NRICM102, a TCM Formula, Attenuates COPD-Relevant Inflammatory Lung Injury in Mice by Improving Pulmonary Function and Reversing Immune Dysregulation

Abstract

Background: Chronic obstructive pulmonary disease (COPD) is a progressive inflammatory lung disorder with limited effective therapies. NRICM102, a traditional multi-herbal formulation originally developed for COVID-19, exhibits anti-inflammatory and immunomodulatory potential. Objectives: The aim of this study was to investigate the therapeutic efficacy of NRICM102 in a COPD-relevant inflammatory lung injury mice model. Methods: Mice were exposed to lipopolysaccharide (LPS) and benzo[a]pyrene (B[a]P) to induce chronic airway inflammation and structural lung damage and treated with NRICM102 (1.5–3.0 g/kg) or dexamethasone. Lung function, histopathology, transcriptomic profiling, and protein expression of key inflammatory markers were assessed. Results: NRICM102 significantly restored LPS+B[a]P-induced enhanced pause (Penh) and arterial oxygen saturation (aO₂%), similar to the effect of dexamethasone. Histological analysis revealed marked alveolar damage, inflammatory cell infiltration, and fibrosis in the model group, all of which were significantly attenuated by NRICM102 in a dose-dependent manner, with high-dose (3.0 g/kg) treatment showing pronounced structural preservation. Transcriptomic profiling revealed that NRICM102, particularly at 3.0 g/kg, partially reversed COPD-associated gene expression patterns, characterized by reduced activation of cytokine signaling, chemokine activity, and antigen presentation pathways. GO, DO, and KEGG enrichment analyses indicated selective modulation of immune-related pathways, with high-dose NRICM102 affecting genes involved in adaptive immunity and cytokine receptor interactions, including a subset of 150 reverted genes. Immunofluorescence analysis confirmed dose-dependent reductions in key inflammatory, immune, and mucus-related markers, including IL-1β, NLRP3, Muc5ac, and MMP12 expression. Conclusions: NRICM102 confers significant protective effects against COPD-relevant inflammatory lung injury by improving pulmonary function, preserving lung architecture, and selectively modulating immune and inflammatory pathways. These results provide preclinical evidence supporting the potential of NRICM102 to modulate inflammation and immune responses associated with COPD-related pathology, although further studies are needed to establish its therapeutic relevance.

1. Introduction

Chronic respiratory diseases, particularly chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis, represent significant global health concerns, contributing substantially to morbidity and mortality worldwide [1]. COPD alone affects over 200 million individuals globally, and causes more than 3 million deaths annually [2]. The environmental factors such as fine particulate matter (PM2.5), vehicle exhaust, and pollutants from combustion processes have been implicated in the high prevalence and severity of these diseases [3,4,5].

COPD is characterized by chronic airway inflammation, irreversible airflow limitation, and structural lung changes such as bronchitis, bronchiolitis, and emphysema [6]. Clinically, patients experience persistent respiratory symptoms, including dyspnea, cough, and sputum production [7]. The pathogenesis of COPD involves an exaggerated immune response to inhaled irritants such as cigarette smoke, triggering the recruitment of neutrophils and macrophages, which leads to sustained inflammation and oxidative stress [6,8,9]. Repeated exposure impairs macrophage function, disrupts epithelial integrity, and causes tissue remodeling. In addition, COPD increases both the number and size of goblet cells, leading to mucus overproduction that obstructs airflow and impairs mucociliary clearance. Mucus overproduction by goblet cells obstructs airways, while destruction of alveolar walls results in emphysema, reducing surface area for gas exchange and leading to breathlessness and hypoxemia [10]. Additionally, airway remodeling, including smooth muscle hypertrophy, fibrosis, and loss of elasticity, leads to airflow obstruction and lung hyperinflation, which compromise diaphragm function and reduce breathing efficiency over time. In this study, we employed a three-week protocol of repeated exposure to lipopolysaccharide (LPS) and benzo[a]pyrene (B[a]P) to induce COPD-relevant inflammatory lung injury in mice, inducing airway inflammation and lung tissue damage [11,12]. While this model recapitulates key pathological features such as airway inflammation, immune dysregulation, epithelial injury, and impaired pulmonary function, it does not fully capture hallmark aspects of human COPD, including long-term disease progression, established emphysema, small airway remodeling, or irreversible airflow limitation resulting from chronic exposure. Accordingly, we refer to this model as a COPD-relevant inflammatory lung injury model to accurately reflect its scope.

Current pharmacological treatments, including bronchodilators and corticosteroids, provide limited effectiveness, particularly in slowing disease progression or reversing structural lung injury [6]. Additionally, prolonged corticosteroid use is associated with adverse systemic effects such as osteoporosis, diabetes, immune suppression, muscle wasting, hypertension, and increased infection risk [8], underscoring the need for safer and more comprehensive therapeutic approaches [13]. Recently, traditional herbal medicines have gained renewed interest due to their broad pharmacological actions and favorable safety profiles. NRICM102, a Taiwanese multi-herbal formula originally developed for COVID-19-related pulmonary complications, has shown anti-inflammatory, immunomodulatory and antifibrotic effects through modulation of cytokine signaling and immune regulation [14,15,16].

In this study, we investigated the potential effects of NRICM102 in a COPD-relevant inflammatory lung injury mouse model induced by repeated exposure to LPS and B[a]P. The objective of this study was to evaluate the protective effects of NRICM102 and to explore the associated molecular mechanisms that may contribute to its efficacy in this model.

2. Results

2.1. Effect of NRICM102 on Pulmonary Function in an LPS+B[a]P-Induced COPD-Relevant Inflammatory Lung Injury Mouse Model

To assess the effects of NRICM102 on COPD-relevant inflammatory lung injury, we employed an LPS and B[a]P-induced COPD-relevant inflammatory lung injury mouse model [12,15]. Mice were exposed to LPS and B[a]P once weekly for three weeks to induce chronic airway inflammation and structural lung damage. NRICM102 and dexamethasone were administered orally during the final five days of each weekly cycle (Experimental schedule shown in Figure 1A). No mortality or abnormalities in behavior, activity, or general appearance were observed in any group throughout the 21-day study period. Pulmonary function was evaluated on day 21 by measuring enhanced pause (Penh) and arterial oxygen saturation (aO₂%). Penh was significantly increased in the LPS+B[a]P group compared with sham controls (Figure 1B). NRICM102 treatment at doses of 1.5 and 3.0 g/kg effectively restored the LPS+B[a]P-induced elevation in Penh. Dexamethasone, used as a positive control [17], produced a comparable reduction in Penh. On the other hand, arterial oxygen saturation (aO₂%) was significantly reduced by LPS+B[a]P exposure (Figure 1C), indicative of compromised gas exchange due to alveolar inflammation and tissue damage. NRICM102 administration restored aO₂% levels in a dose-dependent manner. Doses of 1.5 and 3.0 g/kg completely normalized oxygen saturation to control levels, reflecting enhanced alveolar-capillary function and reduced inflammation. Dexamethasone also restored oxygen saturation effectively, confirming the model’s validity and serving as a pharmacological comparator. Together, these results indicate that NRICM102 ameliorates respiratory dysfunction in this LPS and B[a]P-induced COPD-relevant inflammatory lung injury mouse model.

2.2. Effect of NRICM102 on Lung Histopathology in a COPD-Relevant Mouse Model

To evaluate the protective effect of NRICM102 in COPD-relevant lung injury, we performed a histological assessment to determine the lung tissue structure, a hallmark of COPD pathology. Quantitative histological assessment demonstrated significantly elevated pathology scores in the COPD-relevant model group, characterized by pronounced alveolar damage, inflammatory cell infiltration, and fibrosis [18]. Treatment with NRICM102 significantly improved lung tissue integrity in a dose-dependent fashion (Figure 2). Dexamethasone treatment also significantly improved lung structure in COPD-relevant model. Notably, high-dose NRICM102 (3.0 g/kg) treatment markedly restored lung structure toward levels observed in saline-treated controls and showed a significant effect compared with dexamethasone treatment. Collectively, these findings indicate the superior efficacy of NRICM102 in preserving lung architecture in COPD-relevant pulmonary injury (Figure 2).

2.3. Transcriptomic Insights into NRICM102’s Protective Effects in a COPD-Relevant Lung Injury Model

2.3.1. Effect of NRICM102 on Global Gene Expression and Transcriptomic Clustering

To explore the potential mechanisms underlying NRICM102’s protective effects against LPS+B[a]P-induced COPD-relevant lung injury, we performed transcriptomic profiling using RNA-seq. Frist, principal component analysis (PCA) revealed distinct clustering of the COPD-relevant model from controls (Figure 3A), indicating substantial transcriptomic shifts. Both high-dose (N102H, 3.0 g/kg) and low-dose (N102L, 1.5 g/kg) NRICM102, as well as dexamethasone (DEX, 2 mg/kg), formed separate clusters from the COPD-relevant group, suggesting treatment-induced transcriptional changes. The dexamethasone group showed strong separation from the COPD cluster, while N102H shifted closer to the control group, indicating partial transcriptomic restoration. In contrast, N102L remained more distant from controls and closer to the COPD-relevant group, reflecting a weaker effect and highlighting dose dependency. The differentially expressed genes (DEGs) were then visualized using volcano plots and clustered in a heatmap (Figure 3B,C). Compared to controls, the COPD-relevant group showed marked gene dysregulation, with 1062 genes upregulated and 535 downregulated. N102H downregulated 201 genes and upregulated 26, indicating a strong but selective modulatory effect. N102L exhibited a more balanced regulation, downregulating 160 and upregulating 167 genes, suggesting milder, yet consistent, transcriptomic modulation. Dexamethasone exhibited the most pronounced transcriptomic reversal, downregulating 791 and upregulating 522 genes, closely aligning with the control profile.

2.3.2. Transcriptomic Changes in Lung Tissue Induced by LPS and B[a]P in a COPD-Relevant Model

Next, we further analyzed transcriptomic differences between the COPD-relevant and sham groups to validate the LPS+B[a]P-induced COPD-relevant model in lung tissue. The DEGs were subsequently subjected to functional classification and enrichment analysis using Gene Ontology (GO), Disease Ontology (DO), and the Kyoto Encyclopedia of Genes and Genomes (KEGG). GO enrichment revealed that significant upregulation of immune- and inflammation-related pathways, including leukocyte migration (GO:0050900), myeloid leukocyte migration (GO:0097529), cell chemotaxis (GO:0060326), leukocyte chemotaxis (GO:0030595), neutrophil migration (GO:1990266), neutrophil chemotaxis (GO:0030593), adaptive immune response (GO:0002250), positive regulation of cytokine production (GO:0001819), and regulation of inflammatory response (GO:0050727) (Figure 4A,B). In the cellular component (CC) category, DEGs were enriched in the external side of plasma membrane (GO:0009897), major histocompatibility complex (MHC) protein complex (GO:0042611), and extracellular matrix (GO:0031012), while the molecular function (MF) category highlighted chemokine activity (GO:0008009), chemokine receptor binding (GO:0042379), and cytokine activity (GO:0005125) (Table S1 in the Supplementary Information). DO enrichment analysis showed that the DEGs in the COPD-relevant model are significantly associated with a range of lung and immune-related diseases, sharing the signature of immune activation, chemotaxis, and extracellular remodeling, aligning with classic COPD pathophysiology (Figure 4C, Table S2 in the Supplementary Information). KEGG pathway enrichment analysis revealed significant upregulation of immune-related pathways in the COPD-relevant group, including cytokine-cytokine receptor interactions (mmu04060), phagosome (mmu04145), chemokine signaling pathway (mmu04062), and antigen processing and presentation (mmu04612) (Figure 4D, Table S3 in the Supplementary Information), highlighting the activation of inflammatory and immune pathways in the LPS+B[a]P-induced COPD-relevant model. In contrast, downregulated pathways involved the calcium signaling pathway (mmu04020), cGMP-PKG signaling pathway (mmu04022) and cAMP signaling pathway (mmu04024), focal adhesion (mmu04510), axon guidance (mmu04360), and circadian entrainment (mmu04713) (Figure 4E, Table S3 in the Supplementary Information), indicating impairment in epithelial barrier function, smooth muscle regulation, and systemic circadian homeostasis. Together, GO, DO, and KEGG analyses reveal that LPS+B[a]P exposure activates immune and inflammatory pathways while suppressing regulatory and structural functions, providing a comprehensive transcriptomic signature of chronic inflammation, immune dysregulation, and tissue remodeling in COPD-relevant lung injury.

2.3.3. Expression Transcriptomic Shifts Induced by NRICM102

To explore the protective effects of NRICM102, we analyzed DEGs between the high-dose NRICM102 (N102H) and COPD-relevant groups. The most significantly enriched BPs in GO enrichment analysis include adaptive immune response (GO:0002250), regulation of lymphocyte activation (GO:0051249), leukocyte cell-cell adhesion (GO:0007159), regulation of leukocyte cell-cell adhesion (GO:1903037), positive regulation of immune effector process (GO:0002699), T cell activation (GO:0042110), antigen receptor-mediated signaling pathway (GO:0050851), and regulation of immune response (GO:0050776) (Figure 5A,B). In the CC category, enriched terms included the external side of the plasma membrane (GO:0009897), MHC protein complex (GO:0042611), and immunological synapse (GO:0001772). The MF category showed enrichment of MHC class II receptor activity (GO:0032395), immune receptor activity (GO:0140375), and peptide antigen binding (GO:0042605) (Table S4 in the Supplementary Information). These results indicate that N102H treatment may be associated with changes in immune-related pathways and T cell signaling, which may reflect potential immunomodulatory effects in COPD-relevant lungs. DO enrichment analysis further showed that N102H downregulated genes associated with lung disease, obstructive lung disease, and COPD (Figure 5C, Table S5 in the Supplementary Information). KEGG pathway analysis also showed that N102H modulates key immune signaling pathways, particularly those involved in adaptive immunity and cytokine regulation. Enriched pathways included cytokine-cytokine receptor interaction (mmu04060), Th17 cell differentiation (mmu04659), Th1 and Th2 cell differentiation (mmu04658), and viral protein interaction with cytokine and cytokine receptor (mmu04061) (Figure 5D,E, Table S6 in the Supplementary Information). Together, these results suggest that N102H treatment may influence adaptive immune pathways and reduce the expression of COPD-related genes, potentially contributing to the mitigation of immune dysregulation in COPD-relevant lungs.

2.3.4. Identification and Functional Analysis of Shared DEGs Reversed by N102H Treatment in a COPD-Relevant Model

To further investigate how N102H reverses gene expression changes in COPD-relevant model, we performed an integrative analysis of DEGs from the groups “COPD vs. Sham” and “N102H vs. COPD” comparisons. Among the DEG comparisons, 150 genes were found to be reverted by N102H, showing expression changes opposite to the COPD-relevant model (Figure 6A, Table S7 in the Supplementary Information). Of these, 140 genes were upregulated in COPD-relevant and downregulated by N102H, while 10 genes were downregulated in COPD-relevant and upregulated by N102H. Additionally, one gene exhibited same-directional changes in both comparisons. These reverted genes are visualized in a heatmap (Figure 6B). GO enrichment analysis of the reverted DEGs revealed significant enrichment in BPs related to immunoglobulin-mediated immune response and B cell-mediated immunity (Figure 6C, Table S8 in the Supplementary Information). Notably, KEGG pathway analysis showed that these genes were primarily involved in cytokine-cytokine receptor interaction (mmu04060), chemokine signaling pathway (mmu04062), and viral protein interaction with cytokine and cytokine receptor (mmu04061) (Figure 6D,E, Table S9 in the Supplementary Information). Pathview visualizations of the cytokine-cytokine receptor interaction and viral protein interaction with cytokine-cytokine receptor pathways for the “COPD vs. Sham” and “N102H vs. COPD-relevant” comparisons are presented in Figures S1–S4 in the Supplementary Information. These results indicate that N102H treatment is associated with changes in genes and pathways involved in cytokine signaling and adaptive immune responses, which may reflect a partial reversal of COPD-associated immune dysregulation in the LPS-B[a]P-induced COPD-relevant model.

2.3.5. Expression Transcriptomic Shifts Induced by Dexamethasone

We next examined and validated the transcriptomic effects of the corticosteroid dexamethasone in the COPD-relevant model. The results of the GO enrichment analysis are presented as an emaplot (Figure 7A). The top 15 enriched BPs included myeloid leukocyte migration, leukocyte migration, cell chemotaxis, positive regulation of cytokine production, leukocyte chemotaxis, T cell activation, neutrophil migration, leukocyte cell–cell adhesion, granulocyte migration, myeloid leukocyte activation, muscle contraction, regulation of lymphocyte activation, myofibril assembly, regulation of leukocyte cell–cell adhesion, and the muscle system process (Figure 7B, Table S10 in the Supplementary Information). Downregulated BPs were dominantly associated with inflammatory cell trafficking and activation with strong statistical significance, suggesting robust suppression of inflammatory gene programs by dexamethasone. In contrast, the upregulated BPs were predominantly related to muscle structure and development. The GO enrichment results suggest that dexamethasone treatment suppressed immune-related pathways that are typically elevated in COPD, especially those involving leukocyte and neutrophil migration, hallmarks of chronic lung inflammation. DO enrichment analysis further indicated that dexamethasone downregulated gene sets linked to lung diseases, including COPD (Figure 7C, Table S11 in the Supplementary Information). KEGG pathway analysis revealed that dexamethasone strongly suppressed inflammatory and immune-related pathways activated in COPD, including cytokine-cytokine receptor interaction (mmu04060) and viral protein interaction with cytokine receptors (mmu04061) (Figure 7D, Table S6 in the Supplementary Information). Conversely, it upregulated pathways including cytoskeleton organization, calcium signaling, dilated and hypertrophic cardiomyopathy, and cardiac muscle contraction (Figure 7E, Table S12 in the Supplementary Information), which are associated with muscle structure and function, particularly in cardiac and smooth muscle. These results showed that dexamethasone strongly suppresses pro-inflammatory and immune-related signaling, and the down-regulation of cytokine signaling and immune activation pathways aligns with the known anti-inflammatory mechanism of corticosteroids like dexamethasone. Collectively, these results confirm dexamethasone’s anti-inflammatory effects and its ability to mitigate immune activation, particularly within adaptive and regulatory pathways.

Gene set enrichment analysis (GSEA) using GO terms from the MSigDB C5 mouse gene sets revealed that the LPS+B[a]P-induced COPD-relevant model strongly activated adaptive immune and lymphocyte signaling pathways (Figure 8A, Table S13 in the Supplementary Information). Dexamethasone effectively suppressed these immune pathways, consistent with its established role as a corticosteroid (Figure 8B). High doses of N102 (N102H, 3.0 g/kg) exhibited effects similar to dexamethasone in suppressing immune activation, with additional modulation of signaling and nuclear processes, indicating a multi-target protective mechanism (Figure 8C). Low-dose NRICM102 (N102L, 1.5 g/kg) downregulated immune activation and cytokine production in the COPD-relevant model, exhibiting effects similar to dexamethasone and N102H, though less potent (Figure 8D). Its modulation of cytokine-mediated responses suggests a primarily anti-inflammatory mechanism, focused on dampening inflammatory output rather than upstream immune signaling.

2.4. Effect of NRICM102 on Inflammatory and Immune-Related Protein Expression in COPD-Relevant Lung Injury

We next examined protein-level changes in key pathways reversed by NRICM102, focusing on cytokine signaling, including interleukin-1 beta (IL-1β), inhibin beta (Inhba), and guanylate binding protein 2b (Gbp2b); inflammatory activation, including IL-1β, NOD-like receptor protein 3 (NLRP3), and cluster of differentiation 14 (CD14); neutrophil chemotaxis and innate immunity, including lymphocyte antigen 6 family member G (Ly6g), interferon-activated gene 202B (Ifi202b), immunoglobulin heavy chain G (Ighg), histocompatibility 2, M region locus 2 protein (H2-M2), CD14, Gbp2b, and Inhba; adaptive immune response, including protein arginine methyltransferase 8 (Prmt8) and Ifi202b; and mucin production, including mucin 5AC (Muc5ac) and calcium-activated chloride channel regulator 1 (Clca1). Immunofluorescence analysis showed an elevated expression of IL-1β, Inhba, and Muc5ac in the bronchial epithelium of COPD-relevant lungs (Figure 9A), reflecting increased inflammation, epithelial remodeling, and mucus hypersecretion. NRICM102 reduced these markers in a dose-dependent manner, with dosages of 1.5 and 3.0 g/kg restoring expression levels to those comparable to the dexamethasone treatment groups. Similarly, NRICM102 also dose-dependently reduced the expression of Clca1, H2-M2, and CD14, markers of mucus secretion, antigen presentation, and innate immune activation, respectively (Figure 9B), as well as Prmt8, Ly6g, and Ifi202b, associated with oxidative stress, neutrophil infiltration, and cytotoxic inflammation (Figure 9C). It also suppressed NLRP3, Ighg, and MMP12, linked to inflammasome activation and B cell activity (Figure 9D), and decreased Gbp2b, markers of antigen-presenting cell activity (Figure 9E). Moreover, expression levels at 1.5 and 3.0 g/kg were comparable to those achieved with dexamethasone, indicating robust immunomodulatory effects. Together, these results suggest that NRICM102 effectively attenuated COPD-associated inflammatory and immune responses at the protein level, with high-dose treatment achieving therapeutic effects comparable to dexamethasone.

3. Discussion

COPD is characterized by progressive airflow limitation and chronic inflammation, with limited therapeutic options capable of reversing the underlying pathophysiology [6,19]. While corticosteroids such as dexamethasone offer symptomatic relief, their long-term use is associated with significant adverse effects. There is growing interest in exploring multi-target botanical formulations for their immunomodulatory potential in COPD [20,21,22]. In the present study, we investigated the therapeutic efficacy of NRICM102, a traditional botanical formula [16,23], against LPS and B[a]P-induced COPD-relevant lung injury in mice.

First, we confirmed that as a COPD-relevant model, LPS+B[a]P repeated exposure induced hallmark features of COPD, including increased airway ventilatory indices (including Penh), reduced arterial oxygen saturation, and lung tissue destruction. NRICM102 administration dose-dependently restored pulmonary function, with the 1.5 and 3.0 g/kg doses significantly reducing respiratory resistance and normalizing oxygen saturation to levels comparable to healthy controls. These improvements closely paralleled those observed with dexamethasone, highlighting the potent therapeutic potential of NRICM102 in maintaining pulmonary function. Second, histological analysis showed that NRICM102 markedly preserved alveolar structure, reduced inflammatory infiltration, and attenuated fibrotic remodeling, particularly at the highest dose, which exhibited similar efficacy to dexamethasone. Third, we performed a comprehensive analysis of the effect of NRICM102 on the transcriptomic profiling of lung tissues to elucidate the molecular mechanisms underlying these protective effects. PCA clustering revealed that NRICM102, especially at high doses, induced a global transcriptomic shift toward the control group, indicating partial reversal of COPD-associated gene expression signatures. DEGs analysis demonstrated that while the COPD-relevant model displayed extensive immune activation and suppression of regulatory pathways, NRICM102 selectively modulated a subset of genes associated with immune regulation and inflammation. High-dose NRICM102 (N102H) prominently downregulated genes involved in cytokine signaling, chemokine activity, and antigen presentation, while upregulating regulators of adaptive immunity. GO enrichment analyses further revealed that NRICM102 reversed COPD-associated dysregulation of key biological processes, particularly those related to leukocyte activation, T cell signaling, and cytokine-cytokine receptor interaction. DO analysis indicated that NRICM102 significantly suppressed genes linked to lung and immune diseases, including COPD. Notably, a subset of 150 genes were found to be reverted by NRICM102, suggesting a gene-specific restoration effect. These genes were enriched in immune-regulatory functions, particularly B cell-mediated immunity and cytokine signaling, both of which are crucial in the chronic inflammatory milieu of COPD. Finally, we assessed the key proteins involved in pathways associated with COPD using immunofluorescence staining. COPD-induced increases in key markers of inflammation (IL-1β, NLRP3), epithelial remodeling (Muc5ac, Clca1), innate immunity (CD14, Ly6g, Gbp2b), and adaptive responses (Ighg, Prmt8) were all attenuated by NRICM102 in a dose-dependent manner. These observations also provided validation at the protein level through immunofluorescence, thereby reinforcing the findings obtained from the transcriptomic analysis. Collectively, our results indicate that NRICM102 is associated with changes in innate and adaptive immune-related pathways, attenuation of inflammasome activation, and preservation of lung structure and function. This multi-target mechanism aligns with the complex pathology of COPD, where excessive immune activation and tissue remodeling play central roles. While our findings suggest potential immunomodulatory effects, further studies are required to directly demonstrate effects on immune reprogramming or homeostasis restoration.

The significant improvement in pulmonary function observed with NRICM102 is evidenced by improved respiratory resistance and arterial oxygen saturation, indicating enhanced airway openness and gas exchange. The bell-shaped dose–response observed in Penh measurements suggests an optimal therapeutic dose at 1.5 g/kg, providing maximal therapeutic benefit without unnecessary suppression of baseline lung function. This finding underscores the potential of NRICM102 as an effective steroid-sparing treatment for chronic lung inflammation.

The KEGG pathway analysis of upregulated DEGs in the comparison of “COPD-relevant vs. Sham” highlights a strong activation of immune and inflammatory pathways. The most significantly enriched pathway is cytokine-cytokine receptor interaction, reflecting robust transcriptional activation of cytokines and chemokines that mediate immune cell recruitment and intercellular signaling, key features of chronic inflammation in COPD. Notably, the enrichment of antigen processing and presentation and complement cascades further supports the involvement of both innate and adaptive immune responses. Additional upregulated pathways include phagosome, osteoclast differentiation, and chemokine signaling, indicating enhanced phagocytic activity, immune cell maturation, and sustained immune activation. Interestingly, several pathways associated with infectious and autoimmune diseases, such as tuberculosis, rheumatoid arthritis, leishmaniosis, and graft-versus-host disease, are also enriched, suggesting that the inflammatory landscape in this COPD-relevant model mirrors patterns seen in both chronic infections and immune dysregulation. Overall, these upregulated pathways reflect a transcriptome in COPD-relevant model characterized by immune cell infiltration, heightened inflammatory signaling, and potential auto-inflammatory features, all of which contribute to the persistent lung tissue damage and remodeling seen in this disease.

On the other hand, the KEGG pathway enrichment results from the “N102H vs. COPD-relevant” comparison is highly consistent with the findings from both GO and DO analyses, collectively pointing to a focused immunomodulatory effect of N102H. KEGG analysis highlights significant enrichment in pathways such as cytokine-cytokine receptor interaction, Th1, Th2, and Th17 cell differentiation, and T cell receptor signaling, all of which are critical to adaptive immune function. These results closely align with GO terms enriched in the same comparison, including adaptive immune response, regulation of lymphocyte activation, and T cell activation.

Furthermore, the DO analysis identified disease terms such as lung disease, obstructive lung disease, and chronic obstructive pulmonary disease, indicating that N102H-modulated genes are directly relevant to COPD pathophysiology. The convergence across GO, KEGG, and DO strongly suggests that N102H treatment acts through reprogramming adaptive immunity, particularly T cell-related signaling and regulation, rather than broadly suppressing inflammation. This coordinated shift may be associated with the effects of N102H on immune-related pathways and could contribute to mitigating disease-associated immune changes in COPD-relevant inflammatory lung injury.

Overall, transcriptomic analysis revealed that NRICM102 exerts a nuanced effect, partially restoring gene expression toward baseline levels. Unlike the broad suppression observed with dexamethasone, NRICM102 appears to selectively modulate inflammatory and pathogenic gene networks. However, it remains to be determined whether these transcriptomic changes translate into durable functional improvements and how NRICM102 achieves such molecular selectivity.

The COPD-relevant lung injury model displayed substantial activation of T and B cell-mediated immune responses. Specifically, increased expression of T cell receptor signaling genes (Zap70, Cd3g, and Lck) and elevated levels of MHC class II antigen presentation genes (H2-Ab1, Cd74, and H2-Eb1) indicated enhanced antigen-specific immune activity. Additionally, heightened expression of immunoglobulin genes (Ighg, Ighv, Igkv) highlighted an overactive B cell response characterized by increased antibody production, consistent with chronic immune activation seen in COPD (Tables S14–S17 in the Supplementary Information). NRICM102 treatment, particularly at the medium dose (N102M), effectively attenuated these exaggerated immune signatures by downregulating genes involved in antigen presentation, T cell activation, and antibody responses. This modulation suggests that NRICM102 selectively suppresses pathological immune responses without causing generalized immunosuppression. Furthermore, although the anti-inflammatory IL-10 signaling pathway (including Stat3, Socs3, and Il10ra genes) was upregulated in the COPD-relevant model, likely as a compensatory response, NRICM102 treatment normalized rather than further suppressed this pathway, demonstrating a balanced and refined immunomodulatory action.

Immunofluorescence imaging further confirmed that NRICM102 reduced expression of pathological markers including IL-1β, Muc5ac, NLRP3, Ighg, and MMP12, reflecting reduced epithelial stress, mucus production, and immune activation. Notably, decreased expression of MMP12 and NLRP3 suggests protective effects against alveolar degradation and emphysematous progression, crucial for maintaining lung integrity and function in COPD [24]. Furthermore, MMP-12 and glutathione S-transferase has been associated with a decline in lung function as well as an increased risk of COPD [25,26]. Despite these promising cellular-level findings, challenges remain in translating these outcomes from animal models to human patients. Future studies should focus on validating these cellular and molecular effects in human tissue samples or clinical trial contexts.

From a translational perspective, the observed efficacy of NRICM102 in reversing transcriptomic and proteomic alterations in COPD-relevant model supports its potential as a complementary or alternative therapeutic agent. Importantly, the immunoregulatory signature of NRICM102, especially its effects on T cell signaling and antigen processing, distinguishes it from traditional anti-inflammatory drugs and supports its potential role in restoring immune homeostasis in chronic airway diseases. The ability to modulate cytokine networks and immune cell activation without the broad immunosuppressive effects of corticosteroids makes NRICM102 a promising candidate for further clinical development. Analysis of enriched immune-related pathways revealed that NRICM102 effectively modulates cytokine signaling, viral response, and T/B cell activation pathways. Notably, NRICM102 restored immune balance without activating cardiovascular or muscle dysfunction pathways, common adverse effects associated with prolonged corticosteroid use [27,28]. While these findings are promising, long-term safety studies are essential to confirm the absence of subtle or delayed adverse effects during extended use of NRICM102. Previous reports demonstrate that NRICM102 effectively alleviates severe COVID-19 symptoms through immune modulation in ICU patients [14,16,29] and provides protective effects against bongkrekic acid-induced toxicity in animal models [30]. Nevertheless, further research is necessary to validate the efficacy and safety of NRICM102 across diverse and complex clinical scenarios, including acute COPD exacerbations.

Despite the robust protective effects observed in this study, several limitations should be acknowledged. First, the LPS+B[a]P protocol represents a COPD-relevant inflammatory lung injury model rather than a true chronic COPD model. Compared with long-term cigarette smoke-induced models, the LPS+B[a]P-induced COPD-relevant model does not exhibit progressive alveolar destruction, small airway remodeling, or irreversible airflow limitation. LPS+B[a]P-induced COPD-relevant model represents an acute-to-subacute COPD-relevant inflammatory lung injury associated with environmental and microbial insults, rather than a typical chronic COPD model. Therefore, extrapolation of these findings to established COPD should be made with caution. Second, the pulmonary function assessment in the present study relied primarily on non-invasive whole-body plethysmography. Although widely used for longitudinal evaluation in murine studies, these measurements provide only indirect estimates of airway physiology and do not fully substitute for invasive lung mechanics. Third, transcriptomic analysis indicated that NRICM102 partially restores gene expression toward baseline. However, functional enrichment analyses, including GO, KEGG, DO, and GSEA, represent patterns-recognition or pathway-level associations rather than direct evidence of causality. Future studies are needed to identify causal nodes and determine whether NRICM102 directly modulates specific immune pathways or indirectly reflects reduced tissue injury and inflammation. Fourth, immunofluorescence analyses were conducted on whole-lung tissue, which limits cell-type-specific resolution and precludes definitive attribution of observed molecular changes to distinct immune or structural cell populations. Finally, this study did not evaluate long-term efficacy, durability of response, or safety under prolonged administration.

4. Materials and Methods

4.1. Animal and COPD-Relevant Mice Model

All animal experiments were approved by the Animal Research Committee of the National Research Institute of Chinese Medicine (Approval No. NRICM-IACUC-114-9121-3; Approval Date: 19 February 2022) and conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). Male C57BL/6 mice (6–8 weeks old) were obtained from BioLASCO Taiwan Co. (Taipei, Taiwan) and housed in ventilated cages under standardized environmental conditions (22 °C, ~60% relative humidity, 12 h light/12 h dark cycle). Animals were provided ad libitum access to purified water and a standard diet, and bedding was replaced regularly. The animals were acclimated to the housing environment for 1–2 weeks before the start of the study. Animals were randomly assigned to six groups (n = 5 per group): sham, LPS+B[a]P-induced COPD-relevant model with vehicle, three COPD-relevant model groups receiving NRICM102 at 0.75, 1.5, or 3.0 g/kg, and a positive control group receiving dexamethasone at 2.0 mg/kg. For COPD-relevant model induction, mice received intranasal LPS (20 µg/mouse; Cat. No. L6529, Sigma-Aldrich, St. Louis, MO, USA) on Day 1, followed by intratracheal B[a]P (1.0 mg/mouse; Cat. No. B1760, Sigma-Aldrich, St. Louis, MO, USA) on Day 2 of each week for three consecutive weeks, modeling chronic airway inflammation and structural lung alterations [12]. NRICM102 and dexamethasone (Cat. No. HY-14648, MedChemExpress, Monmouth Junction, NJ, USA) were administered orally at doses of 0.75, 1.5, or 3.0 g/kg, and 2.0 mg/kg, respectively, once daily for five consecutive days in each weekly cycle over the three-week period. Control mice received saline containing 0.1% DMSO according to the same schedule and administration route. On day 21, pulmonary function was assessed non-invasively using the FinePointe Whole Body Plethysmography (WBP) system (Buxco Research Systems, St. Paul, MN, USA), measuring respiratory parameters such as enhanced pause (Penh) and arterial oxygen saturation (aO₂%) [31,32]. To reduce potential confounders, experiments were conducted as follows: (1) animals were monitored daily for any abnormalities in behavior, activity, or general appearance; (2) repeated administrations were carried out at consistent intervals; (3) measurements were taken in a consistent order and at similar times for all animals. Body weight data (day 0~21) are summarized in Table S18. No pre-defined exclusion criteria were applied. At the end of the study, mice were humanely euthanized for tissue collection. Animals were first anesthetized with isoflurane (2–3% induction, 1.5–2% maintenance; Cat. No. I-110, Sigma-Aldrich, St. Louis, MO, USA) delivered via a calibrated vaporizer. Euthanasia was then performed using a gradual-fill CO₂ displacement method (20–30% chamber volume/min) with an additional 2–3 min of exposure to ensure death, in accordance with AVMA 2020 guidelines. All animals survived until the end of the experimental schedule, and all data points were included in the analysis. Lung tissues underwent histological evaluation using hematoxylin and eosin (H&E), Masson’s trichrome staining, immunohistochemical (IHC) staining for inflammatory and fibrotic markers, and next-generation sequencing (NGS) for transcriptomic analysis.

To minimize pain and distress, mice undergoing intratracheal administration or other invasive handling were anesthetized (e.g., isoflurane inhalation) to ensure loss of consciousness during procedures and were monitored during recovery and daily thereafter (activity, posture, grooming/respiration, body weight, and signs of distress). Predefined humane endpoints were applied, and animals meeting criteria (e.g., significant weight loss, persistent respiratory distress, or severe lethargy) were promptly removed and humanely euthanized. Standard husbandry (ad libitum food/water and environmental enrichment) was maintained, and the 3Rs were followed by refining procedures and minimizing animal numbers through study design and statistical planning.

4.2. Preparation of NRICM102

The herbal ingredients used in the NRICM102 formulation were sourced from certified traditional Chinese medicine (TCM) pharmacies at Taichung Veterans General Hospital, Taiwan. The formulation comprises ten medicinal plant components: Houttuynia cordata, Wolfiporia extensa, Trichosanthes kirilowii, Artemisia capillaris, Scutellaria baicalensis, Polygonatum odoratum, Glycyrrhiza glabra, Magnolia officinalis, Pinellia ternata, and Aconitum carmichaelii. The identity of each herb was verified by Dr. Chia-Ching Liaw, curator of the NRICM herbarium, and corresponding voucher specimens (NRICM-LC2020012-2021) have been archived at the National Research Institute of Chinese Medicine (NRICM), Taipei. To prepare NRICM102, prescribed quantities of each herbal ingredient were combined—ranging from 7.5 to 37.5 g per component—and decocted in 1200 mL of water. The mixture was reduced to approximately 300 mL through simmering and subsequently lyophilized, yielding a dry extract (17.7 ± 0.5 g per batch). Chemical profiling of the extract was performed using high-performance liquid chromatography (HPLC) on a Shimadzu Nexera-i LC-2050C 3D system with a COSMOSIL 5C₁₈-AR-II column (ID. 4.6 mm × 250 mm). The mobile phase consisted of distilled water containing 0.3% phosphoric acid and acetonitrile, applied under a gradient elution (0–60 min: 0–100% ACN). Analytical parameters included a flow rate of 1.0 mL/min, injection volume of 10 μL, column temperature of 40 °C, and sample concentration of 10 mg/mL. Key phytochemicals were further purified by column chromatography and structurally confirmed through NMR spectroscopy and high-resolution electrospray ionization mass spectrometry. Finally, the HPLC fingerprint of NRICM102 with the assignment of 22 compounds is shown in Figure 10.

4.3. Hematoxylin and Eosin (H&E) and Masson’s Trichrome Staining

Lung tissues were harvested at the indicated time points, gently perfused with phosphate-buffered saline (PBS), and fixed in 10% neutral buffered formalin for at least 24 h. Fixed tissues were then dehydrated through a graded ethanol series, cleared in xylene, and embedded in paraffin. Paraffin blocks were sectioned at a thickness of 4–5 μm using a rotary microtome and mounted on glass slides. For hematoxylin and eosin (H&E) staining, sections were deparaffinized in xylene, rehydrated through graded ethanol to water, and stained with hematoxylin, followed by eosin according to standard protocols. Stained sections were dehydrated, cleared, and coverslipped for histopathological evaluation.

Masson’s trichrome (MTC) staining was performed on adjacent sections using a standard trichrome staining protocol to assess collagen deposition and fibrotic changes. Briefly, rehydrated sections were sequentially stained with Weigert’s iron hematoxylin, Biebrich scarlet–acid fuchsin, phosphomolybdic/phosphotungstic acid, and aniline blue, followed by differentiation, dehydration, and mounting.

Histopathological evaluation and scoring: H&E- and Masson’s trichrome-stained lung sections were evaluated independently by two investigators who were blinded to the experimental groups. For each mouse, two non-adjacent paraffin sections were analyzed. From each section, five randomly selected, non-overlapping microscopic fields were captured under identical conditions using a digital imaging system (Motic DSAssistant, 4K; Motic China Group Co., Ltd., Xiamen, China). For H&E staining, inflammatory changes were scored using a semi-quantitative scale from 0 to 3 based on the extent and severity of peribronchiolar, perivascular, and alveolar inflammatory cell infiltration (0, none; 1, mild; 2, moderate; 3, severe) [33].

For Masson’s trichrome staining, collagen deposition and fibrotic remodeling were assessed using the Ashcroft scoring system, as originally described by Ashcroft et al. [34] and subsequently standardized by Hübner et al. [35]. Scores from all fields were averaged to generate a single value for each mouse.

4.4. RNA Sequencing and Bioinformatics

For transcriptomic analysis, freshly excised lung tissues were rapidly snap-frozen in liquid nitrogen and stored at −80 °C until use. Total RNA was extracted immediately from collected lung tissues using TRIzol reagent (Cat. No. 15596018, ThermoFisher, Thermo Fisher Scientific, Waltham, MA, USA) according to standard protocols. RNA quality and concentration were assessed using a SimpliNano™ spectrophotometer (Biochrom, Analytik Jena/Biochrom Ltd., Cambridge, MA, USA), while integrity was verified through capillary electrophoresis using a Qsep100 analyzer (BiOptic Inc., New Taipei City, Taiwan). Sequencing libraries were prepared with the KAPA mRNA HyperPrep Kit (Roche, Switzerland) following the manufacturer’s instructions, and sequencing was performed on an Illumina NovaSeq 6000 platform to generate paired-end reads. Raw sequence quality was initially evaluated with FastQC and summarized using MultiQC. Subsequently, adapter sequences and low-quality reads were removed using Trimmomatic (v0.38). Clean reads were mapped to the reference genome via HISAT2 (v2.1.0), and gene-level counts were obtained using FeatureCounts (v2.0.0). Differential expression analysis was carried out using DESeq2 (v1.26.0) or DEGseq (v1.40.0), selected according to data characteristics and experimental design. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed using the clusterProfiler R package (v3.14.3). Analytical methods were consistently applied across all biological replicates, ensuring robust and reproducible results. Gene Ontology (GO) enrichment analysis was performed using annotations from the Gene Ontology Consortium (accessed 4 October 2024). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway information was retrieved from the KEGG database (accessed 4 October 2024). Disease Ontology (DO) analysis was conducted using disease terms curated from the Disease Ontology database (accessed 4 October 2024). Gene set enrichment analysis (GSEA) was performed using MSigDB C5 mouse gene sets (accessed 4 October 2024). The reference genome and gene annotation files (GRCm38/mm10) were obtained from the Ensembl database (accessed 4 October 2024).

4.5. Immunohistochemical Staining

Lung tissues were embedded in OCT compound, snap-frozen, and sectioned at a thickness of 10 µm. Sections were air-dried, fixed in cold acetone for 10 min at −20 °C, and rinsed with PBS. After permeabilization with 0.1% Triton X-100 for 10 min, sections were blocked with 5% normal goat serum for 1 h at room temperature. Primary antibodies against inflammatory and tissue remodeling markers, including IL-1β (1:100, Cat. No. 503501, BioLegend), Inhba (1:100, Cat. No. DF12283, Affinity Biosciences), Muc5ac (1:200, Cat. No. V2198, NSJ Bioreagents), Prmt8 (1:100, Cat. No. 83283, Cell Signaling), Ly6g (1:100, Cat. No. GTX40912, GeneTex), Ifi202b (1:50, Cat. No. SC-166253, Santa Cruz), Clca1 (1:100, Cat. No. DF13501, Affinity Biosciences), H2-M2 (1:100, Cat. No. NBP3-15581, Novusbio), CD14 (1:200, Cat. No. Ab182032, Abcam), NLRP3 (1:100, Cat. No. IR92-388, iREAL), Ighg (1:500, Cat. No. DCABH-5058, Creative Diagnostics), MMP12 (1:50, Cat. No. SC-390284, Santa Cruz), and Gbp2b (C1:100, at. No. ARP97832_P050, Aviva Systems Biology) were applied overnight at 4 °C. Details of the primary antibodies are provided in the Supplementary Information. Following PBS washes, sections were incubated for 1 hr at room temperature in the dark with Alexa Fluor-conjugated secondary antibodies specific to the primary antibody species, donkey anti-rabbit, donkey anti-mouse, or donkey anti-rat (Cat. Nos. A31571, A31573, A78945, respectively; ThermoFisher, MA, USA), each at a 1:500 dilution. Nuclei were counterstained with DAPI (Cat. No. D9542, Sigma-Aldrich, St. Louis, MO, USA), and slides were mounted using antifade mounting medium. Fluorescent images were captured using a Zeiss LSM780 confocal microscope under standardized imaging conditions. Semi-quantitative analyses were conducted by selecting 3–5 representative, non-overlapping fields from peribronchial and alveolar regions, independently assessed by two blinded observers. Immunofluorescence intensities were quantified with ImageJ v. 1.54p, Zen 2011 v. 1.0 (Carl Zeiss), or AlphaEase FC v. 4.0 (Alpha Innotech), converting fluorescence channels to grayscale and measuring mean intensity within defined regions of interest (ROIs). Background signals were corrected using secondary-only controls, and data were normalized relative to sham-treated controls. Adjacent sections were subjected to histopathological analysis using hematoxylin and eosin (H&E) and Masson’s trichrome staining according to standard protocols to assess inflammation, alveolar damage, and fibrosis. Tissue damage was quantified using ImageJ analysis of staining intensity and fibrotic area coverage, alongside a standardized semi-quantitative scoring (0–3) for alveolar destruction, inflammatory cell infiltration, and fibrosis severity. Scores from two blinded observers were averaged across three fields per section for each sample. This histopathological approach follows validated protocols commonly used in murine models of chronic pulmonary injury [18].

4.6. Statistical Analysis

Statistical analysis was performed using GraphPad Prism (version 9.0). Normality was evaluated using the Shapiro–Wilk test. Data that met normality assumptions are presented as mean ± SEM and were analyzed using one-way ANOVA with Tukey’s post hoc test. When normality was not satisfied (Figure 1B), data are presented as median (interquartile range) and were analyzed using ANOVA on ranks (Kruskal–Wallis test) followed by Dunn’s multiple-comparisons test. Statistical significance was set at p < 0.05.

5. Conclusions

Collectively, the findings suggest NRICM102 exerts protective effects in a COPD-relevant inflammatory lung injury model, indicating that NRICM102 may serve as a promising botanical intervention for COPD and related inflammatory lung diseases. Transcriptomic analysis reveals that NRICM102 may influence multiple pathological features associated with COPD. Collectively, these findings indicate that NRICM102 confers protective effects in a COPD-relevant inflammatory lung injury model, suggesting its potential relevance to COPD and related inflammatory lung diseases. Transcriptomic analyses together with immunohistochemical and immunofluorescence staining suggest that NRICM102 may modulate several COPD-associated pathological processes, supporting its consideration as a potential steroid-sparing therapeutic candidate. However, these results should be interpreted within the limitations of the experimental model. Further studies are required to evaluate its efficacy in clinically relevant settings, to define long-term safety and optimal dosing regimens, and to assess potential interactions with existing COPD therapies. In addition, further investigation of each bioactive constituent of NRICM102 may provide mechanistic insight and inform the development of targeted strategies for inflammatory and fibrotic lung diseases.