Fig 1.
Schematic flowchart of the study design.
Table 1.
Effect of CUR on physiological parameters of rats with CKD induced by 5/6Nx.
Fig 2.
CUR ameliorated kidney function and fibrosis in 5/6 Nx rats.
(A) The serum of BUN, Scr, and 24H-P levels were detected by commercial kits. (B) Change of Kidney pathology: representative image of Masson, PAS, and Sirius red staining of renal sections (Masson and PAS, scale bar = 100 μm; Sirius red, scale bar = 100 μm). (C) Quantitative analysis of fibrotic area based on Masson staining. (D) Quantitative analysis of tubular injury based on PAS staining. (E) The levels of FN, ACTA2, and COL1A1 were determined by RT-PCR in the kidney. (F) Kidney expression of TGF-β/Smad signaling pathway assayed by Western blot in different groups. (G) Quantitative analysis of Fig 2F. **P <0.01, Compared with shams; #P < 0.05, ##P < 0.01, compared with 5/6Nx+CUR group, NS means no significant difference. Nx: Nephrectomy, CUR: curcumin, BUN: urea nitrogen, Scr: Serum creatinine, 24H-P: 24h urine protein quantitation, PAS: periodic acid-Schiff, FN: fibronectin, ACTA2: alpha-SMA, COL1A1: Collagen I, RT-PCR: Reverse transcriptase-polymerase chain reaction, TGF-β: Transforming growth factor-β.
Fig 3.
CUR inhibited inflammation in 5/6 Nx rats.
(A) The serum and renal LPS levels were measured by ELISA. (B) The levels of IL-1β, IL-6, and TNF-α by RT-PCR in the kidney. (C) The renal MCP-1 levels were measured by ELISA. (D)The levels of MCP-1 by RT-PCR in kidney tissues. (E) Kidney expression of TLR-4, p‐IκB‐α, IκB‐α, p-NF-κB p65, and NF- κB p65 displayed by Western Blot strip chart. (F) Quantitative analysis of Fig 3E. **P <0.01, compared with the shams; #P < 0.05, ##P < 0.01, compared with 5/6Nx+CUR group. Nx: Nephrectomy, CUR: curcumin, LPS: Lipopolysaccharides, ELISA: enzyme linked immunosorbent assay, IL−1β: interleukin −1β, IL-6: interleukin-6, TNF-α: tumor necrosis factor-α, MCP-1: Monocyte chemoattractant protein-1,RT-PCR: Reverse transcriptase-polymerase chain reaction, TLR4: Toll-like receptor 4, NF-κB p65: Nuclear transcription factor-kappa B p65, p-NF-κB p65: Phospho-Nuclear transcription factor-kappa B p65.
Fig 4.
CUR improved intestinal barrier function in 5/6 nephrectomy rats.
(A, B) Representative image of H&E and PAS staining of ileum sections (scale bar = 500 μm). (C, D) Representative image of H&E and PAS staining of colon sections (scale bar = 100 μm). (E, F) ileum and colon histopathology score. (G)The levels of inflammatory factors by RT-PCR in the ileum. (H) The levels of Ocln, Cldn1, and ZO-1 by RT-PCR in the ileum. (I, J) Western blot analysis of occludin, claudin-1, and ZO-1 protein in ileum tissues. (K, L) Western blot analysis of occludin, claudin-1 and ZO-1 protein in colon tissues.*P <0.05, **P <0.01, compared with the shams; #P < 0.05, ##P < 0.01, compared with the 5/6Nx+CUR group.Nx: Nephrectomy, CUR: curcumin, H&E: hematoxylin-eosin, PAS: periodic acid-Schiff, IL−1β: interleukin −1β,IL-6: interleukin-6, TNF-α: tumor necrosis factor-α, Ocln: Occludin; Cldn1: claudin-1,ZO-1: zonula occludens-1,RT-PCR: Reverse transcriptase-polymerase chain reaction.
Fig 5.
CUR altered the structural composition of gut microbiota in 5/6 nephrectomy rats.
(A) Flower plots diagram. (B) Rarefaction curve measuring the amount of sequencing data. (C) Observed features, Chao 1, Shannon, and Simpson indexes in ɑ‐diversity analysis. (D, E) Analysis of β-diversity of gut microbiome in different groups. PCA of gut microbiota between all groups (D), PCoA between all groups(E). (F) The species composition at the phylum level. (G) The species composition at the order level. (H, I) The species composition and relative abundance at the genus level. (J) Heat maps of the relative abundance of species composition at the genus level. *P <0.05, **P < 0.01, compared with shams; #P < 0.05, ##P < 0.01, compared with the 5/6Nx+CUR group, NS means no significant difference. Nx: Nephrectomy, CUR: curcumin, PCA: Principal Component Analysis, PCoA: Principal Co-ordinates Analysis.
Fig 6.
Functional analysis of the microbial community.
(A) Random forest map analysis. (B) Contrast of COG categories data and (C) KEGG pathway results between 5/6Nx and 5/6Nx +CUR group using PICRUST. (D) Correlation analysis of gut microbiota and CKD-related biochemical indicators. *P <0.05, **P < 0.01, compared to the 5/6Nx group. Nx:Nephrectomy, CUR: curcumin, COG: a cluster of orthologous groups, KEGG: Kyoto Encyclopedia of Genes and Genomes.
Fig 7.
Protective effects of CUR administration on metabolites derived from microbiota.
(A) PCA analysis in different groups. (B) OPLS-DA analysis between sham and 5/6Nx groups (left) and 5/6Nx and 5/6Nx+CUR groups (right). (C) The permutation test for the OPLS-DA model was generated from the serum profile of all samples. (D) The Splot-OPLS-DA analysis of the sham and 5/6Nx group. (E) Volcanic maps of metabolites in sham and 5/6Nx group. (F-G) Relative abundance of representative differential serum metabolites in different groups. (H) Three metabolites in the 5/6Nx and 5/6Nx+CUR groups were analyzed based on PLS-DA ROC curves, associated AUC, and 95% CI. (I) Summary of KEGG pathway analysis of 5/6Nx vs 5/6Nx+CUR. (J) CUR down-regulated differential metabolite KEGG pathway. (K) CUR up-regulated differential metabolite KEGG pathway. *P < 0.05, **P < 0.01, compared with shams; #P < 0.05, ##P < 0.01, compared with the 5/6Nx+CUR group; NS means no significant difference. Nx:Nephrectomy, CUR: curcumin, PCA: principal component analysis, OPLS-DA: Orthogonal projection to latent structure-discriminant analysis, PLS-DA: Partial Least Squares Discriminant Analysis, ROC: Receiver Operation Characteristic, AUC: Area Under the Curve, CI: Confidence Interval, KEGG: Kyoto Encyclopedia of Genes and Genomes.
Fig 8.
Association analysis of differential metabolites with gut microbiota and physiological indices separately.
(A) Correlation analysis of plasma differential metabolites and gut microbiota at the genus level. (B) Correlation analysis of plasma differential metabolites and CKD-related biochemical indicators. Red represents a positive association, and blue represents a negative association. *P < 0.05, **P < 0.01. CKD: chronic kidney disease.