Spirulina Crude Protein Promotes the Migration and Proliferation in IEC-6 Cells by Activating EGFR/MAPK Signaling Pathway

Abstract

Spirulina is a type of filamentous blue-green microalgae known to be rich in nutrients and to have pharmacological effects, but the effect of spirulina on the small intestine epithelium is not well understood. Therefore, this study aims to investigate the proliferative effects of spirulina crude protein (SPCP) on a rat intestinal epithelial cells IEC-6 to elucidate the mechanisms underlying its effect. First, the results of wound-healing and cell viability assays demonstrated that SPCP promoted migration and proliferation in a dose-dependent manner. Subsequently, when the mechanisms of migration and proliferation promotion by SPCP were confirmed, we found that the epidermal growth factor receptor (EGFR) and mitogen-activated protein (MAPK) signaling pathways were activated by phosphorylation. Cell cycle progression from G0/G1 to S phase was also promoted by SPCP through upregulation of the expression levels of cyclins and cyclin-dependent kinases (Cdks), which regulate cell cycle progression to the S phase. Meanwhile, the expression of cyclin-dependent kinase inhibitors (CKIs), such as p21 and p27, decreased with SPCP. In conclusion, our results indicate that activation of EGFR and its downstream signaling pathway by SPCP treatment regulates cell cycle progression. Therefore, these results contribute to the research on the molecular mechanism for SPCP promoting the migration and proliferation of rat intestinal epithelial cells.

Keywords: EGFR signaling pathway; MAPK signaling pathway; cell cycle; intestinal epithelial cells; spirulina.

Figure 1

Electrophoresis profiles of SPCP. Crude protein extracted from spirulina (50 μg/mL) was applied to a 15% polyacrylamide gel and stained with Coomassie Brilliant Blue staining for protein. M, protein standard marker.

Figure 2

When IEC-6 cells were confluent in 6-well plates, uniform scratches were made using a sterilized tip. Then cells were serum-starved for 4 h and then treated with spirulina crude protein (SPCP) for 24 h. After washing with phosphate-buffered saline (PBS), the cells were photographed under a microscope at 100× magnification. Migration was assessed as the distance of movement between 0 h and 24 h, as measured using ImageJ software. The results presented are the means ± SD of three independent experiments. * p < 0.05 indicates a significant difference from the control group.

Figure 3

Effects of SPCP on the proliferation of IEC-6 cells. IEC-6 cells were seeded in 96-well plates at a density of 1 × 10⁴ cells/well. After the cells attached, they were serum-starved for 4 h and then treated with SPCP at the indicated concentrations for 24 h. The viability of cells was examined using the MTS assay. The results presented are the means ± SD of three independent experiments. ** p < 0.01 indicates a significant difference from the control group.

Figure 4

Effect of SPCP treatment on EGFR and EGFR adaptor protein expression in IEC-6 cells. (A) Protein expression levels of EGFR and p-EGFR were assessed through western blot analysis. (B) Protein expression levels of Shc, Grb2, and Sos1 were assessed through western blot analysis. GAPDH was used as an internal standard. The results are presented as means ± SD of three independent experiments. * p < 0.05 indicates a significant difference from the control group.

Figure 5

Effect of SPCP treatment on ERK/MAPK protein expression in IEC-6 cells. The protein expression levels of Ras, Raf-1, p-Raf-1, MEK-1, p-MEK-1, ERK1/2, and p-ERK1/2 in IEC-6 cells were assessed through western blot analysis. GAPDH was used as an internal standard. The results are presented as means ± SD of three independent experiments. * p < 0.05 indicates a significant difference from the control group.

Figure 6

Effect of SPCP treatment on cell cycle progression in IEC-6 cells. IEC-6 cells were treated with SPCP at the indicated concentrations for 24 h, then fixed and stained with PI. Cell cycle analysis was performed using a BD FACSVerse flow cytometer, and the percentages of cells in the G0/G1, S, and G2/M phases were analyzed using the ModFit LT 2.0 program. The results are presented as means ± SD of three independent experiments. * p < 0.05 indicates a significant difference from the control group.

Figure 7

Effect of SPCP treatment on the expression of cell cycle regulatory proteins in IEC-6 cells. (A) Effect of SPCP treatment on cyclin and cyclin-dependent kinase (Cdk) protein expression in IEC-6 cells. The protein expression levels of cyclin D1, cyclin E, Cdk2, Cdk4, Cdk6, and p-Rb in IEC-6 cells were determined through western blot analysis. (B) Effect of SPCP treatment on cyclin-dependent kinase inhibitor (CKI) protein expression in IEC-6 cells. The protein expression levels of p21 and p27 in IEC-6 cells were assessed through western blot analysis. GAPDH was used as an internal standard. The results are presented as means ± SD of three independent experiments. * p < 0.05 indicates a significant difference from the control group.