Determination of the effect of pyocyanin and Saccharomyces boulardii on gut microbiota and TLR4/MyD88/NF-κB and NLRP3 signaling pathways in sepsis induced by cecal ligation and puncture in rats

Abstract

Sepsis is a life-threatening systemic inflammatory condition marked by a dysregulated host response to infection. The intestinal microbiota plays a pivotal role in maintaining immune homeostasis and epithelial barrier integrity, whereas dysbiosis significantly contributes to the pathogenesis of sepsis. This study investigates the effects of the Pseudomonas aeruginosa-derived metabolite Pyocyanin and the probiotic Saccharomyces boulardii (S. boulardii) on microbial composition and the TLR4/MyD88/NF-κB/NLRP3 signaling axis in a cecal ligation and puncture (CLP)-induced rat model of sepsis. The experimental design assessed the synergistic or antagonistic effects of single and combined treatments using molecular, microbiome, and immunohistochemical parameters to evaluate histopathological damage and microbial ecological dynamics. Seven experimental groups were established following CLP. Intra-abdominal Pyocyanin (10 nmol/g) and oral probiotic (10⁶ CFU/kg) treatments were administered either individually or in combination. Focused Ion Beam - Scanning Electron Microscopy (FIB-SEM) analyses revealed that the amorphous structure of Pyocyanin interacted with the surface of S. boulardii. Western blot analysis showed a 2.3-fold increase in TLR4/NF-κB expression in the CLP group (p ≤ 0.05), which synergistically rose to 4.5-fold with Pyocyanin (p ≤ 0.001), whereas probiotic treatment decreased expression levels by 35%. According to 16 S rRNA sequencing, Pyocyanin reduced α-diversity by increasing Lactobacillaceae abundance to 32.66% (Shannon index: 3.598 vs. 4.433 in control), while S. boulardii enhanced β-diversity by elevating Coriobacteriaceae (5.85%) and Prevotellaceae (10.63%) levels (Tables 2, 3 and 4). PCoA confirmed 41.7% Bray-Curtis dissimilarity between groups at the species level (PERMANOVA R²=0.38, p = 0.002). Histopathologically, severe hepatocyte necrosis (73.2 ± 6.1%, p = 0.0022) and a 4.2-fold increase in hepatic TGF-β expression were observed in the CLP group, whereas epithelial barrier damage was significantly attenuated in the probiotic groups. Immunofluorescence analysis revealed that combined treatment reduced Caspase-8 and TLR4 expression by 28% compared to Pyocyanin alone (p ≤ 0.05). In conclusion, S. boulardii supported microbiota homeostasis by suppressing TLR4/NF-κB signaling, whereas Pyocyanin exacerbated the inflammatory response via NLRP3 activation. These findings provide molecular evidence supporting probiotic-assisted immunomodulatory strategies in sepsis therapy.

Keywords: Saccharomyces boulardii; Gut microbiota; Pyocyanin; Sepsis; Western blot.

Fig. 1

SEM image, A S. boulardii cells exhibiting typical oval morphology with smooth and intact surfaces, indicative of healthy yeast structure. B Pyocyanin-treated sample showing disrupted surface topography, membrane irregularities, and potential signs of cellular stress or damage, suggesting the cytotoxic effects of pyocyanin exposure. C S. boulardii + Pyocyanin, where partial preservation of cellular integrity can be observed. Although some surface alterations are present, the morphology appears relatively more preserved compared to the pyocyanin-only group, indicating a possible protective or modulatory role of S. boulardii against pyocyanin-induced damage

Fig. 2

Relative protein expression levels as determined by Western blot analysis in the following experimental groups: Control (Group 1), CLP (Group 2), Pyocyanin (Group 3), S. boulardii (Group 4), CLP + Pyocyanin (Group 5), CLP + S. boulardii (Group 6), and CLP + Pyocyanin + S. boulardii (Group 7). All data are presented as fold changes relative to the control group (target protein/β-actin ratio normalized to 1.0). Values are expressed as mean ± standard deviation (SD). Groups labeled with different letters indicate statistically significant differences (p ≤ 0.05), as determined by one-way ANOVA followed by Tukey’s HSD post hoc test

Fig. 3

Relative protein expression levels determined by Western blot analysis across the experimental groups: Control (Group 1), CLP (Group 2), Pyocyanin (Group 3), S. boulardii (Group 4), CLP + Pyocyanin (Group 5), CLP + S. boulardii (Group 6), and CLP + Pyocyanin + S. boulardii (Group 7). Data are presented as fold change relative to the control group (normalized target protein/β-actin ratio = 1.0). Values represent mean ± standard deviation (SD). Statistical analysis was performed using one-way ANOVA followed by Tukey’s HSD post hoc test. Groups labeled with different letters indicate statistically significant differences (p ≤ 0.05)

Fig. 4

PCoA plot analysis result of bacterial 16 S rRNA gene at species and family level. Control (Group 1), CLP (Group 2), Pyocyanin (Group 3), S. boulardii (Group 4), CLP + Pyocyanin (Group 5), CLP + S. boulardii (Group 6), and CLP + Pyocyanin + S. boulardii (Group 7)

Fig. 5

Diversity curve plot of bacterial 16 S rRNA gene at species level. Control (Group 1), CLP (Group 2), Pyocyanin (Group 3), S. boulardii (Group 4), CLP + Pyocyanin (Group 5), CLP + S. boulardii (Group 6), and CLP + Pyocyanin + S. boulardii (Group 7)

Fig. 6

Dilution curve plot of bacterial 16 S rRNA gene. Control (Group 1), CLP (Group 2), Pyocyanin (Group 3), S. boulardii (Group 4), CLP + Pyocyanin (Group 5), CLP + S. boulardii (Group 6), and CLP + Pyocyanin + S. boulardii (Group 7)

Fig. 7

Taxonomic distributions at the species level result

Fig. 8

From left to right→↓, Liver, Intestine and Spleen tissue, Control group (A), CLP group (B), Pyocyanin group (C), S. boulardii group (D), CLP + Pyocyanin group (E), CLP + S. boulardii group (F), CLP + Pyocyanin + S. boulardii group (G), H&E, Bar: 10 mm

Fig. 9

Statistical analysis data of histopathological results seen in liver tissue. Degeneration, Necrosis, Inflammation (* p = 0.0108** p = 0.0022)

Fig. 10

Statistical analysis data of histopathological results in intestinal tissue. Degeneration, Necrosis, Inflammation (* p = 0.0108** p = 0.0022)

Fig. 11

Statistical analysis data of histopathological results observed in spleen tissue. Degeneration, Necrosis, Inflammation (* p = 0.0108** p = 0.0022)

Fig. 12

From left to right→↓, Liver, Intestine and Spleen tissue, Control group (A), CLP group (B), Pyocyanin group (C), S. boulardii group (D), CLP + Pyocyanin group (E), CLP + S. boulardii group (F), CLP + Pyocyanin + S. boulardii group (G), IHC-P, Bar: 10 mm

Fig. 13

Statistical analysis data of immunohistochemical results in liver, intestine, and spleen tissue from left to right→. TGFβ expression level (* p = 0.0108** p = 0.0022)

Fig. 14

From left to right→↓, Liver, Intestine and Spleen tissue, Caspase-8 expression (FITC), TLR4 expression (Texas Red), immunofluorescence (IF), bar: Bar:50 mm

Fig. 15

Statistical analysis data of immunofluorescence results in liver tissue. Degeneration, Necrosis, Inflammation, TGFβ expression level, Caspase-8 expression level, TLR4 expression level (* p = 0.0108** p = 0.0022)

Fig. 16

Statistical analysis data of immunofluorescence results in intestinal tissue. Caspase-8 expression level, TLR4 expression level (* p = 0.0108** p = 0.0022)

Fig. 17

Statistical analysis data of immunofluorescence results in spleen tissue. Caspase-8 expression level, TLR4 expression level (* p = 0.0108** p = 0.0022)