Salvianolic Acid B Inhibits ERK and p38 MAPK Signaling in TGF-β1-Stimulated Human Hepatic Stellate Cell Line (LX-2) via Distinct Pathways

Abstract

Salvianolic acid B (SA-B) is water-soluble component of Radix Salvia miltiorrhiza. The previous work indicated that SA-B can inhibit MAPK and Smad signaling in activated hepatic stellate cells (HSCs) to perform anti-fibrotic activity Lv et al. 2010. However, some studies have shown that there is cross-talk between MAPK and Smad in certain cell types. Thus, the anti-fibrotic action of SA-B may be through the cross-talk. In order to clarify the mechanism of SA-B further, we knocked down Smad in LX-2 cells (SRV4) via RNAi, and then added TGF-β1, and PD98059 or SB203580 and SA-B. The levels of p-MEK and p-p38 were inhibited by SA-B in SRV4 independent of TGF-β1. The expression of Col I and α-SMA in SRV4 could be reduced by SA-B independent TGF-β1. SB203580 had not significant effect on p-MEK in SRV4 stimulated by TGF-β1. The levels of p-MEK in SRV4 were not increased significantly after TGF-β1 stimulation. PD98059 had no effect on the levels of p-p38 in SRV4 irrespective of TGF-β1. In conclusion, SA-B inhibits the synthesis of Col I in LX-2 cells independent of TGF-β1 stimulation, and the anti-fibrotic effect of SA-B is due to direct inhibition of p38 signaling and inhibition the cross-talk of Smad to ERK signaling.

Figure 1

Inhibition of Smad4 in LX-2 transfected with Pol II miR RNAi expression vectors (SRV1, SRV2, SRV3, and SRV4). (a) Smad4 mRNA expression levels detected by real time PCR 72 h after selection with blasticidin. Results were normalized to Smad4 expression in “Negative control” using the 2^−ΔΔCt method (where Ct is threshold cycle). **Significant difference versus negative control (n = 3, P < 0.01). On the right side of the figure is shown the electrophoresis of PCR product. (b) Smad4 protein expression levels detected after selection with blasticidin, using the Western blot. Blotting with anti-β-actin antibody was conducted as a protein loading control. Quantification of the intensity of bands calibrated to the intensity of total protein bands (means ± SD). **Significant difference versus negative control (n = 3, P < 0.01).

Figure 2

The effects of SA-B on p38 MAPK pathway via Inhibition of ERK and Smad signaling. (a) P38 phosphorylation in LX-2 cells. The levels of phosphorylated p38 protein were determined by Western blot using anti-phospho-p38 antibodies. The levels of total p38 protein were determined by Western blot using anti-p38 antibodies. Quantification of the intensity of bands calibrated to the intensity of total protein bands (means ± SD). ^##Significant difference versus Control, Negative control, and SRV4 (n = 3, P < 0.01); ^■■■Significant difference versus SM4 + TGF (n = 3, P < 0.001); ^▲▲▲Significant difference versus TGF (n = 3, P < 0.001); ***Significant difference versus SM4 + TGF + PD (n = 3, P < 0.001). (b) α-SMA level in LX-2. The levels of α-SMA protein were determined by Western blot using anti-α-SMA antibodies. Blotting with anti-GAPDH antibodies was conducted as a protein loading control. Quantification of the intensity of bands calibrated to the intensity of total protein bands (means ± SD). ^##Significant difference versus Control, Negative control, and SRV4 (n = 3, P < 0.01); ^▲▲Significant difference versus TGF (n = 3, P < 0.01); ^⋄⋄⋄Significant difference versus SM4 + TGF (n = 3, P < 0.001), ^▵▵▵Significant difference SM4 + TGF + PD and SM4 + TGF + SA-B (n = 3, P < 0.001); ***Significant difference versus SM4 + TGF + SA-B, TGF + SA-B + PD (n = 3, P < 0.001). (c) Col. I level in LX-2. The levels of Col. I protein were determined by Western blot using anti-Col. I antibodies. Blotting with anti-GAPDH antibodies was conducted as a protein loading control. ^##Significant difference versus Control, Negative control, and SRV4 (n = 3, P < 0.01); ^▲▲Significant difference versus TGF (n = 3, P < 0.01); ^▵▵Significant difference versus SM4 + TGF (n = 3, P < 0.01); *Significant difference versus SM4 + TGF + PD, SM4 + TGF + SA-B, and TGF + SA-B + PD (n = 3, P < 0.05).

Figure 3

The effects of SA-B on ERK signaling via inhibition of p38 MAPK and Smad signaling. (a) MEK phosphorylation in LX-2 cells. The levels of phosphorylated MEK protein were determined by Western blot using anti-phospho-MEK antibodies. The levels of total MEK protein were determined by Western blot using anti-MEK antibodies. Quantification of the intensity of bands calibrated to the intensity of total protein bands (means ± SD). ^###Significant difference versus Control, Negative control, and SRV4 (n = 3, P < 0.001); **Significant difference versus TGF (n = 3, P < 0.01); ^▲▲▲Significant difference versus SM4 + TGF, SM4 + TGF + SB, and TGF + SA-B + SB (n = 3, P < 0.01). (b) α-SMA levels in LX-2. The levels of α-SMA protein were determined using anti-α-SMA antibodies. Blotting with anti-GAPDH antibodies was conducted as a protein loading control. ^##Significant difference versus Control, Negative control and SRV4 (n = 3, P < 0.01); ^▲▲Significant difference versus TGF (n = 3, P < 0.01); ^⋄⋄Significant difference versus SM4 + TGF (n = 3, P < 0.01); Significant difference versus SM4 + TGF (n = 3, P < 0.001); ***Significant difference versus SM4 + TGF + SB, SM4 + TGF + SA-B, and TGF + SA-B + SB (n = 3, P < 0.001). (c) Col. I level in LX-2. The levels of Col. I protein were determined by Western blot using anti-Col. I antibodies. Blotting with anti-GAPDH antibodies was conducted as a protein loading control. ^##Significant difference versus Control, Negative control, and SRV4 (n = 3, P < 0.01); ^▲▲Significant difference versus TGF (n = 3, P < 0.01); ^◊Significant difference versus SM4 + TGF (n = 3, P < 0.05); ^◊◊Significant difference versus SM4 + TGF (n = 3, P < 0.01); **Significant difference versus SM4 + TGF + SB, SM4 + TGF + SA-B, and TGF + SA-B + SB (n = 3, P < 0.01).

Figure 4

Proposed mechanisms of the anti-fibrotic effects of SA-B on activated HSCs stimulated by TGF-β1.