Piperazine ferulate attenuates high glucose‑induced mesangial cell injury via the regulation of p66Shc

Abstract

Diabetic nephropathy (DN) is a severe microvascular complication of diabetes. Hyperglycemia‑induced glomerular mesangial cells injury is associated with microvascular damage, which is an important step in the development of DN. Piperazine ferulate (PF) has been reported to exert protective effects against the progression of DN. However, whether PF prevents high glucose (HG)‑induced mesangial cell injury remains unknown. The aim of the present study was to investigate the effects of PF on HG‑induced mesangial cell injury and to elucidate the underlying mechanisms. Protein and mRNA expression levels were determined via western blot analysis and reverse transcription‑quantitative PCR, respectively. IL‑6 and TNF‑α levels were measured using ELISA. Reactive oxygen species levels and NF‑κB p65 nuclear translation were determined via immunofluorescence analysis. Apoptosis was assessed by measuring lactate dehydrogenase (LDH) release, as well as using MTT and flow cytometric assays. The mitochondrial membrane potential of mesangial cells was determined using the JC‑1 kit. The results revealed that LDH release were increased; however, cell viability and mitochondrial membrane potential were decreased in the HG group compared with the control group. These changes were inhibited after the mesangial cells were treated with PF. Moreover, PF significantly inhibited the HG‑induced production of inflammatory cytokines and the activation of NF‑κB in mesangial cells. PF also attenuated the HG‑induced upregulation of the expression levels of fibronectin and collagen 4A1. Furthermore, the overexpression of p66^{Src homology/collagen (Shc)} abolished the protective effect of PF on HG‑induced mesangial cell injury. In vivo experiments revealed that PF inhibited the activation of inflammatory signaling pathways, glomerular cell apoptosis and mesangial matrix expansion in diabetic mice. Collectively, the present findings demonstrated that PF attenuated HG‑induced mesangial cells injury by inhibiting p66^Shc.

Keywords: piperazine ferulate; inflammatory cytokine; diabetic nephropathy; apoptosis; p66 Src homology/collagen; mesangial cells.

Figure 1.

PF prevents the mesangial cell injury induce by HG. (A) Structure of PF. (B) Effect of various concentration of HG on cell viability. (C) Effect of various concentration of PF on HG-induced cell damage (HG, 30 mM). (D) Representative flow cytometric results. (E) Statistical analysis of the flow cytometry data. (F) Level of LDH released in the supernatant of cultured cells. Data are expressed as the mean ± SEM, n=3. **P<0.01, ***P<0.001 vs. control group; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. HG group. PF, piperazine ferulate; HG, high glucose; OD, optical density; LDH, lactate dehydrogenase; +PF, HG + PF.

Figure 2.

PF attenuates the production of inflammatory cytokines and fibrosis in mesangial cells induced by HG. (A) Fibronectin mRNA expression. (B) Collagen 4A1 mRNA expression. (C) Level of IL-6 in cell culture supernatant. (D) Level of TNF-α in cell culture supernatant. (E) Immunofluorescence image of NF-κB p65; ×400 magnification. Data are expressed as the mean ± SEM, n=3. *P<0.05, ***P<0.001 vs. control group; ^#P<0.05 vs. HG group. PF, piperazine ferulate; HG, high glucose; +PF, HG + PF.

Figure 3.

PF inhibits the expression of p-p66^Shc in HG-exposed mesangial cells. (A) Western blot analysis of p-p66^shc and total p66^shc. (B) Densitometric analyses of p-p66^shc/p66^shc and (C) p-p66^shc. (D) Western blot analysis of IKKα/β, p-IKKα/β, NF-κB p65 and p-NF-κB p65. (E) Densitometric analyses of p-IKKα/β/IKKα/β and (F) p-NF-κB p65/NF-κB p65. (G) Fluorescence image of ROS detected using dihydroethidium; ×400 magnification. Data are expressed as the mean ± SEM, n=3. *P<0.05, **P<0.01, ***P<0.001 vs. control group; ^#P<0.05, ^###P<0.001 vs. HG group. PF, piperazine ferulate; HG, high glucose; p-, phosphorylated; Shc, Src homology/collagen; +PF, HG + PF.

Figure 4.

PF regulates HG-induced mesangial cell injury by inhibiting p66^shc. (A) Effect of pcDNA-p66^shc (1 ng/µl or 2 ng/µl) on the expression of p66^shc. (B) Western blot analysis verified the validity of the pcDNA-p66^Shc. (C) Level of IL-6 in the cell culture supernatant. (D) Western blot analysis of caspase-3, p-IKKα/β, total IKKα/β, Bcl2, Bax, fibronectin and β-actin. Densitometric analyses of (E) caspase-3, (F) p-IKK/IKK, (G) Bcl2, (H) Bax and (I) fibronectin. (J) Representative image of mesangial cells stained with JC-1 (red for aggregate form of JC-1 and green for monomeric form); ×400 magnification. Data are expressed as the mean ± SEM, n=3. **P<0.01, ***P<0.001 vs. vector group; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. vector + HG group; ^$P<0.01 vs. vector + HG + PF group. PF, piperazine ferulate; HG, high glucose; vector, empty vector control; p-, phosphorylated; Shc, Src homology/collagen.

Figure 5.

PF attenuates kidney injury in diabetic mice. (A) Western blot analysis of p-p66^shc, total p66^shc, IκBα, p-NF-κB p65, total NF-κB p65, p-IKKα/β, total IKKα/β and β-actin. (B) Densitometric analysis of p-p66^shc and total p66^shc. (C) Densitometric analyses of p-p66^shc/p66^shc and p-NF-κB p65/NF-κB p65. (D) Densitometric analyses of IκBα and p-IKKα/β/IKKα/β. (E) Representative image of the TUNEL staining; ×400 magnification; white arrowheads indicate TUNEL-positive cells. Data are expressed as the mean ± SEM, n=3. *P<0.05, **P<0.01 vs. control group; ^#P<0.05, ^##P<0.01 vs. HG group. PF, piperazine ferulate; HG, high glucose; p-, phosphorylated; Shc, Src homology/collagen; +PF, diabetic nephropathy + PF.

Figure 6.

PF attenuates mesangial matrix expansion in diabetic mice. Effect of PF on the mRNA expression levels of (A) collagen 4A1 and (B) fibronectin. (C) Immunohistochemistry images of collagen 4A1 and fibronectin; ×400 magnification. (D) Quantification analyses of immunohistochemistry staining of collagen 4A1. (E) Quantification analyses of immunohistochemistry staining of fibronectin. (F) Masson's trichrome staining; ×400 magnification. (G) Quantification analyses of Masson's trichrome staining. Data are expressed as the mean ± SEM, n=3. *P<0.05, **P<0.01 vs. control group. ^#P<0.05, ^##P<0.01 vs. model group. PF, piperazine ferulate; +PF, diabetic nephropathy + PF.

Figure 7.

Schematic diagram of pathways involved in the protective effects of PF against HG-induced mesangial cell injury. In the diabetic environment, HG induces glomerular mesangial cell injury, which is characterized by an increase in apoptosis, inflammatory cytokine release and mesangial matrix synthesis. PF attenuated HG-induced mesangial cell injury by inhibiting p66^Shc. PF, piperazine ferulate; HG, high glucose; Shc, Src homology/collagen; ROS, reactive oxygen species; DN, diabetic nephropathy.