Inhibition of PAI-1 Activity by Toddalolactone as a Mechanism for Promoting Blood Circulation and Removing Stasis by Chinese Herb Zanthoxylum nitidum var. tomentosum

Abstract

Traditional Chinese medicine has been used to treat a variety of human diseases for many centuries. Zanthoxylum nitidum var. tomentosum is used as an adjuvant to promote blood circulation and remove stasis. However, the mechanisms of improving circulation and other biological activities of Z. nitidum var. tomentosum are still unclear. Plasminogen activator inhibitor-1 (PAI-1) regulates the plasminogen activation system through inhibition of tissue-type and urokinase-type plasminogen activators (tPA and uPA). PAI-1 has been linked to fibrin deposition that evolves into organ fibrosis and atherosclerosis. In the present study, we showed that ethanol extract prepared from Z. nitidum var. tomentosum exhibited PAI-1 inhibitory activity, and identified toddalolactone as the main active component that inhibited the activity of recombinant human PAI-1 with IC₅₀ value of 37.31 ± 3.23 μM, as determined by chromogenic assay, and the effect was further confirmed by clot lysis assay. In vitro study showed that toddalolactone inhibited the binding between PAI-1 and uPA, and therefore prevented the formation of the PAI-1/uPA complex. Intraperitoneal injection of toddalolactone in mice significantly prolonged tail bleeding and reduced arterial thrombus weight in a FeCl₃-induced thrombosis model. In addition, the hydroxyproline level in the plasma and the degree of liver fibrosis in mice were decreased after intraperitoneal injection of toddalolactone in CCl₄-induced mouse liver fibrosis model. Taken together, PAI-1 inhibition exerted by toddalolactone may represent a novel molecular mechanism by which Z. nitidum var. tomentosum manifests its effect in the treatment of thrombosis and fibrosis.

Keywords: PAI-1; PAI-1 inhibitor; fibrinolysis; pharmacology; toddalolactone.

FIGURE 1

Inhibition of PAI-1 by Zanthoxylum nitidum var. tomentosum extract. (A) Preparative HPLC chromatogram of the extract. (B) Inhibition of PAI-1 activity by the HPLC-resolved fractions of the extract (n = 4).

FIGURE 2

Identification of toddalolactone in the active fraction Z. nitidum var. tomentosum extract. (A) Analytical HPLC chromatogram of #27 with detection wavelength of 254 nm. (B) Chromatogram of toddalolactone standard (left) and Peak 4 of #27 of the extract (right) under the same chromatographic analysis condition. Inset: structure of toddalolactone.

FIGURE 3

Effect of toddalolactone on PAI-1 activity. (A) Inhibition of PAI-1 activity by toddalolactone as determined by chromogenic assay. Data are the means ± SEs from four independent experiments. (B) Inhibition of PAI-1/uPA complex formation by toddalolactone. PAI-1 and uPA were incubated without or with the indicated concentrations of toddalolactone and then subjected to SDS-PAGE analysis. (C) Effect of toddalolactone on uPA-mediated lysis of fibrin clots. The fibrin clot was treated with uPA+PAI-1 only () or with 30 μM (), 60 μM (), 125 μM (), 250 μM () and 500 μM () of toddalolactone, or by uPA only (). (D) Effect of toddalolactone on uPA activity in the presence of PAI-1. Data are the means ± SEs from five experiments, each carried out in duplicate. “^∗” and “^∗∗” indicate significantly different from the control (no toddalolactone) at the P < 0.05 and P < 0.01 level, respectively.

FIGURE 4

Effect of toddalolactone on arterial thrombosis induced by FeCl₃. (A) Sham group; (B) FeCl₃-induced mice not given toddalolactone; (C) toddaloctone-treated mice.

FIGURE 5

Effect of toddalolactone on liver fibrosis. (A) Effect of toddalolactone on weight loss in mice as a consequence of CCl₄-induced liver fibrosis. Data are the means ± SEs from five animals. (B) Effect of toddalolactone on the level collagen hydroxyproline induced in mice by CCl₄. Data are the means ± SEs from six animals. “^∗” and “^∗∗” indicate significantly different from the control at the P < 0.05 and P < 0.01 level, respectively.

FIGURE 6

Inhibitory effect of toddalolactone on CCl₄-induced liver fibrosis. (A,D) Healthy control group; (B,F) Model group; (C,F) Toddalolactone treatment group. Images in (A–C) are 100× magnification, whereas images in (D–F) are 400× magnification.