Pinggan Yuyin Qingre formula (平肝育阴清热方) ameliorates meibomian gland dysfunction through the interleukin-17/nuclear factor kappa B pathway based on network pharmacology and experimental validation

doi:10.19852/j.cnki.jtcm.2026.01.016

Abstract

Abstract:

OBJECTIVE: To investigate the pharmacological effects and underlying mechanisms of Pinggan Yuyin Qingre formula (平肝育阴清热方, PGYYQR) in the treatment of meibomian gland dysfunction (MGD) through network pharmacology and in vivo validation.

METHODS: A mouse model of MGD was induced using the stearoyl-coenzyme a desaturase 1 inhibitor, followed by PGYYQR treatment for 2 weeks. MGD sign scoring, hematoxylin and eosin (HE) staining, oil red o (ORO) staining, and serum inflammatory cytokine analysis were conducted to assess the effects of PGYYQR on meibomian gland (MG) function, histopathology, and associated inflammation. Network pharmacology was employed to identify the active compounds and potential targets of PGYYQR. Molecular mechanisms were further investigated using Western blotting, reverse transcription quantitative real-time polymerase chain reaction, and reactive oxygen species (ROS) assays.

RESULTS: PGYYQR treatment significantly reduced the scores of MG orifice obstruction and meibum quality in MGD mice. HE and ORO staining further demonstrated that PGYYQR ameliorated glandular damage and lipid dysfunction. Enzyme-linked immunosorbent assay results revealed that PGYYQR markedly decreased the serum levels of key inflammatory cytokines, including interleukin (IL)-1β, IL-6, and tumor necrosis factor-α. Network pharmacology identified 162 active compounds and 598 target genes in PGYYQR. Among these, IL-6, IL-1β, matrix metalloproteinase-9, and C-X-C motif chemokine ligand 8 were recognized as core targets related to MGD and were mainly enriched in the IL-17/ nuclear factor kappa B (NF-κB) signaling pathway. Further molecular analyses confirmed that PGYYQR significantly inhibited the IL-17/NF-κB axis by downregulating IL-17 expression and reducing phos-phorylated NF-κB p65 levels at both the protein and mRNA levels in MG tissues. PGYYQR also effectively reduced ROS levels in the conjunctival tissues of MGD mice.

CONCLUSION: PGYYQR effectively improves MG function and preserves local tissue morphology in MGD model mice, primarily by suppressing the inflammatory response through coordinated modulation of the IL-17/NF-κB signaling pathway and oxidative stress.

Key words: meibomian gland dysfunction, network pharmacology, interleukin-17, NF-kappa B, signal transduction, reactive oxygen species, Pinggan Yuyin Qingre formula

GAO Yinli, ZHANG Linhua, ZHANG Yaqiang, ZHANG Liuhe, ZHI Nan, LIAN Haihong. Pinggan Yuyin Qingre formula (平肝育阴清热方) ameliorates meibomian gland dysfunction through the interleukin-17/nuclear factor kappa B pathway based on network pharmacology and experimental validation[J]. Journal of Traditional Chinese Medicine, 2026, 46(1): 172-182.

Figures/Tables 4

Figure 1 PGYYQR improves MG function and restores MG morphology in MGD mice A: slit-lamp observation of each group after modeling. obstruction of MG orifices (indicated by the purple arrow), increased viscosity of meibum (indicated by the red arrow); B: slit-lamp observation of each group after intervention with PGYYQR; C: MG orifice obstruction score (n = 10); D: meibum quality score (n = 10); E: histological changes of MG tissues with HE staining (× 100); F: lipid deposition in MG tissues with ORO staining (× 100 ); A1, B1, E1, F1: control group; A2, B2, E2, F2: model group; A3, B3, E3, F3: PGYYQR group. Control group: no treatment; model group: treated with A939572 solution (10 mg/kg); PGYYQR group: treated with A939572 solution (10 mg/kg) and PGYYQR decoction. PGYYQR: Pinggan Yuyin Qingre formula; MG: meibomian gland; MGD: meibomian gland dysfunction; HE: hematoxylin and eosin; ORO: oil red o; ac: acinar cell; du: ductal lumen. Data are presented as mean ± standard deviation. Post-intervention comparisons were analyzed by the Wilcoxon rank-sum test due to uniform values (standard deviation = 0) in the model group. aP < 0.05, bP < 0.01 vs model group

Figure 2 PGYYQR alleviates the inflammatory response in MGD mice A: levels of inflammatory cytokines in ocular serum measured by ELISA (n = 10); A1: IL-1β levels; A2: IL-6 levels; A3: TNF-α levels; B: histological observation of inflammatory changes in MG tissues with HE staining (× 400). Abnormal MG duct wall thickening (indicated by the black arrow), inflammatory cell infiltration (indicated by the red arrow); B1: control group; B2: model group; B3: PGYYQR group. Control group: no treatment; model group: treated with A939572 solution (10 mg/kg); PGYYQR group: treated with A939572 solution (10 mg/kg) and PGYYQR decoction. PGYYQR: Pinggan Yuyin Qingre formula; MGD: meibomian gland dysfunction; ELISA: enzyme-linked immunosorbent assay; IL-1β: interleukin-1β; IL-6: interleukin-6; TNF-α: tumor necrosis factor-α; MG: meibomian gland; HE: hematoxylin and eosin. Data are presented as mean ± standard deviation. Comparisons between groups were conducted using one-way analysis of variance with Tukey’s post hoc test. aP < 0.01 vs control group; bP < 0.01, cP < 0.05 vs model group.

Figure 3 Functional enrichment analysis of the potential targets of PGYYQR for the treatment of MGD A: GO enrichment analysis; B: KEGG enrichment analysis. PGYYQR: Pinggan Yuyin Qingre formula; MGD: meibomian gland dysfunction; GO: gene ontology; BP: biological process; CC: cellular component; MF: molecular function; KEGG: kyoto encyclopedia of genes and genomes.

Figure 4 PGYYQR suppresses the IL-17/NF-κB axis and reduces oxidative stress A: IL-17 levels in ocular serum measured by ELISA (n = 10); B: protein expression levels of p-NF-κB p65, NF-κB p65 in MG tissues (n = 3); B1: protein band; B2: quantitative analysis of p-NF-κB p65 protein expression normalized to GAPDH; B3: quantitative analysis of NF-κB p65 protein expression normalized to GAPDH; C: mRNA expression of NF-κB p65 in MG tissues (n = 3); D: ROS fluorescence intensity in conjunctival tissues (n = 10). Control group: no treatment; model group: treated with A939572 solution (10 mg/kg); PGYYQR group: treated with A939572 solution (10 mg/kg) and PGYYQR decoction. PGYYQR: Pinggan Yuyin Qingre formula; IL-17: interleukin-17; NF-κB: nuclear factor kappa B; ELISA: enzyme-linked immunosorbent assay; p-NF-κB p65: phospho-nuclear factor kappa B p65; MG: meibomian gland; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; ROS: reactive oxygen species. Data are presented as mean ± standard deviation. Comparisons between groups were conducted using one-way analysis of variance with Tukey’s post hoc test. aP < 0.01, cP < 0.05 vs control group; bP < 0.01, dP < 0.05 vs model group; eP > 0.05 between groups.

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