Paeoniflorin Prevents Intestinal Barrier Disruption and Inhibits Lipopolysaccharide (LPS)-Induced Inflammation in Caco-2 Cell Monolayers
Abstract
Inflammatory bowel disease (IBD) is closely associated with bacterial infection and the disruption of the intestinal barrier. Paeoniflorin (PF), a bioactive compound from Paeonia lactiflora Pallas plants, has been reported to exhibit anti-inflammatory effects. However, the impact of PF on intestinal barrier function and its underlying molecular mechanisms has not been clarified.
In this study, PF significantly increased transepithelial electrical resistance (TEER), decreased FITC-dextran permeability, and restored the expression of tight junction proteins, including occludin, ZO-1, and claudin-5, in LPS-induced Caco-2 cells. Treatment with PF suppressed LPS-induced expression of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and matrix metalloproteinase-9 (MMP-9). Furthermore, PF suppressed nuclear factor kappa B (NF-κB) activation through stimulation of the Nrf2/HO-1 signaling pathway.
These findings demonstrate that PF inhibits LPS-induced intestinal barrier disruption and inflammatory responses, suggesting its potential as a therapeutic agent for IBD.
Introduction
The intestinal epithelium functions as a barrier that separates the intestinal mucosa from the lumen environment. It consists of proliferating and differentiating intestinal epithelial cells (IECs) forming a monolayer. Damage to tight junctions (TJs) in IECs contributes to IBD progression by increasing intestinal permeability.
Tight junction proteins act as sealing complexes between adjacent IECs, regulating paracellular water, ion, and solute transport, while preventing immunogenic macromolecule entry. The most important proteins include ZO-1, occludin, and claudins. Upregulation of these proteins can reduce intestinal permeability. Conversely, disruption of their expression leads to intestinal barrier impairment.
Lipopolysaccharide (LPS), a bacterial component, plays a key role in triggering systemic and intestinal inflammation. Studies show that LPS increases TJ permeability and disrupts intestinal barrier function. Persistent inflammation amplifies barrier disruption and enhances pro-inflammatory cytokine release, generating a vicious cycle.
Several natural compounds demonstrate protective effects on intestinal barrier dysfunction. For example, vitamin A reverses LPS-induced damage; extracts of Boswellia serrata and Curcuma longa increase TEER values; probiotics combined with herbal extracts protect against inflammation-induced TEER decreases; and 6-gingerol restores barrier function while suppressing inflammation.
Paeoniflorin (PF), a major compound isolated from Paeonia lactiflora, exhibits anti-inflammatory, antioxidant, and immunoregulatory activities. However, its specific effects on intestinal barrier inflammation remain unknown. Using human Caco-2 cells as a model, we investigated the protective effects of PF on barrier function under LPS-induced conditions and explored the underlying mechanisms.
Materials and Methods
Materials
PF was purchased from Solarbio (Beijing, China). LPS was obtained from Sigma-Aldrich. Cell culture media and fetal bovine serum were obtained from GIBCO. Antibodies against tight junction proteins, HO-1, COX-2, MMP-9, Nrf2, and NF-κB were purchased from commercial suppliers.
Cell Culture
Caco-2 cells were grown in DMEM/F12 medium containing 10% FBS and 1% penicillin-streptomycin at 37 °C in 5% CO2. Media were replaced every 2 days. Cells were starved in serum-free medium for 24 h before experiments.
Cell Viability Assay
The cytotoxicity of PF was tested using a CCK-8 assay. Caco-2 cells were treated with LPS (10 μg/mL) or PF at 10, 50, 100, or 150 μM for 24 h or 48 h. Viability was measured using absorbance at 450 nm.
TEER and Permeability Assays
Barrier integrity was assessed by measuring TEER and FITC-dextran flux across Caco-2 monolayers cultured on transwell inserts. TEER values were normalized to membrane surface area, while permeability was assessed by measuring FITC-dextran transport.
RT-PCR and Western Blotting
RT-PCR was used to measure mRNA levels of TNF-α, IL-6, COX-2, iNOS, occludin, ZO-1, and claudin-5. Western blotting was performed to analyze protein expression of tight junction proteins, COX-2, iNOS, MMP-9, NF-κB, IκBα, Nrf2, and HO-1.
Immunofluorescence
Cells were fixed, blocked, and incubated with primary antibodies for occludin and p65 (NF-κB subunit), followed by fluorescently labeled secondary antibodies. Nuclei were counterstained with DAPI.
siRNA Transfection
Nrf2 siRNA and control siRNA were transfected into cells using Lipofectamine RNAiMAX according to the manufacturer’s instructions to assess the role of Nrf2 in PF-mediated effects.
Statistical Analysis
Data were presented as mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test was used to assess statistical significance (p < 0.05).
Results
Dose-Effect of PF on Caco-2 Cell Viability
Cell viability assays showed no significant cytotoxicity at concentrations ≤150 μM PF after 24 h or 48 h. Only at 150 μM after 48 h did PF slightly reduce viability. Therefore, doses of 10, 50, and 100 μM were used in subsequent experiments.
PF Inhibits LPS-Induced Inflammation in Caco-2 Cells
LPS stimulation increased mRNA and protein levels of IL-6, TNF-α, iNOS, and COX-2. PF significantly reversed these increases in a dose-dependent manner. PF suppressed both transcription and translation of these inflammatory mediators.
PF Prevents Loss of Tight Junction Proteins
LPS significantly decreased TEER values and increased FITC-dextran permeability. PF treatment restored TEER and reduced permeability. Western blot and immunofluorescence confirmed that PF restored occludin, ZO-1, and claudin-5 expression reduced by LPS.
PF Suppresses NF-κB Activation
LPS stimulation caused IκBα degradation and increased nuclear NF-κB p65 expression. PF inhibited NF-κB nuclear translocation and also suppressed MMP-9 expression. Immunofluorescence confirmed reduced NF-κB nuclear localization with PF pretreatment.
PF Activates Nrf2/HO-1 Pathway
PF increased Nrf2 and HO-1 protein expression under LPS stimulation. Nrf2 siRNA knockdown abrogated this effect, reversed improvements in TEER and permeability, and abolished PF-mediated downregulation of NF-κB target genes. This demonstrated that PF exerts anti-inflammatory and barrier-protective effects through Nrf2-mediated inhibition of NF-κB signaling.
Discussion
IBD, including ulcerative colitis and Crohn’s disease, is a chronic, relapsing inflammatory disorder that remains difficult to treat effectively with current therapies. Excessive side effects and limited efficacy highlight the need for new therapeutic approaches.
The present study demonstrates that PF attenuates intestinal inflammation and barrier dysfunction in Caco-2 cells. PF improved cell integrity by restoring tight junction protein expression and protecting barrier function. It significantly inhibited LPS-induced NF-κB activation while activating the Nrf2/HO-1 signaling pathway. Importantly, Nrf2 knockdown abolished the protective effects, confirming that the anti-inflammatory properties of PF are dependent on this pathway.
Damage to tight junction proteins contributes to increased intestinal permeability and IBD progression. Our findings establish that PF counteracts TJ disruption by LPS and thus preserves barrier function. In addition, PF inhibited pro-inflammatory cytokine production and restored epithelial resistance.
The results suggest that PF may represent a novel therapeutic candidate capable of suppressing intestinal inflammation by blocking NF-κB activation and activating Nrf2/HO-1 signaling.
Conclusion
This study demonstrates that paeoniflorin significantly inhibits LPS-induced intestinal barrier disruption in Caco-2 cells. By suppressing NF-κB signaling and activating the Nrf2/HO-1 pathway, PF restored tight junction protein levels, reduced inflammation, and strengthened barrier integrity. These results indicate that PF is a promising therapeutic candidate for the prevention and treatment of intestinal inflammation NSC 178886 and IBD.