Forsythoside A Alleviates High Glucose-Induced Oxidative Stress and Inflammation in Podocytes by Inactivating MAPK Signaling via MMP12 Inhibition

Abstract

Background: Podocyte injury serves an important role during the progression of diabetic nephropathy (DN). The aim of this study was to investigate the effects of forsythoside A (FA) on high glucose (HG)-induced podocyte injury and to identify the possible mechanisms.

Methods: MPC-5 podocytes were cultured under HG conditions. After exposure to different doses of FA, cell viability and apoptosis were respectively evaluated with CCK-8 assay and flow cytometry. Then, the levels of oxidative stress-related markers and inflammatory factors were examined by corresponding kits. Western blot analysis was employed to detect the expression of Nox2, Nox4, COX-2, iNOS and matrix metalloproteinases 12 (MMP12). Subsequently, MMP12 was overexpressed to assess whether the effects of FA on HG-stimulated podocyte injury were mediated by MMP12 and MAPK signaling.

Results: Results indicated that FA dose-dependently elevated cell viability, reduced cell apoptosis in HG-induced MPC-5 cells. Additionally, FA significantly inhibited oxidative stress, which could be certified by decreased content of malondialdehyde (MDA), enhanced activities of superoxide dismutase (SOD) and catalase (CAT), and downregulated expression of Nox2 and Nox4. Moreover, notably reduced levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 were observed in FA-treated MPC-5 cells under HG conditions, accompanied by decreased COX-2 and iNOS expression. Remarkably, FA suppressed MMP12 expression in a dose-dependent manner, and the effects of FA on MPC-5 cells exposed to HG were partially counteracted by MMP12 overexpression. Mechanically, FA inactivated the expression of phospho-ERK (p-ERK), p-p38 and p-JNK, which was restored after MMP12 overexpression.

Conclusion: These findings demonstrate a protective mechanism of FA by inactivating MAPK signaling via MMP12 inhibition in HG-induced podocyte injury, providing a promising therapeutic candidate for the treatment of DN.

Keywords: apoptosis; diabetic nephropathy; high glucose; inflammation; oxidative stress.

Figure 1

FA treatment intensified cell viability and suppressed cell apoptosis in HG-induced podocytes. (A) The chemical structural formula of FA. (B) Cell viability was tested with CCK-8 assay after treatment with different doses of FA. (C) Cell viability was evaluated using CCK-8 assay in the presence or absence of FA under HG condition. (D) The activity of LDH was determined by means of LDH assay kit. (E and F) Cell apoptosis was assessed by TUNEL assay. *P<0.05, **P<0.01, ***P<0.001.

Figure 2

FA treatment significantly alleviates oxidative stress and inflammation in HG-induced MPC-5 cells. (A–C) The levels of MDA, SOD and CAT were tested by means of corresponding commercially available kits. (D) Western blot analysis was employed to examine the expression of Nox2 and Nox4. (E–G) The concentrations of TNF-α, IL-1β and IL-6 were evaluated with ELISA kits. (H) The expression of COX-2 and iNOS was determined with Western blotting. *P<0.05, **P<0.01, ***P<0.001.

Figure 3

FA reduced MMP12 expression level in HG-induced MPC-5 cells. (A and B) The expression of MMP12 was respectively evaluated using Western blotting and RT-qPCR. (C and D) Western blot assay and RT-qPCR were used to detect MMP12 level after transfection. *P<0.05, ***P<0.001.

Figure 4

MMP12 overexpression partially reversed the impact of FA treatment on cell viability and cell apoptosis in HG-induced podocyte. (A) Cell viability was tested with CCK-8 assay. (B) LDH activity was measured using LDH assay kit. (C and D) TUNEL assay was adopted for evaluation of cell apoptosis. *P<0.05, **P<0.01, ***P<0.001.

Figure 5

MMP12 upregulation conspicuously abrogated the inhibitory effects of FA treatment on oxidative stress and inflammation in HG-induced MPC-5 cells. (A–C) The levels of MDA, SOD and CAT were examined using the corresponding commercially available kits. (D) The expression of Nox2 and Nox4 was determined with Western blotting. (E–G) The concentrations of TNF-α, IL-1β and IL-6 were assessed by ELISA kits. (H) The expression of COX-2 and iNOS was measured with Western blotting. *P<0.05, **P<0.01, ***P<0.001.

Figure 6

FA ameliorated HG-induced podocyte injury by inactivating MAPK signaling via MMP12 inhibition. The expression of p-ERK, p-p38 and p-JNK was determined with Western blot analysis. *P<0.05, **P<0.01, ***P<0.001.