Reduction in microbiota-derived short-chain fatty acids contributes to the pathogenesis of pulmonary arterial hypertension

Abstract

Background

Pulmonary arterial hypertension (PAH) is a progressive and fatal cardiopulmonary disorder, with growing evidence implicating proinflammatory gut dysbiosis in its pathogenesis. Fast growing broiler chickens (Gallus gallus) spontaneously develop PAH with histopathological features that closely resemble those of the human disease, providing a robust translational model.

Methods

Gut microbiota composition in PAH-afflicted broilers was compared to that of healthy controls to identify disease-associated microbial alterations. Microbiota depletion was achieved using a broad-spectrum antibiotic cocktail, and oral supplementation with calcium acetate, a short-chain fatty acid (SCFA) salt, was administered to assess therapeutic potential. Pulmonary cytokine expression was measured to evaluate inflammation.

Results

PAH-afflicted broilers exhibited gut microbial alterations similar to those observed in human patients, characterized by a reduction in bacterial genera involved in the production of anti-inflammatory metabolites, particularly SCFAs, and an increase in arginine- and tryptophan-producing taxa. Microbiota depletion selectively enriched SCFA-producing bacteria and prevented the onset of PAH. Calcium acetate supplementation significantly mitigated disease progression and reduced pulmonary expression of proinflammatory cytokines.

Conclusions

These findings establish a causal relationship between microbiome-derived metabolites and pulmonary vascular remodeling, supporting SCFA-based interventions as a promising therapeutic strategy for PAH.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12931-026-03591-9.

Introduction

Pulmonary arterial hypertension (PAH) is a progressive cardiopulmonary disease in humans characterized by extensive obliterative changes in small to midsized pulmonary arterioles, leading to a progressive increase in pulmonary arterial resistance and, ultimately, right ventricular failure and mortality [1, 2]. The pathophysiology of PAH involves complex mechanisms at the cellular and molecular level, including endothelial dysfunction, smooth muscle cell proliferation, and perivascular inflammation. Despite the availability of FDA-approved treatments such as endothelin receptor antagonists (ERAs), phosphodiesterase type-5 inhibitors (PDE5i), and prostacyclin analogs, PAH remains an incurable disease (Management of Pulmonary Arterial Hypertension).

There is increasing evidence that inflammation and immune dysregulation play a critical role in driving pulmonary vascular remodeling that leads to PAH [3–6]. Perivascular inflammation has been shown to correlate with the extent of pulmonary vascular remodeling [3]. Furthermore, PAH patients exhibit increased circulating levels of inflammatory cytokines that significantly impact disease outcomes [7, 8]. Moreover, findings in experimental animal models further substantiate the involvement of inflammation in PAH [9–11].

Emerging evidence increasingly links immune dysregulation and perivascular inflammation in PAH to alterations in the gut microbiome, known as gut dysbiosis. In patients with PAH, gut dysbiosis is marked by a proinflammatory microbial signature, characterized by a reduction in bacterial genera that produce anti-inflammatory short-chain fatty acids (SCFAs), accompanied by a decrease in circulating SCFA levels [12, 13]. This pattern of dysbiosis is also observed in experimental PAH models, where pro-inflammatory microbial species prevail, and beneficial probiotics are diminished [14, 15].

Recent findings by Su et al. provide compelling evidence for a causal relationship between gut dysbiosis and PAH, as demonstrated through Mendelian randomization analysis [16]. Consistent with these findings, preclinical studies using the Sugen/hypoxia rat model show that antibiotic-induced depletion of the gut microbiome attenuates pulmonary vascular remodeling and prevents the development of PAH [17]. However, it is important to note that the Sugen/hypoxia rat model does not fully recapitulate the pathogenesis of PAH [18]. Therefore, it is essential to employ more clinically relevant animal models to confirm the causal relationship between gut dysbiosis and PAH and to evaluate the potential effectiveness of microbiota-based therapeutic approaches in managing the disease.

Broiler chickens (Gallus gallus), a breed of meat-producing poultry, are inherently susceptible to PAH (also known as ascites syndrome or pulmonary hypertension syndrome) [19]. The histological changes observed in the affected birds closely mimic the human PAH phenotype, including media hypertrophy, intimal hyperplasia, plexiform lesions and perivascular inflammation, providing an excellent model of spontaneous human disease [19–26]. Our recent investigations have presented compelling evidence indicating that chronic inflammation is involved in the pathogenesis of PAH in this avian model [27, 28].

This study aimed to delineate gut microbiome alterations associated with broilers with PAH and to evaluate the therapeutic potential of microbiota-directed interventions. Metagenomic profiling of PAH-affected birds revealed a pronounced depletion of SCFA-producing bacterial taxa. Notably, administration of a broad-spectrum antibiotic regimen, which promoted the selective resurgence of SCFA-producing microbes, significantly attenuated PAH incidence. Moreover, both fecal microbiota transplantation (FMT) and oral supplementation with calcium acetate [Ca(OAc)₂], a bioavailable SCFA salt, conferred robust protection against PAH development under experimentally induced hypertensive conditions.

Materials and methods

Animal ethics

The animal experiments followed the National Guidelines for the Ethical Review of Laboratory Animal Welfare and were reviewed and approved by the Ethics Committee of Zhejiang University (ZJU20170554).

Reagents

Sodium chloride, formaldehyde, and antibiotics (ampicillin, vancomycin, neomycin, and metronidazole) were purchased from various suppliers, including Sinopharm Group (Shanghai, China) and Macklin Biochem (Shanghai, China). NucleoSpin^® Tissue DNA extraction kit from Gene Co., Ltd (Guangzhou, China). Metagenomic library construction kits and Nextera XT indexing kits were sourced from Takara Bio Inc. (Dalian, China) and Illumina Inc. (CA, USA). In contrast, NEBNext Ultra II DNA library preparation kits were obtained from New England Biolabs (MA, USA). Recombinant chicken VEGF, bFGF, IGF, and TNFα were prepared in our laboratory. Chicken peripheral blood mononuclear cell separation solution and chicken endothelial progenitor cell (EPC) culture medium were purchased from Tianjin Haoyang Biological Products Technology (Tianjin, China). High-glucose DMEM was obtained from Fude Biotechnology (Hangzhou, China). Rabbit anti-NLRP3 and MLKL were purchased from HuaBio (Hangzhou, China); Rabbit anti-caspase-1 was obtained from Wanlei Biotechnology (Shenyang, China).

Gut microbiome profiling

Animal grouping

Fertilized eggs from an Arbor Acres-strain commercial line were disinfected and hatched in our laboratory. Following hatching, the birds were reared under standard thermal conditions with continuous light in the animal experimental facility at the Center for Veterinary Sciences, Zhejiang University. At day 15, they were randomly assigned to one of three experimental groups: untreated birds (Control), birds exposed to sub-thermoneutral temperatures (PAH), and birds exposed to sub-thermoneutral temperatures with antibiotic treatment (PAH + AT), with 24 birds in each group.

Antibiotic treatments

Starting at day 15, birds in the PAH + AT group were treated with an antibiotic cocktail consisting of ampicillin, neomycin, metronidazole, and vancomycin [17]. The antibiotics were added to the drinking water at final concentrations of 1 g/L each for ampicillin, neomycin, and metronidazole, and 500 mg/L for vancomycin. Antibiotic treatment continued throughout the experimental period.

Experimental PAH model

At 18 days of age, birds in the PAH and PAH + AT groups were subjected to sub-thermoneutral temperatures to expedite the onset of pulmonary arterial hypertension (PAH), as outlined in our prior work [29]. Exposure to cool temperatures has been shown to trigger PAH, likely by increasing metabolic demand, which elevates cardiac output and pulmonary blood flow, resulting in increased pulmonary arterial pressure [30]. In brief, the ambient temperature within the poultry house was gradually reduced by 2 °C per day, from 28 °C to 14 °C, and maintained at 14 °C until 39 days of age.

Right-to-total ventricular weight (RV/TV) ratio and PAH Morbidity

Beginning on day 18, birds were monitored daily for clinical signs of disease. Any bird exhibiting depression, labored breathing (gasping), and/or abdominal distension was humanely euthanized by cervical dislocation. The heart was immediately removed, and the ventricles were dissected and weighed to calculate the right-to-total ventricular weight ratio (RV/TV ratio) as an index of pulmonary hypertension. Birds with an RV/TV ratio ≥ 0.25 were classified as having pulmonary arterial hypertension (PAH) [31–33]. The RV/TV ratios of birds that died during the experimental period were also recorded. At day 39, all remaining birds were euthanized. PAH morbidity was defined as the percentage of birds in each group that developed PAH during the experimental period.

Fecal sample collection

At the end of the experiment, 6 birds with RV/TV ratios < 0.25 were selected from each of the Control and PAH+AT groups, while 6 birds with RV/TV ratios > 0.25 were selected from the PAH group for fecal sample collection. In addition, 6 birds from the PAH group with RV/TV ratios < 0.25, defined as PAH-resistant birds, were also selected for sampling. Immediately after euthanasia, luminal contents from the distal ileum and cecum were aseptically collected and transferred into sterile tubes. All samples were snap-frozen in liquid nitrogen (–196 °C) and stored until further processing.

Bacterial genomic DNA extraction and 16S rRNA gene amplicon sequencing

Bacterial genomic DNA was extracted using the NucleoSpin Tissue DNA Kit (Macherey-Nagel, Germany) following the manufacturer’s protocol. A two-step PCR amplification was performed to target the V3–V4 hypervariable region of the 16S rRNA gene [29]. In the first round, amplicons were generated using the 16 S (V3–V4) Metagenomic Library Construction Kit for NGS (Takara Biomedical Technology, Dalian, China) with primers 341 F (5′-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG-3′) and 806R (5′-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA G-3′). The resulting PCR products were used as templates for a second-round indexing PCR with the Nextera XT Index Kit (Illumina, San Diego, CA, USA). Sequencing libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, MA, USA), according to the manufacturer’s instructions. Paired-end sequencing (2 × 250 bp) was conducted on the Illumina MiSeq platform (Illumina, San Diego, CA, USA). Genome sequencing was performed at the Magigene Technology Co., Ltd (Guangzhou, China). Subsequent data processing was performed.

Fecal microbiota transplantation (FMT)

Animal grouping

Chicks were hatched and reared under the same conditions as previously described until 18 days of age. At that point, birds were divided into four groups, with 18 in each group: untreated birds (Control), birds exposed to sub-thermoneutral temperatures (PAH model), birds exposed to sub-thermoneutral temperatures and administered a defined microbiota transplant consisting of a cultured Coprobacter isolate (PAH+Coprobacter), and birds exposed to sub-thermoneutral temperatures and administered a conventional fecal microbiota transplant derived from donor feces (PAH + FMT). All birds had ad libitum access to feed and water.

Coprobacter transplantation

Anaerobic bacterial strains were isolated from the intestinal contents of PAH + AT birds by plating on Columbia blood agar and incubating under anaerobic conditions (90% N₂, 5% H₂, 5% CO₂) at 37 °C. Taxonomic identification was performed through 16 S rRNA gene sequencing, confirming one isolate as a member of the Coprobacter genus. Starting 1 week after the initiation of cool temperature exposure, broilers in the PAH+Coprobacter group received a daily oral gavage of 150 µL phosphate-buffered saline (PBS) containing 3 × 10⁸ colony-forming units (CFU) of the Coprobacter isolate for 2 weeks.

Conventional fecal microbiota transplantation (FMT)

Fresh fecal samples were aseptically collected from PAH + AT birds on the day of euthanasia. A total of 13 g of feces was homogenized in 20 mL of sterile phosphate-buffered saline (PBS) using sterile silica beads at 45 Hz for 1 min. The homogenate was then filtered through a 75 μm cell strainer to remove particulate matter, yielding approximately 15 mL of clarified fecal suspension. Beginning 7 days after initiation of cool temperature exposure, birds in the PAH + FMT group received a daily oral gavage of 150 µL of the fecal filtrate for 2 weeks.

Calcium acetate treatments

Animal grouping

PAH was induced as in the previous experiment. Birds were randomly divided into four groups: Control (reared under standard conditions), PAH (exposed to PAH-inducing conditions), PAH+25 mM OAc⁻ (exposed to PAH-inducing conditions with 25 mM acetate treatment), and PAH+75 mM OAc⁻ (exposed to PAH-inducing conditions with 75 mM acetate treatment), with 30 birds in each group.

Acetate treatment

Beginning on the first day of exposure to sub-thermoneutral temperatures, calcium acetate [Ca(OAc)₂] was administered via drinking water to the PAH+25 mM OAc⁻ and PAH+75 mM OAc⁻ groups, achieving final concentrations of 25 mM and 75 mM, respectively. Treated birds had continuous ad libitum access to the supplemented drinking water for 2 weeks. Water consumption was comparable across all groups, suggesting that calcium acetate administration via drinking water did not alter fluid intake.

Tissue sampling

After each experiment, birds were humanely euthanized. The whole left lung was removed immediately and cut in the transverse plane at the major rib indentations (costal sulci). For the histological study, one interrib division from the middle of each lung was collected and fixed in 4% (w/v) formaldehyde. The apical regions of all the sampled lungs were rapidly frozen in liquid nitrogen and then stored at -80 °C.

Lung histological analyses

The formaldehyde-fixed, paraffin-embedded lung tissues were cut at 5–7 μm thickness. Plexiform lesions were counted on H&E-stained sections, and lesion density (number of lesions per section/cm² per section) was calculated as described previously [23, 34]. Verhoeff-van Gieson staining was performed to facilitate the identification of the elastic lamina of pulmonary arterioles. The media wall thickness of the vessels with an outer diameter between 25 and 100 μm was analysed at 400× magnification. Relative medial thickness (RMT) was expressed as medial thickness/external radius [20].

Immunohistochemical staining

Immunohistochemistry (IHC) for α-smooth muscle actin (α-SMA) was used to assess muscularization in pulmonary arterioles. Briefly, lung paraffin sections were deparaffinized, rehydrated, and subjected to antigen retrieval with Tris-EDTA buffer (pH 9.0). Sections were incubated overnight at 4 °C with a 1:50 dilution of mouse anti-α-SMA antibody, followed by incubation with a secondary antibody. DAB (3,3′-Diaminobenzidine) was used for visualization, and the sections were counterstained with hematoxylin.

Pulmonary arterioles in terminal bronchioles and alveolar areas were evaluated for muscularization, with vessels classified as non-muscularized (NM; muscle layer surrounding < 50% of the vessel), partially muscularized (PM; muscle layer surrounding > 50% but < 100% of the vessel), or fully muscularized (FM; muscle layer surrounding 100% of the vessel). The percentage of vessels in each muscularization category was quantified.

Western blot

Total protein was extracted from lung tissue lysates and separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA). Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 30 min at room temperature, then incubated overnight at 4 °C with primary antibodies. After washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. Immunoreactive bands were visualized using an enhanced chemiluminescence (ECL) detection system (FDbio Science, Hangzhou, China).

In vitro experiment

Early endothelial progenitor cells (eEPCs) were isolated and treated with tumor necrosis factor (TNF)-α according to previously established protocols from our laboratory [26]. Briefly, peripheral blood was collected from the wing vein of 4-week-old broilers, and mononuclear cells were isolated by density gradient centrifugation. Cells were seeded at a density of 1 × 10⁷ cells per well in 6-well culture plates and maintained at 39 °C with 5% CO₂ in EPC culture medium (TBD Science, Tianjin, China) supplemented with 10% fetal bovine serum, 100 ng/mL vascular endothelial growth factor (VEGF), two ng/mL basic fibroblast growth factor (bFGF), and two ng/mL insulin-like growth factor (IGF). After 48 h, non-adherent cells were removed and replaced with fresh medium. Following an additional 48 h of culture, cells were stimulated with TNF-α at a final concentration of 50 ng/mL. For the acetate intervention, calcium acetate [Ca(OAc)₂] was added to the culture medium at final concentrations of 0.05, 0.5, or 5 mM. After 6 days of culture, the cells were used for further experiments.

Statistical analysis

All data are expressed as mean ± standard deviation (SD), unless otherwise indicated. Normality of data distribution was assessed using the Shapiro–Wilk test. For comparisons between two groups, Student’s t-test was employed. One-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test was used to compare differences among multiple groups. For data sets with small sample sizes or non-normal distributions, the non-parametric Mann–Whitney U test was applied. The incidence of PAH was analyzed using the chi-squared (χ²) test. Statistical analyses were performed using SPSS version 22.0 for Windows (IBM Corp., Armonk, NY, USA). A P value of < 0.05 was considered statistically significant.

Results

Antibiotic-induced microbiota shifts enrich SCFA-producing bacteria in the gut microbiome and attenuate PAH development

Previous studies have shown that disruption of the gut microbiota using a broad-spectrum antibiotic cocktail attenuates PAH in the Sugen/hypoxia rat model [17]. To evaluate whether microbiota modulation similarly influences PAH progression in our avian model, broiler chickens were treated with the same antibiotic regimen and exposed to sub-thermoneutral temperatures to induce PAH. Remarkably, antibiotic treatment significantly prevented the development of PAH, as evidenced by reduced PAH morbidity compared to the PAH model group (Fig. 1A), supporting a causal role for the gut microbiota in PAH pathogenesis. These findings are consistent with prior observations in rodent models [17].

Fig. 1.

Modifying gut microbiota alleviated PAH’s morbidity and histologic features. A Cumulative PAH morbidity. B Right-to-total ventricle ratio (RV/TV) of birds in Control, PAH, and PAH+AT groups. PAH: birds exposed to low temperature; PAH+AT: birds exposed to low temperature following the antibiotic treatment. *P<0.05, ***P<0.001

To confirm that the development of PAH is associated with gut microbial shifts, we selected birds from the Control, PAH and PAH+AT groups and the luminal contents from their distal ileum and cecum were subjected to 16S rRNA gene amplicon sequencing. Notably, none of the birds selected from the Control and PAH+AT had an RV/TV above 0.25, indicative of normal pulmonary artery pressure, while those from the PAH group demonstrated disease progression (Fig. 1B). Microbial profiles across the groups were compared using linear discriminant analysis (LDA), which identified 47 bacterial taxa with significant differences in abundance (LDA score > 3.0; Fig. 2A). We also compared overall community changes using alpha and beta diversity indices (Supplementary Material).

Fig. 2.

LDA of the microbial community between groups. A Linear discriminant effect size (LEfSe) analysis was performed (logarithmic LDA score threshold ≥ 3) on all three groups (Control, PAH, and PAH + AT). Linear discriminant effect size (LEfSe) analysis was performed (logarithmic LDA score threshold ≥ 3) between (B) Control and PAH groups, (C) Control and PAH + AT groups, and D PAH and PAH + AT groups. PAH: birds with induced PAH; PAH.AT: birds exposed to PAH-inducing conditions followed by antibiotic treatment

Further taxonomic analysis using the Linear Discriminant Analysis Effect Size (LEfSe) approach, with a threshold LDA score > 3.6, revealed distinct microbial signatures between experimental groups (Fig. 2B, C, D). The PAH + AT group exhibited a significantly higher relative abundance of several bacterial genera, including Alistipes, Fusobacterium, Coprobacter, Elusimicrobium, and Enterobacter, compared to the PAH group. Among these, Alistipes (phylum Bacteroidota) is a Gram-negative, anaerobic genus known for its anti-inflammatory properties in the gut, primarily through the production of SCFAs [35, 36]. Fusobacterium has also been reported to produce SCFAs, particularly acetate and butyrate [37]. Similarly, Coprobacter is capable of producing SCFAs [38]. In contrast, the PAH group was enriched in Bacteroides, Faecalibacterium, Lachnoclostridium, and Parabacteroides. Notably, Faecalibacterium contributes to arginine biosynthesis [39], and Bacteroides is involved in tryptophan metabolism [40].

In context, these findings suggest that PAH development is associated with a compositional shift in the gut microbiota toward genera involved in amino acid biosynthesis and a concomitant reduction of beneficial SCFA-producing taxa.

Similarity in gut microbiota alteration between the broiler PAH model and human patients

At the phylum level, the analysis shows that Bacteroidota is the most abundant phylum in the feces of broilers, followed by Firmicutes, Fusobacteriota, Desulfobacterota, and Proteobacteria (Fig. 3A). The ratio of Firmicutes to Bacteroidetes (F/B) is a marker of gut microbiota dysbiosis. The F/B ratio in the PAH + AT group was significantly higher than in the other groups (Fig. 3B), indicating that antibiotic treatment altered the gut microbiota composition. There was no significant difference in the F/B ratios between the control and PAH birds, and this is the same as the report on microbiota changes in human patients [41].

Fig. 3.

Microbiota composition changes at phylum level in groups. A Gut microbial composition changes at the phylum level. B Ratio of Firmicutes to Bacteroidetes (F/B) in each group. PAH: birds exposed to low temperature; PAH + AT: birds exposed to low temperature following the antibiotic treatment; PAH.NC: PAH-resistant birds in the PAH + AT group. **P < 0.01

Microbiota-based interventions, including FMT and Coprobacter supplementation, attenuate PAH development

To further validate that alterations in the gut microbiota mediated the protective effects observed with antibiotic treatment, we performed conventional FMT using donor microbiota from the PAH + AT group. Recipient broiler chickens received FMT following one week of exposure to cold temperatures to induce PAH. Notably, FMT treatment significantly reduced the RV/TV ratio compared to the untreated PAH group (Fig. 4A). It was also associated with a lower incidence of PAH (Fig. 4B). To assess further its impact on the PAH phenotype, histological analysis of pulmonary vascular remodeling was conducted. FMT notably attenuated the medial thickening of small pulmonary arteries (diameter 50–100 μm), as evidenced by reductions in both WA/TA ratio and RMT (Fig. 4D, E). In addition, FMT markedly prevents the formation of plexiform lesions (Fig. 4G), a pathological hallmark of advanced PAH. Collectively, these findings support a causal role for the gut microbiota in modulating PAH development.

Fig. 4.

The effects of transplantation of Coprobacter and FMT on the development of PAH in broiler chickens. A The effects of Coprobacter transplantation and fecal microbiota transplantation (FMT) on the heart index of broilers (n = 18). B The effects of Coprobacter transplantation and FMT on the incidence of PAH in broilers. C Pulmonary arterioles (outer diameter < 200 μm, Verhoeff-stained) in control, PAH, PAH + Coprobacter, and PAH + FMT groups of broilers. D The effects of Coprobacter transplantation and FMT on the wall area to total area (WA/TA) ratio of pulmonary arterioles in broilers (n = 6). E The effects of Coprobacter transplantation and FMT on the relative medial thickness (RMT) of pulmonary arterioles in broilers (n = 6). F Morphology of plexiform lesions in the lungs of PAH group chicken(H&E stained). G The effects of Coprobacter transplantation and FMT on plexiform lesion (PL) density in broilers (n = 6). PAH: birds with induced PAH; PAH+Coprobacter: birds exposed to PAH-inducing conditions followed by Coprobacter administration; PAH + FMT: birds exposed to PAH-inducing conditions followed by FMT administration. *P < 0.05, **P < 0.01, and ***P < 0.001

To directly evaluate the protective role of SCFA-producing taxa, we conducted a parallel experiment in which Coprobacter was administered to broiler chickens. Similar to conventional FMT, Coprobacter supplementation effectively prevented the development of PAH (Fig. 4), further reinforcing the contribution of SCFA-producing bacteria to disease attenuation.

Ca(OAc)2 treatment confers protection against PAH

Building on the above findings, we investigated whether exogenous SCFAs could protect against PAH development. To this end, calcium acetate [Ca(OAc)₂], an SCFA salt, was administered during PAH induction. In line with previous studies demonstrating the protective effect of butyrate against hypoxia-induced PAH in rats [42], supplementation with Ca(OAc)₂ at both 25 mM and 75 mM in drinking water significantly attenuated PAH. The Ca(OAc)₂-treated birds exhibited reduced PAH morbidity (Fig. 5B), lower RV/TV ratios (Fig. 5C), diminished medial thickening of small pulmonary arterioles (Fig. 5E), and fewer plexiform lesions relative to the untreated PAH model (Fig. 5F), indicating a robust protective effect.

Fig. 5.

Protection effect of Ca(OAc)2 treatment in the process of PAH development. Cumulative PAH (A) mortality and (B) morbidity. C Right-to-total ventricle ratio (RV/TV) of birds in Control, PAH, and acetate treatment groups. D Representative photomicrographs of muscular pulmonary arterioles from Control, PAH, and acetate treatment groups (Verhoeff’s stain). E Relative medial thickness (RMT) was calculated. At least 3 arterioles with complete inner and external elastic laminas (external diameters of 25–100 μm) were randomly selected from the lung section of each bird for analysis. Data are presented as a scatter plot with mean ± SD. The scatter plot contains ≥ 3 measurements from the same bird. F Plexiform lesion density in the lung. G-J Lung samples from Control, PAH, and acetate treatment groups were collected at week 3 post-treatment, homogenized, and subjected to Western blot analysis with anti- IL-1β, anti-tumor necrosis factor (TNF)-α, and anti-interleukin (IL)-8. Tubulin was used as the equal loading control. PAH: birds with induced PAH; PAH+25mM OAc−: birds exposed to PAH-inducing conditions with 25 mM acetate treatment; PAH+75mM OAc−: birds exposed to PAH-inducing conditions with 75 mM acetate treatment. *P < 0.05, **P < 0.01, and ***P < 0.001

To further characterize the effects of Ca(OAc)₂ on the PAH phenotype, we analyzed the expression of proinflammatory cytokines in lung tissue by immunoblotting. Birds in the PAH model group exhibited elevated levels of key inflammatory mediators, including tumor necrosis factor (TNF)-α (Fig. 5I), interleukin (IL)-1β (Fig. 5H), and IL-8 (Fig. 5J), compared to the control group. In contrast, Ca(OAc)₂ treatment significantly reduced the expression of these cytokines. These findings support the presence of an inflammatory component in the pathogenesis of PAH and suggest that the protective effect of SCFA supplementation may involve, at least in part, the attenuation of pulmonary inflammation.

Inhibition of pyroptosis and necroptosis underlies the anti-inflammatory effects of Ca(OAc)₂

Pyroptosis and necroptosis are the two modalities of inflammatory cell death pathways that contribute to the release of proinflammatory mediators into the extracellular environment, thereby amplifying tissue inflammation and injury [43]. To investigate whether Ca(OAc)₂ protects cells from inflammatory cell death, we utilized a TNFα-stimulated eEPC model. This model was selected based on our previous findings that chronic inflammation in PAH drives the trans-differentiation of eEPCs into macrophage-like cells, contributing to vascular lesion formation [27]. There was no significant effect on eEPCs cell viability after 6 days of treatment with TNFα and Ca(OAc)₂, either alone or in combination (P > 0.05) (Fig. 6A). Microscopic observations revealed that continuous TNFα stimulation caused a morphological change in eEPCs from spindle-shaped to round cells with multiple protrusions resembling macrophages. Ca(OAc)₂ reversed these morphological changes in eEPCs in a dose-dependent manner (Fig. 6B). Following chronic TNFα stimulation, the level of mature IL-1β, a hallmark of pyroptotic cell death [44], was markedly increased. We also observed a significant upregulation of key pyroptosis-related proteins, including NLRP3 and cleaved caspase-1. As anticipated, treatment with Ca(OAc)₂ led to a substantial reduction in the expression of these proteins cells (Fig. 6C-G). In parallel, TNFα exposure elevated levels of phosphorylated mixed lineage kinase domain-like protein (p-MLKL), a key effector of necroptosis [45], which was also attenuated by Ca(OAc)₂ treatment (Fig. 6H, I). Together, these findings suggest that Ca(OAc)₂ confers protection against inflammatory cell death by suppressing both pyroptosis and necroptosis.

Fig. 6.

Ca(OAc)₂ inhibits the activation of the inflammasome pathway and expression of inflammatory factors in eEPCs induced by TNFα. A Effects of TNFα and Ca(OAc)₂ treatment alone and in combination for 6 days on eEPCs cell viability as assessed by CCK-8 (n = 3). B Morphological changes in eEPCs after 6 days of treatment with TNFα and Ca(OAc)₂ alone and in combination. Changes in protein levels of (C) IL-1β, (E) NLRP3, (E) Caspase-1, and (H) p-MLKL in eEPCs after 6 days of stimulation with TNFα and Ca(OAc)₂ as detected by Western blot. Gray value analysis results for (D) IL-1β (F) NLRP3 and (G) cleaved Caspase-1, and (I) p-MLKL (n = 3).*P < 0.05,  ***P < 0.001

Discussion

In the present study, we found that dysbiosis of gut microbiota, particularly the reduction in the abundance of SCFA- producing bacteria, may play a significant role in the development of PAH. Additionally, we demonstrated that the gut dysbiosis observed in the broiler PAH model closely resembles that reported in human patients. Our findings confirm that SCFA-Ca(OAc)₂ has a preventive effect against the progression of PAH and suggest that it helps alleviate inflammation by modulating lytic cell death.

Our study is the first to establish a direct link between gut microbiota modulation and protection against PAH in a non-mammalian model. While the gut–lung axis has gained increasing attention in mammalian studies, the broiler chicken model offers unique advantages for investigating microbiota-PAH interactions. Unlike rodent models, fast-growing broilers are highly susceptible to PAH [22, 24, 26, 46, 47], developing plexiform-like lesions pathologically similar to those observed in human PAH [22, 29, 34, 48, 49]. This spontaneous disease phenotype enables the study of microbiota-driven disease progression and therapeutic intervention in a physiologically relevant setting. Consistent with our results, other researchers have also found that PAH significantly reduced the microbial diversity, including alpha diversity, richness, and evenness, and altered the composition of intestinal flora [12, 14, 15, 17, 50, 51]. Firmicutes and Bacteroidetes are the most abundant bacterial phyla affecting host physiology in both humans and animals. An imbalanced F/B ratio is also a vital characteristic of gut dysbiosis. Recently, Callejo et al. have described that the F/B ratio was higher in Su/Hx rats as compared with control rats [15]. Also, the MCT-induced PAH rats showed an increased F/B ratio compared to control rats [50], suggesting that the F/B ratio can serve as an indicator of PAH in rats. In our study, however, there was no difference in the F/B ratios between the control and PAH birds, and this is the same as the report on human patients [41]. The discrepancy suggests that the form of expression in gut microbiota dysbiosis differs among different animals. What is more important, the broiler model may be more suitable as a comparative medical model of PAH, not only in histomorphology but also in the intestinal microbiome.

In the PAH group, we also observed a decrease in the gut bacteria involved in the production of SCFAs. This decrease is also reflected in the development of PAH in both human patients and experimental animal models. Kim et al. have recently reported that the abundance of butyric and propionate-producing bacteria, such as Coprococcus and Butyrivibrio were decreased in PAH patients compared with those in control subjects [12]. Callejo et al. demonstrated that both acetate-producing bacteria and the level of serum acetate were decreased in PAH rats [15]. Similarly, the abundance of propionate-producing bacteria such as Akkermansia and Bacteroides was reduced in sugen/hypoxia rats compared to the control group [17]. More importantly, SCFA-producing taxa dominated the gut microbiota of PAH-resistant broilers modified by antibiotics. Among the identified taxa, Alistipes was the most abundant and is recognized as an acetate producer [52]. For this reason, acetate was prioritised as the main metabolite of interest. Short-chain fatty acids (SCFAs) produced by gut microbiota can be absorbed into the bloodstream from the gastrointestinal tract and have physiological effects on various tissues, including the lungs and cardiovascular system [53–56]. These SCFAs inhibit inflammatory responses, which occur through reduced adhesion of leukocytes to endothelial cells. This effect is mediated by the downregulation of the expression of VCAM-1 and ICAM-1 caused by SCFAs [57, 58]. Based on this evidence, we concluded that a lack of SCFAs can lead to the progression of PAH.

Modification of the gut microbiota by using antibiotics suppressed the progression of PAH in our study, but the microbial imbalance caused by it cannot be ignored. The side effects limit the use of antibiotics in PAH to a certain extent, so other harmless interventions should be considered first. Probiotics and/or FMT are a potent way to alter a patient’s intestinal microbiota, which is currently used clinically to ameliorate certain diseases [59, 60]. We have found a positive therapeutic effect of Coprobacter transplantation and FMT on PAH in broilers, similar to a series of studies conducted by Wedgwood and Sharma et al. [51, 61]. Coprobacter is known to produce the SCFAs propionic and acetic acids, both of which have documented anti-inflammatory and barrier-protective functions, including the ability to suppress TNF-α–induced NF-κB activation and reduce pro-inflammatory cytokine release [62], which provides a mechanistic rationale for the protective effects we observed following Coprobacter transplantation. Although several studies have indicated that FMT exerts therapeutic effects in various inflammation-related diseases [63–65], Li et al. [66] found that only 47% of the human gut microbiota could be re-established in the mouse gut, with low reproducibility of microbial composition shifts even with a similar diet. This underscores the importance of optimizing donor-recipient compatibility in transplantation therapy. Considering differences in broiler strains and rearing conditions, the efficacy of Coprobacter transplantation and FMT in the clinical prevention and treatment of PAH requires verification across diverse production environments. Compared to interventions like Coprobacter transplantation and FMT, correcting abnormal gut microbiota metabolites may be a therapeutic strategy applicable across various PAH models, which is reflected in the preventive effect of calcium acetate on two types of PAH animals. Similar to our study, Karoor et al. [42] validated the protective effect of butyrate in a rat model of hypoxic PH, and Liu et al. [67] also found that a new synthetic drug of dichloroacetate can alleviate SuHx-induced PAH. These findings suggest that SCFAs offer similar protective effects across different PAH models, hinting at SCFAs’ potential therapeutic applications for human PAH in comparative medicine. Due to variations in gut microbiota composition in broilers reared under different conditions, FMT treatment cannot guarantee effectiveness in PAH. Thus, correcting abnormal gut microbiota metabolites, especially SCFAs, may be key to clinical prevention and treatment of PAH in broilers. In broiler production, SCFAs are commonly used as harmless feed additives [68], significantly increasing broiler feed intake and meat yield, which enhances growth performance and nutritional status [69, 70]. Research on SCFAs’ role in maintaining broiler immune homeostasis primarily focuses on gut health. Studies show that SCFAs increase intestinal villus height and goblet cell numbers, inhibit the colonization of harmful bacteria, and promote the proliferation of probiotics [71, 72]. Additionally, SCFAs positively influence overall immune homeostasis in poultry, enhancing both specific and nonspecific immune functions in broilers [68].

Some directions still need to be further explored in the present study. Firstly, previous studies have reported that cold stress promotes dysbiosis characterized by reduced SCFA-producing bacteria [73]. Similar shifts were observed in our PAH-induced birds, suggesting that part of the microbial and immunological alterations may be attributable to cold-induced physiological stress rather than PAH alone. However, the protective effects of acetate/FMT observed in our study, despite the cold challenge, indicate that modulation of the gut microbiota provides benefit beyond the baseline effects of cold stress [74]. Secondly, some predominant bacteria can serve as biomarkers of normal or disease for clinical diagnosis. For example, gut microbiota analysis can predict the incidence of PAH in human patients with 85% accuracy [12]. In our study, we also screened biomarkers via LEfSe analysis alone. However, further experiments are needed to determine whether these biomarkers can serve as diagnostic markers for PAH. Thirdly, rigorous experimental studies should be conducted to ascertain the causative role of gut dysbiosis and altered circulating microbiome metabolites in the pathogenesis of PAH. Lastly, although our findings support a protective role for SCFAs in PAH, circulating SCFA levels were not quantified following calcium acetate administration. SCFAs are known to modulate immune cell activation through binding to G-protein-coupled receptors, including GPR41 and GPR43 [75]. Therefore, future studies should assess both systemic SCFA concentrations and the expression or activation of SCFA-associated receptors to determine whether these pathways mediate the observed protective effects. Such analyses would help establish a more direct mechanistic link between gut-derived metabolites, host receptor signaling, and pulmonary vascular remodeling.

Conclusion

This study suggests that gut dysbiosis, characterised by a decrease in short-chain fatty acid (SCFA)-producing bacteria, may contribute to the development of PAH. Using a broiler model, we found that modifying gut microbiota through antibiotics, SCFA supplementation, and FMT significantly reduces PAH progression and pulmonary vascular remodeling. The broiler model effectively mimics human PAH due to similarities in gut microbiota changes, making it a valuable tool for exploring microbiome-related mechanisms and potential therapies. Future studies should investigate the translational potential of these findings in human patients with PAH, with a focus on optimizing microbiota-targeted therapies to enhance clinical outcomes.

Authors’ contributions

HI: Writing – original draft, Visualization, Formal analysis. RL: Methodology, Investigation, Conceptualization, Experimentation. QHL: Software, Data Curation. CZZ: Validation, Methodology. XT and YTY: Writing – review & editing, Supervision, Resources, Project administration, Conceptualization. All authors read and approved the final manuscript.

Funding

This study was supported by the National Natural Science Foundation of China (Project No. 31872444).

Data availability

All relevant data and materials are stored at the Veterinary Pathology Laboratory of Zhejiang University and can be obtained from the corresponding author upon reasonable request. Uncropped images for all blots are provided in the supplementary file 2.

Declarations

Ethics approval and consent to participate

All experimental protocols conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and were approved by the Animal Ethics Committee of Zhejiang University. Additionally, all animals were handled with care and euthanized humanely during the study.

Consent for publication

Not Applicable.

Competing interests

The authors declare no competing interests.