Traditional Chinese Medicine, Ziyin-Mingmu Decoction, Regulates Cholesterol Metabolism, Oxidative Stress, Inflammation and Gut Microbiota in Age-related Macular Degeneration Models

Abstract

Background: Age-related macular degeneration (AMD) is the commonest cause of retinal disorders in the aged population. Ziyin-Mingmu decoction (ZD) has been widely used to treat AMD patients over thousands of years, however the underlying functional mechanisms of ZD are largely elusive. In this study, we aim to elucidate the therapeutic mechanisms of ZD in AMD models.

Methods: An in vivo AMD mouse model and an in vitro AMD model were established. Cholesterol level in mouse tissues was measured. Expression of antioxidant genes and proinflammatory cytokines in mouse tissues and in human retinal pigment epithelial (RPE) cells were detected using biochemical approaches. Gut microbiota community and functional pathways were analysed using bioinformatics approach. Compounds in ZD were identified using HPLC/MS.

Results: High fat diet (HFD)-fed mice had significantly higher levels of cholesterol in the retina, RPE, liver and serum, and markedly decreased expression of cholesterol metabolism-associated genes in those tissues, compared to mice fed with normal diet. Similarly, expression of antioxidant and inflammation genes was dysregulated in HFD-fed mouse tissues. ZD treatment reversed these HFD-induced pathological effects. HFD also altered the composition of cecum bacterial communities and associated metabolic pathways, which returned to control levels by ZD. In vitro assays showed that H₂O₂ significantly increased oxidative stress and enhanced expression of proinflammatory cytokines. Co-treatment with ZD significantly counteracted these changes. HPLC/MS identified 105 compound in water extracted ZD and most are polyphenols.

Conclusion: Our data suggests that protection of ZD against AMD is possibly through mitigating cholesterol level, oxidative stress and inflammation, and modulating gut microbiota by polyphenols.

Keywords: age-related macular degeneration; cholesterol; gut microbiota; inflammation; oxidative stress; ziyin-mingmu decoction.

Fig. 1

Effect of ZD treatment on cholesterol level in the RPE, retina, liver and serum. Data was analysed with one-way ANOVA, followed by Bonferroni multiple comparison test and is displayed as mean ± SE. CD, control diet; HFD, high-fat- diet; RPE, retinal pigment epithelial cells; ZD, Ziyin-Mingmu decoction. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns: no significance.

Fig. 2

Effect of ZD treatment on expression of cholesterol trafficking and metabolism associated genes in the RPE (A), retina (B) and liver (C). Cycle threshold (CT) values of targeted genes were normalised to Gapdh gene then analyzed by 2^−ΔΔCt formula. The fold changes of individual genes were analysed with one-way ANOVA, followed by Bonferroni multiple comparison test and displayed as mean ± SE. CD, control diet; HFD, high-fat diet; RPE, retinal pigment epithelial cells; ZD, Ziyin-Mingmu decoction. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns: no significance.

Fig. 3

Effect of ZD treatment on expression of antioxidant genes, catalase, glutathione peroxidase 1 (Gpx1) and superoxide dismutase (Sod1) in RPE (A), retinas (B) and liver (C). Cycle threshold (CT) values of targeted genes were normalised to Gapdh gene then analyzed by 2⁻.^ΔΔCt formula. The fold changes of individual genes were analysed with one-way ANOVA, followed by Bonferroni multiple comparison test and displayed as mean ± SE. CD, control diet; HFD, high-fat- diet; RPE, retinal pigment epithelial cells; ZD, Ziyin-Mingmu decoction. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Fig. 4

Effect of ZD treatment on proinflammatory genes, Il-1β (A) and Tnfα (B) in RPE, retina and liver. Cycle threshold (CT) values of targeted genes were normalised to Gapdh gene then analyzed by 2^−ΔΔCt formula. The fold changes of individual genes were analysed with one-way ANOVA, followed by Bonferroni multiple comparison test and displayed as mean ± SE. CD, control diet; HFD, high-fat diet; RPE, retinal pigment epithelial cells; ZD, Ziyin-Mingmu decoction. ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns: no significance.

Fig. 5

Difference of the phyla relative abundance difference (A) and Heatmap representation (B) in cecum samples of three experimental groups. The cecal content of control group (CL); High-fat- diet (HFD) group and High-fat- diet + ZD treatments (HFD + ZD) group.

Fig. 6

Bacterial community diversities, α-diversity (A) and β-diversity (B), among the three experimental groups. α -diversity: the Simpson and Shannon metrics revealed significant differences in the bacterial community diversity among three groups, with a p value < 0.05 considered statistically significant (a). β-diversity: Principal Coordinate Analysis (PCoA), generated using Bray–Curtis, and Jaccard Index, highlight distinct microbiome composition associated with each group. Control animals (red) display a distinct pattern, HFD animals (red) and HFD + ZD (green).

Fig. 7

Significant differences in bacterial MetaCyc pathways of HFD compared with CL and HFD + ZD. (A) pathways that were significantly reduced and (b) pathways that were significantly increased in HFD. The relative abundance of MetaCyc pathways involved reducing cofactor-dependent and energy-support metabolisms (A) and enhancing the inflammation (B) in HFD compared with CL and HFD + ZD. A p value of < 0.05 was considered statistically significant.

Fig. 8

Effect of ZD treatment on antioxidant capacity in human retinal pigment epithelial cells. (A) Production of reactive oxygen species (ROS) and malondialdehyde (MDA), Catalase (CAT) activity, and glutathione level in untreated and treated cells were measured using commercial kits. (B) MRNA levels of antioxidant genes in untreated and treated cells measured by qRT-PCR. Data was analysed with one-way ANOVA, followed by Bonferroni multiple comparison test, and displayed as means ± SE. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns: no significance.

Fig. 9

Effect of ZD treatment on expression of proinflammatory cytokines (A) Secreted IL-1β, IL-6, IL-8 and TNFα from untreated and treated cells measured by ELISA. (B) MRNA levels of Il-1β, Il-6, Il-8 and Tnfα in untreated and treated cells examined by qRT-PCR. Data was analysed with one-way ANOVA, followed by Bonferroni multiple comparison test, and displayed as means ± SE. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns: no significance.

Fig. 10

(A) Base peak chromatograms of ZD extract obtained in negative ionisation mode. (B) Base peak chromatograms of ZD extract obtained in positive ionisation mode.

Fig. 11

The underlying mechanism of Ziyin-Mingmu decoction (ZD) against age related macular degeneration (AMD) is possibly via regulating cholesterol metabolism, suppressing oxidative stress and inflammation in the retina and liver, and modulating gut microbiota by the functional compounds, mainly polyphenols, identified in the water extract of ZD.