Shikonin extracted from medicinal Chinese herbs exerts anti-inflammatory effect via proteasome inhibition

Abstract

Shikonin, extracted from medicinal Chinese herb (Lithospermum erythrorhizo), was reported to exert anti-inflammatory and anti-cancer effects both in vitro and in vivo. We have found that proteasome was a molecular target of shikonin in tumor cells, but whether shikonin targets macrophage proteasome needs to be investigated. In the current study, we report that shikonin inhibited inflammation in mouse models as efficiently as dexamethasone. Shikonin at 4 μM reduced the Lipopolysaccharides (LPS)-mediated TNFα release in rat primary macrophage cultures, and blocked the translocation of p65-NF-κB from the cytoplasm to the nucleus, associated with decreased proteasomal activity. Consistently, shikonin accumulated IκB-α, an inhibitor of NF-κB, and ubiquitinated proteins in rat primary macrophage cultures, demonstrating that the proteasome is a target of shikonin under inflammatory conditions. Shikonin also induced macrophage cell apoptosis and cell death. These results demonstrate for the first time that proteasome inhibition by shikonin contributes to its anti-inflammatory effect. The novel finding about macrophage proteasome as a target of shikonin suggests that this medicinal compound has great potential to be developed into an anti-inflammatory agent.

Fig. 1. Shikonin inhibits LPS- and xylene-mediated inflammation in mouse models in vivo

A. Kunming mouse model of auricle swelling: Mice were pretreated with various doses of shikonin (i.p injection of 0.5, 1, 4 mg/kg, respectively) or dexamethasone (2.5 mg/kg) for 30 min, then 50 μl of xylene was scraped evenly to the same site of one ear, and 30 min later the weight difference of both ears were compared in different groups. (Means+S.D., n=10). *P<0.05, compared with DM control group. B. NIH mouse model of LPS-mediated acute inflammation. NIH mice were pretreated with shikonin as indicated in A for 90 min. After i.p injection of LPS (35 mg/kg) for 30 min, i.v injection of Evans blue and i.p injection of acetic acid was performed. 30 min later peritoneal fluid was collected for absorbance assay of Evans blue. OD_{590 nm} was expressed as means + S.D. (n =10). *P < 0.05, compared with DM control group.

Fig. 2

Shikonin inhibits LPS-induced TNFα production in rat primary macrophage cultures. Rat primary macrophage cultures were pretreated with either solvent DMSO (DM) or indicated concentrations of shikonin for 1.5 h and then exposed to LPS (1 μg/ml). At 2 and 4 h after LPS treatment, TNFα level in cultured supernatants was determined by ELISA kit. Values represent means ± S.D. (n=3). *P<0.05, compared with DM group; ^#P<0.05, compared with LPS group.

Fig. 3

Shikonin inhibits NF-kB translocation from the cytoplasm to the nucleus in rat primary macrophage cultures. Rat primary macrophage cultures were plated on 8-well chamber slides and preincubated with vehicle (DM) or 1, 4 μmol/L of shikonin for 1.5 h, followed by stimulation with LPS (1 μg/ml) for 4 h. The cells were immunostained by NF-κBp65 antibody and the nuclear was stained with Hochest33342. Typical fluorescent images by Confocal microscopy were shown. Red signal stands for NF-κBp65 positive and Blue signal means nuclear. Shi1 and Shi4 indicates LPS + shikonin (1 μM) or LPS + shikonin (4 μM), respectively.

Fig. 4. Shikonin accumulates IκB-α and ubiquitinated proteins in rat primary macrophage cultures and specifically inhibits the proteasomal chymotrypsin-like activity

A. Cell lysate (10 μg protein) was incubated with indicated doses of shikonin in a Tris–HCL reaction system for 90 min, followed by measurement of peptidase assay with specific fluorescent proteasome substrates. Each column represents Means + S.D. of three repeats. PSI means MG132 (10 μM). Tryp-like means Trypsin-like, Cas-like means Caspase-like and CT-like means Chymotrypsin-like activity. B. Rat macrophage cells were pretreated with 1, 4 μmol/L of shikonin for 1.5 h and subsequently treated with LPS (1 μg/ml) for 6 h. Protein levels of IκB-α and Ub-prs were determined by Western blotting. Typical images were shown. GAPDH was used as loading control. C. Rat macrophage cells were treated with shikonin (2 μmol/L), LPS (1 μg/ml) alone or combination of the two agents for 2 h and 4 h, ubiquitinated-proteins. and IκB-α were detected by Western blotting. Typical images were shown. GAPDH was used as a loading control.

Fig. 5. Shikonin induced cell death in rat primary macrophage cultures

A. As in Fig. 4C, the supernatant was collected and LDH release was detected. The average is from the results of three repeats. B. As in Fig. 4B, macrophages were collected and labelled with Annexin V and Propidium idodide (PI) for cell death assay by flow cytometry. Typical flow images were shown. C. As described in Fig. 4B, the supernatants were collected for LDH assay. Each column represents Means + S.D. of three repeats.