthesis of histone H1 kinase, a vital enzyme for cell cycle progression. Furthermore, L-mimosine has been shown to specifically inhibit the expression of cyclin D1, a key cell cycle regulator, and, notably, to up-regulate the protein levels of p27, a potent cyclin-dependent kinase inhibitor that acts to arrest the cell cycle. Earlier studies have consistently demonstrated that incubation with L-mimosine leads to a discernible increase in the population of cells arrested in the G0/G1 phases, as evidenced in human prostate carcinoma DU145 cells. More directly relevant to our current investigation, it has been previously established that the L-mimosine-induced decrease in eIF3a expression leads to an elevated translation of p27 protein, an event that precedes and contributes to the observed cell cycle arrest. Importantly, the regulatory interplay between eIF3a and proteins such as p27 in controlling protein expression has been specifically implicated in mediating the broader functions of eIF3a in cellular proliferation.
Given the accumulating evidence strongly implicating a potential and indeed critical role for eIF3a in the intricate pathogenesis of pulmonary fibrosis, alongside its established involvement in the proliferation and differentiation of pulmonary fibroblasts, the present study was meticulously designed. Our primary objective was to comprehensively assess whether L-mimosine possesses the capacity to inhibit the development and progression of bleomycin-induced pulmonary fibrosis in a well-established rat model. More specifically, a key focus of this investigation was to meticulously explore the precise effects of L-mimosine on the expression profiles of eIF3a and p27, unraveling their potential involvement in its therapeutic actions. Furthermore, we aimed to quantify the impact of L-mimosine on collagen accumulation, a direct measure of fibrotic severity, and to thoroughly characterize its influence on the cellular proliferation of primary cultured pulmonary fibroblasts, providing critical insights into its cellular mechanisms of action.
Materials And Methods
Reagents And Materials
The foundational reagents and materials critical for the execution of this comprehensive study were procured from reputable commercial sources to ensure high quality and reproducibility. L-Mimosine, specifically derived from Koa hoale seeds, was obtained from Sigma (St. Louis, MO, Cat. no.: M0253). Bleomycin, a potent inducer of pulmonary fibrosis, was supplied by Nippon Kayaku Co. Ltd (Tokyo, Japan). Transforming growth factor-beta 1 (TGF-β1), a key profibrotic cytokine utilized in *in vitro* experiments, was purchased from PeproTech (New Jersey, USA). For histological assessment of collagen deposition, the Masson’s trichrome stain kit was acquired from Nanjing KeyGEN Biotech (Nanjing, China). The BrdU cell proliferation assay kit, a standard tool for measuring DNA synthesis, was provided by Roche (Mannheim, Germany). Dulbecco’s Modified Eagle’s Medium (DMEM), a fundamental cell culture medium, was obtained from GIBCO (New York, NY, USA). Oligonucleotide primers, essential for gene expression analysis, were custom-synthesized and purchased from Shanghai Sangon Biological Engineering Co. Ltd. (Shanghai, China). The PrimeScript reverse transcription reagent Kit and SYBR Premix Ex Taq, crucial components for quantitative polymerase chain reaction (qPCR), were procured from TaKaRa Biotechnology Co., Ltd. (Dalian, China). Primary antibodies specific for p27 and eIF3a were sourced from Cell Signaling (Boston, MA, USA). Primary antibodies directed against alpha-smooth muscle actin (α-SMA), collagen I, and collagen III, key markers of fibrosis, were purchased from Abcam (Hong Kong, China). Finally, the antibody for Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), utilized as a loading control, was obtained from Santa Cruz (CA, USA) with the catalog number sc-137179 at a dilution of 1:2000.
Animals
For the *in vivo* experimental paradigm, healthy male Sprague-Dawley (SD) rats, aged between 6 and 8 weeks and weighing approximately 180 to 220 grams, were meticulously sourced from Nanjing Qinglongshan Experimental Animal Company (certificate No: SCXK (jun) 2007-012; Nanjing, China). All animal experiments were conducted with the utmost adherence to ethical guidelines and established standards for animal welfare, specifically in accordance with the rigorous principles outlined in the US National Institutes of Health Guide for the Care and Use of Laboratory Animals. Furthermore, the entire experimental protocol received explicit approval from the Medicine Animal Welfare Committee of Wannan Medical College, China, underscoring the commitment to humane and responsible animal research.
Animal Experiments
A total of forty-eight rats were systematically allocated into four distinct experimental groups through a process of random assignment, ensuring twelve animals were present in each group to provide sufficient statistical power. The experimental groups were constituted as follows: The first group, designated as the control group, comprised SD rats that underwent an intraperitoneal anesthetic induction using sodium pentobarbital (P3761, 30 mg/kg; Sigma), followed by a singular intratracheal instillation of 0.9% saline solution. This served as a baseline control, mimicking the procedural aspects without inducing fibrosis. The second group, referred to as the bleomycin (BLM) group, consisted of rats similarly anesthetized intraperitoneally with sodium pentobarbital, after which they received a single intratracheal instillation of bleomycin at a dose of 5 mg per kilogram of body weight, dissolved in 1 mL of saline. This group served as the positive control for the induction of pulmonary fibrosis. The third and fourth groups represented the bleomycin-treated with L-mimosine groups, where rats were administered L-mimosine at two distinct dosages: 25 and 50 mg per kilogram of body mass per day, respectively. The bleomycin dose of 5 mg/kg was carefully selected based on its proven efficacy in inducing pulmonary fibrosis in previous studies, including our own earlier work. The specific L-mimosine concentrations (25 and 50 mg/kg) were determined through extensive pilot studies conducted by our research team to identify optimal therapeutic ranges. To prepare L-mimosine for administration, it was initially dissolved in Tris-buffer, adjusted to a pH of 8.9. Subsequently, the pH of this solution was precisely adjusted to 7.2 using 1 N hydrochloric acid. Prior to each administration, this stock solution was freshly diluted with 0.9% physiological saline to achieve the desired working concentration. L-mimosine was administered daily via subcutaneous injection, commencing on day 1 and continuing consistently until day 28 following the initial bleomycin or saline treatment, which was designated as day 0. On day 29, all rats across all groups were humanely sacrificed by exsanguination. The extent of pulmonary fibrosis development was rigorously assessed post-mortem through detailed lung histology, following methodologies previously established in the literature.
Lung Tissue Histology And Masson’s Trichrome Staining
For the meticulous light microscopic investigation of lung tissue pathology, the right lung tissues were carefully harvested and immediately prepared. They were fixed by inflation, a technique that preserves lung architecture, using freshly prepared 4% paraformaldehyde in phosphate-buffered saline (PBS) at a pH of 7.4. This fixation process was allowed to proceed for a duration of 24 hours. Following adequate fixation, the tissues were then dehydrated and embedded in paraffin wax, enabling the preparation of thin sections. Tissue sections, precisely 5 micrometers in thickness, were cut longitudinally from the apex to the bottom of the right lung, ensuring comprehensive representation of the entire organ. These sections were then subjected to differential staining procedures. Initially, they were stained with hematoxylin and eosin (H&E), a conventional histological stain that provides general morphological information and highlights cellular structures, allowing for an overall assessment of lung pathology. Subsequently, Masson’s trichrome stain was employed, a specialized histological technique specifically utilized for the demonstration of collagen deposition, which is a hallmark of fibrotic tissue. With Masson’s trichrome, collagen fibers are distinctly stained in a vibrant blue color, while cell nuclei appear dark red or purple, and the cytoplasm of cells stains red or pink, enabling clear visualization and quantification of fibrotic areas. The entire staining procedure was carried out strictly in accordance with the manufacturer’s instructions provided by KeyGEN Biotech (Nanjing, China). To ensure objectivity and minimize potential bias, all histological assays, including interpretation and scoring, were performed by investigators who were blinded to the specific intervention each animal received.
Cell Experiments
Primary rat pulmonary fibroblasts were meticulously prepared from the lung tissue of male 10-week-old healthy Sprague-Dawley rats, utilizing a well-established trypsin digestion method as previously described. These isolated cells were then cultured under controlled conditions at 37 degrees Celsius with an atmosphere of 5% carbon dioxide, immersed in Dulbecco’s Modified Eagle’s Medium supplemented with 20% fetal bovine serum, 100 units per milliliter of penicillin, and 100 micrograms per milliliter of streptomycin to support robust growth and prevent contamination. The identity of the cultured fibroblasts was rigorously confirmed through immunofluorescence staining using an antibody specific for Vimentin (ab8978, 1:50; Abcam, Hong Kong, China), a definitive marker for mesenchymal cells. For all subsequent experimental procedures, fibroblasts between passages 3 and 6 were selected to ensure cellular stability and minimize variations that can arise from prolonged passaging.
Two distinct series of experiments were meticulously designed to address specific research questions. The first series aimed to comprehensively investigate the impact of L-mimosine on the proliferation of these primary fibroblasts and to explore its mechanistic correlation with the eIF3a/p27 signaling pathway. For this series, the cells were systematically divided into six different experimental groups. The first group served as the control, where cells were incubated with double distilled water, which acted as the solvent for TGF-β1, for a duration of 24 hours. The second group, the TGF-β1 group, involved incubating cells with TGF-β1 at a concentration of 5 nanograms per milliliter for 24 hours to induce a profibrotic response. The subsequent three groups (iii-v) comprised cells pre-treated with varying concentrations of L-mimosine (1, 10, or 100 micromolar) for 1 hour, immediately followed by exposure to TGF-β1 (5 nanograms per milliliter) for 24 hours. This setup allowed for the evaluation of L-mimosine’s dose-dependent inhibitory effects. The final group (vi) involved pre-treatment with L-mimosine at 100 micromolar for 1 hour, followed by incubation with double distilled water for 24 hours, serving as a specific L-mimosine-only control.
The second series of experiments was specifically designed to elucidate the precise role of the eIF3a/p27 signaling pathway in the context of TGF-β1-induced proliferation of rat fibroblasts. For this series, cells were divided into four distinct groups. The first group, the control, involved incubation with double distilled water for 24 hours. The second group, the TGF-β1 group, received TGF-β1 (5 nanograms per milliliter) for 24 hours. The third group, designated as +Scrambled, involved pre-transfection of cells with a non-targeting, small interfering RNA negative control for eIF3a for 24 hours, prior to treatment with TGF-β1 (5 nanograms per milliliter) for an additional 24 hours. This group served as an essential control for non-specific effects of the transfection process. The fourth group, designated as +eIF3a siRNA, involved pre-transfection of cells with a specific small interfering RNA targeting eIF3a for 24 hours, before subsequent treatment with TGF-β1 (5 nanograms per milliliter) for 24 hours. Following these treatments, comprehensive cell proliferation assays were performed across all groups. Additionally, the expression levels of collagen I, collagen III, α-SMA, and eIF3a were rigorously analyzed to assess the molecular consequences of these interventions. The selection of a 24-hour duration for TGF-β1 treatment was based on prior pilot studies that demonstrated optimal induction of fibrotic markers within this timeframe.
Small Interfering RNA Transfection
To specifically manipulate the expression of the eIF3a gene, small interfering RNA (siRNA) was meticulously designed and synthesized by GenePharma CO (Shanghai, China). The sequences chosen for the rat eIF3a siRNA were as follows: sense strand, 5′-GGCCAAACAAGUUGAACAA-3′; and antisense strand, 5′-UUGUUCAAC UUGUUUGGCC-3′. Fibroblasts, when they reached a confluence of approximately 70% to 80% in their culture dishes, were subjected to the transfection procedure using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA), a widely recognized and efficient lipid-based delivery system for nucleic acids. The efficacy of the transfection process, specifically the degree to which eIF3a gene expression was suppressed, was rigorously evaluated. This assessment was performed by measuring both eIF3a messenger RNA (mRNA) expression levels using real-time polymerase chain reaction (PCR) and eIF3a protein expression levels through Western blot analysis. These dual measurements ensured a comprehensive understanding of the knockdown efficiency. Following a 24-hour incubation period after transfection, the cells were then deemed ready and subsequently utilized for the various downstream experiments as outlined in the previous sections.
Cell Proliferation Assays
To accurately and comprehensively quantify cell proliferation, two distinct and complementary methodologies were employed, building upon our previously established protocols. The first method involved the direct measurement of DNA synthesis, a hallmark of actively dividing cells, which was assessed using the BrdU (bromodeoxyuridine) marking technique. BrdU is a synthetic nucleoside that is incorporated into newly synthesized DNA, allowing for its detection. The second method involved the analysis of the cell cycle distribution, providing insights into the proportions of cells in different phases of growth and division, which was performed using flow cytometry. This dual-approach ensured a robust and reliable assessment of cellular proliferative activity.
qPCR Analysis
The quantitative polymerase chain reaction (qPCR) experiments were meticulously planned, executed, and reported in strict accordance with the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, ensuring transparency, reproducibility, and high scientific rigor. Total RNA was comprehensively extracted from two distinct sources: whole left lung tissue and primary cultured lung fibroblasts, utilizing the highly efficient TRIzol reagent (Invitrogen). Following RNA extraction, a precisely measured amount of RNA, ranging from 0.2 to 0.5 micrograms, was subjected to a reverse transcription reaction using the PrimeScript reverse transcription reagent Kit (DRR037S; TaKaRa, Dalian, China) to synthesize complementary DNA (cDNA). Quantitative analysis of the changes in gene expression levels was then performed using SYBR Premix Ex Taq (DRR42OA; TaKaRa) on an ABI 7300 system. The PCR cycling conditions were optimized to ensure efficient and specific amplification: an initial incubation step at 95 degrees Celsius for 15 seconds, followed by 40 cycles, each consisting of a denaturation phase at 95 degrees Celsius for 5 seconds and an annealing/extension phase at 60 degrees Celsius for 31 seconds. The specific primer sequences utilized for the amplification of target genes were as follows: for p27, forward 5′-CAGAATCATAAGCCCCTGGA-3′ and reverse 5′-TCTGCGAGTCAGGCATTTG-3′; for eIF3a, forward 5′-TCAAGTCGCCGGGACGATA-3′ and reverse 5′-CCTGTCATCAGCACGTCTCCA-3′; for α-SMA, forward 5′-CTATTCCTTCGTGACTACT-3′ and reverse 5′-ATGCTGTTATAGGTGGTT-3′; for collagen I, forward 5′-CCAACTGAA CGTGACCAAAAACCA-3′ and reverse 5′-GAAGGTGCTGGGTAGGGAAGTAGGC-3′; for collagen III, forward 5′-ATTCTGCCACCCTGAACTCAAGAGC-3′ and reverse 5′-TCCATGTAGGCAATGCTGTTTTTGC-3′. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was employed as a robust internal reference gene for normalization, with its primer sequences being: forward 5′-TGGCCTCCAAGGAGTAAGAAAC-3′ and reverse 5′-GGCCTCTCTCTTGCTCTCAGTATC-3′. Data analysis was meticulously performed using the comparative Ct method, also known as the 2-ΔΔCt method, implemented through the ABI software, ensuring accurate quantification of relative gene expression.
Western Blot Analysis
For the comprehensive assessment of protein expression, total protein was meticulously extracted from both whole left lung tissue samples and the cultured primary lung fibroblasts. This extraction was performed using RIPA buffer, which was carefully supplemented with 0.1% phenylmethylsulfonyl fluoride (PMSF) to inhibit protease activity and preserve protein integrity. Following protein quantification, equal amounts of protein from each individual sample, precisely 50 micrograms, were carefully loaded and separated by size using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoretic separation, the resolved proteins were then efficiently transferred from the gel onto polyvinylidene fluoride (PVDF) membranes, which provide a stable matrix for antibody binding.
Subsequently, these membranes were incubated overnight at a consistent temperature of 4 degrees Celsius with specific primary antibodies, carefully chosen to detect the target proteins of interest. The primary antibodies used were: anti-eIF3a (catalog number #3411, diluted 1:1000) and anti-p27 (catalog number #3698, diluted 1:1000), both procured from Cell Signaling (Boston, MA, USA). Additionally, primary antibodies against α-SMA (catalog number ab5694, diluted 1:2000), collagen I (catalog number ab34710, diluted 1:1000), and collagen III (catalog number ab7778, diluted 1:1000) were obtained from Abcam (Hong Kong, China). For loading control purposes, an antibody against GAPDH (catalog number sc-137179, diluted 1:2000) was sourced from Santa Cruz (CA, USA). After the primary antibody incubation, the membranes were thoroughly washed and then incubated with appropriate horseradish peroxidase (HRP)-coupled secondary antibodies. Specifically, goat anti-mouse (sc-2005, 1:2000) or goat anti-rabbit (sc-2030, 1:5000) secondary antibodies from Santa Cruz (CA, USA) were utilized, depending on the host species of the primary antibody. The resulting chemiluminescence signals, indicative of protein presence and abundance, were detected and captured using the EasySee Western Blot Kit (Beijing TransGen Biotech, Beijing, China). For quantitative analysis, the densitometry of the protein bands was meticulously performed using Image J 1.43 software, a widely used image processing program developed by the National Institutes of Health, allowing for a precise determination of relative protein expression levels.
Statistical Analysis
All quantitative results obtained from the various experiments were rigorously presented as means plus or minus the standard errors of the mean (SEM), providing a clear indication of data variability and reliability. Statistical analysis was meticulously conducted using Analysis of Variance (ANOVA), a powerful statistical test suitable for comparing means across three or more groups. Following a significant ANOVA result, post-hoc multiple comparisons were performed using the Newman-Student-Keuls test. This specific post-hoc test is designed to identify which specific pairs of group means are significantly different from each other while controlling for the family-wise error rate. A result was considered to be statistically significant if the calculated P-value was less than 0.05, indicating a high probability that the observed differences were not due to random chance.
Results
Effect of L-Mimosine on Survival Percent in Bleomycin-Induced Pulmonary Fibrosis Rats
An initial critical assessment of L-mimosine’s overall impact on the health and survival of rats challenged with bleomycin was conducted. During the entire experimental period, no mortality was observed among the rats assigned to the control group, indicating the baseline safety of the procedures without fibrotic induction. In stark contrast, within the bleomycin-induced pulmonary fibrosis group, a significant proportion of animals succumbed, with four rats dying between day 2 and day 9 post-bleomycin instillation, highlighting the severe lethality associated with this experimental model of fibrosis. In the groups receiving L-mimosine treatment, while some mortality was still recorded, there was a discernible improvement in the survival profile. Specifically, in the L-mimosine group administered at 25 milligrams per kilogram of body mass per day, four rats died, but their deaths occurred later, between day 3 and day 14. Similarly, in the higher dose L-mimosine group, receiving 50 milligrams per kilogram of body mass per day, three rats died, with these fatalities occurring even later, between day 4 and day 16. Although specific quantitative data for the survival curve are not presented here, a qualitative observation indicated a clear and marked shift to the right in the survival curve for the L-mimosine-treated groups when compared to the bleomycin-only group, suggesting an extended survival duration and an overall protective effect conferred by L-mimosine treatment.
Effect of L-Mimosine on Lung Histological Alteration and Alpha-SMA Expression in Bleomycin-Induced Pulmonary Fibrosis Rats
Histopathological examination of lung tissues confirmed the successful induction of pulmonary fibrosis by bleomycin, consistent with findings from previous studies. After a four-week period following bleomycin exposure, the lung architecture of affected rats exhibited significant and profound disturbances in the normal alveolar structure. This was characterized by a marked thickening of the interalveolar septa, which are the thin walls between air sacs, and a dense interstitial infiltration by both inflammatory cells and fibroblasts. Myofibroblasts are widely recognized as the principal effector cells driving the progression of pulmonary fibrosis. A key identifying characteristic of these activated myofibroblasts is their robust expression of alpha-smooth muscle actin (α-SMA), a contractile protein typically associated with muscle cells but aberrantly expressed by fibroblasts during fibrotic processes. In alignment with established literature, bleomycin administration dramatically upregulated the expression of α-SMA, observed at both the messenger RNA (mRNA) and protein levels, within the lung tissue of the affected rats. Crucially, treatment of these rats with L-mimosine significantly ameliorated all these detrimental effects induced by bleomycin, leading to a substantial improvement in lung histology and a significant reduction in α-SMA expression (P < 0.05). This suggests that L-mimosine effectively counteracts the myofibroblast differentiation and activity that are central to fibrotic pathology.
Effect of L-Mimosine on Lung Collagen Accumulation in Bleomycin-Induced Pulmonary Fibrosis Rats
Masson's trichrome staining, a specialized histological technique for visualizing collagen, provided clear and compelling evidence of the profound structural damage and excessive extracellular matrix deposition in the lungs of bleomycin-challenged rats. The instillation of bleomycin resulted in severe distortion of the normal lung structure, accompanied by a pronounced accumulation of collagen fibers, which stained distinctly blue, throughout the lung parenchyma. In stark contrast, rats treated with saline maintained a well-alveolized, normal lung histology, indicative of healthy tissue. Beyond the visual histological alterations, bleomycin also led to a significant upregulation in the expression of both collagen I and collagen III, observed at both the mRNA and protein levels, within the lung tissue of rats. These collagen types are major components of fibrotic scar tissue. Importantly, all these bleomycin-induced effects, encompassing both the histological accumulation of collagen and the molecular upregulation of collagen gene expression, were significantly alleviated by the therapeutic administration of L-mimosine (P < 0.05). This direct demonstration of reduced collagen burden underscores L-mimosine's potent antifibrotic capacity.
Effect of L-Mimosine on Expression of eIF3a and p27 in Lungs from Bleomycin-Induced Pulmonary Fibrosis Rats
Building upon established knowledge regarding the role of eukaryotic translation initiation factor 3a (eIF3a) in cellular processes, where its suppression has been shown to inhibit malignant phenotypes in cancer cells and its overexpression promotes cellular transformation, our previous research had already highlighted eIF3a's significant involvement in bleomycin-induced pulmonary fibrosis. Consistent with our earlier findings, the administration of bleomycin in the current study dramatically increased the expression of eIF3a, evident at both the mRNA and protein levels, within the lung tissues of the rats. Furthermore, extending these observations, we made a critical new discovery: the expression of p27, a known cell cycle inhibitor, was conspicuously and significantly decreased in the lungs of rats afflicted with bleomycin-induced pulmonary fibrosis. Importantly, the therapeutic intervention with L-mimosine profoundly reversed all these bleomycin-induced molecular dysregulations. Specifically, L-mimosine treatment significantly attenuated the bleomycin-induced upregulation of eIF3a expression and concomitantly restored the bleomycin-induced downregulation of p27 expression (P < 0.05). These findings suggest that L-mimosine exerts its antifibrotic effects, at least in part, by modulating the expression balance of eIF3a and p27 within the fibrotic lung environment.
Effect of L-Mimosine on Proliferation of Pulmonary Fibroblasts Induced by TGF-Beta 1
Transforming growth factor-beta 1 (TGF-β1) is a well-established and crucial cytokine in the pathogenesis of fibrotic diseases, known for its potent ability to induce the excessive proliferation and accumulation of pulmonary fibroblasts, concurrently promoting the synthesis and deposition of collagen. Our *in vitro* experiments rigorously investigated the effects of L-mimosine on these TGF-β1-induced profibrotic responses in cultured pulmonary fibroblasts. As clearly demonstrated, exposure of these primary pulmonary fibroblasts to TGF-β1 at a concentration of 5 nanograms per milliliter for 24 hours significantly increased the percentage of cells actively engaged in DNA synthesis and cell division, as indicated by an increased proportion of cells in the S + G2 phase of the cell cycle and heightened BrdU incorporation. Furthermore, TGF-β1 exposure led to a noticeable upregulation of α-SMA, a marker of myofibroblast differentiation, at both the mRNA and protein levels. Importantly, L-mimosine treatment, administered at varying concentrations of 1, 10, and 100 micromolar, demonstrated a dose-dependent and significant inhibitory effect on the TGF-β1-induced proliferation of pulmonary fibroblasts and the associated upregulation of α-SMA expression (P < 0.05). In contrast, L-mimosine administered alone at the highest concentration of 100 micromolar, in the absence of TGF-β1, had no significant effect on either fibroblast proliferation or α-SMA expression, confirming its specific antagonistic action against fibrotic stimuli rather than a general cytotoxic effect.
Effect of L-Mimosine on Expression of Collagen I and Collagen III Induced by TGF-Beta 1 in Cultured Pulmonary Fibroblasts
Pulmonary fibroblasts are recognized as the primary cellular source of collagen within the lung, and under specific pathological conditions such as fibrosis, they undergo activation and significantly ramp up their production of this critical extracellular matrix protein. Our *in vitro* investigations further explored the impact of L-mimosine on collagen production by these fibroblasts when challenged with a profibrotic stimulus. As unequivocally shown, exposure of cultured pulmonary fibroblasts to TGF-β1 at a concentration of 5 nanograms per milliliter for 24 hours resulted in a significant increase in the expression of both collagen I and collagen III. These increases were observed at both the messenger RNA and protein levels, indicating enhanced transcriptional activity and subsequent protein synthesis of these key fibrotic markers. Remarkably, the co-treatment with L-mimosine, across the tested concentration range of 1, 10, and 100 micromolar, conspicuously inhibited the TGF-β1-induced upregulation of both collagen I and collagen III expression (P < 0.05). Consistent with observations on cell proliferation, L-mimosine at 100 micromolar when administered alone, without TGF-β1, exerted no significant effect on the basal expression levels of collagen I and collagen III, reinforcing its specific anti-fibrotic action.
Effect of L-Mimosine on TGF-Beta 1-Induced Expression of eIF3a and p27 in Cultured Pulmonary Fibroblasts
Previous research has established a mechanistic link where the suppression of eukaryotic translation initiation factor 3a (eIF3a) expression by L-mimosine leads to an increased translation of p27, thereby contributing to cell cycle arrest. Building upon this, our current study specifically investigated the intricate interplay between L-mimosine, eIF3a, and p27 within the context of TGF-β1-induced fibrotic responses in cultured pulmonary fibroblasts. We discovered that the application of exogenous TGF-β1 to these cells resulted in a marked upregulation of eIF3a expression, while simultaneously causing a significant downregulation of p27 expression. Critically, we observed that L-mimosine, at concentrations of 1, 10, and 100 micromolar, profoundly reversed these TGF-β1-induced molecular alterations. It effectively counteracted the TGF-β1-mediated upregulation of eIF3a expression and significantly restored the TGF-β1-induced downregulation of p27 expression, with these effects being evident at both the mRNA and protein levels (P < 0.05). Similar to previous findings, L-mimosine administered alone at 100 micromolar, in the absence of TGF-β1, had no discernible effect on the baseline expression of either eIF3a or p27, again underscoring its role in specifically counteracting fibrotic stimuli.
To definitively ascertain the causative role of eIF3a in mediating the TGF-β1-induced alterations in p27 expression within pulmonary fibroblasts, we strategically employed a highly specific small interfering RNA (siRNA) approach targeting eIF3a. In our preliminary optimization studies, we evaluated the efficacy of three distinct siRNA sequences designed to target eIF3a to establish a robust eIF3a knockdown in pulmonary fibroblasts. The results of this pilot study revealed that transfection with the third specific siRNA sequence for 24 hours yielded the most optimal efficiency in inhibiting eIF3a expression. Consequently, this highly effective third siRNA sequence was chosen for all subsequent experiments. As unequivocally demonstrated by our findings, the eIF3a siRNA successfully inhibited the TGF-β1-induced upregulation of eIF3a expression, confirming its efficacy. Most importantly, and providing strong support for the proposed pathway, we observed that eIF3a siRNA reversed the TGF-β1-induced downregulation of p27 expression (P < 0.05). This direct genetic manipulation therefore confirms that eIF3a is a critical upstream regulator influencing p27 expression in these fibrotic cellular responses.
Effect of eIF3a Knockdown on TGF-Beta 1-Induced Cell Proliferation and Expression of Alpha-SMA, Collagen I, and Collagen III in Cultured Pulmonary Fibroblasts
Expanding upon the critical findings regarding eIF3a's role in p27 regulation, the present study further investigated the broader functional consequences of eIF3a knockdown on key fibrotic processes induced by TGF-β1 in cultured pulmonary fibroblasts. Our results clearly indicated that the specific eIF3a siRNA significantly inhibited the proliferative effect exerted by TGF-β1 on pulmonary fibroblasts. This inhibitory effect was evidenced by a notable decrease in BrdU incorporation, reflecting reduced DNA synthesis, and a corresponding reduction in the percentage of cells residing in the S+G2 phase of the cell cycle, indicating a slowdown in cell division. In accordance with these findings, the eIF3a siRNA also effectively suppressed the TGF-β1-induced upregulation of α-SMA expression, a key marker of myofibroblast differentiation. Furthermore, the genetic knockdown of eIF3a similarly inhibited the TGF-β1-induced increase in the expression of collagen I and collagen III, which are central components of the extracellular matrix accumulated in fibrotic conditions. These inhibitory effects on α-SMA, collagen I, and collagen III were observed at both the messenger RNA and/or protein levels (P < 0.05), providing comprehensive evidence of eIF3a's direct involvement in promoting fibrotic characteristics and highlighting its potential as a therapeutic target in the context of pulmonary fibrosis.
Discussion
Pulmonary fibrosis stands as a relentlessly progressive and severe pathological condition primarily characterized by the pathological and excessive proliferation of fibroblasts, which are key cellular architects of connective tissue, and the subsequent unbridled deposition of extracellular matrix components. This aberrant biological process invariably leads to a profound disruption of the normal lung tissue architecture and a severe compromise of its vital function, ultimately culminating in respiratory failure. Despite the critical and life-threatening nature of this disease, particularly in its idiopathic form, which remains a fatal respiratory illness in humans, our current understanding of its underlying mechanisms remains incomplete. Furthermore, the efficacy of existing therapeutic interventions for idiopathic pulmonary fibrosis has been largely unsatisfactory, leaving a significant unmet medical need. Consequently, there is an urgent and imperative requirement for the identification and development of novel and more effective therapeutic strategies to combat this devastating disease.
Among the diverse family of eukaryotic initiation factors (eIFs), which are central to the regulation of protein synthesis, eIF3 distinguishes itself as the largest and most intricate initiation factor complex. It is composed of a remarkable thirteen distinct subunits, systematically designated from eIF3a to eIF3m. Within this elaborate complex, eIF3a, also commonly known as p170, represents the largest subunit. Its significance extends beyond its structural role, as research has increasingly implicated eIF3a in fundamental cellular processes, notably including tumorigenesis. Compelling evidence from numerous studies has consistently demonstrated that the targeted suppression of endogenous eIF3a expression can effectively inhibit the acquisition of malignant phenotypes in human cancer cells.
Conversely, the deliberate overexpression of ectopic eIF3a has been shown to actively promote the malignant transformation of mammalian cells, firmly establishing its oncogenic potential. Beyond its direct role in cellular transformation, it has been robustly demonstrated that eIF3a expression levels correlate with the prognosis of human cancer patients. Specifically, an upregulation of eIF3a in lung cancer patients has been linked to their responsiveness to platinum-based chemotherapy regimens and has been shown to contribute to an increased sensitivity to cisplatin, suggesting its utility as a predictive biomarker and a potential therapeutic target in oncology. Our own prior investigations have significantly expanded this understanding by revealing a crucial role for eIF3a in the pathogenesis of bleomycin-induced pulmonary fibrosis. We previously demonstrated that eIF3a is critically involved in the bleomycin-induced fibrotic process, as well as in the transforming growth factor-beta 1 (TGF-β1)-induced proliferation and differentiation of pulmonary fibroblasts, with these effects being mediated, at least in part, via the ERK1/2/eIF3a pathway. In the current study, these earlier findings are further corroborated and reinforced. We observed that bleomycin administration dramatically increased eIF3a expression in the lungs of rats, and similarly, exogenous TGF-β1 markedly upregulated eIF3a expression in cultured pulmonary fibroblasts. These consistent results provide strong additional confirmation that eIF3a is indeed intricately involved in the regulation of fibroblast proliferation, a central driver of pathological progression in pulmonary fibrosis.
Transforming growth factor-beta 1 (TGF-β1) is unequivocally recognized as one of the most pivotal and potent profibrotic cytokines, playing a central role in the initiation and progression of fibrotic diseases across various organs, including the lung. This versatile cytokine is produced by a diverse array of cell types, including but not limited to macrophages, epithelial cells, and fibroblasts themselves, creating a complex autocrine and paracrine regulatory network. Clinical research has consistently revealed that TGF-β1 is significantly upregulated in both the lung tissue and the circulating plasma of patients afflicted with pulmonary fibrosis, underscoring its relevance in human disease. Furthermore, experimental studies involving the overexpression of active TGF-β1 have been shown to directly induce pulmonary fibrosis *in vivo*, a condition characterized by extensive and aberrant deposition of extracellular matrix proteins, including collagen, fibronectin, and elastin, alongside the pervasive emergence of cells exhibiting a myofibroblast phenotype, which are highly contractile and extracellular matrix-producing cells. Recent mechanistic investigations have indicated that the TGF-β1-induced proliferation observed in NIH3T3 cells, a commonly used fibroblast cell line, might be mechanistically linked to a concurrent downregulation of p27 (Kip1) expression. p27 is a crucial cell cycle inhibitor that fundamentally determines the transition point from the quiescent G0/G1 phase into the proliferative S phase of the cell cycle.
As a member of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors, p27 functions as a critical negative regulator of the protein kinase CDK2/cyclin E complex, effectively blocking the progression of the cell cycle at the G0/G1 phase, thus preventing uncontrolled cell division. Physiologically, the cellular levels of p27 are typically elevated during the G0/G1 phases of the cell cycle, maintaining cellular quiescence. However, upon exposure to mitogenic stimuli, p27 undergoes rapid proteasomal degradation, thereby relieving its inhibitory effect and allowing the unimpeded action of CDK2/cyclin E to promote robust cell proliferation.
Crucially, it has been previously established that the intricate regulation of protein expression, including that of p27, by eIF3a is integrally involved in mediating the broader functions of eIF3a in cellular proliferation. Based on this compelling body of evidence, we put forth the hypothesis that the pathogenesis of bleomycin-induced pulmonary fibrosis and the specific TGF-β1-induced proliferation and differentiation of pulmonary fibroblasts are mechanistically mediated via a coordinated action of the eIF3a/p27 signaling pathway. Our current findings provide substantial support for this hypothesis. We observed that bleomycin dramatically increased the expression of eIF3a while simultaneously downregulating the expression of p27 in the lungs of rats, mirroring the cellular environment conducive to fibrosis. Similarly, in *in vitro* settings, exogenous TGF-β1 markedly upregulated eIF3a expression and concomitantly decreased p27 expression in cultured pulmonary fibroblasts. To definitively confirm the causal role of the eIF3a/p27 pathway in mediating TGF-β1-induced cell proliferation in pulmonary fibroblasts, we developed and employed a highly specific eIF3a siRNA.
Remarkably, in the present study, the targeted knockdown of eIF3a gene expression effectively reversed the TGF-β1-induced downregulation of p27 expression. Concurrently, eIF3a knockdown also abrogated the TGF-β1-induced proliferation of fibroblasts and reversed the upregulation of α-SMA, collagen I, and collagen III, which are all hallmarks of fibrotic progression. These comprehensive results provide robust confirmation that the eIF3a/p27 pathway is indeed intricately involved in the regulation of fibroblast proliferation within the context of pulmonary fibrosis in rats. While these *in vitro* and *in vivo* findings are highly compelling, it is important to acknowledge that the precise and multifaceted roles of the eIF3a/p27 pathway in the complex development of pulmonary fibrosis *in vivo* warrant further rigorous and in-depth investigation, particularly within clinical settings to validate its translational potential.
L-mimosine, a naturally occurring plant amino acid, has been widely recognized for its ability to reversibly arrest mammalian cell cycles, specifically at the late G1 phase. Prior studies, notably those utilizing prostate DU145 carcinoma cells, have consistently demonstrated that incubation with L-mimosine leads to a significant increase in the population of cells arrested in the G0/G1 phases, a phenomenon that is subsequently associated with an upregulation of p27 protein levels. Furthermore, it has been previously elucidated that the L-mimosine-induced decrease in eIF3a expression results in an elevated translation of p27, an event that precedes and contributes to the observed cell cycle arrest. In the present study, these established cellular mechanisms were directly linked to the *in vivo* therapeutic effects observed in the bleomycin-induced pulmonary fibrosis model. Our detailed histological examinations revealed that L-mimosine treatment profoundly alleviated the pathological thickening of the interalveolar septa and reduced the dense interstitial infiltration by inflammatory cells and fibroblasts, indicative of a significant reduction in the fibrotic burden.
Concomitantly, Masson's trichrome staining, a specific marker for collagen, unequivocally demonstrated that L-mimosine treatment substantially reduced the pathological accumulation of collagen within the lungs of rats suffering from bleomycin-induced pulmonary fibrosis. At the molecular level, L-mimosine treatment effectively decreased the bleomycin-induced overexpression of eIF3a, α-SMA, collagen I, and collagen III. Crucially, it also upregulated the expression of p27, directly counteracting the molecular drivers of fibrosis, and subsequently attenuated the overall progression of bleomycin-induced pulmonary fibrosis. Extending these *in vivo* observations to a controlled cellular environment, we further discovered that L-mimosine remarkably attenuated the excessive proliferation of cultured pulmonary fibroblasts, along with their induced expression of α-SMA, collagen I, and collagen III, all of which were potently stimulated by TGF-β1. This profound inhibitory effect of L-mimosine on fibroblast activation and extracellular matrix production was consistently accompanied by a parallel inhibition of eIF3a expression and a concomitant increase in p27 expression. These convergent findings across both *in vivo* and *in vitro* models provide compelling evidence, strongly suggesting that L-mimosine inhibits the progression of bleomycin-induced pulmonary fibrosis in rats primarily through the intricate and crucial eIF3a/p27 signaling pathway.
In summary, the collective body of evidence generated from this comprehensive investigation strongly indicates that the demonstrable antifibrotic effect of L-mimosine in rats afflicted with bleomycin-induced pulmonary fibrosis is closely linked to its antiproliferative activity. This critical antiproliferative action, which helps to curb the excessive fibroblast expansion characteristic of fibrosis, is mechanistically mediated via the eIF3a/p27 signaling pathway. These groundbreaking findings collectively point to L-mimosine as a promising and potential candidate compound that could significantly enhance or complement current therapeutic approaches for pulmonary fibrosis. However, it is also important to acknowledge certain challenges associated with L-mimosine. Previous reports have indicated that L-mimosine exhibits some degree of toxicity and notably poor solubility in both fat and water, suggesting that its direct application as a therapeutic drug may be limited due to suboptimal "druggability" characteristics. Therefore, recognizing these limitations while still appreciating its potent antifibrotic mechanism, our ongoing research efforts are now focused on the strategic development of novel derivatives of L-mimosine. The ultimate hope is to synthesize new and improved inhibitors of eIF3a that possess enhanced pharmacological properties, including better solubility and reduced toxicity, which would pave the way for their future rigorous basic and clinical study in the challenging field of pulmonary fibrosis.
Acknowledgments
This research endeavor received crucial financial support from the Natural Science Foundation of Anhui Province, under grant number 1408085QH168, and from the National Natural Science Foundation of China, under grant number 81273512. The generous support from these funding bodies was instrumental in enabling the successful completion of this study.