Advanced glycosylation end products inhibit the proliferation of bone-marrow stromal cells through activating MAPK pathway

Abstract

Background: The purpose of present study was to explore the mechanism of nuclear factor-kappa B (NF-κB), phosphatidylinositol 3-kinase (PI3K)/protein kinase B(PKB/Akt) and mitogen-activated protein kinase (MAPK) signaling pathways after intervention of advanced glycosylation end products (AGEs) on rat bone-marrow stromal cells (BMSCs).

Methods: Prepare and identify AGEs. BMSCs were isolated from 16 SD rats and cultured with different concentration of AGEs. Cell viability was detected by cell counting kit-8 (CCK-8). BMSCs were cultured with AGEs (0.25 mg/ml) for 30 min, 12 h, 24 h, 72 h and 120 h. In addition, BMSCs were cultured with AGEs, AGEs + JNK inhibitor and AGEs + P38 inhibitor for 24 h and 48 h, respectively. Western blotting and RT-PCR were used to determine the protein and mRNA expression levels, respectively.

Results: Cell viability of BMSCs was significantly correlated with concentration and effect time of AGEs (P < 0.05), and the most appropriate concentration was 0.25 mg/ml. AGEs stimulation significantly increased the protein expression levels of NF-κB p65, JNK, p38 (P < 0.05), decreased IκB (P < 0.05), but had no effect on the protein expression of Akt in BMSCs (P > 0.05). At the mRNA level, JNK and p38 inhibitors significantly reduced the levels of NF-κB p65, p38 and JNK, increased IκB (P > 0.05), but had no effect on Akt in BMSCs (P > 0.05). At the protein level, JNK and p38 inhibitors notably decreased the expression of NF-κB p65, p38, p-JNK, P-IκB and JNK (P < 0.001), and increased IκB (P < 0.05).

Conclusion: Advanced glycosylation end products can inhibit the proliferation of bone-marrow stromal cells through activating MAPK pathway.

Keywords: Advanced glycosylation end products; Bone-marrow stromal cells; MAPK pathway; Proliferation.

Fig. 1

AGEs and rat BMSCs. A AGEs and BSA. B Fluorescence intensity. C BMSCs of primary generation. D BMSCs of third generation. Images (100× magnification) are representative images of three independent samples

Fig. 2

Effect of AGEs on cell viability of BMSCs. A Effects of different concentrations of AGEs on the differentiation of BMSCs. B Effects of different concentrations of AGEs on the differentiation of BMSCs for 24 h. C Effects of different concentrations of AGEs on the differentiation of BMSCs for 48 h. D Effects of different concentrations of AGEs on the differentiation of BMSCs for 72 h. E Effects of action time and concentration of AGEs on cell vitalities of BMSCs. Images (100× magnification) are representative images of three independent samples. Data are expressed as means ± SD. ***P < 0.001; ^{a, b, c, d, e}P < 0.001 vs. control group (ANOVA, F = 20.8–157.7)

Fig. 3

Effect of AGEs on mRNA expression levels of factors in MAPK signaling pathway. Data are expressed as means ± SD (n = 3). ***P < 0.001 vs. control group

Fig. 4

Effect of AGEs on protein expression levels of factors in MAPK signaling pathway. Data are expressed as means ± SD. (n = 3). **P < 0.05 vs. control group. a 30 min; b 12 h; c 24 h; d 72 h; e 120 h

Fig. 5

Effect of JNK and P38 inhibitors on AGEs in mRNA expression levels of factors in MAPK signaling pathway. Data are expressed as means ± SD (n = 3). **P < 0.05 vs. control group

Fig. 6

Effect of JNK inhibitor on AGEs in protein expression levels of factors in MAPK signaling pathway. Data are expressed as means ± SD (n = 3). **P < 0.001 vs. control group

Fig. 7

Effect of P38 inhibitor on AGEs in protein expression levels of factors in MAPK signaling pathway. Data are expressed as means ± SD (n = 3). **P < 0.001 vs. control group