RSC96 Schwann Cell Proliferation and Survival Induced by Dilong through PI3K/Akt Signaling Mediated by IGF-I

Abstract

Schwann cell proliferation is critical for the regeneration of injured nerves. Dilongs are widely used in Chinese herbal medicine to remove stasis and stimulate wound-healing functions. Exactly how this Chinese herbal medicine promotes tissue survival remains unclear. The aim of the present study was to investigate the molecular mechanisms by which Dilong promote neuron regeneration. Our results show that treatment with extract of Dilong induces the phosphorylation of the insulin-like growth factor-I (IGF-I)-mediated phosphatidylinositol 3-kinase/serine-threonine kinase (PI3K/Akt) pathway, and activates protein expression of cell nuclear antigen (PCNA) in a time-dependent manner. Cell cycle analysis showed that G(1) transits into the S phase in 12-16 h, and S transits into the G(2) phase 20 h after exposure to earthworm extract. Strong expression of cyclin D1, cyclin E and cyclin A occurs in a time-dependent manner. Small interfering RNA (siRNA)-mediated knockdown of PI3K significantly reduced PI3K protein expression levels, resulting in Bcl(2) survival factor reduction and a marked blockage of G(1) to S transition in proliferating cells. These results demonstrate that Dilong promotes the proliferation and survival of RSC96 cells via IGF-I signaling. The mechanism is mainly dependent on the PI3K protein.

Figure 1

IGF-I-mediated PI3K/Akt signal pathway activation time course for Schwann cell treated with Dilong extract. RSC96 cells were treated with 125 μg ml⁻¹ Dilong extract for different times as indicated, and the protein expression of IGF-I was determined by western blot. α-tubulin was used as a loading control.

Figure 2

Dilong extract induce the expression of PCNA and promote G₁ progression. RSC96 cells were stimulated for 24 h with 125 μg ml⁻¹ Dilong extract. The protein expression of PCNA was determined by western blot. (a) α-tubulin was used as a loading control. Cell cycle distribution was analyzed using flow cytometry. (b) Data (percentage of cells in the indicated phases) are presented as means ± SD of three independent measurements. ^a P < .05, ^b P < .01, ^c P < .001, ^d P < .0001 versus the control group.

Figure 3

Dilong extract induces the expression of proteins involved in the cell cycle in a time-dependent manner. RSC96 cells were stimulated for 24 h in the presence of 125 μg ml⁻¹ Dilong extract. The protein expression of cell cycle regulatory proteins cyclin A, cyclin D1 and cyclin E were determined by western blot. α-tubulin was used as a loading control.

Figure 4

PI3K knockdown inhibited Dilong extract-induced survival and proliferation. Schwann cell was transiently transfected with 100 nM PI3K siRNA for 8 h before Dilong extract treatment. After incubation with 125 μg ml⁻¹ Dilong extract for 24 h, cells were harvested and analyzed by immunoblotting using antibodies against anti-PI3K antibody and Bcl₂. (a) α-tubulin was used as a loading control. Cell cycle distribution was analyzed by flow cytometry. (b) Data (percentage of cells in the indicated phases) are the means ± SD of three independent measurements. ^#compared to control group, P = .066; *compared to Dilong-treated group, P = .037.

Figure 5

Schematic model of the survival and proliferative effects of Dilong extract on RSC96 Schwann cell. Stimulation of Schwann cell with Dilong extract activate IGF-I signaling, leading to upregulation of the PI3K/Akt pathway and activation of the cell cycle regulatory proteins cyclin D1, E and A, resulting in the survival and proliferation of RSC96 Schwann cell. Dotted lines indicate the hypothetical molecular mechanism of the bioactive compound present in Dilong powder.