LTA

Bacterial pathogen-associated molecular patterns (PAMPs), specifically lipopolysaccharide (LPS) from Gram-negative bacteria (E. coli, P. aeruginosa) and lipoteichoic acid (LTA) from Gram-positive bacteria (S. aureus, S. epidermidis), activate the TLR-4 and TLR-2 signalling pathways in keratinocytes. This activation leads to uncontrolled inflammation and cellular damage. Inflammatory skin disorders, such as atopic dermatitis and chronic wounds, are often exacerbated by these pathways.

This study elucidates the protective mechanisms of kaempferol, a natural flavonoid, against LPS + LTA-induced cytotoxicity and inflammation in HaCaT keratinocytes and a murine model of systemic inflammation.

In vitro, HaCaT cells were pretreated with kaempferol before exposure to LPS/LTA, followed by assessments of viability (MTT), apoptosis (Annexin V/PI), cytokine levels (ELISA), mRNA expression (qPCR), and protein expression (western blot). For in vivo studies, BALB/c mice were divided into five groups: (1) saline control (i.p.); (2) LPS/LTA (i.p.); and (3-5) kaempferol pretreatment (25, 50, 75 mg/kg, oral) before LPS/LTA challenge. Inflammatory markers were evaluated via ELISA, histopathology (H&E and Picro-Sirius red staining), and western blot.

Kaempferol significantly reduced apoptosis and suppressed the secretion of pro-inflammatory cytokines, including IL-6, IL-1β, and TNF-α. Mechanistically, it inhibited the phosphorylation of NF-κB p65 and MAPK (p38/JNK) by downregulating TLR-2 and TLR-4 expression in both HaCaT keratinocytes and an LPS + LTA-induced systemic model. These actions resulted in reduced liver inflammatory foci and collagen deposition. Furthermore, kaempferol interfered with MyD88-dependent signalling, lowered ROS overproduction, and inhibited IRF3 activity.

Kaempferol differentially modulates TLR-2/TLR-4 crosstalk, thereby mitigating bacterial toxin-induced inflammation in both HaCaT keratinocytes and systemic model. This modulation was evidenced by a reduction in inflammatory foci and TLR activation in liver tissues, suggesting a novel therapeutic approach for skin inflammation. Furthermore, its dual TLR antagonism and cytoprotective effects could potentially surpass those of synthetic inhibitors, thereby enhancing its translational potential for treating TLR-mediated dermatological conditions.

The microbial metabolite indole not only affects gut microbiome function but also regulates intestinal homeostasis and the immune response. Indole is primarily located in the intestinal tract; however, its effects on duck intestinal epithelial cells (IECs) under lipoteichoic acid (LTA) stimulation remain unknown. In this study, primary duck IECs were isolated, and their identity was verified using immunofluorescence staining and specific gene expression profiling. The proliferative activity of IECs was detected using the CCK-8 method. The viability of IECs under varying LTA concentrations was detected using the MTT assay. The mRNA levels of TNF-α and IL-1β under varying LTA concentrations and of TNF-α, IL-6, TGF-β1, claudin-1, and ZO-1 under varying indole concentrations were detected using quantitative real-time polymerase chain reaction (qRT-PCR). The expression levels of NF-κB-Rel and NF-κB-p65 in IECs subjected to a combination of LTA (40 μg/mL, 24 h) and indole (50 mg/L, 24 h) were detected using qRT-PCR and western blotting, respectively. We observed a reduction in IEC viability with increasing LTA concentrations. In vitro, LTA (40 μg/mL, 24 h) treatment significantly induced the mRNA expression of TNF-α and IL-1β in IECs (p < 0.001). However, indole (50 mg/L, 24 h) treatment significantly reduced their expression (p < 0.01), whereas it significantly increased mRNA levels of TGF-β1, claudin-1, and ZO-1 (p < 0.01) under LTA (40 μg/mL, 24 h) treatment. The mRNA expression of NF-κB-Rel significantly decreased in the LTA + indole group (p < 0.001) compared to that in the LTA group. Grayscale value analyses revealed that the LTA + indole group had lower phosphorylated-NF-κB-p65/NF-κB-p65 ratios for the total and cytoplasmic protein fractions of IECs than those of the LTA group (p < 0.001). In conclusion, indole reduces the inflammatory response in LTA-treated duck IECs by regulating the NF-κB signaling pathway, which may also maintain the function of the intestinal epithelial barrier by enhancing the mRNA expression of genes encoding tight junction proteins (claudin-1 and ZO-1) in ducks.