Age-related macular degeneration (AMD) is an incurable visual disorder, which is the third highest cause of blindness in developed countries, with only cataracts and glaucoma as more prevalent eye diseases. Approximately 200 million individuals aged over 50 years are estimated to be affected (1). AMD is chronic in nature and worsens as it progresses from the early and intermediate stages to the late stage. Late AMD is subdivided into dry and wet forms. The dry form is most common and is characterized by the presence of abnormal deposits (drusen) underneath the retinal pigment epithelial (RPE) layer and RPE atrophy. Wet AMD is usually the cause of severe symptoms, such as vision loss, and can cause symptoms to develop in a much shorter timeframe, normally weeks to months. Wet AMD is defined by chronic neovascularization, when new blood vessels, induced by vascular endothelial growth factor (VEGF), grow under the RPE layer and break into the Bruch’s membrane, resulting in bleeding and sudden loss of vision (2). There is no effective treatment for dry AMD, though antioxidants have shown subtle benefits (3).In contrast, anti-VEGF therapy is effective for wet AMD; however, many patients experience incomplete responses, including persistent exudation, hemorrhage, and ongoing lesion fibrosis (4). AMD is a complex disease, associated with environmental and genetic risk factors, and multiple signaling pathways are involved (5).

Cholesterol plays a critical role in the maintenance of cellular structure and function and is associated with various disorders, including AMD (6). Cholesterol has been reported to enrich drusen and form sub-RPE crystals in patients with wet AMD, implicating dysregulation of cholesterol trafficking and metabolism (7, 8). Many studies have demonstrated that cholesterol metabolism and transport genes, apolipoprotein E (APOE), hepatic lipase (LIPC), CETP (cholesteryl ester transfer protein), and ATP Binding Cassette Subfamily A Member 1 (ABCA1) are associated with AMD pathogenesis and progression (9, 10). Knockout of cholesterol efflux-related genes (e.g., Apoe and Abca1) and cholesterol metabolism genes such as Cyp27a1 and Cyp46a1 cause retinal pathology in mice (11–14). We also found that the translocator protein, TSPO, mediated RPE cholesterol efflux, and loss of TSPO caused intracellular accumulation of cholesterol in human and mouse RPE cells (15, 16). High intake of dietary cholesterol has been reported to be associated with an increase in the risk of developing AMD (17–19). A high cholesterol-containing diet causes AMD pathological features in rabbits (20). All these findings suggest cholesterol is involved in AMD pathogenesis and that lowering cholesterol is a therapeutic strategy for patients with AMD.

Traditional Chinese Medicine has been used to treat both dry and wet AMD for thousands of years in China (2). Over 196 prescriptions have been described for treating AMD, among them, Qijudihuang pill (QP), which is regularly prescribed to patients with AMD. QP also shows therapeutic effects in other ocular disorders, such as diabetic retinopathy, retinitis pigmentosa, glaucoma, and dry eye disease (21, 22). QP is made of eight medicinal herbs, namely, Fructus lycii, Moutan Cortex, Dioscoreae Rhizoma, Corni Fructus, Poria, Alismatis Rhizoma, Flos Chrysanthemi Indici, and Rehmanniae Radix Praeparata. Using a network pharmacology approach, over 134 active compounds have been identified in QP and 72 candidate targets predicted, some of which were reported to have a role in retinal disorders (21). However, the underlying protective mechanisms of QP against ocular diseases, particularly AMD, are not explored.

In the present study, we treated high-fat diet-fed mice with QP and investigated changes in cholesterol metabolism, expression of antioxidant and inflammation genes, and gut microbiome.

Although QP has been used to treat AMD in China for a long period, the underlying functional mechanism has not been investigated. In the current study, we treated a high-fat diet-fed mouse model of AMD and found QP decreased HFD-induced elevated levels of cholesterol in the RPE/choroid, retina, liver, and serum, upregulated expression of antioxidant genes, and suppressed expression of pro-inflammatory genes in the RPE/choroid and retinas of high-fat diet-fed animals, as well as modulated gut microbial composition.

Cholesterol accumulates in the drusen of patients with AMD and can be enzymatically or non-enzymatically oxidized into oxysterols (6). Loss of oxysterol-producing enzymes or cholesterol transporters causes retinal degeneration in rodents. HFD exacerbates AMD pathological features in Apoe (involved in cholesterol efflux) knockout mice (31). HFD has also been shown to induce oxidative stress, inflammation, and dysbiosis, resulting in accelerated photoreceptor degeneration in rd10 mice (a retinitis pigmentosa mouse model) (32). In the present study, we also demonstrated that feeding with HFD caused increased cholesterol levels, decreased expression of antioxidant genes, and upregulated expression of inflammatory mediators in examined tissues and that these effects were reversed by QP treatment. Network pharmacology predicted that QP contains multiple active compounds and potential protein targets, of which the top ones are involved in inflammation (22). Therefore, it is supposed that there are multiple signaling pathways associated with QP’s protective effect in HFD-fed mice, which requires further investigation.

Excess cholesterol in the retina and RPE is reversibly transported back to the liver and converted to bile acids, principally by mitochondrial Cyp27A1 (33) and Cyp7A1 (34) located in the endoplasmic reticulum. The products are conjugated by taurine or glycine prior to transport into the gall bladder and their release into the duodenum is stimulated by feeding. The majority of the secreted BA is reabsorbed following deconjugation in the ileum to complete the BA enterohepatic circulation, while a small proportion arrives in the colon (35). Gut bacteria metabolize bile acids by a wide variety of reactions including deconjugation, re-conjugation, dihydroxylation, and oxidation (36, 37). The diversity of gut bacteria and their metabolic capacity to act on BAs determines effects on host systems via a multitude of BA-activated signal receptors that regulate hepatic cholesterol metabolism by affecting the expression of enzymes causing their formation, including the two Cyps (37).

When mice are provided with a high-fat diet there are multiple effects on cholesterol metabolism, including conversion to bile acids. These alter the spectrum of bacteria in the gut, and thus the signaling through bile acids, to affect the host including on the gut-brain axis (38). Indeed, HFD causes widespread changes in microbiota communities, leading to dysbiosis associated with obesity, type 2 diabetes, and AMD as well as a number of other health-compromising conditions (39–41). Those effects were observed in this study at the phylum level with the frequently observed increase of the ratio of Bacteroidetes compared to Firmicutes. The changes can also be seen in terms of beta diversity with the PCoA revealing a clear separation between the CD and HFD groups. Furthermore, analysis with the LEfSe tool revealed an HFD-associated increase in abundance of a variety of taxa previously commented on by ourselves and others (23, 42–44). At the same time, other taxa were decreased by this diet. For instance, Anaerotruncus was increased and has been associated with HCC and non-alcoholic steatohepatitis (45), whereas beneficial f-S27-4 was decreased (23, 43).

One objective of this project was to ascertain whether QP has an effect on gut microbiome and thus whether the observed changes play a mechanistic contribution to potential therapeutic effects. Even while the HFD was continued, QP, at least in part, reversed the effects observed with HFD alone. Thus, the spectrum of phyla reverted more towards that seen in CD as illustrated by a decrease of Bacteroidetes/Firmicutes, a movement of the HFD+QP group towards CD in PCoA plots, and a more restricted change in abundances of taxa in LEfSe, both for those that increased and those that decreased in abundance. The number and identity of taxa that were either increased or decreased in HFD+QP vs. CD were significantly diminished compared to when HFD alone was compared to CD.

Changes in the composition of microbial communities will have effects on global metabolic capacity and thus on interaction with the host. We examined the extent and nature of such potential changes with PICRUSt2. On a global scale, using diversity measures, clear changes induced by HFD and partially reversed by QP were apparent in PCoA plots, changes that closely parallel those seen when considering taxa alone. In terms of individual pathways increased by HFD, those generating pro-inflammatory effectors were notable. These included the synthesis of enterobactin, polymixin B, and lipopolysaccharide, each of which was increased by HFD but reversed when QP was included in the diet. We propose that the increase of IL-1B and TNF-a in the liver, RPE/choroid, and retina are also associated with these changes occurring in the gut microbiota due to HFDs. Likewise, we propose that the decrease of anti-oxidative enzymes catalase, GPX, and SOD1 is a response to changes in the abundance of bacterial taxa, their associated metabolic capacity, and nature. For instance, Enterobactin is a siderophore and derivative of 2,3 dihydroxy N-benzoylserine lactone that chelates primarily ferric iron and is produced by Gram-negative bacteria (46). Since iron is a requirement of host and microbes, an imbalance of iron uptake will impair host systems, and enterobactin has been shown to promote bacterial colonization (30) and interfere with host immune responses (47). Polymixins are microbially-produced antibiotics that illustrate two effects of the HFD. The production of polymixin antibiotics in the gut provides one mechanism leading to the remodeling of bacterial communities and indeed the original interest in these compounds arose as they provided a prospect for the treatment of multidrug-resistant organisms (48). Aligning with this mechanism of remodeling are bacteria, such as Pseudomonas, that have resistance to polymixin (49). However, the drugs also produced marked nephrotoxicity, illustrating the second point that the change of bacterial communities results in the formation of toxic compounds, causing harm to the host. The mode of action of polymixins is thought to be by interaction with phospholipids, causing membrane disruption including macrophages, and stimulating the production of IL-1β (50). An alternative theory suggests that polymixins induce oxidative stress caused by the formation of reactive oxygen species (51), which resonates with altered oxidative resistance in RPE. Resistant bacteria occur in Escherichia, Klebsiella, Salmonella, Shigella, Enterobacter, and Citrobacter (48), and have altered LPS structures that block the interaction with antibiotics (52). Other possible contributors to intestinal inflammation are changes to purinogenic pathways as found in inflammatory bowel disease (53). These pathways were found to be lowered by the HFD but raised to control levels when QP was administered.

While the complex effects of HFD on bacteria and their interaction with the host are starting to be understood, QP has not been previously explored at the levels described here. We have started to characterize the component chemicals present in the QP plant extract (in preparation) and established the principle components. How these might interact with the host and bacteria demands future investigation but one possibility is that they act with the promiscuous nuclear hormone receptor pregnane-X receptor (PXR) located in the liver and gut enterocytes. PXR regulates the expression of many Phase 1 and Phase 2 enzymes and thus affects the metabolism of cholesterol to BAs, for instance, the induction of SULT2A, which conjugates BAs prior to export, and of Mrp2 and Oatp2, the export components (54).

TCM preparations have widely shown lipid-lowering function in dyslipidemia (55, 56). For targeting cholesterol, TCM can inhibit intestinal absorption of cholesterol, suppress endogenous cholesterol synthesis, promote cholesterol reverse transport and excretion, and regulate the expression of cholesterol homeostasis-associated transcription factors (55, 56). Berberine, a major functional compound of many medicinal herbs, has been shown to lower blood cholesterol in atherogenic-diet-fed rats at least partly via the inhibition of intestinal cholesterol absorption (57). TCM Jiang-Zhi-Ning, containing four Chinese herbs, can significantly lower cholesterol, triglyceride, and low-density lipoprotein-cholesterol in hyperlipidemic rats, partly by inhibiting the expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the rate-limiting enzyme for cholesterol synthesis (58). TCM FufangZhenzhuTiaoZhi has been shown to decrease plasma cholesterol in hyperlipidemic rats via downregulating HMGCR expression and increasing expression and activity of 7-α hydroxylase (CYP7A1, the rate-limiting enzyme for bile acid synthesis) (59). In the present study, our data suggested that the effect of QP in lowering cholesterol is possibly through the promotion of cholesterol metabolism, transport, and excretion. It will be worth investigating whether QP inhibits cholesterol intestinal absorption and endogenous synthesis. Additionally, there are reports about the hepatotoxicity of TCM and natural products in animal models and human patients (60, 61), so it may be necessary to examine whether there is any potential toxicity of QP in animal models and patients with AMD.

This study aimed to investigate the relationship between HFD and AMD and start to unravel the mechanisms whereby QP may provide relief from the condition. HFD was shown to exacerbate lipid accumulation in the RPE and retina and associated inflammation and oxidative stress. Changes in the gut microbiota, their metabolic capacity, and signaling between the host and microbes will have contributed to these pathological conditions. We propose that the ability of QP to reverse these effects is related to mechanisms acting to lower cholesterol levels, oxidative stress, and inflammation, and to modulate gut microbiota; and these will be investigated in future studies.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

The animal study was approved by The Animal Welfare Committee, Glasgow Caledonian University. The study was conducted in accordance with the local legislation and institutional requirements.

XS: Conceptualization, Funding acquisition, Writing – original draft, Writing – review & editing. YC: Investigation, Writing – original draft. KSI: Formal Analysis, Writing – original draft. XL: Investigation, Writing – original draft. AW: Investigation, Writing – original draft. YW: Investigation, Writing – original draft. XDY: Investigation, Writing – original draft. XZ: Formal Analysis, Writing – original draft. ZT: Project administration, Writing – original draft. ZH: Project administration, Writing – original draft. JAC: Formal Analysis, Writing – original draft, Writing – review & editing.