Apigenin Alleviates Autoimmune Uveitis by Inhibiting Microglia M1 Pro-Inflammatory Polarization

Abstract

Purpose: Apigenin is a natural small molecule compound widely present in various vegetables and fruits. Recently, Apigenin was reported to inhibit lipopolysaccharide (LPS)-simulated microglial proinflammatory activation. Considering the important role of microglia in retinal disorders, we wonder whether Apigenin could exert a therapeutic effect on experimental autoimmune uveitis (EAU) through reprogramming retinal microglia to a beneficial subtype.

Methods: EAU was induced in C57BL/6J mice by immunization with interphotoreceptor retinoid-binding protein (IRBP)651-670, followed by intraperitoneal administration of Apigenin. Disease severity was assessed based on clinical and pathological scores. In vivo, Western blotting was used to quantify protein levels of classical inflammatory factors, microglial M1/M2 markers and the tight junction protein of the blood-retinal-barrier (BRB). Immunofluorescence was used to determine the Apigenin's efficacy on microglial phenotype. In vitro, Apigenin was added in LPS and IFN-γ stimulated human microglial cell line. Western blotting and Transwell assays were used to analyze the phenotype of microglia.

Results: In vivo, we found that Apigenin significantly reduced the clinical and pathological scores of EAU. The protein levels of inflammatory cytokines were significantly decreased in retina, and BRB disruption was ameliorated after Apigenin treatment. Meanwhile, Apigenin inhibited microglia M1 transition in EAU mice retina. In vitro functional studies showed that Apigenin decreased LPS and IFN-γ-induced microglial inflammatory factor production and M1-activation via the TLR4/MyD88 pathway.

Conclusions: Apigenin can ameliorate retinal inflammation in IRBP induced autoimmune uveitis through inhibiting microglia M1 pro-inflammatory polarization via TLR4/MyD88 pathway.

Figure 1.

Apigenin ameliorates inflammation in EAU. (A) Representative pictures of slit lamp photography at d14 of each group (white arrow, conjunctival or ciliary congestion). (B) The clinical scores of anterior segments from d7 to d14 (*P < 0.05, the EAU+API group vs. the EAU group by Mann-Whitney U analysis, median ± interquartile, n = 4). (C) Representative fundus images of posterior pole at d14 of each group. (D)The fundus clinical scores at d14 (*P < 0.05, **P < 0.01 vs. the EAU group by Kruskal–Wallis, median ± interquartile, n = 5). (E) Representative H&E staining sections of the retina at day 14 of each group (black arrow, retinal folding; red arrow, vasculitis; green arrow, granulomatous formation) (Scale bars: 100 µm). (F) Pathological scores at day 14 according to Caspi's criteria (*P < 0.05, **P < 0.01 vs. the EAU group by Kruskal–Wallis, median ± interquartile, n = 5). (G) Representative images of Western blotting results for inflammatory cytokines in whole retinal extracts and corresponding statistics (mean ± SD; *P < 0.05, **P < 0.01, n = 3; one-way ANOVA). (H) Representative image of Western blotting results for BRB function and corresponding statistic (mean ± SD; *P < 0.05, n = 3; one-way ANOVA).

Figure 2.

Apigenin inhibits microglial M1 pro-inflammatory polarization in EAU. (A) Representative images of western blotting results for microglial M1/M2 marker in whole retinal extracts and corresponding statistics (mean ± SD; *P < 0.05, **P < 0.01, n = 3; one-way ANOVA). (B) Representative immunostaining images of microglia (IBA1, red) and M1 marker (CD74, green) in retinal flat mounts, nuclei were stained with DAPI (Scale bars: 100 µm). (C) Histogram summarizing the percentage of CD74⁺IBA1⁺/IBA1⁺ cells in different groups; three fields per each sample were chosen for analysis (mean ± SD; ***P < 0.001, n = 3; one-way ANOVA).

Figure 3.

Apigenin reduced ameboid microglia and microglia migration in EAU mice retina. (A) Representative immunostaining images of microglia (IBA1, green) in retinal flat mounts (n = 3, scale bars: 100 µm). (B) Representative immunostaining images of microglia (IBA1, green) in retinal paraffin sections, nuclei were stained with DAPI (n = 3, scale bars: 100 µm).

Figure 4.

Apigenin inhibits inflammatory cytokines production and M1 polarization in HMC3 cell line. (A) CCK8 assays were carried out to test drug toxicity on HMC3 cell lines both under physiological condition and pathological condition. (B) RT-qPCR to analyze mRNA level for each inflammatory cytokine. (C) Representative images of Western blotting results for inflammatory cytokines and corresponding statistics. (D) Representative images of Western blotting results for microglial M1/M2 markers and corresponding statistics. (E) Wound healing assays to test microglia mobility and corresponding statistics (scale bars: 200 µm). (F) Transwell assays to test microglia migration ability and corresponding statistics (scale bars: 200 µm). All above data show as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant; n = 3; one-way ANOVA.

Figure 5.

Apigenin inhibits microglial M1 polarization via TLR4/MyD88 signaling. (A) Representative images of Western blotting results for TLR4/MyD88 signal and corresponding statistics. (B) Representative images of western blotting results for Nrf2/HO-1 signal and corresponding statistics. Above data show as mean ± SD; *P < 0.05, **P < 0.01; ns, not significant; n = 3; one-way ANOVA.