Rapeseed protein-derived antioxidant peptide RAP alleviates renal fibrosis through MAPK/NF-κB signaling pathways in diabetic nephropathy

Figures

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  Figure 1. RAP ameliorates biochemical indices, serum TGF-β1 levels, and oxidative stress parameters in diabetic mice. Notes: The relative amount of (A) 24-hour albuminuria, (B) serum creatinine, (C) blood urea nitrogen, (D) triglyceride, (E) TGF-β1, (F) CAT, (G) MDA, and (H) SOD in the serum of mice. Data are presented as the mean ± SD, n=8. ##p<0.01 and ###p<0.001 versus control mice; *p<0.05, **p<0.01, and ***p<0.001 versus DM. Abbreviations: CAT, catalase; DM, diabetic model; MDA, malondialdehyde; RAP-H, high-dose RAP; RAP-L, low-dose RAP; SOD, superoxide dismutase; TGF-β1, transforming growth factor-β1.

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  Figure 2. RAP inhibits the glomerular hypertrophy and glomerular basement membrane thickening in diabetic mice. Notes: (A) Kidney sections were stained with PASM and H&E staining as well as immunohistochemical staining of CTGF and TGF-β1 (original magnifications, ×400). The staining of (B) CTGF and (C) TGF-β1 in mouse kidney were quantified. The mRNA levels of (D) CTGF and (E) TGF-β1 were analyzed by reverse transcriptase polymerase chain reaction. Data are presented as the mean ± SD, n=8. ###p<0.001 versus control mice; *p<0.05, **p<0.01, and ***p<0.001 versus DM. Abbreviations: CTGF, connective tissue growth factor; DM, diabetic model; IHC, immunohistochemistry; PASM, periodic acid-silver methenamine; RAP-H, high-dose RAP; RAP-L, low-dose RAP; TGF-β1, transforming growth factor-β1.

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  Figure 3. RAP alleviates collagen deposition, ECM accumulation, and α-SMA expression in diabetic mice. Notes: (A) Representative renal sections were stained with Masson-trichrome and Sirius red along with immunohistochemical staining of FN and α-SMA (original magnifications, ×400). Quantitative analysis of the results for fibrotic area stained with (B) Masson and (C) Sirius red in mouse kidneys. The positive areas of (D) FN and (E) α-SMA staining were quantified. (F) The protein and mRNA expressions of (G) FN and (H) α-SMA were measured by Western blot and reverse transcriptase polymerase chain reaction. Data are presented as the mean ± SD, n=8. ###p<0.001 versus control mice; *p<0.05, **p<0.01, and ***p<0.001 versus DM. Abbreviations: DM, diabetic model; FN, fibronectin; IHC, immunohistochemistry; RAP-H, high-dose; RAP-L, low-dose RAP; α-SMA, α-smooth muscle actin.

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  Figure 4. RAP alleviates mitogen-activated protein kinase and nuclear factor κB signaling pathways in diabetic mice. Notes: (A) The expression levels of phosphorylated ERK1/2, p38, and p65 were determined by immunohistochemical staining (original magnifications, ×400). The staining of (B) p-ERK1/2, (C) p-p38, and (D) p-p65 were quantified. (E) ERK1/2, p38, p65, and their phosphorylation levels were measured by Western blot analysis. (F, G and H) Densitometric quantification of phosphorylated ERK1/2 and p38 to total ERK1/2 and p38, respectively, and p-p65 to GAPDH are summarized. Data are presented as the mean ± SD, n=8. ###p<0.001 versus control mice; *p<0.05, **p<0.01, and ***p<0.001 versus DM. Abbreviations: DM, diabetic model; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IHC, immunohistochemistry; RAP-H, high-dose RAP; RAP-L, low-dose RAP.

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  Figure 5. RAP inhibits HG-induced ECM accumulation in cell culture medium and MCs proliferation. Notes: (A) MCs were cultured with NG DMEM or HG DMEM or HG DMEM with different concentrations of RAP (10, 50, 100, 150, and 200 μM), respectively. (B) MCs were treated with NG DMEM cultured in the presence or absence of indicated concentrations of RAP and cell viability was measured by MTT. (C and D) The levels of COL4 and FN in the supernatant of MCs were determined by enzyme-linked immunosorbent assay analysis. Data are presented as the mean ± SD. #p<0.05 and ###p<0.001 versus NG; *p<0.05, **p<0.01, and ***p<0.001 versus HG. Abbreviations: COL4, collagen 4; ECM, extracellular matrix; FN, fibronectin; HG, high glucose; MCs, mesangial cells; NG, normal glucose.

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  Figure 6. RAP prevents HG-induced intracellular ECM accumulation and mitogen-activated protein kinase/nuclear factor κB signaling pathways in mesangial cells. Notes: (A) The protein expressions of FN, TGF-β1, CTGF, and α-SMA were measured by Western blot. The mRNA levels of (B) FN, (C) TGF-β1, (D) CTGF, and (E) α-SMA in mesangial cells were assessed by reverse transcriptase polymerase chain reaction. (F) The phosphorylation of ERK1/2, p38, and p65 in mesangial cells was examined by Western blot. (G–I) Densitometric quantification of phosphorylated ERK1/2 and p38 to total ERK1/2 and p38, respectively, or p-p65 compared to α-tubulin was summarized. Data are presented as the mean ± SD. ##p<0.01 and ###p<0.001 versus NG; *p<0.05, **p<0.01, and ***p<0.001 versus HG. Abbreviations: CTGF, connective tissue growth factor; ECM, extracellular matrix; FN, fibronectin; HG, high glucose; NG, normal glucose; α-SMA, α-smooth muscle actin; TGF-β1, transforming growth factor-β1.