Berberine Inhibits the Inflammatory Response Induced by Staphylococcus aureus Isolated from Atopic Eczema Patients via the TNF-α/Inflammation/RAGE Pathways

Abstract

Atopic eczema patients exhibit high levels of Staphylococcus aureus (S. aureus) skin colonization. S. aureus can stimulate macrophages and the expression of proinflammatory cytokines. Berberine (BBR), an alkaloid, attenuates S. aureus toxin production. This study investigated if BBR suppressed bacterial growth and inflammatory response induced by eczema-patient-derived S. aureus using murine macrophage (RAW 264.7) and human monocyte cell lines (U937). RAW 264.7 and U937 were treated with BBR at different concentrations and stimulated with heat-killed S. aureus (ATCC #33591) or S. aureus derived from severe eczema patients (EC01-EC10), who were undergoing topical steroid withdrawal, for 24 h. TNF-α protein levels were determined by ELISA, gene expression by qRT-PCR, cell cytotoxicity by trypan blue excursion, and reactive oxygen species (ROS) levels by fluorometric assay. BBR showed a bacteriostatic effect in S. aureus (ATCC strain #33591 and clinical isolates (EC01-EC10) and suppressed TNF-α production in RAW 264.7 and U937 cells exposed to heat-killed S. aureus (ATCC and clinical isolates) dose-dependently without any cell cytotoxicity. BBR (20 µg/mL) suppressed >90% of TNF-α production (p < 0.001), downregulated genes involved in inflammatory pathways, and inhibited S. aureus ROS production in U937 and RAW 264.7 cells (p < 0.01). BBR suppresses S. aureus-induced inflammation via inhibition of TNF-α release, ROS production, and expression of key genes involved in the inflammatory pathway.

Keywords: S. aureus; TNF-α; atopic dermatitis; berberine; inflammation.

Figure 1

Inhibitory effect of BBR on clinical S. aureus isolates. Clinical S. aureus strains (EC01–EC10) were cultured in the presence of BBR at different concentrations (32 μg/mL, 64 μg/mL, 128 μg/mL, 256 μg/mL, and 512 μg/mL) for 18–20 h. MIC was determined by measuring absorbance at 625 nm. BBR showed dose-dependent inhibition of bacteria growth in clinical strains. (A) EC01; (B) EC02; (C) EC03; (D) EC04; (E) EC06; (F) EC07; (G) EC0; and (H) EC10. Green–without BBR; Blue–with BBR. Data represent mean ± SD. (N = 3 replicates; * p > 0.05; ** p > 0.01; *** p < 0.001 vs. 0).

Figure 2

Dose-dependent effect of BBR on ATCC strain heat-killed S. aureus. RAW 264.7 cells were cultured in the presence and absence of ATCC strain (#33591) HKS (MOI, 1:2) and BBR at different concentrations (2.5 μg/mL, 5 μg/mL, 10 μg/mL, and 20 μg/mL) for 24 h. (A) BBR pre-treatment showed a dose-dependent inhibition of TNF-α levels following HKS stimulation. (B) Cell viability measured by trypan blue exclusion showed no cell cytotoxicity across all groups. (C,D) Similar results were observed in U937 following BBR treatment. (A,C); Green- without BBR; Blue- with BBR). Data represent mean ± SD. (N = 3–6 replicates; ^### p < 0.001 vs. untreated; *** p < 0.001 vs. HKS).

Figure 3

Dose-dependent effect of BBR on clinical isolates of heat-killed S. aureus. U937 cells were cultured in the presence and absence of clinical isolates of HKS (1 × 10⁶ CFU/mL) and BBR at different concentrations. BBR treatment (2.5 µg/mL, 5 μg/mL, 10 μg/mL, and 20 μg/mL) showed a dose-dependent inhibition of TNF-α production in the presence of heat-killed clinical S. aureus isolates (A–H) EC01, EC02, EC03, EC04, EC06, EC07, EC09, EC10. Green- without BBR; Blue- with BBR. TNF-α levels were measured by ELISA. Data represent mean ± SD. (N = 3–5 replicates; ^### p < 0.001 vs. untreated; ** p < 0.01; *** p < 0.001 vs. HKS).

Figure 4

No cell cytotoxicity was observed with BBR, and heat-killed S. aureus. U937 cells were cultured in the presence and absence of clinical isolates of HKS (1 × 10⁶ CFU/mL) and BBR at different concentrations. BBR treatment (2.5 µg/mL, 5 μg/mL, 10 μg/mL, and 20 μg/mL) showed no cell cytotoxicity at any concentration for clinical strains (A–H) EC01, EC02, EC03, EC04, EC06, EC07, EC09, EC10. Green- without BBR & HKS; Blue- with BBR & HKS. Cell cytotoxicity was evaluated by trypan blue exclusion. Data represent mean ± SD. (N = 3 replicates).

Figure 5

Effect of BBR on gene expression associated with TNF, AGE-RAGE, and inflammatory pathways. U937 cells were cultured in the presence in BBR (20 μg/mL) and ATCC (#33591) HKS (MOI, 2:1) for 24 h. Cells were harvested, and gene expression analysis was performed by qRT-PCR. There was a significant decrease in expression of (A) TNF-α; (B) IL-1β, (C) IL-6; (D) PTGS2; (E) CASP3; (F) MAPK1; (G) AKT; (H) MAPK3 in the presence of BBR. Green- without BBR & HKS; Orange- with HKS & without BBR; Blue- With HKS & BBR. Data represent mean ± SD. (N = triplicate culture. ^# p < 0.05; ^## p < 0.01; ^### p < 0.001 vs. untreated; * p < 0.05; *** p < 0.001 vs. HKS).

Figure 6

Effect of BBR on ROS production: Intracellular reactive oxygen species (ROS) levels were measured using the DCFDA/H2DCFDA-Cellular ROS assay kit (Abcam, MA) as per the manufacturer’s instructions. U937 pre-treated with BBR (20 μg/mL) showed reduced ROS production as compared to untreated cells stimulated with clinical S. aureus strains. (A) EC01; (B) EC02; (C) EC03; (D) EC04; (E) EC06; (F) EC07, (G) EC09; and (H) EC10. Green- without BBR & HKS; Orange- with HKS & without BBR; Blue- With HKS & BBR. Data represent mean ± SD. (N = triplicate culture. ^### p < 0.001 vs. untreated; ** p < 0.01; *** p < 0.001 vs. HKS).