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. 2022 Nov 2:13:1040846.
doi: 10.3389/fmicb.2022.1040846. eCollection 2022.

Gut microbiota dysbiosis as an inflammaging condition that regulates obesity-related retinopathy and nephropathy

Jie Li  1 , Jun-Lin Lv  1 , Xin-Yue Cao  1 , Hai-Ping Zhang  2 , Yu-Jun Tan  1 , Ting Chu  3 , Li-Li Zhao  4 , Zhong Liu  4 , Yu-Shan Ren  1
Affiliations

Gut microbiota dysbiosis as an inflammaging condition that regulates obesity-related retinopathy and nephropathy

Jie Li et al. Front Microbiol. .

Abstract

Diabetes-specific microvascular disease is a leading cause of blindness, renal failure and nerve damage. Epidemiological data demonstrated that the high morbidity of T2DM occurs as a result of obesity and gradually develops into serious complications. To date, the mechanisms that underlie this observation are still ill-defined. In view of the effect of obesity on the gut microflora, Leprdb/db mice underwent antibiotic treatment and microbiota transplants to modify the gut microbiome to investigate whether microbes are involved in the development of diabetic nephropathy (DN) and/or diabetic retinopathy (DR). The mouse feces were collected for bacterial 16S ribosomal RNA gene sequencing. Cytokines including TNF-α, TGF-β1, IFN-γ, IL-1β, IL-6, IL-17A, IL-10, and VEGFA were detected by enzyme-linked immunosorbent assay (ELISA), flow cytometry, real-time PCR and immunofluorescent assay. Eyes and kidney were collected for histopathological assay. Intestinal permeability was also detected using Evans Blue. The results showed that obesity influenced metabolic variables (including fast/fed glucose, insulin, and triglyceride), retinopathy and nephropathy, and the gut microbiota. Obesity mainly reduced the ratio of Bacteroidetes/Firmicutes and influenced relative abundance of Proteobacteria, Actinobacteria, and Spirochetes. Obesity also increased intestinal permeability, metabolic endotoxemia, cytokines, and VEGFA. Microbiota transplants confirm that obesity aggravates retinopathy and nephropathy through the gut microbiota. These findings suggest that obesity exacerbates retinopathy and nephropathy by inducing gut microbiota dysbiosis, which further enhanced intestinal permeability and chronic low-grade inflammation.

Keywords: diabetic nephropathy; diabetic retinopathy; gut microbiota (GM); inflammaging; obesity.

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Conflict of interest statement

Authors L-LZ and ZL were employed by Lunan Pharmaceutical Group Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
The effect of obesity on metabolic variables exacerbated retinopathy and nephropathy. (A) Schematic representation of experimental timeline where half of the mice (Leprdb/db and Leprdb/m) start a regular diet (RD) at 6 weeks and later half of these receive neomycin (AB) treatment from the age of 12 weeks until killing at week 36. (B) Weight gain of AB-treated mice compared to control mice; Error bars represent mean ± S.D. n = 8 in per group. *P < 0.05. (C–G) The effect of obesity on metabolic variables including Blood glucose (C,D), insulin (E,F), and triacylglycerol (G) levels after 16 h of fasting (C,E), and in the random fed state (D,F) in mice with or without AB-treatment. n = 8 in per group. *P < 0.05. (H–K) HE staining was performed to detect vessels morphological changes of retinal sections (H, Upper) followed by quantitation of the thickness of NFL (I) and GCL + IPL (J). HE (H, Middle) and Periodic Acid-Schiff (PAS) (H, Bottom) staining was performed to detect glomerulus morphological changes of kidney sections followed by quantitation of the glomerular area (K). Error bars represent mean + S.D. n = 8 in per group. *P < 0.05.
FIGURE 2
FIGURE 2
Obesity induced gut microbiota dysbiosis. Representative charts of relative abundance of bacterial phyla (A), class (B), order (C), family (D) and genus (E) in gut microbiota of Leprdb/db and Leprdb/m mice treated with neomycin or not, and relative proportion per group of different phyla (F–J). n = 8 in per group. *P < 0.05.
FIGURE 3
FIGURE 3
Obesity augments gut permeability, metabolic endotoxemia, and systemic inflammation. (A) Concentration of gut-absorbed Evans Blue in serum 24 h after oral administration in db/db and db/m mice, treated with vehicle or with antibiotic. n = 8 in per group. *P < 0.05. (B) Activation of PRRs induced by stimulation with serum isolated from db/db and db/m mice and receiving vehicle or neomycin for 3 weeks. *P < 0.05. (C–I) Serum cytokine profiles determined by ELISA assay of TGF-β1 (C), IL-10 (D), TNF-α (E), IFN-γ (F), IL-1β (G), IL-6 (H), and IL-17A (I) in db/db and db/m mice, treated with vehicle or with antibiotic. n = 8 in per group. *P < 0.05. (J–Q) IFN-γ (J,K), IL-10 (L,M), IL-17A (N,O), and TNF-α (P,Q) levels were assayed by flow cytometry. n = 8 in per group. *P < 0.05.
FIGURE 4
FIGURE 4
Obesity promoted local inflammation in retina and kidney. Representative immunofluorescence images of retina (A, Left) and kidney (A, Right) tissues stained with VEGFA, TGF-β1, IL-10, TNF-α, IFN-γ, IL-1β, IL-6, and IL-17A antibodies in db/db and db/m mice, which were treated with vehicle or with antibiotic. The relative quantitative analyses are presented in (B). Bar = 50 μm. Data are expressed as the means ± S.D., n = 8 in per group. *P < 0.05.
FIGURE 5
FIGURE 5
Obesity exacerbated DR, DN, and inflammaging condition through gut microbiota. (A) Schematic representation of microbiotal transfer experiments where recipient mice (db/db) were gavaged with a suspension of fecal pellets in PBS from donor mice (db/m) or vehicle. The experimental timeline describing preparation of mice for microbiotal transfers where mice received 7 days of antibiotics (neomycin and ampicillin) at 6 weeks of age. Starting at 8 weeks of life, db/db mice received weekly microbiotal transplants from db/m donor mice until killing at week 36. (B) Body weight, blood glucose, insulin, and triacylglycerol levels after 16 h of fasting, and in the random fed state in mice with or without microbiotal transfers. n = 8 in per group. *P < 0.05. (C) HE staining was performed to detect vessels morphological changes of retinal sections (C, Upper), HE (C, Middle) and PAS (C, Bottom) staining was performed to detect glomerulus morphological changes of kidney sections. (D) Quantitation of the thickness of NFL(D, Upper), GCL + IPL (D, Middle) and the glomerular area (D, Bottom) in db/db mice with or without microbiotal transfers were followed. Error bars represent mean + S.D. n = 8 in per group. *P < 0.05. (E,F) Representative charts of relative abundance of bacterial phyla, class, order, family (E) and genus in gut microbiota of db/db mice with or without microbiotal transfers, and relative proportion per group of different phyla (F). n = 8 in per group. *P < 0.05.
FIGURE 6
FIGURE 6
Microbiotal transfers ameliorated gut permeability, metabolic endotoxemia, and inflammaging condition in obesity mice. (A,B) Concentration of gut-absorbed Evans Blue (A) in serum 24 h after oral administration and activation of PRRs (B) of db/db mice with or without microbiotal transfers. n = 8 in per group. *P < 0.05. (C–I) Serum cytokine profiles of db/db mice with or without microbiotal transfers determined by ELISA assay of TGF-β1 (C), IL-10 (D), TNF-α (E), IFN-γ (F), IL-1β (G), IL-6 (H), and IL-17A (I). n = 8 in per group. *P < 0.05. (J,K) IFN-γ, IL-10, IL-17A, and TNF-α levels were assayed by flow cytometry. n = 8 in per group. *P < 0.05. (L,M) Representative immunofluorescence images of retina and kidney tissues stained with VEGFA, TGF-β1, IL-10, TNF-α, IFN-γ, IL-1β, IL-6, and IL-17A antibodies in db/db mice with or without microbiotal transfers (L). The relative quantitative analyses were presented in (M). Bar = 50 μm. Data are expressed as the means ± S.D., n = 8 in per group. *P < 0.05.
FIGURE 7
FIGURE 7
Obesity-induced gut microbiota dysbiosis increases intestinal permeability, metabolic endotoxemia, and systemic and local inflammation and ultimately contributes to retinopathy and nephropathy. The ratio of two dominant phyla in intestinal flora, Bacteroidetes and Firmicutes significantly decreases in obesity (db/db) mice compared to normal (db/m) mice, with a relative increase in Firmicutes in obesity mice and decrease in Bacteroidetes. This dysbiosis is accompanied by heightened intestinal permeability, which allows increased translocation of pathogen-associated molecular patterns (PAMPs) (endotoxemia). Recognition of these microbe-specific molecules by pattern recognition receptors (PRRs) present on innate immune cells triggers synthesis and excretion of cytokines causing chronic systemic low-grade inflammation. Ultimately, this exacerbates retinopathy and nephropathy.
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