Luteolin alleviates Herpes Simplex Keratitis by inhibiting inflammatory responses via suppressing the PTGS2/NF-κB signaling pathway

Abstract

Objective: Herpes Simplex Keratitis (HSK) is a leading cause of infectious-related blindness, primarily driven by corneal inflammation and dysregulated immune response. Luteolin (LUT), a flavonoid with anti-inflammatory and antiviral properties, has the potential to target key signaling pathways and provide therapeutic benefits. This study aimed to investigate the mechanisms through which LUT alleviates HSK.

Methods: LUT's potential targets in HSK were identified using a systemic biology approach. Protein-Protein Interaction (PPI) analysis was performed to determine key hub genes. Molecular docking was used to assess LUT's binding affinity to PTGS2 and other key proteins. HSK mice were treated with LUT, and corneal opacity, edema, and thickness were then evaluated using slit-lamp microscopy and Optical Coherence Tomography. Inflammatory cytokine levels were quantified by quantitative Real-Time Polymerase Chain Reaction PCR (qRT-PCR), while PTGS2/NF-κB pathway activation, PTGS2 expression, and NF-κB phosphorylation levels were assessed by Western Blotting (WB).

Results: LUT was found to regulate 30 HSK-related proteins, with AKT1, TNF, and PTGS2/NF-κB identified as key nodes. Furthermore, molecular docking confirmed a strong binding to PTGS2 (-9.7 kcal/mol). LUT treatment significantly reduced corneal opacity and edema, restoring corneal thickness to near-normal levels. RT-qPCR revealed downregulation of inflammatory cytokines, and WB analysis showed decreased PTGS2 expression and NF-κB phosphorylation, confirming LUT's role in attenuating corneal inflammation via inhibiting the PTGS2/NF-κB signaling pathway.

Conclusions: LUT alleviates HSK by modulating the PTGS2/NF-κB signaling pathway, reducing inflammation and corneal damage, and highlighting its therapeutic potential for ocular inflammatory diseases.

Keywords: Herpes Simplex Keratitis; LUT; PTGS2/NF-κB; Traditional Chinese Medicine; inflammatory response.

Figure 1

Frequency of Chinese herbs used in Traditional Chinese Medicine (TCM) prescription for Herpes Simplex Keratitis (HSK).

Figure 2

Relationship among TCM core herbs for HSK, active ingredients of herbs, and protein targets of active ingredients. Note: Orange indicates TCM core herbs for HSK; Yellow indicates active ingredients of herbs; Blue indicates protein targets of active ingredients.

Figure 3

Core protein targets of TCM prescription for HSK. A: Venn Diagram of TCM core herb targets and HSK-related disease targets. B: Protein-protein interaction network (PPI) of 30 intersection target genes. C: Degree value of the top 5 core target genes in the PPI network.

Figure 4

GO and KEGG functional enrichment analysis. A: GO Functional Enrichment Analysis. B: KEGG Functional Enrichment Analysis. Note: G0: gene ontology BP: Biological Process; CC: Cellular Component; MF: Molecular Function.

Figure 5

Molecular docking model diagram of proteins and active compounds. Note: AKT1: Serine/threonine-protein kinase; EGFR: Epidermal growth factor receptor; BCL2: B-cell lymphoma 2; PTGS2: Prostaglandin-endoperoxide synthase; SRC: Non-receptor tyrosine kinase.

Figure 6

Luteolin (LUT) alleviated HSK in mice. A: Slit-lamp photographs showing corneal opacity over time in HSK and HSK+LUT groups; B: Comparison of corneal opacity scores between HSK and HSK+LUT groups at Day 1, 3, 5; C: Corneal thickness measured by RT-OCT; D: Corneal thickness measurement. ns: P>0.05; *, **: P<0.01; ***: P<0.001.

Figure 7

LUT alleviated HSK by suppressing NF-κB signaling and inflammatory responses in mouse cornea. A: The expression of NF-κB pathway proteins was significantly decreased in corneal tissues following LUT treatment, as assessed by western blotting. HSK+LUT: Group treated with LUT. Protein levels were normalized to β-actin (n=3). B: RT-qPCR results of inflammatory factors. C: Slit-lamp photographs and corresponding clinical scoring. HSK+BAY: Group treated with Bay inhibitor. D: RT-OCT and corneal thickness measurement. Data are presented as mean ± SD (n=3). ns: P>0.05; *: P<0.05; **: P<0.01; ***: P<0.001.

Figure 8

LUT alleviated HSK through PTGS2/ NF-κB pathway. A: Western blot analysis of PTGS2 expression in animal model treated with LUT. B: Western blot analysis of NF-κB pathway in cells with PTGS2 silencing by siRNA. Vehicle-Con: Control group treated with vehicle. si-PTGS2: cells treated with PTGS2-targeting siRNA. C: Slit-lamp photograph showing corneal condition under different treatment groups. FK+HSK: Group treated with FK inhibitor; FK+LUT: Group treated with FK inhibitor and LUT treatment. D: RT-OCT and corneal thickness measurement. ns: P>0.05; *: P<0.05; **: P<0.01; ***: P<0.001.