SCM-198 ameliorates pulmonary arterial hypertension by modulating gut microbiota in rats

Introduction

Pulmonary arterial hypertension (PAH) is a chronic and progressive vascular disease characterized by elevated pulmonary arterial pressure and extensive vascular remodeling, ultimately leading to right heart failure and high mortality (1,2). Vascular aging, marked by endothelial dysfunction, oxidative stress, and chronic inflammation, is now recognized as a critical driver of vascular remodeling and a key pathogenic mechanism underlying PAH and other age-related cardiopulmonary disorders (3). Although current therapies, including prostacyclin analogs, endothelin receptor antagonists, and phosphodiesterase-5 inhibitors, provide clinical benefits by alleviating symptoms, they generally fail to reverse established vascular remodeling and are often associated with adverse effects (4,5). Given the pivotal role of vascular aging in PAH, microbiota-driven mechanisms present a promising avenue for therapeutic exploration. In recent years, gut microbiota has emerged as a major modulator of vascular health. The bidirectional “gut-lung axis” highlights how alterations in microbial composition may contribute to pulmonary inflammation and vascular remodeling (6-8). Dysbiosis of gut microbiota has been linked to endothelial dysfunction, systemic inflammation, and oxidative stress, which are central to the pathogenesis of vascular aging (9,10). Moreover, studies have shown that microbial metabolites such as short-chain fatty acids (SCFAs) and trimethylamine N-oxide (TMAO) directly impact vascular tone and inflammation (11,12).

SCM-198, an active alkaloid extracted from Leonurus japonicus Houtt., has demonstrated potent anti-inflammatory, antioxidant, and anti-apoptotic effects in cardiovascular and neurovascular diseases (13). However, its role in pulmonary vascular aging remains largely unexplored. Current evidence suggests that SCM-198 may exert vasoprotective effects through direct cellular mechanisms, including the regulation of oxidative stress, apoptosis, and endothelial function (14).

SCM-198 is known for its antioxidant, anti-apoptotic, and endothelial-protective effects, yet its role in pulmonary vascular aging remains unclear. While the influence of SCM-198 on gut microbiota remains to be elucidated, the gut-lung axis is increasingly recognized as a key pathway in pulmonary vascular health. In this study, we hypothesize that SCM-198 may attenuate pulmonary vascular aging by reducing vascular remodeling and potentially influencing the gut microbiota. Using an MCT-induced PAH rat model and 16S ribosomal RNA (rRNA) sequencing, we explored the therapeutic potential of SCM-198 through the gut-lung axis. We present this article in accordance with the ARRIVE reporting checklist (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1363/rc).

Methods

Animals

A total of 30 specific pathogen-free (SPF)-grade male Sprague-Dawley (SD) rats (weighing 140–150 g) were obtained from the Guangzhou Saiye Biotechnology Co., Ltd. [license No. SCXK (yue)-2020-0055]. Rats were randomly assigned to four groups: control group (CON, n=6), monocrotaline (MCT)-induced model group (n=8), low-dose (LD) SCM-198 treatment group (MCT + SCM-198 50 mg/kg, n=8), and high-dose (HD) SCM-198 treatment group (MCT + SCM-198 100 mg/kg, n=8). All animals were housed in an SPF facility under controlled conditions (temperature 25±1 °C, humidity 50%±5%, 12-h light/dark cycle) and acclimatized for one week prior to the start of the experiment. All experimental procedures were approved by the Animal Ethics Committee of Macau University of Science and Technology (approval No. AL006/DICV/DIS/2021), in compliance with institutional guidelines for the care and use of animals. A protocol was prepared before the study without registration.

Establishment of animal models

To establish a rat model of PAH, all animals except those in the control group received a single intraperitoneal injection of 2% MCT at a dose of 50 mg/kg. One week after MCT administration, rats in the treatment groups were given SCM-198 daily by oral gavage at the designated doses for three consecutive weeks, while rats in the CON and MCT groups received an equal volume of normal saline. Throughout the experiment, animals were maintained under standard conditions with free access to food and water.

Histological evaluation of pulmonary arteries

Rats were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally), after which the heart and lungs were harvested. The left lung was perfused with 4% paraformaldehyde via the trachea and fixed in the same solution for at least 24 hours. Tissues were then processed through a graded ethanol dehydration series, cleared with xylene, embedded in paraffin, and sectioned longitudinally at 4 µm thickness. Slides were dried on a warming plate, deparaffinized with xylene, stained with hematoxylin and eosin (H&E), dehydrated again, cleared, and mounted using neutral resin. Lung tissue sections were observed under a light microscope. Pulmonary arterioles with external diameters ranging from 150 to 300 µm were selected for imaging under identical magnification. The luminal area and total cross-sectional area of the vessels were measured using ImageJ software, and medial wall thickness ratio (MWT) was calculated as:

MWT=Totalvesselarea−LumenareaTotalvesselarea⋅100%[1]

To assess right ventricular hypertrophy, the right ventricle (RV) was dissected from the left ventricle plus septum (LV + S) after removing the atria. Tissues were dried with filter paper and weighed separately. The right ventricular hypertrophy index (RVHI) was calculated as follows:

RVHI=RV/(LV+S)[2]

16S rRNA gene amplification

Fecal samples from the CON (C), MCT (M), and SCM-198 treatment groups (LD and HD) were collected and immediately stored at −80 °C. Genomic DNA was extracted following standard protocols. The V3–V4 hypervariable region of the bacterial 16S rRNA gene was amplified using barcoded universal primers. Polymerase chain reaction (PCR) was performed using the KAPA HiFi Hot Start Ready Mix (2×) (TaKaRa Bio Inc., Shiga, Japan) along with polyacrylamide gel electrophoresis (PAGE)-purified primers: forward (CCTACGGGNGGCWGCAG) and reverse (GACTACHVGGGTATCTAATCC). Each PCR reaction consisted of 2 µL of DNA template (10 ng/µL), 1 µL of each primer (10 µM), and 15 µL of 2× PCR mix, totaling 19 µL. Amplification was carried out in a thermal cycler (Applied Biosystems 9700, Foster City, CA, USA) using the following conditions: initial denaturation at 95 °C for 3 min; five cycles of 95 °C for 30 s, 45 °C for 30 s, and 72 °C for 30 s; followed by 20 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s; with a final extension at 72 °C for 5 min. PCR products were analyzed by electrophoresis on 1% agarose gels prepared in TBE buffer, stained with ethidium bromide, and visualized under ultraviolet (UV) illumination.

Library preparation and sequencing

The purified PCR products were subjected to library construction using the Ion Plus Fragment Library Kit (Thermo Scientific, Waltham, MA, USA; catalog No. 4471252), according to the manufacturer’s instructions. The quality and concentration of the resulting libraries were evaluated using a Qubit^® 2.0 Fluorometer (Thermo Scientific). Libraries passing quality control were pooled in in equimolar amounts and sequenced on the Illumina NovaSeq 6000 platform to generate paired-end reads with lengths ranging from 400 to 600 bp.

Sequence data processing

Raw reads were preprocessed to remove low-quality sequences and adapter contamination using Trimmomatic (v0.33), followed by primer removal via Cutadapt (v1.9.1). After merging paired-end reads, potential chimeric sequences were eliminated using the UCHIME algorithm (v8.1) implemented in USEARCH (v10). The remaining high-quality sequences were retained for downstream analysis. Amplicon sequences were denoised or clustered into representative features, referred to as operational taxonomic units (OTUs) or amplicon sequence variants (ASVs), depending on the analysis workflow. Taxonomic assignments were made based on a reference database, and community composition was profiled across multiple taxonomic levels (from phylum to species).

Diversity and community structure analysis

Alpha diversity was estimated using richness and evenness metrics, including abundance-based coverage estimator (ACE), Chao1, Shannon, and Simpson indices. To evaluate sampling depth and species richness, rarefaction and rank-abundance plots were constructed. Beta diversity was analyzed to compare microbial composition across groups. Community dissimilarity was visualized using principal coordinate analysis (PCoA), principal component analysis (PCA), and non-metric multidimensional scaling (NMDS) based on Bray-Curtis or UniFrac distance metrics. Unweighted Pair Group Method with Arithmetic Mean (UPGMA) and boxplot visualizations were also employed to assess inter-group variation.

Differential abundance and functional profiling

Intergroup differences in microbial taxa were evaluated at various classification levels. Linear discriminant analysis effect size (LEfSe) was applied to detect significantly enriched taxa as potential biomarkers.

Network pharmacology analysis

The two-dimensional (2D) structure of SCM-198 was obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/) (CID: 161464). Potential targets of SCM-198 were predicted using the SwissTargetPrediction database (http://swisstargetprediction.ch/) and PharmMapper database (https://www.lilab-ecust.cn/pharmmapper/index.html). The potential protein targets identified by PharmMapper were converted to gene symbols via the UniProt database (https://www.uniprot.org/). PAH-related targets were retrieved from the OMIM database (https://www.omim.org/) and GeneCards database (https://www.genecards.org/) using the keyword “pulmonary arterial hypertension”. Common targets between SCM-198 and PAH were visualized using Venny 2.1 (https://bioinfogp.cnb.csic.es/tools/venny/). These intersecting genes were used for further functional enrichment and network construction. Common targets were uploaded to the STRING database (https://string-db.org/) to construct a protein-protein interaction (PPI) network with a confidence threshold >0.4, and disconnected nodes were hidden. The PPI network data were imported into Cytoscape software (v3.9.1). Node degree values were calculated using the “NetworkAnalyzer” tool. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed for common targets using the Hiplot platform (https://hiplot.com.cn/home/index.html) with a significance threshold of P<0.05. Results were visualized as bubble plots, displaying the top 20 significantly enriched biological processes (BPs), molecular functions (MFs), and signaling pathways.

Statistical analysis

All statistical analyses were conducted using R (v4.2.0) and GraphPad Prism 8.0. Depending on the data distribution, either Student’s t-test or one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used to compare groups. For non-normally distributed data, non-parametric tests such as the Wilcoxon rank-sum test were applied. A P value less than 0.05 was considered statistically significant.

Results

SCM-198 alleviates pulmonary arterial remodeling in MCT-induced PAH rats

To assess the therapeutic effect of SCM-198 on pulmonary vascular remodeling, histological changes in pulmonary arterioles were examined by H&E staining. As shown in Figure 1A, the control group exhibited normal vascular morphology with a thin and regular medial layer. In contrast, the MCT group showed significant thickening of the vascular wall and reduced luminal area, indicating severe pulmonary arterial remodeling. Notably, treatment with SCM-198 at both 50 and 100 mg/kg mitigated these pathological changes, with the HD group demonstrating a more pronounced improvement in vessel wall structure.

Figure 1 SCM-198 attenuates pulmonary vascular remodeling in MCT-induced PAH rats. (A) Representative H&E-stained images of pulmonary arterioles (external diameter 150–300 μm) from each group. The MCT group exhibits significant medial thickening compared to the control group, while SCM-198 treatment alleviates vascular remodeling in a dose-dependent manner. Scale bar =50 μm. (B) Quantification of PVR as MWT% among groups. Data are presented as mean ± SD (n=6–8). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test; P<0.05 was considered significant. (C) HW/BW across four groups: control, model (MCT-induced PAH rats), and SCM-198 treatment groups (50 mg/kg, 100 mg/kg). Statistical analysis was performed using one-way ANOVA followed by post hoc tests. Data are presented as mean ± SD (n=6); P<0.05, P<0.01. (D) Quantitative analysis of RVHI across experimental groups. The MCT-induced model group exhibited a significant increase in RVHI compared to the control group, indicating the development of right ventricular hypertrophy. Treatment with SCM-198 at both 50 and 100 mg/kg effectively reduced RVHI, with the HD group showing a more pronounced reduction. Data are expressed as mean ± SD (n=3). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. P<0.05 was considered statistically significant. ANOVA, analysis of variance; H&E, hematoxylin and eosin; HD, high dose; HW/BW, heart weight to body weight ratio; LV + S, left ventricle plus septum; MCT, monocrotaline; MWT%, medial wall thickness percentage; PAH, pulmonary arterial hypertension; PVR, pulmonary vascular remodeling; RV, right ventricle; RVHI, right ventricular hypertrophy index; SD, standard deviation.

Quantitative analysis of pulmonary arterial wall thickness (Figure 1B) revealed a significant increase in the MWT% in the MCT group compared to the control group (P=0.001). Administration of SCM-198 significantly reduced MWT% in a dose-dependent manner, with the HD SCM-198 group showing the most significant attenuation compared to the MCT group (P=0.002).

Besides, we also detected the heart weight and body weight of all rats. Our results showed that SCM-198 relieved MCT-induced cardiac hypertrophy (Figure 1C). The heart weight to body weight ratio (HW/BW) was significantly elevated in the MCT-induced model group compared to the control group (P=0.001), reflecting right ventricular hypertrophy due to pulmonary hypertension. Treatment with SCM-198 at 50 and 100 mg/kg effectively reduced the HW/BW ratio. Notably, the HD group (100 mg/kg) showed a more pronounced reduction compared to the model group (P<0.001) and the LD group (P=0.03), indicating a dose-dependent protective effect of SCM-198 against cardiac hypertrophy.

In addition, right ventricular hypertrophy was assessed using the RVHI. MCT administration significantly elevated RVHI compared to the control group (P<0.001). SCM-198 treatment reduced RVHI in a dose-dependent manner, with the 100 mg/kg group showing a more pronounced reduction compared to both the model group (P<0.001) and the 50 mg/kg group (P=0.006) (Figure 1D).

SCM-198 modulates gut microbiota composition at multiple taxonomic levels in a PAH rat model

In order to explore the mechanism of SCM-198 in treating PAH induced by MCT, we conducted 16 s RNA sequencing and analyzed the sequencing results. At the phylum level (Figure 2A), the gut microbiota was predominantly composed of Firmicutes, Bacteroidota, and Proteobacteria. Compared with the control group (C), the MCT-induced model group (M) exhibited a decreased abundance of Bacteroidota and an increased abundance of Actinobacteriota. In both SCM-198 treatment groups (LD and HD), Bacteroidota levels were elevated, while Actinobacteriota levels were declined. At the family level (Figure 2B), the relative abundance of Erysipelotrichaceae was increased, while Muribaculaceae was decreased in the M group compared with C. These trends were reversed in the LD and HD groups following SCM-198 treatment. At the genus level (Figure 2C), unclassified_Muribaculaceae was reduced in the M group and increased in the LD and HD groups. Lactobacillus abundance was elevated in the M group but decreased after treatment. Unclassified_Lachnospiraceae was reduced in M, remained low in LD, and increased in HD.

Figure 2 Gut microbial composition at different taxonomic levels. (A) Microbial composition at the phylum level. Compared with the control group [C], the MCT-induced model group [M] exhibited a reduction in Bacteroidota and an increase in Actinobacteriota. These changes were reversed in the SCM-198 treatment groups (LD and HD). (B) Relative abundance of fecal microbiota at the family level. The M group showed elevated levels of Erysipelotrichaceae and reduced levels of Muribaculaceae compared to C. SCM-198 treatment (LD and HD) mitigated these changes. (C) Relative abundance of dominant bacterial genera. In the M group, unclassified_Muribaculaceae decreased and was restored after SCM-198 treatment. Lactobacillus increased in M and declined in LD and HD. Unclassified_Lachnospiraceae was reduced in M and recovered in HD. C, control group; LD and HD, SCM-198 low- and high-dose treatment groups, respectively; M, MCT-induced model group; MCT, monocrotaline.

Alpha diversity analysis of gut microbiota

Alpha diversity indices were employed to evaluate microbial richness (Chao1, ACE) and diversity (Shannon, Simpson) across the four groups (Figure 3). Although no statistically significant differences were detected (P>0.05), a decreasing trend in diversity and richness was observed in the MCT-induced model group compared to the control group. Both LD and HD SCM-198 treatment groups exhibited an upward trend in diversity indices, particularly in Shannon and Chao1, suggesting a potential tendency toward microbiota restoration.

Figure 3 Alpha diversity indices of gut microbiota in each group. (A) Simpson index, (B) Shannon index, (C) Chao1 index, and (D) ACE index. No statistically significant differences were observed among groups (P>0.05). However, the model group [M] showed a decreasing trend in microbial richness and evenness compared to the control group [C]. Both SCM-198 treatment groups (LD and HD) exhibited a tendency toward recovery, particularly in Shannon and Chao1 indices. ACE, abundance-based coverage estimator; C, control group; LD and HD, SCM-198 low- and high-dose treatment groups, respectively; M, MCT-induced model group; MCT, monocrotaline.

Beta diversity analysis of gut microbiota

PCoA and NMDS were conducted to evaluate beta diversity among the four experimental groups based on gut microbial composition. As shown in Figure 4A, PCoA revealed a clear separation among the control, model, and SCM-198 treatment groups along the first two principal coordinates (PC1: 40.43%, PC2: 16.46%), indicating substantial differences in community structure. Notably, the model group (MCT-induced) deviated markedly from the control group, whereas both the LD and HD SCM-198 groups exhibited partial restoration toward the control cluster, suggesting a dose-responsive modulatory effect of SCM-198 on gut microbiota. Consistently, NMDS analysis (Figure 4B) demonstrated distinct clustering among groups, with a stress value of 0.1083, indicating a reliable ordination. These findings further support that SCM-198 treatment ameliorated MCT-induced dysbiosis and contributed to the reestablishment of gut microbial homeostasis.

Figure 4 Beta diversity analysis of gut microbiota. (A) PCoA based on the Weighted UniFrac distance reveals distinct clustering of microbial communities among groups. Notably, LD and HD treatment groups show a partial shift toward the control group, indicating microbiota restoration. (B) NMDS plot (stress =0.1083) further confirms intergroup differences in microbial composition. LD and HD, SCM-198 low- and high-dose treatment groups, respectively; NMDS, non-metric multidimensional scaling; PCoA, principal coordinate analysis.

LEfSe analysis reveals group-specific microbial signatures in response to SCM-198

LEfSe analysis identified key discriminatory taxa across groups. At the genus level (g__), Clostridium_sensu_stricto_1 was significantly enriched in the model group, potentially reflecting dysbiotic expansion linked to PAH pathology. In contrast, Fusobacterium was predominantly associated with the LD treatment group, while the HD group was characterized by the enrichment of Candidatus_Saccharimonas and Lachnospiraceae_NK4A136_ group. At the family level (f__), Fusobacteriaceae and Clostridiaceae were elevated in the model group, consistent with their known roles in inflammation and microbial imbalance. Conversely, Saccharimonadaceae was enriched in the HD group, suggesting a treatment-associated shift in community structure. At the phylum level (p__), Fusobacteriota was notably enriched in the LD group. Collectively, these taxa may serve as microbial indicators of PAH progression as well as targets modulated by SCM-198 intervention (Figure 5A,5B).

Figure 5 LEfSe analysis of gut microbiota among groups. (A) Cladogram generated from LEfSe analysis highlights taxa with significant differential abundance (LDA score >2.0) among Control (red), Model (purple), LD (blue), and HD (green) groups. (B) Histogram of LDA scores (log10) shows the most discriminative taxa contributing to group differences. LD and HD, SCM-198 low- and high-dose treatment groups, respectively; LDA, linear discriminant analysis; LEfSe, linear discriminant analysis effect size.

Network pharmacological analysis results

In network pharmacology, a total of 343 potential targets of SCM-198 were predicted via PharmMapper and SwissTargetPrediction databases, while 9,477 PAH-related targets were retrieved from OMIM and GeneCards databases. Venn analysis identified 71 overlapping targets (Figure 6A), suggesting their critical role in the therapeutic effects of SCM-198 against PAH. The PPI network of 71 common targets was constructed using the STRING database. Degree analysis revealed the top 10 core targets: Akt1, Mmp9, Pparg, Egfr, Esr1, Hsp90aa1, Kdr, Agt, Mapk14, and Prkaca (Figure 6B). Notably, Akt1 (degree =36) and Mmp9 (degree =30) exhibited the highest connectivity, indicating their potential roles as hub genes in SCM-198-mediated PAH regulation.

Figure 6 Target screening and core network analysis of SCM-198 for pulmonary arterial hypertension therapy. (A) Venn diagram of drug-disease target IntersectionPotential targets of SCM-198 (blue circle, n=343) predicted by the PharmMapper and SwissTargetPrediction databases, and PAH-related targets (yellow circle, n=9,477) retrieved from the OMIM and GeneCards databases. Intersection analysis using the Venny online tool (v2.1) identified 71 shared targets (overlap region). (B) Core targets in the PPI network. The PPI network of shared targets was constructed via the STRING database, and node connectivity (degree) was analyzed with Cytoscape software (v3.9.1). PPI, protein-protein interaction.

To further elucidate the potential molecular mechanisms by which SCM-198 exerts its therapeutic effects on PAH, GO functional annotation and KEGG pathway enrichment analyses were performed on the 71 overlapping targets. The GO enrichment analysis revealed that, at the BP level (Figure 7A), these targets were significantly involved in pathways such as “response to peptide”, “wound healing”, “cellular response to oxidative stress”, “reactive oxygen species metabolic process”, and “protein kinase B signaling”. This suggests that SCM-198 may alleviate PAH by modulating oxidative stress and key signal transduction pathways. In terms of MF (Figure 7B), the targets were enriched in activities such as “endopeptidase activity”, “protein serine/threonine kinase activity”, and “oxidoreductase activity”, indicating that SCM-198 might influence proteolysis and redox enzyme function. The cellular component (CC) analysis demonstrated that these targets were primarily located in structures such as the “vesicle lumen” and “membrane raft”, suggesting involvement in vesicle transport and membrane-associated processes (Figure 7C). KEGG pathway analysis further indicated that the targets were significantly enriched in key signaling cascades, including the “PI3K-Akt signaling pathway”, “lipid and atherosclerosis”, “FoxO signaling pathway”, “insulin signaling pathway”, and “HIF-1 signaling pathway” (Figure 7D). These pathways are closely related to vascular remodeling, oxidative stress, and metabolic regulation. Collectively, these findings suggest that SCM-198 may exert its therapeutic effects on PAH through multi-target and multi-pathway regulation of vascular function and metabolic homeostasis.

Figure 7 GO and KEGG enrichment analyses of SCM-198 targets in PAH. (A) BP; (B) MF; (C) CC; and (D) KEGG pathways. Key enrichments include oxidative stress responses, kinase activities, vesicle-related structures, and signaling pathways such as PI3K-Akt and HIF-1. Circle size indicates gene count, color reflects adjusted P values. BP, biological process; CC, cellular component; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; MF, molecular function; PAH, Pulmonary arterial hypertension.

Discussion

PAH is a progressive and life-threatening disorder characterized by maladaptive remodeling of the pulmonary arterial tree, leading to increased vascular resistance, elevated right ventricular afterload, and ultimately right heart failure (15). In recent years, growing evidence has demonstrated that the gut microbiota plays a critical role in the development and progression of PAH by modulating immune responses, inflammatory mediators, and microbial metabolites, thereby forming a pathogenic network centered on the gut-lung axis (7). A previous study revealed that PAH patients exhibit a proinflammatory gut microbial imbalance and a marked reduction in anti-inflammatory metabolites, such as SCFAs and secondary bile acids, suggesting that the gut microbiota may serve as a novel therapeutic target for PAH intervention (16).

In this study, an MCT-induced rat model of PAH was established, followed by oral administration of SCM-198 at low and high doses to assess its therapeutic potential on pulmonary vascular remodeling and gut microbiota modulation. Histological analysis using H&E staining revealed significant thickening of the pulmonary arterial medial wall in the MCT group, which was dose-dependently attenuated by SCM-198, indicating its protective effect on the pulmonary vasculature. To explore the gut-lung axis as a potential mechanism, fecal samples were subjected to 16S rRNA gene sequencing. Alpha diversity indices (Shannon, Chao1) showed a declining trend in the MCT group and partial restoration in SCM-198-treated groups, suggesting that microbial diversity might be improved. Beta diversity analyses using PCoA and NMDS clearly separated the MCT group from controls, while the microbial communities in SCM-198-treated rats clustered closer to the control group, indicating partial restoration of dysbiotic structure. Taxonomic profiling revealed decreased abundance of Bacteroidota and increased Actinobacteriota and Erysipelotrichaceae in the MCT group, along with reductions in beneficial taxa such as Muribaculaceae and unclassified_Muribaculaceae. Conversely, potentially pro-inflammatory genera including Lactobacillus, Allobaculum, and Clostridium_sensu_stricto_1 were elevated in disease states. These alterations were largely reversed following SCM-198 treatment. LEfSe analysis identified Fusobacterium and Clostridium_sensu_stricto_1 as disease-enriched biomarkers, while Candidatus_Saccharimonas and Lachnospiraceae_NK4A136_group were enriched in the HD treatment group. At the phylum level, Actinobacteriota was significantly enriched in the MCT-induced PAH model group (Model) compared to the control group (C), consistent with previous findings suggesting its involvement in inflammation and metabolic dysregulation in PAH (17). In contrast, Bacteroidota, known for its SCFA-producing capacity, was markedly reduced in the Model group, aligning with earlier reports of its depletion in PAH and related cardiopulmonary conditions (18). At the family level, Muribaculaceae was significantly decreased in the Model group. As a symbiotic taxon closely associated with SCFA biosynthesis and intestinal homeostasis, its reduction indicates a loss of anti-inflammatory and barrier-supportive microbial components, reflecting microbiota imbalance under pathological conditions (19). Conversely, Erysipelotrichaceae was markedly elevated; this family has been implicated in pro-inflammatory responses and metabolic dysregulation in various disease models, suggesting a shift toward a pro-inflammatory microbial profile in PAH (20). In parallel, Prevotellaceae, another SCFA-associated family, was significantly reduced, potentially diminishing microbial contributions to host metabolic and immune regulation (21). At the genus level, Ligilactobacillus was significantly downregulated. This genus participates in tryptophan metabolism and indole-3-acetic acid (IAA) production, which activates the aryl hydrocarbon receptor (AhR) pathway. Its reduction may impair gut-lung axis-mediated immune modulation, thereby promoting inflammation (22). Unclassified_Muribaculaceae also declined significantly and displayed dynamic changes across different PAH models, potentially modulating amino acid metabolism and inflammatory signaling pathways involved in disease progression (20). Similarly, unclassified_Lachnospiraceae was significantly decreased; notably, Lachnospiraceae bacterium GAM79 was the only taxon significantly associated with PAH status (log₂ fold change =−1.59, P<0.001). Given its role in SCFA production, this reduction may compromise butyrate-mediated anti-inflammatory and vasoprotective mechanisms, contributing to vascular remodeling and chronic inflammation (23). Finally, Allobaculum was markedly upregulated in the PAH model. This genus exhibited negative correlations with several host metabolites—including pyridoxamine, sebacic acid, and dibutyl phthalate—implicated in inflammation, oxidative stress, and vascular dysfunction, suggesting its involvement in PAH pathogenesis through microbiota-metabolite interactions (18).

Collectively, these findings indicate that PAH is associated with a distinctive pro-inflammatory microbial shift coupled with metabolic disruption, highlighting the gut microbiota as a potential modulator of disease progression. In this context, strategies aimed at restoring microbial homeostasis may hold therapeutic potential. SCM-198, a bioactive compound with known anti-inflammatory and endothelial-protective properties, demonstrated significant effects not only on attenuating pulmonary vascular remodeling but also on partially correcting gut microbial dysbiosis in an MCT-induced PAH rat model. Specifically, SCM-198 treatment restored the abundance of SCFA-producing and anti-inflammatory taxa such as Bacteroidota and Muribaculaceae, while reducing the presence of pro-inflammatory genera including Clostridium_sensu_stricto_1 and Allobaculum. These shifts are crucial, as they suggest rebalancing of the gut-lung axis, which may influence systemic immune tone and pulmonary vascular integrity.

This study revealed that SCM-198 alleviates PAH through multi-target and multi-pathway regulation, as demonstrated by network pharmacology analysis, which complements its role in gut microbiota-lung axis modulation. The PPI network identified Akt1 and Mmp9 as key regulatory nodes. Akt1, as a pivotal component of the PI3K-Akt signaling pathway, is extensively involved in endothelial cell survival, anti-apoptotic processes, and oxidative stress resistance. Prior studies have confirmed the central role of PI3K-Akt signaling in PAH pathogenesis, particularly in regulating oxidative stress and vascular remodeling (24). Moreover, Mmp9 plays a critical role in vascular remodeling and inflammation, with elevated expression frequently observed in PAH (25,26). Thus, SCM-198 may alleviate vascular remodeling and inflammatory responses by modulating Akt1 activity and suppressing Mmp9 expression.

Importantly, the PI3K-Akt pathway not only regulates vascular functions but is also intricately linked to the gut microbiota. It was reported that alterations in PI3K-Akt and NRF2/HO-1 signaling pathways in a pulmonary fibrosis model were accompanied by reduced gut microbiota diversity, suggesting a tight association between oxidative stress signaling and gut dysbiosis (27). Consistent with our gut microbiota sequencing results, SCM-198 improved microbial diversity, increased beneficial Bacteroidota, and decreased potentially pathogenic Clostridium_sensu_stricto_1, potentially synergizing with oxidative stress suppression to attenuate PAH progression. Emerging evidence suggests that metabolites derived from gut microbiota can modulate oxidative stress and influence PI3K-Akt and FoxO signaling pathways, thereby improving vascular function and attenuating vascular remodeling and inflammation (28). Moreover, oxidative stress plays a pivotal role in the progression of cardiovascular diseases, including PAH, by activating PI3K-Akt and FoxO signaling, which promotes vascular smooth muscle cell proliferation and vascular remodeling (29). Therefore, SCM-198 likely exerts therapeutic effects through a gut microbiota-PI3K-Akt-oxidative stress axis.

There are several limitations in this study. First, only male rats were used, which may limit the generalizability of the findings. MCT has been shown to induce PAH more reliably in males, whereas females exhibit relative resistance to MCT-induced vascular injury due to sex-related hormonal and molecular differences (30). While this approach enhances reproducibility and consistency in disease modeling, it does not capture sex-specific variations in PAH pathogenesis. Future studies including both sexes and employing alternative models that induce PAH in a sex-independent manner will be essential to provide a more comprehensive understanding of disease mechanisms and therapeutic responses. Second, although the MCT-induced PAH model is widely used and well established, it does not fully replicate the multifactorial pathogenesis of human PAH, which involves genetic, immune, and environmental factors. Moreover, MCT induces systemic toxicity and inflammation that affect multiple organs, making it difficult to isolate the specific contribution of gut microbial changes to PAH progression. As such, the observed microbial alterations and inflammatory responses may partly reflect generalized toxicity rather than gut-specific interactions. More selective models will be needed in future studies to better clarify the causal role of the gut-lung axis in PAH. Third, the composition of gut microbiota in rats differs considerably from that in humans, which limits the direct clinical translatability of our microbial findings. In addition, the resolution of 16S rRNA sequencing constrains taxonomic discrimination and cannot distinguish between functionally distinct strains within the same genus. Finally, while SCM-198 attenuated vascular remodeling and improved gut microbial composition in the MCT model, its general anti-inflammatory and antioxidant properties suggest that the observed therapeutic effects may, in part, be attributable to mitigation of MCT-induced toxicity rather than direct modulation of PAH pathophysiology. Therefore, although our results provide mechanistic insights into the vascular and microbial pathways potentially influenced by SCM-198, their translational significance should be interpreted with caution given the inherent limitations of the MCT model.

Conclusions

In summary, this study underscores the therapeutic potential of SCM-198 in PAH through a dual mechanism involving both gut-lung axis modulation and multi-target pharmacological regulation. Network pharmacology analysis identified key molecular targets, including Akt1 and Mmp9, which are central to PI3K-Akt signaling, oxidative stress responses, and vascular remodeling. These findings complement the observed microbial remodeling, wherein SCM-198 restored gut microbial diversity and enriched beneficial taxa such as Bacteroidota while reducing pro-inflammatory genera like Clostridium_sensu_stricto_1. By simultaneously modulating gut microbiota composition and targeting vascular signaling pathways, SCM-198 exerts systemic benefits that extend beyond local vascular effects. This integrative approach reveals a mechanistic link between gut microbiota modulation and the regulation of key vascular signaling pathways, particularly PI3K-Akt and FoxO, in the pathophysiology of PAH. Such coordination may attenuate inflammatory responses, oxidative stress, and vascular remodeling, thus offering SCM-198 as a promising candidate for adjunctive PAH therapy. This highlights the potential of microbiota-targeted pharmacological strategies to restore gut-lung axis homeostasis and mitigate microbe-derived inflammatory signaling.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1363/rc

Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1363/dss

Peer Review File: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1363/prf

Funding: This work was supported by the National Key Research and Development Program of China (No. 2022YFE0209700, to Y.Z.Z.), Macau Science and Technology Development Fund (FDCT) (0012/2021/AMJ, 0001/2024/RDP, 0001/2024/AKP, 0092/2022/A2, 0144/2022/A3, to Y.Z.Z.), Shenzhen-Hong Kong-Macao Science and Technology Fund (Category C: SGDX20220530111203020, to Y.Z.Z.), and the National Natural Science Foundation of China (No. 82204393, to Z.L.).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1363/coif). Y.Z.Z. reports receiving funding support from the Macau Science and Technology Development Fund (FDCT) (0012/2021/AMJ, 0001/2024/RDP, 0001/2024/AKP, 0092/2022/A2, 0144/2022/A3), Shenzhen-Hong Kong-Macao Science and Technology Fund (Category C: SGDX20220530111203020), and the National Key Research and Development Program of China (No. 2022YFE0209700). Z.L. reports receiving funding support from the National Natural Science Foundation of China (No. 82204393). The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All experimental procedures were approved by the Animal Ethics Committee of Macau University of Science and Technology (approval No. AL006/DICV/DIS/2021), in compliance with institutional guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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