Luteolin, a natural flavonoid, inhibits methylglyoxal induced apoptosis via the mTOR/4E-BP1 signaling pathway

Abstract

Methylglyoxal (MG) accumulation has been observed in human cerebrospinal fluid and body tissues under hyperglycaemic conditions. Recent research has demonstrated that MG-induces neuronal cell apoptosis, which promotes the development of diabetic encephalopathy. Our previous animal study has shown that luteolin, a natural flavonoid, attenuates diabetes-associated cognitive dysfunction. To further explore the neuroprotective properties of luteolin, we investigated the inhibitive effect of luteolin on MG-induced apoptosis in PC12 neuronal cells. We found that MG inhibited cell viability in a dose-dependent manner and induced apoptosis in PC12 cells. Pretreatment with Luteolin significantly elevated cell viability, reduced MG-induced apoptosis, inhibited the activation of the mTOR/4E-BP1 signaling pathway, and decreased pro-apoptotic proteins, Bax, Cytochrome C as well as caspase-3. Furthermore, we found that pretreatment with the mTOR inhibitor, rapamycin, significantly reduced the expression of the pro-apoptotic protein Bax. Therefore, these observations unambiguously suggest that the inhibitive effect of Luteolin against MG-induced apoptosis in PC12 cells is associated with inhibition of the mTOR/4E-BP1 signaling pathway.

Figure 1

Luteolin (Lut) prevented MG-induced decrease in cell viability in a dose-dependent manner. (A) MTT assays were performed to detect viability of PC12 cells treated with MG (0.1–2.0 mM) for 12 h, 24 h and 36 h. Results are expressed relative to control and are presented as means ± SD of three independent experiments, each performed in triplicate. ^* p < 0.05, ^** p < 0.01 vs control group. (B) Representative photographs of cell morphology of PC12 cells treated with MG (0.1–2.0 mM) for 36 h. Morphological changes of PC12 cells were observed by phase-contrasted microscopy. (C) Chemical structure of Lut. (D) Morphological changes of PC12 cells, pretreated with Lut for 3 h, followed by MG (0.5 mM) for 36 h. Data are presented as means ± SD of three independent experiments, each performed in triplicate. ^# p < 0.01 vs control group; ^* p < 0.05 and ^** p < 0.01 vs MG group. (E) Representative images of Lut-induced protection against MG-induced cytotoxicity. Cells were observed by phase-contrast microscopy.

Figure 2

Luteolin (Lut) inhibited MG-induced cell apoptosis. Apoptosis assessment of PC12 cells treated with MG (0.1–2.0 mM) for 36 h. Lut protected PC12 cells against MG-induced apoptosis. (A) Cells were stained by fluorescent annexin V and propidium iodide (PI) and then examined for apoptosis by flow cytometry. (B) The percent of Annexin V positive cells following increasing concentrations of MG. Data are representatives from three independent experiments and the percentages of different populations were labeled in the figures. ^* p < 0.05 and ^** p < 0.01 vs control group. (C) Nuclear fragmentation was assessed by nuclei staining with Hoechst 33358. MG (0.1–2 mM) increased the apoptosis in PC12 cells. (D) Cells were pretreated with Lut (1, 5, 10 μM) for 3 h, followed by 0.5 mM MG exposure for 36 h. Morphological apoptosis was determined by Hoechst 33258 staining.

Figure 3

Luteolin (Lut) inhibited the activation of mTOR-4E-BP1 induced by MG. PC12 cells were treated with the Lut (1, 5 and 10 μM) for 3 h, followed by incubating with 0.5 mM MG for 24 h. The activation of mTOR (A and C) and 4E-BP1 (B and D) was determined by western blot and immunofluorecence staining. Data are presented as means ± SD of three independent experiments, each performed in triplicate. ^# p < 0.01 vs control group; ^* p < 0.05 and ^** p < 0.01 vs MG group. The full-length blots/gels are presented in Supplementary Figs S4 and S5.

Figure 4

Luteolin (Lut) inhibited MG-induced overexpression of Bax, Cyt C and caspase-3. The cells were pretreated with Lut (1, 5, 10 μM) for 3 h, followed by 0.5 mM MG administration for an additional 36 h. Bax (A) and Cyt C (B) were determined by western blotting analysis and the band densities were normalized with β-actin. Data are presented as means ± SD of three independent experiments, each performed in triplicate. ^# p < 0.01 vs control group; ^* p < 0.05 and ^** p < 0.01 vs MG group. The expression of Bax (C), Cyt C (D) and caspase-3 (E) was analyzed by immunofluorescent staining. Histograms show the quantification of the fluorescence intensity of the corresponding proteins. ^# p < 0.01 vs control group; ^* p < 0.05 vs MG group. The full-length blots/gels are presented in Supplementary Figs S6 and S7.

Figure 5

A proposed model of molecular targets of Luteolin (Lut) in preventing MG-induced apoptosis. Our study found that Lut prevents MG-induced apoptosis by decreasing protein phosphorylation of mTOR, and 4E-BP1 in PC12 cells. Furthermore, our study also confirmed the mTOR/4E-BP1 signaling pathway modulates apoptosis and regulates the expression of the pro-apoptotic protein Bax in PC12 cells. This suggests that Lut prevented MG-induced cell apoptosis and decreased the expression of the pro-apoptotic Bax protein and Cyt C and casepase-3 through inhibiting mTOR/4E-BP1 signaling pathway.