Synergistic Effect of Ferulic Acid and Z-Ligustilide, Major Components of A. sinensis, on Regulating Cold-Sensing Protein TRPM8 and TPRA1 In Vitro

Abstract

Angelica sinensis has been used to attenuate cold-induced cutaneous vasospasm syndrome, such as Raynaud's disease and frostbite, in China for many years. Ferulic acid (PubChem CID: 445858) and Z-ligustilide (PubChem CID: 529865), two major components extracted from Angelica sinensis, had been reported to inhibit vasoconstriction induced by vasoconstrictors. In this study, the pharmacological interaction in regulating cold-induced vascular smooth muscle cell contraction via cold-sensing protein TRPM8 and TRPA1 was analyzed between ferulic acid and Z-ligustilide. Pharmacological interaction on inhibiting [Ca(2+)]i influx evoked by TRPM8 agonist WS-12 or TRPA1 agonist ASP 7663 as well as cold-induced upregulation of TRPM8 was determined using isobolographic analysis. The isobolograms demonstrated that the combinations investigated in this study produced a synergistic interaction. Combination effect of two components in inhibiting RhoA activation and phosphorylation of MLC20 induced by WS-12 or ASP 7663 was also being quantified. These findings suggest that the therapeutic effect of Angelica sinensis on cold-induced vasospasm may be partially attributed to combinational effect, via TRPM8 and TPRA1 way, between ferulic acid and Z-ligustilide.

Figure 1

Contribution of Ca²⁺ store to TRPM8 and TRPA1 agonist-evoked Ca²⁺ signal. (a) Images of field of HASMC before (left) and during (right) 50 μM WS-12 (upper) and 50 μM ASP7663 (down) application. (b) WS-12 50 μM or ASP 50 μM-evoked calcium signal was measured in the absence or presence of ryanodine 30 μM or 2-APB 100 μM or xestospongin C 5 μM.  ^∗Significant difference between control groups (P < 0.05).

Figure 2

Effects of ferulic acid and Z-ligustilide on [Ca²⁺]_i entry responses to TRPM8 or TRPA1 agonist with absence of CICR. Inhibition of HASMC response to WS-12 50 μM (a) or ASP 7663 50 μM (b) by ferulic acid (red symbols and line) or Z-ligustilide (blue symbols and line) with absence of CICR. Sigmoidal log concentration-response curve was drawn. Results were presented as a percentage of the peak [Ca²⁺]_i entry level measured in the presence of 50 μM WS-12 or ASP 7663 50 μM. Each value was derived from 3 individual experiments with a total of 30–80 cells. HASMC were quiescent in serum-free medium for 24 hours before experiments.

Figure 3

Isobolograms of combination of ferulic acid with Z-ligustilide in inhibiting [Ca²⁺]_i response to agonists. The individual doses of ferulic acid and Z-ligustilide to achieve 90% (straight line) inhibition (Fa = 0.90), 75% (hyphenated line) inhibition (Fa = 0.75), and 70% (crosses) inhibition (Fa = 0.70) were plotted on the x-axes and y-axes. Combination index (CI) values calculated by Calcusyn software are represented by points above (indicating antagonism) or below the lines (indicating synergism).

Figure 4

Inhibition effects of ferulic acid, Z-ligustilide, or their combination on GTP-RhoA level and phosphorylation of MLC₂₀ with absence or presence of WS-12 or ASP 7663. HASMC were stimulated by ferulic acid or Z-ligustilide alone (a), 50 μM WS-12 (b), or 50 μM ASP 7663 (c) for 10 minutes in the absence or presence of ferulic acid, Z-ligustilide, or their combination. Data are expressed in % of GTP-RhoA expression normalized to total RhoA expression or phosphorylation of MLC₂₀ normalized to total MLC₂₀ expression. Immunofluorescence images of phosphorylation of MLC₂₀ were also captured. Bars are mean values from 3 individual samples.  ^∗Significant difference compared to control (P < 0.05).  ^#Significant difference compared to ferulic acid group or Z-ligustilide group (P < 0.05).

Figure 5

Effect of ferulic acid, Z-ligustilide, or their combination on cold-induced upregulation of TRPM8. (a) and (b) show effects of different concentration of ferulic acid and Z-ligustilide on inhibiting trpm8 gene expression. (c) shows isobolograms of combination of ferulic acid with Z-ligustilide. Combination index (CI) values calculated by Calcusyn software were all below points, which indicate synergism.  ^∗Significant difference between control group versus cold-stimulation group.  ^∗∗Significant difference between groups versus cold-stimulation group.