Ferulic Acid, an Angelica sinensis-Derived Polyphenol, Slows the Progression of Membranous Nephropathy in a Mouse Model

Abstract

Membranous nephropathy (MN) is a leading cause of adult nephrotic syndrome but lacks adequate treatment. Different extracts of Angelica sinensis (AS) and one of its active compounds, ferulic acid (FA), were used to evaluate the therapeutic effects in a MN mouse model. The MN model was grouped into three subgroups: no treatment (N-T), treatment at induction of MN (Pre-T), and treatment after full-blown MN (Post-T). The results showed that the methanol (ME) layer of AS extract exhibited a therapeutic effect on MN-induced proteinuria. The ME layer-enriched compound, FA, improved the hypoalbuminemia, hyperlipidemia, and proteinuria in both Pre-T and Post-T groups. Ferulic acid also reduced the formation of oxidative protein products and increased the synthesis of antioxidant enzymes in groups Pre-T and Post-T. Regarding angiogenesis factors, the antiangiogenic factors in renal glomeruli were increased in group N-T, but, after FA treatment, only one of the antiangiogenic factors, thrombospondin-1, showed a significant decrease. Furthermore, the expression of Th2 predominant showed significant decrease in both Pre-T and Post-T groups when compared to that of N-T group. In summary, FA retarded the progression of MN, and the mechanisms involved the regulation of oxidative stresses, angiogenic and antiangiogenic factors, and attenuation of Th2 response.

Figure 1

Differential extraction of Angelica sinensis (AS).   The powder of AS was extracted sequentially with acetone, methanol, and water. The extracts were concentrated under reduced pressure to yield an acetone extract (AS-AE), methanol extract (AS-ME), and water-soluble extract (AS-WE). The layer of AS-ME was chromatographed on an Amberlite XAD-4 column and gave 4 separated fractions, including H₂O fraction, 30% MeOH/H₂O fractions, 50% MeOH/H₂O fraction, and 100% MeOH. The 100% MeOH fraction was further chromatographed on Lobar RP-18 to get purified ferulic acid.

Figure 2

Ferulic acid attenuated the severity of renal histopathological changes in MN mouse model. At the end of the experiment, kidney samples were collected for the histopathological examination. Representative sections from renal tissue in NC ((a), (e), and (i)), N-T ((b), (f), and (j)), Pre-T ((c), (g), and (k)), and Post-T ((d), (h), and (l)) groups were performed with H&E staining ((a) through (d)), IgG immunofluorescence staining ((e) through (h)), and colloidal iron staining ((i) through (l)), original magnifications: 400x.

Figure 3

Ferulic acid reduced the level of advanced oxidation protein products (AOPPs) and myeloperoxidase (MPO) in MN mouse model. At the end of the experiment, serum and renal cortical tissues were collected, and renal cortical tissues were processed by sonicator in a ratio of per mg tissue in 5 μL phosphate buffer. The formation of oxidation protein was determined by detecting the AOPPs levels in serum (a) and renal cortical tissues (b). Paraffin-embedded mouse kidney sections were examined their expression of MPO by immunohistochemical staining in NC ((c) and (g)), N-T ((d) and (h)), Pre-T ((e) and (i)) and Post-T ((f) and (j)) groups. Original magnifications: 400x ((c), (d), (e), and (f)) and 40x ((g), (h), (i), and (j)) (*P < 0.05; **P < 0.01).

Figure 4

Ferulic acid increased the expression level of superoxide (SOD), glutathione (GPx), and catalase (CAT) in MN. At the end of the experiment, renal cortical tissues were processed by sonicator in a ratio of per mg tissue in 5 μL phosphate buffer for detecting the level of antioxidant enzymes SOD, GPx, and CAT. The renal cortical level of SOD (a), GPx (b), and CAT (c) was determined in NC, N-T, Pre-T, and Post-T groups (*P < 0.05; **P < 0.01).

Figure 5

Ferulic acid regulated the differential expression of angiogenic and antiangiogenic factors in MN mouse model.  At the end of the experiment, kidney samples of different groups were harvested and freshly isolated the glomeruli according to the Materials and Methods. Total protein lysate was extracted from isolated glomeruli and separated in 10% SDS page by electrophoresis, and further blotting by anti-VEGF (a), TSP-1 (b), and K5 (c) antibodies. The bar chart represented the semiquantification level of the expression density (target/actin) (Left Panel) (*P < 0.05; **P < 0.01). Immunofluorescence staining was also undertaken to exam the expression level and location of VEGF, TSP-1, and K5 in NC and N-T, original magnifications, 400x (Right Panel).

Figure 6

Ferulic acid changes the pattern of Th1/Th2 immune response. At the end of the experiment, serum samples were collected for determining the serum IgG1 and IgG2a level. The quantification of IgG1 and IgG2a was using a commercialized ELISA kit and analyzed in spectrometer. Data were calculated by regression analysis. Each symbol represented an individual sample and the intercept, R ² of the regression lines, interval of confidence, and P value were further determined in NT (a), Pre-T (b), and Post-T (c) groups.