The etiology and pathogenesis of PF, particularly IPF, are largely undetermined, but notable advancements and consensus have been achieved in the field. IPF is generally believed to result from aberrant wound healing responses following damage to alveolar epithelial cells (AECs). Normally, AECs exhibit sophisticated and efficient repair mechanisms, with wound healing progressing through four distinct stages: coagulation/clotting, inflammatory cell migration, fibroblasts migration/proliferation/activation, and tissue remodeling and degradation (Raghu et al., 2022a; Spagnolo et al., 2021). However, factors such as cigarette smoking, subclinical infections, environmental pollutants, occupational exposures, chronic microaspiration of gastric content, abnormal lung microbiota composition, and genetic predisposition may cause repeated AEC injury (Bonella et al., 2023). This repeated injury leads to aberrant repair processes, excessively activating various inflammatory responses, repair pathways, and signaling pathways, resulting in the secretion of numerous cytokines.

Complex bidirectional interactions between epithelial cells, mesenchymal cells, and ECM subsequently induce a series of cellular events, including fibroblasts differentiation into myofibroblasts, migration, proliferation, and activation of AECs and fibroblasts, inhibition of apoptosis, and persistent abnormal ECM secretion. The continuous ECM deposition in the lung interstitium between alveoli and capillaries leads to irreversible structural changes and lung damage (Raghu et al., 2022a; Spagnolo et al., 2021). Within the lung epithelial tissue, alveolar epithelial type I (AT1) cells are the primary mediators of gas exchange, occupying most of the epithelial surface. Alveolar type II epithelial (AT2) cells act as progenitor cells for AT1, capable of differentiating into AT1 cells to renew and repair alveolar epithelial tissue while secreting surfactant, aiding in metabolic and immune functions. The differentiation of AT2 into AT1 is critical for maintaining normal lung function (Liu et al., 2022a; Moss et al., 2022; Herrera et al., 2018).

Alveolar epithelial wound repair is a complex, coordinated process. Persistent imbalance or injury at any stage of tissue repair can lead to fibrosis, progressing to various lung diseases. AT2 cells senescence, apoptosis, abnormal differentiation, and reduced abundance can impair alveolar epithelial repair and significantly contribute to PF. In IPF, AT2 cells senescence, apoptosis, abnormal differentiation, and decreased abundance are prevalent in the gas exchange regions of the lungs. Factors such as endoplasmic reticulum stress, mitochondrial dysfunction, and telomere shortening within these cells impede their ability to repair damaged epithelium effectively (Moss et al., 2022; Yao et al., 2021). Additionally, repetitive alveolar epithelial micro-injury, preventing AT2 cells from differentiating into AT1, results in abnormal alveolar epithelial regeneration and repair, increasing mechanical tension, which activates TGF-β signaling in AT2 cells (Moss et al., 2022). The aberrant TGF-β signaling induces the epithelial-mesenchymal transition (EMT), so that they acquire the characteristics of migration and secretion of ECM, directly promoting fibrosis (Wu et al., 2020) (Figure 1).

The excessive transformation of fibroblasts into myofibroblasts during IPF progression is another critical factor. Under normal conditions, fibroblasts maintain ECM homeostasis. However, during acute injury, chronic inflammation, or repeated micro-injury, fibroblasts lose this balance and transform into myofibroblasts for repair. Myofibroblasts, characterized by enhanced proliferation and migration capabilities, secrete numerous cytokines and ECM, expressing proteins like alpha-smooth muscle actin (α-SMA), contributing to damage repair (Hinz and Lagares, 2020; Younesi et al., 2024). In IPF, excessive myofibroblast proliferation, apoptosis evasion, and invasiveness drive disease progression. Epithelial cell-secreted TGF-β promotes myofibroblast differentiation and proliferation, leading to further ECM deposition and accelerating disease progression. Inhibiting myofibroblast ECM secretion and contraction, as well as fibroblast-to-myofibroblast transformation (FMT), is a pivotal therapeutic approach for organ fibrosis (Hinz and Lagares, 2020; Mayr et al., 2024).

ECM deposition is a hallmark of IPF, reflecting an imbalance between increased ECM synthesis and dysregulated degradation that culminates in irreversible scarring. At the molecular level, ECM degradation is mainly mediated by MMPs, the zinc-dependent endopeptidases secreted by macrophages, fibroblasts, NK cells, and neutrophils following tissue injury (Greenlee et al., 2007). Fibrotic scar tissues, primarily composed of cross-linked type I collagen fibers, is initially degraded by collagenases (e.g., MMP-1, MMP-2, MMP-8, MMP-13, MMP-14, MMP-15, and MMP-16), followed by gelatinases (e.g., MMP-2 and MMP-9) and other proteases (Sang, 1998; Karsdal et al., 2016). Beyond ECM degradation, MMPs also play critical roles in regulating inflammation and cellular behaviour by modulating growth factors, cytokines, chemokines, and cell surface receptors, thereby orchestrating a complex network of signaling events that ultimately drive IPF progression (Karsdal et al., 2016).

In IPF, dysregulated MMP expression and function drive disease progression through diverse mechanisms. For instance, MMP-3 promotes EMT by activating Wnt/β-catenin and TGF-β signaling pathways, while simultaneously inducing fibroblast proliferation and collagen deposition, as demonstrated by reduced fibrosis in MMP-3-knockout mice following bleomycin challenge (Pardo et al., 2016; Roque et al., 2020). Similarly, MMP-7, which is highly expressed in IPF lung tissue and plasma, is secreted by damaged AECs and drives fibrosis through multiple mechanisms, including cleaving basement membrane components such as E-cadherin and integrin β4, activating bioactive molecules including osteopontin and TGF-β, and promoting fibroblast activation and collagen deposition (Richards et al., 2012). Consequently, MMP-7 deficiency attenuates fibrosis, and its serum levels have emerged as a potential diagnostic biomarker for IPF (Richards et al., 2012; White et al., 2016).

Beyond these direct profibrotic actions, MMPs also modulate the fibrotic microenvironment through complex feedback loops with other cellular components. For example, MMP-9, produced by both AECs and Thy-1-negative fibroblasts, not only activates TGF-β to sustain a profibrotic cycle but also enhances fibroblast migration and collagen deposition (Kobayashi et al., 2014), thereby contributing to abnormal alveolar bronchiolization. Meanwhile, the circulating fibrocytes—CD45⁺/collagen⁺ bone marrow-derived cells—exploit the proteolytic activity of MMP-2, MMP-8, and MMP-9 to degrade ECM barriers, facilitating their migration into lung tissue and subsequent differentiation into myofibroblasts (Kobayashi et al., 2014; Espindola et al., 2021). Furthermore, MMPs critically modulate inflammation and immune responses that fuel fibrosis progression. Elevated MMP-8 levels in IPF bronchoalveolar lavage fluid (BALF) exacerbate neutrophil-mediated inflammation and fibrosis by cleaving anti-inflammatory factors like IL-10. Similarly, MMP-28 (Epilysin) regulates macrophage polarization, with knockout mice showing reduced M2 macrophage infiltration and attenuated fibrosis (Roque et al., 2020). These findings collectively underscore the multifaceted roles of MMPs in IPF pathogenesis, functioning not merely as ECM modifiers but as dynamic regulators of cellular crosstalk, immune responses, and signaling pathways that collectively drive fibrotic progression.

However, paradoxically, certain MMPs demonstrate context-dependent antifibrotic potential, suggesting protective roles under specific conditions. For instance, although MMP-1 efficiently degrades collagen, it is predominantly expressed in AECs during IPF, where it promotes epithelial repair and inhibits apoptosis—effects suppressed by the dominant profibrotic mediator TGF-β (Sang, 1998; Richards et al., 2012; Kobayashi et al., 2014; Link et al., 2025). Similarly, MMP-19 exerts protective effects by inhibiting fibroblast activation through prostaglandin E₂ (PGE₂)-mediated pathways and preserving non-fibrotic lung regions (Yu et al., 2012). These findings underscore the multifaceted roles of MMPs in IPF, warranting careful consideration in the development of targeted therapies.

Collectively, MMPs drive IPF by activating TGF-β, altering ECM biomechanics, and sustaining myofibroblast survival, making them therapeutic targets. However, their functional heterogeneity and organ-specific roles demand highly specific strategies. Challenges include safety concerns (e.g., impaired wound healing from long-term MMP inhibition) and functional redundancy, where MMP-2 or MMP-12 might compensate for MMP-9 inhibition, necessitating multi-target inhibitors or combination therapies (Greenlee et al., 2007; Sang, 1998; Karsdal et al., 2016; Pardo et al., 2016; Roque et al., 2020; White et al., 2016; Kobayashi et al., 2014; Espindola et al., 2021; Sun et al., 2020; Lin et al., 2023; Khan et al., 2024). Future research should integrate multi-omics to dissect dynamic MMP networks and advance precision-targeted or combinatorial approaches to improve IPF treatment.

HSP47 (Serpin H1) is indispensable for the efficient secretion, processing, fibril assembly, and deposition of collagen in the ECM. As an endoplasmic reticulum (ER)-resident, collagen-specific molecular chaperone, HSP47 binds to the Yaa-Gly-Xaa-Arg-Gly motif within procollagen triple helices via hydrophobic and hydrophilic interactions. This binding prevents misfolding and aggregation of procollagen and facilitates its transport to the Golgi apparatus (Okuno et al., 2021; Bellaye et al., 2021).

Clinical and preclinical evidence supports a critical role for HSP47 in fibrogenesis. Lung tissues from IPF patients show significantly elevated HSP47 expression, which positively correlates with collagen deposition (Okuno et al., 2021; Khan and Däinghaus, 2024). In bleomycin-induced murine models of pulmonary fibrosis, both pharmacological inhibition and siRNA-mediated silencing of HSP47 reduce collagen accumulation and improve lung architecture and function (Bellaye et al., 2021; Sakamoto et al., 2023). Moreover, elevated serum HSP47 levels in various interstitial lung diseases, including acute interstitial pneumonia, further underscore its potential as a biomarker for fibrotic progression (Sakamoto et al., 2023).

Furthermore, HSP47’s fibrogenic role is not isolated but intricately linked to key profibrotic signaling cascades, particularly those mediated by TGF-β. TGF-β1 upregulates HSP47 expression through the MAPK signaling pathway, thereby promoting excessive collagen synthesis (Khan and Däinghaus, 2024; Ito and Nagata, 2017). HSP47 stabilizes collagen to increase ECM stiffness, which further amplifies TGF-β signaling. This establishes a self-perpetuating cycle that exacerbates fibroblast activation and fibrotic progression. Additionally, the inflammatory cytokine IL-1β synergizes with TGF-β to activate heat shock factor 1 (HSF1), which transcriptionally upregulates HSP47, forming a pro-fibrotic positive feedback loop (Sakamoto et al., 2023). These findings collectively indicate that targeting HSP47—either directly or via its upstream regulatory pathways—may effectively disrupt pathological collagen metabolism, offering novel therapeutic opportunities for IPF management.

The NF-κB pathway is a critical mediator of inflammation and is aberrantly activated in various diseases, including breast, lung, and colon cancers (Baker et al., 2011). When AECs are damaged by oxidative stress, infection, or mechanical injury, NF-κB can be triggered via pattern recognition receptors (e.g., TLR4) or pro-inflammatory factors (TNF-α, IL-1β). This activation enhances the expression of TNF-α, IL-6, CCL2, and MCP-1, leading to intensified local inflammation and exacerbation of fibrosis development (Dong et al., 2022). Additionally, immune cells (e.g., macrophages, neutrophils) activated by NF-κB secrete cytokines (e.g., TNF-α, IL-1β), which further stimulate NF-κB in AECs, amplifying the inflammatory cascade in IPF (Baker et al., 2011; Li et al., 2017b).

Identifying NF-κB inhibitors is therefore crucial for antifibrotic therapy, for which multiple NPs have shown promise. For example, phycocyanin (Li et al., 2017b) and β-carboline alkaloids found in Xuelingzhi (Cui et al., 2019) can both inhibit NF-κB, thereby suppressing the recruitment and activation of inflammatory cells. Additionally, juglanin, extracted from crude “Polygonum aviculare”, has also been shown to inhibit the NF-κB pathway (Dong and Yuan, 2018). Similarly, phycocyanin can inhibit the abnormal activation of AECs through the TLR2-MyD88-NF-κB signaling mediated by TLR2 (Li et al., 2017b), thus reducing the secretion of ECM components and pro-inflammatory cytokines/chemokines, such as IL-6, CCL2, and CXCL1 (Li et al., 2017a). Furthermore, β-carboline alkaloids from snow fungus exhibit anti-inflammatory activity by inhibiting NF-κB signaling (Cui et al., 2019). These natural compounds demonstrate considerable potential in modulating NF-κB signaling, thereby offering therapeutic benefits in attenuating inflammation-driven fibrotic progression in IPF.

The TGF-β signaling pathway is a principal therapeutic target in IPF, as it is prominently upregulated and activated during fibrotic progression. Specifically, TGF-β1/Smad signaling significantly contributes to IPF pathogenesis by facilitating the activation, proliferation, and differentiation of epithelial cells and collagen-producing myofibroblasts, ultimately exacerbating ECM deposition and fibrosis development (Wu et al., 2021; Peng et al., 2022). In murine fibrosis models, overexpressed TGF-β binds to type II TGF-β receptor (TβRII) forming a heterodimer with TβRI, leading to excessive activation and phosphorylation of Smad2/3 proteins. Activated Smad2/3 then translocate into the nucleus, initiating transcriptional regulation of numerous fibrotic genes, triggering fibroblast phenotype transformation, promoting EMT, and significantly enhancing ECM accumulation (Peng et al., 2022; Derynck and Zhang, 2003; Massagué, 2012). Furthermore, TGF-β also activates the PI3K-Akt and Erk pathways, facilitating EMT, collagen accumulation, and inflammatory responses, further exacerbating fibrosis development (Wu et al., 2021).

Considering the crucial pathological role of the TGF-β/Smad pathway, the therapeutic potential of various natural products in IPF treatment has been explored and reported. For example, compounds such as Biochanin-A (BCA) (Andugulapati et al., 2020), Inonotus sanghuang extract (ISE) from ethyl acetate fraction (Su et al., 2021), salvianolic acid B (Liu et al., 2016), total ginsenosides (TG) (Yang et al., 2019), Dendrobium officinale (Chen et al., 2018), the amino acid EZY-1 (Yu et al., 2019), and low molecular weight fucoidan (LMWF) (Wu et al., 2021) were shown to effectively inhibit this signaling in bleomycin-induced mouse models of pulmonary fibrosis. Additionally, in vitro studies confirm that LMWF (Wu et al., 2021) alleviates EMT and fibrotic phenotype by simultaneously blocking TGF-β/Smad and PI3K/Akt pathways in A549 cells, indicating multifaceted therapeutic effects. Furthermore, tanshinone IIA (Tan IIA) (Feng et al., 2020a), derived from traditional Chinese medicine Salvia miltiorrhiza (Danshen), robustly suppresses TGF-β1/Smad signaling, inhibiting collagen I, collagen III, and α-SMA expression, effectively reducing fibrosis severity in a silica-induced pulmonary fibrosis model using Wistar rats. In another example, triptolide (Lin et al., 2023) exhibited antifibrotic effects in a mouse model of chest ion irradiation-induced lung injury. Moreover, trichocarboline A, a β-carboline alkaloid from the deep-sea fungus Trichoderma MCCC 3A01244 (Hao et al., 2022), was further tested to found to reduce pulmonary fibrosis by downregulating Smad2 and Smad3 phosphorylation, thereby directly inhibiting TGF-β/Smad signal transduction in human lung fibroblast cell lines (HFL1). These findings collectively underscore the cross-species and multi-model efficacy of natural products in targeting the TGF-β/Smad pathway, highlighting their promise as therapeutic agents for IPF.

AMPK is an energy sensor activated under energy-deficient conditions to regulate cellular metabolism (Hardie et al., 2012). Upon activation, it inhibits energy-consuming pathways and promotes energy-generating processes, thereby maintaining cellular homeostasis. Additionally, AMPK also promotes autophagy, facilitating the clearance of damaged organelles and proteins—an essential process for delaying or preventing fibrosis (El-Horany et al., 2023). In the bleomycin-induced mouse model of pulmonary fibrosis, the AMPK activator metformin reverses established fibrosis by suppressing TGF-β1-driven collagen and FN production, enhancing mitochondrial biogenesis, restoring myofibroblast susceptibility to apoptosis, and promoting autophagy-mediated ECM degradation (Rangarajan et al., 2018; Kheirollahi et al., 2019). Moreover, heterophyllin B, isolated from Radix Pseudostellariae, ameliorates fibrosis by activating AMPK, which in turn suppresses the TGF-β1/Smad2/3 signaling and downregulates STING expression. These actions collectively reduce collagen deposition and restoring energy metabolism (Shi et al., 2022).

During IPF pathogenesis, persistent oxidative damage contributes to epithelial injury, fibroblast activation, and ECM deposition (El-Horany et al., 2023; Hybertson et al., 2011), while the Nrf2 signaling pathway plays a crucial role in mitigating oxidative stress. In vivo and in vitro studies demonstrate that activation of Nrf2 reduces fibrotic progression by upregulating antioxidant enzymes such as heme oxygenase-1 (HO-1) and NAD(P)H quinone dehydrogenase 1 (NQO1), thereby countering reactive oxygen species (ROS) accumulation in lung tissues (Dong et al., 2022; Zhang et al., 2018). Notably, Nrf2 activation inversely regulates NADPH oxidase 4 (NOX4), a key ROS-generating enzyme implicated in TGF-β-induced myofibroblast differentiation and epithelial cell apoptosis (Liu et al., 2016). This antagonistic relationship highlights Nrf2’s critical role in regulating oxidative stress and restraining the fibrotic cascade in IPF (Hybertson et al., 2011).

Building on these findings, several NPs have been shown to exert antifibrotic effects by activating the Nrf2 pathway in vitro and in vivo. For instance, Salvianolic acid B was shown to attenuate paraquat-induced pulmonary fibrosis by restoring the Nrf2/NOX4 redox balance, where Nrf2 activation suppressed NOX4 expression and downstream TGF-β1/Smad3 signaling (Liu et al., 2016). And tanshinone IIA (Tan IIA) (Feng et al., 2020a), from S. miltiorrhiza, enhances Nrf2-mediated expression of antioxidant enzymes, thereby reducing fibrosis severity in silica-induced pulmonary fibrosis models. Similarly, geraniol (Tavares et al., 2021), a component of essential oil from Cymbopogon winterianus, activates Nrf2, leading to downregulation of pro-oxidant mediators and attenuation of fibrotic progression. Additionally, LMWF (Dong et al., 2022) alleviated bleomycin-induced fibrosis through Nrf2-mediated suppression of NOX4 and oxidative stress markers. These findings collectively highlight the therapeutic potential of targeting the Nrf2/NOX4 axis to disrupt oxidative stress-driven fibrotic cascades in IPF, and natural products targeting this pathway offer promising therapeutic potential for mitigating oxidative stress and halting fibrosis progression.

The PI3K/Akt signaling pathway is a central regulator of cell growth, proliferation, and survival. Importantly, elevated PI3K/Akt activity has been observed in IPF lung tissues and in bleomycin-induced pulmonary fibrosis models, underscoring its pathophysiological relevance (Wang et al., 2022a). In the fibrotic lung, activation of PI3K/Akt promotes EMT, enhances fibroblast survival, and stimulates ECM production, thereby exacerbating fibrosis progression (Qian et al., 2018).

Recently, several NPs have been identified to exert antifibrotic effects by suppressing PI3K/Akt signaling. Compounds like Astragaloside IV (Qian et al., 2018), a natural saponin from Astragalus membranaceus, and LMWF (Wu et al., 2021) have been identified as effective inhibitors of the PI3K/Akt signaling cascade in bleomycin-treated mice, thereby exerting a beneficial inhibitory effect on ECM deposition and fibrosis progression. These findings underscore the therapeutic promise of targeting the PI3K/Akt pathway with natural compounds to attenuate fibrosis in IPF.

Altogether, these interconnected pathways—NF-κB, TGF-β1/Smad, AMPK, Nrf2, and PI3K/Akt—underscore the multifactorial nature of IPF pathogenesis. NPs, through their potential to simultaneously modulate multiple pathways, emerge as promising candidates for developing innovative antifibrotic therapies. In the subsequent section, we will systematically examine the anti-pulmonary fibrotic mechanisms of over 20 bioactive compounds derived from both terrestrial (Section 3.1) and marine (Section 3.3) sources and one nature product analogue (Section 3.4). This comprehensive analysis will encompass: (1) experimental models employed in their pharmacological evaluation, including both in vivo animal systems and in vitro cell-based platforms; (2) key pathological markers analyzed in these studies, such as collagen deposition indicators, inflammatory cytokines, and EMT biomarkers; and (3) molecular targets validated through contemporary pharmacological approaches.

Biochanin-A (BCA), an organic isoflavone isolated from the leaves and stems of Trifolium pratense L and many other herbs of Chinese medicine (Yan et al., 2021), has been demonstrated to mitigate PF in various cell lines, including LL29, normal human lung fibroblasts (NHLF), and diseased human lung fibroblasts (DHLF). BCA achieves this by modulating the TGF-β/Smad3 pathway, thereby ameliorating the fibrosis cascade response and inhibiting the expression of fibrosis markers such as FN-I during the EMT. In vitro scratch assays revealed that BCA dose-dependently attenuates TGF-β-induced cell migration. In a bleomycin-induced pulmonary fibrosis model in Wistar rats, a 14-day oral administration of BCA significantly reduced lung inflammation, infiltration of inflammatory cells, expression of inflammatory markers, collagen deposition, and fibrosis markers in lung tissues. BCA treatment also markedly decreased the expression of phospho-smad3, α-SMA, and FN-I, along with histopathological abnormalities in lung tissue (Andugulapati et al., 2020). These findings underscore BCA’s potential application in modulating pathways related to PF and managing disease progression.

Tanshinone IIA (Tan IIA), a lipophilic component derived from S. miltiorrhiza (Danshen), significantly inhibits the EMT process in silica-induced pulmonary fibrosis models by suppressing the TGF-β1/Smad signaling pathway (Feng et al., 2020a). In Wistar rats exposed to silica, intraperitoneal injection of Tan IIA effectively reduced collagen I, collagen III, and α-SMA expression, diminishing the extent of PF. In vitro studies demonstrated that Tan IIA treatment increased nuclear expression of Smad7, inhibited Smad2 and Smad3 phosphorylation by TβR1, and reduced the nuclear accumulation of phosphorylated Smad3, thus impeding ECM biosynthesis and myofibroblast differentiation. Moreover, Tan IIA mitigated oxidative stress induced by silica. Activation of the Nrf2 signaling pathway in A549 and human bronchial epithelial (HBE) cell lines further enhanced Tan IIA’s antifibrotic effects by modulating EMT and TGF-β1/Smad pathway activation (Feng et al., 2020a). Additionally, puerarin (Pue), a compound derived from Sorbus aucuparia, exhibits synergistic effects with Tan IIA in combating fibrosis progression (Xue et al., 2021).

In an IPF mouse model administered bleomycin-A5 intratracheally, Tan IIA and Pue combined therapy significantly reduced the expression of fibrosis markers and macrophage infiltration, improving lung function and survival rates. In vitro experiments in NIH-3T3 cells revealed that the combination treatment downregulated α-SMA expression and inhibited JAK2, STAT3, and STAT1 signaling pathways, reducing fibroblast activation and migration induced by TGF-β1 and IL6, thereby suppressing fibrosis progression through the JAK2-STAT3/STAT1 pathway. Juglanin (Jug, kaempferol-3-O-α-L-arabinofuranoside), a compound isolated from crude “Polygonum aviculare”, inhibits the STING pathway, effectively counteracting TGF-β-induced collagen accumulation. Elevated Sting expression was observed in bleomycin-induced mouse lung tissues, suggesting that bleomycin may induce pulmonary fibrosis through STING activation. Juglanin treatment significantly reduced Sting expression, ameliorating TGF-β-induced collagen accumulation and highlighting its potential application in treating fibrotic diseases (Williams et al., 2020).

β-carboline alkaloids, extracted from Snow Fungus, exhibit anti-inflammatory effects by inhibiting the secretion of cytokines MCP-1, IL-6, TNF-α, and IL-1β in LPS-induced RAW264.7 cells, thereby mitigating inflammation (Cui et al., 2019). These alkaloids regulate the NF-κB/p65 pathway by inhibiting p65 phosphorylation, thereby curbing inflammation initiation. In TGF-β1-induced A549 cells, β-carboline alkaloids prevented the suppression of E-cadherin by TGF-β1, reduced mesenchymal and fibrotic markers such as α-SMA and Vimentin, and slowed the EMT process, thereby impeding PF progression.

Lycorine (LYC), an alkaloid from Amaryllidaceae plants, significantly reduced alveolar collapse and collagen accumulation in bleomycin-induced pulmonary fibrosis in mice, lowering fibrosis marker levels including hydroxyproline (HYP). Flow cytometry revealed that LYC could reverse macrophage reduction in vivo. Real-time PCR demonstrated that LYC blocked bleomycin-induced expression of type I collagen, Acta2, and Fn, and reduced α-SMA and FN levels, improving bleomycin-induced pulmonary fibrosis. LYC also exerted anti-inflammatory effects by inhibiting Caspase-1 cleavage and pro-IL-1β maturation, alleviating fibrosis and inflammation. Molecular docking and SPR analysis showed that LYC inhibited NLRP3 inflammasome activation and pyroptosis by binding to the PYD domain of ASC, suppressing ASC-NLRP3 interaction, thus exhibiting anti-inflammatory effects and improving fibrosis (Liang et al., 2020).

Total ginsenosides (TG), chemical components from ginseng, have demonstrated therapeutic efficacy against IPF. TG treatment improved bleomycin-induced pulmonary fibrosis in mice, reducing interstitial fibrosis, collagen deposition, and alveolar wall destruction. TG downregulated TGF-β1, α-SMA, Smad2, and Smad3 expression while upregulating Smad7, inhibiting fibroblast proliferation and abnormal ECM deposition via the TGF-β1/Smad pathway. Additionally, TG’s protective effect against IPF may involve the MMP system, as TG significantly downregulated MMP-2, MMP-9, and tissue inhibitor of metalloproteinase-1 induced by bleomycin, potentially disrupting basement membrane integrity and mitigating fibroblast invasion (Yang et al., 2019).

Polyphenolic compounds, a diverse class with extensive biological activities, include phenolic acids, flavonoids, stilbenes, lignans, and tannins. These compounds have shown significant efficacy in alleviating fibrotic disease symptoms (Du et al., 2016). Salvianolic acids, notably Salvianolic acid A (Sal A) and Salvianolic acid B (Sal B) from S. miltiorrhiza, exhibit robust antioxidant activity. Nrf2 is critical for cellular protection against oxidative stress, and NOX4, closely linked to the TGF-β1/Smad3 pathway in lung fibroblasts, is essential for regulating the myofibroblast phenotype in IPF. Sal B modulates the Nrf2/Nox4 redox balance and TGF-β1/Smad3 signaling, alleviating oxidative stress in paraquat-poisoned mice and showing potential therapeutic effects on PF (Zhang et al., 2021). Sal B also reduces hydroxyproline (HYP), type I collagen, tissue factor (TF), and related coagulation factors, exerting antifibrotic and anticoagulant effects (Liu et al., 2016). Given Sal’s therapeutic potential, efforts are being made to develop a new inhalable dry powder formulation to enhance bioavailability, which is currently less than 5% orally (Jiang et al., 2021).

Resveratrol (Res), a polyphenolic compound, mitigates oxidative damage and fibrosis by leveraging its antioxidant properties, thereby preventing PF in vivo. MicroRNA-21 (miR-21) amplifies TGF-β1 signaling, exacerbating PF progression. In bleomycin-induced rat models, Res inhibited miR-21 by downregulating c-Jun and c-Fos, blocking the MAPKs/AP-1 pathway (Wang et al., 2018). Res also modulated fibrosis-related proteins FN, COL1 A1, COL3 A1, α-SMA, TIMP-1, and MMP-2, 9, and 13, inhibiting PF progression through the TGF-β/Smad/ERK pathway. Additionally, Res alleviated PM2.5-induced lung inflammation and fibrosis by inhibiting autophagy and NLRP3 inflammasome activity, reducing IL-1β in BEAS-2B cells (Ding et al., 2019). These properties suggest that resveratrol holds promise for PF treatment.

Triptolide, a bioactive diterpene from Tripterygium wilfordii, was shown to downregulate mRNA and protein expression of α-SMA, type I collagen, FN, and vimentin, inhibiting lung fibroblast migration and transformation. Triptolide reduced TGF-β1-induced overexpression of LOX, LOXL1, and LOXL2, as well as total LOX activity, diminishing oxidative stress and altering endopeptidase expression such as MMP2, MMP9, MMP13, and MMP14. Immunofluorescence showed Triptolide inhibited the Integrin-β1-FAK-YAP pathway, reducing nuclear YAP1 content and inhibiting fibrotic gene transcription (Lin et al., 2023). These findings support Triptolide’s potential as an antifibrotic agent. Essential oil from Cymbopogon winterianus (EOCW), containing eugenol, geraniol, and citronellal, has anti-inflammatory and antioxidant properties. Eugenol enhances Nrf2 expression, geraniol inhibits 5-lipoxygenase and nitric oxide, and citronellal reduces COX-2 and prostaglandin-E2 (PGE2) expression. EOCW alleviated pulmonary fibrosis progression in bleomycin-induced mice, likely due to TGF-β expression inhibition (Tavares et al., 2021). These properties make EOCW a candidate for PF treatment, warranting further investigation into its dosage and mechanisms.

Ophiocordyceps lanpingensis polysaccharides (OLP), extracted from the edible fungus Ophiocordyceps lanpingensis, show therapeutic potential against PF. In bleomycin-treated mice, OLP reduced pro-inflammatory protein MCP-1, decreased macrophage recruitment, and downregulated related cytokines and myofibroblast markers like α-SMA. OLP preserved alveolar function and inhibited collagen production, mitigating bleomycin-induced pulmonary fibrosis (Zhou et al., 2020). Dendrobium officinale, an Orchidaceae herb, produces polysaccharides (PDO) with mild anti-inflammatory and strong antifibrotic effects. Oral PDO reduced E-cadherin and α-SMA expression and decreased type I collagen and FN synthesis in bleomycin-treated rats. PDO downregulated Smad2/3 and pSmad2/3 expression, alleviating myofibroblast formation and proliferation by inhibiting TGFβ1-Smad2/3 signaling (Chen et al., 2018). Angelica sinensis polysaccharide (ASP) from Angelica sinensis roots inhibits lncRNA DANCR in bleomycin-treated rat lung tissue and type II alveolar epithelial cells (RLE-6TN), reducing oxidative stress and PF severity. ASP reversed collagen deposition and α-SMA upregulation induced by TGF-β1, and restored E-cadherin expression (Qian et al., 2020). Polysaccharides’ complex composition and mechanisms necessitate further research to validate their pharmacological activity and support drug development.

Heterophyllin B from Radix Pseudostellariae exhibits anti-inflammatory activity by promoting AMPK activation and inhibiting STING, thereby reducing TGF-β1-induced abnormal proliferation of MLE-12 cells and overexpression of STING in bleomycin-treated mice. Heterophyllin B inhibited TGF-β1 and Smad2/3 phosphorylation, and reduced myofibroblast markers α-SMA and COL-1 in lung tissues, demonstrating anti-pulmonary fibrosis effects. It offers significant therapeutic potential, as shown in bleomycin-induced pulmonary fibrosis models treated with oral heterophyllin B (Shi et al., 2022).

LMWF, a distinctive sulphated polysaccharide derived from seaweed through radical degradation, has a molecular weight of approximately 8,100 Da, with fucose as its primary monosaccharide. Wu et al. investigated the antifibrotic effects and mechanisms of LMWF using both in vivo and in vitro models (Wu et al., 2021). Protein blot analyses in lung cancer A549 cells (CCL-185, ATCC, Rockville, MD, US) were conducted via the MTT method. Lung tissues from different experimental groups of mice were collected for pathological evaluation through HE and Masson’s trichrome staining, histological, and immunohistochemical analysis. Serum IL-6 levels were measured using ELISA kits, and bronchoalveolar lavage fluid (BALF) was analyzed for lavage fluid and protein concentrations using BCA kits. Results demonstrated that LMWF treatment in bleomycin-induced pulmonary fibrosis mice decreased TGF-β, TNF-α, and IL-6 expression in lung tissue, mirroring the effects of nintedanib in reducing inflammatory cytokine accumulation. Furthermore, LMWF attenuated phosphorylation activation of the PI3K/AKT signaling pathway induced by TGF-β1 in A549 cells, lowering β-catenin and COL2A1 expression, and preventing EMT. In the TGF-β/Smad pathway, LMWF downregulated TGF-β1, Smad2/3, and Smad4 expression, thereby inhibiting pathway activity and curbing TGF-β1-induced EMT progression. These findings suggest that LMWF has significant therapeutic potential in inhibiting human IPF by mitigating inflammation and EMT processes (Dong et al., 2022; Wu et al., 2021).

Dong et al. also evaluated LMWF extracted from Laminaria japonica via radical degradation, focusing on its effects on bleomycin-treated male C57BL/6J mice and TGF-treated A549 cells, with an additional assessment of its antioxidant properties (Dong et al., 2022). Pathological changes and collagen deposition in lung tissues of bleomycin-induced mice were evident from H&E, Masson’s trichrome, and Sirius red staining. GSH, MDA, and SOD levels in lung tissues were measured using kits, and hydrogen peroxide content was assessed with enzyme biosensors. LMWF treatment reduced MDA and 8-iso-PGF2α levels while increasing SOD, GSH, HO-1, and NQO1 concentrations, thereby preventing oxidative stress damage and attenuating pulmonary fibrosis by enhancing antioxidant enzyme protein expression. In vitro, LMWF regulated antioxidant factors in lung fibrosis mouse lung tissue, reducing oxidative stress and fibrosis. Immunofluorescence, protein blotting, and immunohistochemistry showed that LMWF at 50 mg/kg and 100 mg/kg doses upregulated Nrf-2, HO-1, and NQO1 expression in a dose-dependent manner, inhibiting fibrosis. High doses (100 mg/kg) showed no toxicity in A549 cells, aligning with in vivo results, indicating LMWF’s high safety profile (Dong et al., 2022).

A separate study investigated the effects of fucoidan obtained via enzymatic hydrolysis on the infiltration of inflammatory cells in lung tissues of irradiated 8-week-old male C57BL/6 mice. Fucoidan significantly reduced neutrophil and macrophage infiltration in irradiated lung tissues. Cytokine array analysis and EIA confirmed that fucoidan lowered the expression levels of inflammatory cytokines such as TIMP-1, CXCL1, MCP-1, MIP-2, and IL-1Ra in pleural effusion of irradiated mice. In vivo, these cytokines were linked to radiation-induced lung fibrosis, selectively activating macrophages and neutrophils. Thus, fucoidan administration reduced cytokine expression in pleural effusion and decreased collagen expression in fibroblasts, correlating with reduced inflammatory cell infiltration in lung tissues. This finding suggests potential fucoidan-based therapies for preventing lung fibrosis in clinical settings (Yu et al., 2018). Overall, fucoidan, due to its wide availability and high safety profile, shows promise as a therapeutic agent for treating PF, with no observed toxic side effects at high doses (100 mg/kg) (Dong et al., 2022; Wu et al., 2021).

From the deep-sea fungus Trichoderma MCCC 3A01244, 25 compounds were isolated, among which Trichocarboline A, a β-carboline alkaloid, exhibited significant biological activity. At a 10 μM concentration in HFL1 cell lines, Trichocarboline A demonstrated low cytotoxicity and robustly inhibited TGF-β1-induced collagen accumulation. CCK8 assays showed a notable reduction in cellular collagen deposition. Mechanistic studies indicated that Trichocarboline A downregulated phosphorylation levels of Smad2 and Smad3, suppressing the TGF-β/Smad signaling pathway. This inhibition curtailed TGF-β1-induced expression of FN, proliferating cell nuclear antigen (PCNA), and α-SMA in HFL1 cells, reducing ECM deposition. Trichocarboline A binds to Smad4, hindering TGF-β/Smad pathway signaling, and downregulates transcription of fibrosis-related genes. As a newly identified β-carboline alkaloid, Trichocarboline A emerges as a lead compound for the development of more effective antifibrotic drugs (Hao et al., 2022).

EZY-1, a 16-amino acid peptide derived from the edible seaweed Eucheuma, showed significant inhibitory effects on bleomycin-induced pulmonary fibrosis in mice. Using protein chip analysis and in vitro pull-down combined with LC-MS/MS, potential EZY-1 target proteins were screened. In vitro analysis identified ERK, Akt, raptor, SHP2, PDGFR, β-catenin, and vitronectin as potential targets. EZY-1 notably inhibited Smad2 and Smad3 phosphorylation in lung tissues of bleomycin-induced mice, suggesting interference with the TGF-β/Smad signaling pathway. Additionally, EZY-1 inhibited the phosphorylation of ERK and p38MAPK, blocking IPF progression through inhibition of the TGF-β-activated MAPK signaling pathway. EZY-1 also significantly reduced collagen fiber deposition and proline hydroxylation levels, lowered malondialdehyde (MDA) levels, and increased superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity, offering protection against oxidative damage. It inhibited tyrosine phosphorylation of proteins such as PI3K p85, ERK1, p38MAPK, and c-Abl, and demonstrated anti-fibrotic and anti-inflammatory properties superior to pirfenidone. EZY-1 exhibited high safety with no acute toxicity effects at a 5 g/kg dosage, indicating its promise as a safe and effective drug for IPF treatment (Yu et al., 2019).

Phycocyanin (PC), a light-harvesting protein from Spirulina platensis, was studied by Li et al. in C57 BL/6 wild-type (WT) mice and toll-like receptor (TLR) 2-deficient mice treated with PC for 28 days post-bleomycin exposure. PC significantly reduced PF markers, including hydroxyproline (HYP), vimentin, surfactant-associated protein C (SP-C), fibroblast-specific protein-1 (S100A4), and α-SMA, while increasing E-cadherin and podoplanin expression. PC also reduced early-stage inflammation-related proteins IL-6, TNF-α, and MPO. It improved the pathological state of PF by protecting type I alveolar epithelial cells and inhibiting EMT. In bleomycin-treated mice, PC reduced TLR2 pathway gene expression, with limited effects in TLR2-deficient mice, suggesting that PC mediates antifibrotic effects via the TLR2-MyD88-NF-κB pathway. Future exploration of this pathway may establish PC as a novel antifibrotic agent (Li et al., 2017b).