Total Flavones of Abelmoschus manihot Remodels Gut Microbiota and Inhibits Microinflammation in Chronic Renal Failure Progression by Targeting Autophagy-Mediated Macrophage Polarization

Abstract

Background: Recently, progression of chronic renal failure (CRF) has been closely associated with gut microbiota dysbiosis and intestinal metabolite-derived microinflammation. In China, total flavones of Abelmoschus manihot (TFA), a component of Abelmoschus manihot, has been widely used to delay CRF progression in clinics for the past two decades. However, the overall therapeutic mechanisms remain obscure. In this study, we designed experiments to investigate the renoprotective effects of TFA in CRF progression and its underlying mechanisms involved in gut microbiota and microinflammation, compared with febuxostat (FEB), a potent non-purine selective inhibitor of xanthine oxidase.

Methods: In vivo, the CRF rat models were induced by uninephrectomy, potassium oxonate, and proinflammatory diet, and received either TFA suspension, FEB, or vehicle after modeling for 28 days. In vitro, the RAW 264.7 cells were exposed to lipopolysaccharide (LPS) with or without TFA or FEB. Changes in parameters related to renal injury, gut microbiota dysbiosis, gut-derived metabolites, and microinflammation were analyzed in vivo. Changes in macrophage polarization and autophagy and its related signaling were analyzed both in vivo and in vitro.

Results: For the modified CRF model rats, the administration of TFA and FEB improved renal injury, including renal dysfunction and renal tubulointerstitial lesions; remodeled gut microbiota dysbiosis, including decreased Bacteroidales and Lactobacillales and increased Erysipelotrichales; regulated gut-derived metabolites, including d-amino acid oxidase, serine racemase, d-serine, and l-serine; inhibited microinflammation, including interleukin 1β (IL1β), tumor necrosis factor-α, and nuclear factor-κB; and modulated macrophage polarization, including markers of M1/M2 macrophages. More importantly, TFA and FEB reversed the expression of beclin1 (BECN1) and phosphorylation of p62 protein and light chain 3 (LC3) conversion in the kidneys by activating the adenosine monophosphate-activated protein kinase-sirtuin 1 (AMPK-SIRT1) signaling. Further, TFA and FEB have similar effects on macrophage polarization and autophagy and its related signaling in vitro.

Conclusion: In this study, we demonstrated that TFA, similar to FEB, exerts its renoprotective effects partially by therapeutically remodeling gut microbiota dysbiosis and inhibiting intestinal metabolite-derived microinflammation. This is achieved by adjusting autophagy-mediated macrophage polarization through AMPK-SIRT1 signaling. These findings provide more accurate information on the role of TFA in delaying CRF progression.

Keywords: autophagy; chronic renal failure; gut microbiota; macrophage polarization; microinflammation; total flavones of Abelmoschus manihot.

Figure 1

Effects of TFA and FEB on renal injury. (A) Photomicrographs of periodic acid-Schiff (PAS) staining and Masson’s trichrome staining in the Normal, Model, TFA, and FEB rat groups. Scale bar: 100 μm. (B) Fibrosis area. (C) A WB analysis of α-SMA and collagen I in the kidneys of the Normal, Model, TFA, and FEB rats. The data are expressed as the mean ± SD, (n = 3). ^* P < 0.05, ^** P < 0.01. TFA, total flavones of Abelmoschus manihot; FEB, febuxostat; α-SMA, α-smooth muscle actin; WB, Western blotting; SD, standard deviation.

Figure 2

Effects of TFA and FEB on gut microbiota dysbiosis. (A) Changes in the relative abundance of gut microbiota at the order level in the four rat groups. (B) NMDS analysis for the relative abundance of bacterial OTU in the four rat groups. (C) Unweighted unifrac β-diversity of bacterial community. (D) ANOSIM analysis of categorical variables between the Normal and Model groups, Model and TFA groups, and Model and FEB groups. (E) MetaStat analysis for the relative abundance of significantly altered bacterial taxa at the family and genus levels in the four rat groups. (F) LEfSe for the significant bacterial taxa with LDA scores > 4.0 in the four rat groups. Differences are represented by the color of over-represented taxa: blue indicating the Normal group; green indicating the Model group; yellow indicating the TFA group; and red indicating the FEB group. Circles represent phylogenetic levels from phylum (innermost circle) to genera (outermost circle). (G) Predicted microbial functions using Tax4Fun in the four rat groups. The data are expressed as the mean ± SD, (n = 3). *P < 0.05, **P < 0.01. TFA, total flavones of Abelmoschus manihot; FEB, febuxostat; NMDS, non-metric multi-dimensional scaling; OTU, operational taxonomic unit; ANOSIM, analysis of similarities; LDA, linear discriminant analysis; LEfSe, linear discriminant analysis effect size; SD, standard deviation.

Figure 3

Effects of TFA and FEB on gut-derived metabolites. (A–D) Serum levels of DAO, SRR, d-serine, and l-serine in the Normal, Model, TFA, and FEB rat groups. The data are expressed as the mean ± SD. ^* P < 0.05, ^** P < 0.01. TFA, total flavones of Abelmoschus manihot; FEB, febuxostat; DAO, d-amino acid oxidase; SRR, serine racemase; SD, standard deviation.

Figure 4

Effects of TFA and FEB on microinflammation. (A, B) Serum levels of IL1β and TNF-α in the Normal, Model, TFA, and FEB rat groups. (C) A WB analysis of IL1β, TNF-α, and NF-κB in the kidneys of the Normal, Model, TFA, and FEB rats. The data are expressed as the mean ± SD (n = 3), ^* P < 0.05, ^** P < 0.01. TFA, total flavones of Abelmoschus manihot; FEB, febuxostat; IL1β, interleukin 1β; TNF-α, tumor necrosis factor-α; NF-κB, nuclear factor-κB; WB, Western blotting.

Figure 5

Effects of TFA and FEB on macrophage polarization in vivo. (A, B) Immunofluorescence staining of iNOS and CD163 in the kidneys of the Normal, Model, TFA, and FEB rats. Scale bar: iNOS, 100 μm; CD163, 50 μm. (C–E) Immunohistochemical staining of IL6, CD206, and ARG1 in the kidneys of the Normal, Model, TFA, and FEB rats. Scale bar: 100 μm. *P < 0.05, **P < 0.01. TFA, total flavones of Abelmoschus manihot; FEB, febuxostat; iNOS, inducible nitric oxide synthase; IL6, interleukin 6; ARG1, arginase 1.

Figure 6

Effects of TFA and FEB on macrophage polarization in vitro. (A) A WB analysis of iNOS and IL6 in RAW 264.7 cells exposed to LPS (100 ng/ml) with or without TFA (20 μg/ml) or FEB (100 nM) for 24 h. (B) A WB analysis of CD163, CD206, and ARG1 in RAW 264.7 cells exposed to LPS (100 ng/ml) with or without TFA (20 μg/ml) or FEB (100 nM) for 24 h. The data are expressed as the mean ± SD, (n = 3). ^* P < 0.05, ^** P < 0.01. TFA, total flavones of Abelmoschus manihot; FEB, febuxostat; iNOS, inducible nitric oxide synthase; IL6, interleukin 6; LPS, lipopolysaccharide; ARG1, arginase 1; NS, not significant; WB, Western blotting.

Figure 7

Effects of TFA and FEB on autophagy-mediated macrophage polarization through AMPK-SIRT1 signaling in vivo. (A) Immunohistochemical staining of BECN1 and LC3 in the kidneys of the Normal, Model, TFA, and FEB rats. Scale bar: 100 μm. (B–D) A WB analysis of BECN1, LC3, p-p62, p62, p-mTOR, mTOR, AMPK, and SIRT1 in the kidneys of the Normal, Model, TFA, and FEB rats. The data are expressed as the mean ± SD, (n = 3). ^* P < 0.05, ^** P < 0.01. TFA, total flavones of Abelmoschus manihot; FEB, febuxostat; BECN1, beclin1; p-p62, phosphorylated p62; p-mTOR, phosphorylated mTOR; NS, not significant; WB, Western blotting.

Figure 8

Effects of TFA and FEB on autophagy-mediated macrophage polarization through AMPK-SIRT1 signaling in vitro. (A) A WB analysis of BECN1 and LC3 in RAW 264.7 cells exposed to LPS (100 ng/ml) with or without TFA (20 μg/ml) or FEB (100 nM) for 24 h. (B) A WB analysis of AMPK, and SIRT1 in RAW 264.7 cells exposed to LPS (100 ng/ml) with or without TFA (20 μg/ml) or FEB (100 nM) for 24 h. The data are expressed as the mean ± SD, (n = 3). ^* P < 0.05, ^** P < 0.01. TFA, total flavones of Abelmoschus manihot; FEB, febuxostat; BECN1, beclin1; LPS, lipopolysaccharide; NS, not significant; WB, Western blotting.

Figure 9

Schematic diagram of TFA remodeling gut microbiota and inhibiting microinflammation in CRF progression. TFA, total flavones of Abelmoschus manihot; CRF, chronic renal failure.