Background 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. Objectives 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. Methods 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. Results 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. Conclusion 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. Graphical Abstract

Kaempferol suppresses TLR-2 and TLR-4 coactivation by attenuation of LPS/LTA-induced inflammation and cellular stress

期刊论文
发表日期：

2025年9月5日

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Muhammad Haroon,
Sun Chul Kang

摘要

Background

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.

Objectives

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.

Methods

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.

Results

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.

Conclusion

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.

Graphical Abstract