Gut Microbiota Dysbiosis And Its Role In A Diet-induced Non-human Primate Model Of Age-related Macular Degeneration

Objectives: Age-related macular degeneration(AMD)is a multi-factor neurodegenerative disease,which is an important cause of irreversible blindness in elderly people.With its pathogenesis not fully elucidated,the early prevention and treatment of AMD is important yet difficulty.Epidemiological studies have shown that diet and metabolism could be related to the development of AMD.In recent years,it has been reported that high-calorie diets might promote development of AMD-like changes in mice through gut microbiota.However,at present,these studies are limited to rodents without the primate-specific macula,and the lack of stable nonhuman primate AMD models is the bottleneck of AMD research.The related molecular mechanism of AMD still needs to be further elucidated using non-human primate models.Methods: The crab-eating macaques(Macaca fascicularis)model of AMD was induced by(High sugar and high fat)HSHF diet and multi-group analysis was carried out,and then verified in vivo by C57BL/6J mice and in vitro with cultured cells.The experiments were as follows:(1)Ten young male monkeys were randomly divided into two groups: the HSHF and control groups,and 30 male middle-aged Macaque were also divided into two groups: the HSHF group(N= 25)and control group(N=5)and subjected to feeding of HSHF or normal chow diet for 36 months.Then metagenomic sequencing of Macaque feces,and RNA sequencing of peripheral blood leukocytes and fundus imaging analysis were carried out.(2)33 16-month-old mice were divided into 3 groups: a control group(N = 8),a HSHF group(N= 13)and a HSHF+ AMD macaque fecal microbiota transplantation(FMT)group(N = 12).After 20 weeks of treatment,AMD-like changes of the retina in each group were evaluated.(3)24 8-week-old mice were randomly divided into three groups: control group,laser-induced RNV group,laser-induced RNV+FMT group.At Day 7 the retinal neovascularization was evaluated.Gut microbiota of mice was analyzed using high throughput sequencing of 16 S r RNA gene(16s HTS).(4)THP1 and ARPE19 were cultured in the medium containing AMD macaque serum and assessed for cell proliferation and oil red O staining.Results:(1)After long-term HSHF dietary inductionintervention,hypercholesterolemia was observed in both young and middle-aged monkeys,but only middle-aged monkeys showed AMD pathological changes,which were aggravated with the increase of induction time.(2)The RNA sequencing of peripheral blood leukocytes showed up-regulated inflammation and metabolic pathways,such as glycerol and carbohydrate metabolism.(3)Metagenomic sequencing of gut microbiota showed reduced gut microbiota diversity in AMD monkeys.Probiotics such as Faecalibacterium prausnitzi were significantly reduced in AMD monkeys,while the abundance of potentially harmful microbes such as Escherichia coli was increased.In terms of microbial functions,synthesis of harmful metabolites such as enterocin,putrescine and lipopolysaccharide were up-regulated,while the potential beneficial functions such as synthesis of nitrite reductase,butyric acid kinase and tyrobutyric acid were down-regulated.(4)Transplantation of fecal microbiota from AMD monkeys aggravated AMD-like changes in elderly mice fed with the HSHF diet,such as decolorization and irregular changes in retinal pigment epithelia,increased inflammatory response and metabolism in the retina,such as positive regulation of T cell,macrophage and granulocyte chemotaxis,as well as enhanced fatty acid transport and lipid metabolism in the retina.16 s HTS showed that HSHF diet increased the species diversity.However,the transplantation of AMD fecal microbiota will led to decrease of the species diversity of gut microbiota.(5)After one week of AMD fecal microbiota transplantation,the area of RNV increased and the microflora of small intestine changed significantly,and the abundance of some probiotics decreased significantly.(6)AMD macaque serum increased lipid accumulation in human peripheral blood monocytes and retinal pigment epithelial cells.Conclusions: Long-term HSHF diet can induce AMD pathological manifestations in Macaca fascicularis,in which age is a necessary factor for the occurrence of AMD,while longterm hypercholesterolemia and gut microbiota imbalance may be potential promoting factors.