Shikonin, a component of chinese herbal medicine, inhibits chemokine receptor function and suppresses human immunodeficiency virus type 1

Abstract

Shikonin is a major component of zicao (purple gromwell, the dried root of Lithospermum erythrorhizon), a Chinese herbal medicine with various biological activities, including inhibition of human immunodeficiency virus (HIV) type 1 (HIV-1). G protein-coupled chemokine receptors are used by HIV-1 as coreceptors to enter the host cells. In this study, we assessed the effects of shikonin on chemokine receptor function and HIV-1 replication. The results showed that, at nanomolar concentrations, shikonin inhibited monocyte chemotaxis and calcium flux in response to a variety of CC chemokines (CCL2 [monocyte chemoattractant protein 1], CCL3 [macrophage inflammatory protein 1alpha], and CCL5 [regulated upon activation, normal T-cell expressed and secreted protein]), the CXC chemokine (CXCL12 [stromal cell-derived factor 1alpha]), and classic chemoattractants (formylmethionyl-leucine-phenylalanine and complement fraction C5a). Shikonin down-regulated surface expression of CCR5, a primary HIV-1 coreceptor, on macrophages to a greater degree than the other receptors (CCR1, CCR2, CXCR4, and the formyl peptide receptor) did. CCR5 mRNA expression was also down-regulated by the compound. Additionally, shikonin inhibited the replication of a multidrug-resistant strain and pediatric clinical isolates of HIV in human peripheral blood mononuclear cells, with 50% inhibitory concentrations (IC(50)s) ranging from 96 to 366 nM. Shikonin also effectively inhibited the replication of the HIV Ba-L isolate in monocytes/macrophages, with an IC(50) of 470 nM. Our results suggest that the anti-HIV and anti-inflammatory activities of shikonin may be related to its interference with chemokine receptor expression and function. Therefore, shikonin, as a naturally occurring, low-molecular-weight pan-chemokine receptor inhibitor, constitutes a basis for the development of novel anti-HIV therapeutic agents.

FIG. 1.

Chemical structure of shikonin (C₁₆H₁₆O₅; molecular weight, 288.2994).

FIG. 2.

Effect of shikonin on human monocyte viability. Freshly isolated human monocytes were placed in 96-well plates at 10⁵ cells/well (200 μl) with the indicated concentrations of shikonin. After incubation for 24 h at 37°C in humidified air with 5% CO_2, cell viability was measured by the standard MTS assay and the trypan blue exclusion assay. Data are expressed as the means ± standard errors of the means for quadruplicate samples. *, P < 0.05 compared with the results for the control group (medium only); **, P < 0.01 compared with the results for the control group (medium only).

FIG. 3.

Effect of shikonin on chemokine- and chemoattractant-induced human monocyte chemotaxis. (A) Chemotactic response of monocytes (treated with 0.5 μM shikonin) to C5a (10⁻⁹ M), CCL5 (RANTES; 100 ng/ml), CCL2 (monocyte chemoattractant protein 1; 100 ng/ml), CXCL12 (stromal cell-derived factor 1α; 100 ng/ml), CCL3 (macrophage inflammatory protein 1α; 100 ng/ml), and formylmethionyl-leucine-phenylalanine (fMLP; 10⁻⁹ M); (B) shikonin-inhibited monocyte migration induced by CCL5 (RANTES; 100 ng/ml) in a dose-dependent manner. ***, P < 0.001 compared with the results for the control group (chemokine or chemoattractant only). C.I., chemotactic index.

FIG. 4.

Effect of shikonin on calcium mobilization evoked by chemoattractants. Human monocytes were loaded with a dye (Fura-2) and then stimulated with CCL5 (RANTES; 100 ng/ml), CCL2 (monocyte chemoattractant protein 1; 100 ng/ml), and C5a (10⁻⁹ M), as indicated. The ratio of the fluorescence at a wavelength of 340 to the fluorescence at a wavelength of 380 nm was recorded and calculated by using the FL WinLab program. (A) Calcium mobilization induced by CCL5 (RANTES), CCL2 (monocyte chemoattractant protein 1), and C5a. Shikonin (1 μM) was then added to the cell suspension for 50 s (B), 10 min (C), 20 min (D), and 30 min (E) before stimulation with chemoattractants.

FIG. 5.

Effects of shikonin on chemokine receptor expression on human monocytes/macrophages. Human monocytes in medium supplemented with 20 ng of M-CSF per ml were incubated for 24 h. Shikonin (1 μM) was added to the medium, and the monocytes were incubated for another 24 h. The cells were detached, washed with PBS, and then stained with control MAb or purified anti-human CCR1, CCR2, or FPR MAb, followed by staining with goat anti-mouse IgG (Fc specific)-FITC antibody, or were stained with FITC-conjugated isotype mouse IgG, FITC-conjugated anti-human CCR5 MAb, or anti-human CXCR4 MAb. Stained cells were analyzed on and EPICS profiler (Coulter Corp.). Data are expressed as the percentage of positive cells. The black histograms show the results for the isotype-matched controls.

FIG. 6.

Dose-dependent inhibition of CCR5 surface expression on human monocytes/macrophages by shikonin. Human monocytes were incubated for 46 h in medium supplemented with 20 ng of M-CSF per ml. Shikonin was added to the medium at the desired concentration, and incubation was continued for another 2 h at 37 or 4°C. The cells were detached and washed with PBS and then stained with FITC-conjugated isotype-matched mouse IgG or FITC-conjugated anti-human CCR5 MAb. Stained cells were analyzed on an EPICS profiler (Coulter Corp.). (A) Medium control; (B to G) treatment with increasing concentrations of shikonin (0.1, 0.5, 1, 5, 10, and 50 μM, respectively) at 37°C; (H) treatment with 10 μM shikonin at 4°C. Grey histograms, results for the isotype-matched IgG control; open histograms, results with CCR5 antibody staining. MFI, mean fluorescence intensity.

FIG. 7.

Down-regulation of CCR5 mRNA expression by shikonin. (A) Human monocytes in medium supplemented with 20 ng of M-CSF per ml were incubated for 24 h. Shikonin (1 μM) was added to the medium, and the monocytes were incubated for another 24 h. The cells were detached and washed with PBS. Total cellular RNA was subjected to RT-PCR analysis, as described in Materials and Methods. (B) The relative mRNA levels represent the ratio of the signal intensity from densitometric readings after normalization with β-actin.

FIG. 8.

Suppression of HIV-1 replication in human PBMCs or human macrophages. (A to E) For the RT activity assay, human PBMCs were coincubated with HIV-1 RoJo, Wejo, Teki, SLKA, or MDR-769 for 7 days. HIV-1 RT activity was monitored in vitro by standard RT assays of cell culture supernatants. (F) For p24, macrophages (10⁶ cells/well) cultured for 7 days in 24-well plates were incubated with or without shikonin for 2 h before infection with HIV-1_Ba-L. The cells were then washed to remove unbound virus. Fresh medium containing shikonin was added. The culture medium was replaced twice weekly. Seven days after infection, HIV-1 p24 core antigen levels were measured by enzyme-linked immunosorbent assay. The cell viability was evaluated simultaneously by MTS assays. The data show the percent activity compared to that for the control.