Myricetin induces M2 macrophage polarization to alleviate renal tubulointerstitial fibrosis in diabetic nephropathy via PI3K/Akt pathway

Figure 1 Myricetin alleviated the kidney injuries of diabetic nephropathy mice. A: Timeline of in vivo assay; B: Body weights; C: Blood glucose levels; D: 24 h-microalbumin levels; E: Kidney/body weight ratios; F: Serum creatinine levels; G: Albumin-to-creatinine ratios; H: Creatinine clearance values. ACR: Albumin-to-creatinine ratio. ^bP < 0.05.

Figure 2 Myricetin ameliorated the renal histopathological changes of diabetic nephropathy mice. A: Hematoxylin and eosin staining showing renal tubule damage analysis (left and right) of kidneys; B: Periodic acid-Schiff staining (left) and mesangial matrix analysis (right); C: Kidney injury molecule-1 (KIM-1) and neutrophil gelatinase associated lipocalin (NGAL) levels by reverse transcription-PCR; D: NGAL and KIM-1 Levels determined by western blotting. Scale bar: 100 μm. HE: Hematoxylin and eosin; PAS: Periodic acid-Schiff; KIM-1: Kidney injury molecule-1; NGAL: Neutrophil gelatinase associated lipocalin. ^bP < 0.05.

Figure 3 Myricetin inhibited inflammation factors and fibrosis in the renal tissue of diabetic nephropathy mice. A: Immunofluorescent staining of F4/80 protein; B: Tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, IL-1β, and IL-10 mRNA levels; C: Serum TNF-α, IL-6, IL-1β, and IL-10 Levels; D: Immunofluorescent staining of collagen-1a1 (Col1a1) protein; E: Reverse transcription-PCR analysis of Col1a1 and alpha-smooth muscle actin (α-SMA) mRNAs; F: Western blotting analysis of Col1a1 and α-SMA proteins; G: Masson’s trichrome staining (left) and renal fibrosis analysis (right); H: Sirius-red staining (left) and fibrosis analysis (right). TNF-α: Tumor necrosis factor-alpha; α-SMA: Alpha-smooth muscle actin; IL: Interleukin; Col1a1: Collagen-1a1. ^bP < 0.05.

Figure 4 Myricetin switched the phenotype of macrophages in renal tissues of db/db mice. A and B: Immunofluorescent staining of CD86 protein (A) and CD206 protein. Scale bar: 100 μm. ^bP < 0.05.

Figure 5 High glucose induced the M1 macrophage polarization of RAW 264. 7 cells. A and B: Flow cytometry analysis of RAW 264.7 macrophages labeled with CD86 and CD206 exposed to different concentrations of glucose (A) and to 33.3 mmol/L glucose for different times (B); C and D: Inducible nitric oxide synthase (iNOS) levels induced by different concentrations of glucose (C) and by 33.3 mmol/L glucose for different times (D); E and F: Relative cytokine mRNA (E) and protein (F) levels of iNOS, tumor necrosis factor-alpha, interleukin (IL)-6, IL-1β, IL-10, and arginase-1 after exposure to 33.3 mmol/L glucose for 24 h detected by reverse transcription-PCR (E) and western blotting (F). Arg-1: Arginase-1; TNF-α: Tumor necrosis factor-alpha; IL: Interleukin; iNOS: Inducible nitric oxide synthase; HG: High-glucose. ^bP < 0.05.

Figure 6 Myricetin regulated the polarization of RAW 264. 7 cells to M2-type induced by high glucose. A: In vitro assay; B: Flow cytometry analysis of CD86-labeled and CD206-labeled RAW 264.7 macrophages induced with 33.3 mmol/L glucose and different concentration of myricetin; C: Levels of inducible nitric oxide synthase (iNOS) induced by 33.3 mmol/L glucose and after treatment with 25 μM of myricetin detected by ELISA; D and E: Levels of iNOS, tumor necrosis factor-alpha, interleukin (IL)-6, IL-1β, IL-10 and arginase-1 mRNAs (D) and proteins (E) induced with 33.3 mmol/L glucose and after treatment with 25 μM of myricetin detected by reverse transcription-PCR (D) and western blotting (E). Arg-1: Arginase-1; TNF-α: Tumor necrosis factor-alpha; IL: Interleukin; iNOS: Inducible nitric oxide synthase; HG: High-glucose. ^bP < 0.05.

Figure 7 Identification of potential targets of diabetic nephropathy and myricetin using bioinformatics analysis. A: Venn diagrams; B: Protein-protein interaction network of myricetin and diabetic nephropathy targets, in which edge points represent protein interactions and line thickness indicates data strength; C: Myricetin's key target gene for treating diabetic nephropathy; D: Potential targets for myricetin treatment of diabetic nephropathy from the Gene Ontology algorithm; E: Myricetin treatment pathways from the Kyoto Encyclopedia of Genes and Genomes database; F: Signaling pathway of PI3K-Akt; G and H: The 3D structures of the docking mode of myricetin and PI3K (G) and myricetin and Akt (H); I: Visualization of the binding abilities of myricetin with PI3K and Akt. DN: Diabetic nephropathy.

Figure 8 Myricetin regulated the polarization of RAW 264. 7 cells through the PI3K/Akt signaling pathway in diabetic nephropathy mice. A-D: CD86-labeled and CD206-labeled RAW 264.7 macrophages under the 33.3 mmol/L glucose condition and after 25 μM myricetin treatment and LY294002 administration assessed by flow cytometry (A) and for protein levels of inducible nitric oxide synthase (iNOS), arginase-1 (Arg-1), phosphorylated-Akt (at S473) and Akt by western blotting (B) or mRNA levels of iNOS, tumor necrosis factor-alpha, interleukin (IL)-6, IL-1β, IL-10, and Arg-1 by reverse transcription PCR (C), and for secreted levels of TNF-α,IL-6, IL-1β, and IL-10 by ELISA. Arg-1: Arginase-1; TNF-α: Tumor necrosis factor-alpha; IL: Interleukin; iNOS: Inducible nitric oxide synthase; HG: High-glucose. ^bP < 0.05.

Figure 9 Overview of protective effects of myricetin against diabetic nephropathy. Myricetin can regulate the polarization of macrophages through the PI3K/Akt signaling pathway to ameliorate the injuries of kidneys in mice with diabetic nephropathy. Arg-1: Arginase-1; TNF-α: Tumor necrosis factor-alpha; IL: Interleukin.