Cryptotanshinone inhibits macrophage migration by impeding F-actin polymerization and filopodia extension
Introduction
Dried roots of Salvia miltiorrhiza Bunge (Danshen) have been used in traditional Chinese medicine for the treatment of several pathologies including coronary heart disease, hepatitis, and chronic renal failure (Lu and Foo, 2002). Cryptotanshinone and tanshinone IIA (Fig. 1) are two major tanshinones in this plant. Tanshinone showed a variety of biological activities including anti-inflammation (Kim et al., 2002) and cytotoxicity against human tumor cell lines (Lin and Chang, 2000, Ryu et al., 1997, Yuan et al., 2003). One of the therapeutic objectives in inflammation is to reduce inflammatory cell infiltration. The inappropriate recruitment of leukocytes from blood to the site of infection is an essential process of inflammation and can result in tissue destruction. A variety of products present at the site of inflammation can act as chemotactic agents, including formylmethionyl peptides, platelet activating factor, leukotriene B4, and complement products. Tanshinone IIA was reported to show inhibitory actions on leukocyte chemotactic migration (Gao, 1985, Zhou et al., 1997). Cryptotanshinone was previously observed to possess the most powerful antibacterial activity among tanshinones (Lee et al., 1999) and counteract inflammation through inhibiting cyclooxygenase II activity (Jin et al., 2006) and endothelin-1 expression (Zhou et al., 2006). Nevertheless, there is no report of the effects of cryptotanshinone on inflammatory cell infiltration. In this study, complement 5a (C5a) and macrophage inflammatory protein (MIP-1α) were used to induce cell migration, a crucial determinant of leukocyte trafficking, to evaluate the anti-inflammatory properties of cryptotanshinone.
Redistribution of F-actin fibers and the formation of pseudopodia are important events in cell locomotion (Watts, 1996). Leukocyte migration is mediated by the coordinated activation of the cytoskeleton and associated chemoattractant receptors (Ma et al., 1998, Postma et al., 2004). The major component of the cytoskeleton is actin which, in response to chemotactic stimuli, is rapidly and transiently converted from a monomeric, globular form, G-actin, to a needle-like filamentous form, F-actin (Gupta and Campenot, 1996). Although cell migration cannot be attributed to F-actin polymerization alone, the redistribution of F-actin fibers and the formation of pseudopodia are important events in cell locomotion (Watts, 1996). Thus, we also attempted to characterize the effect of cryptotanshinone on chemoattractant-induced morphological change, F-actin reorganization and pseudopodia formation.
Section snippets
Cell culture conditions
RAW264.7 (American Type Culture Collection, TIB 71, Rockville, MD) macrophage-like cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco BRL, India, USA) supplemented with 10% heat-inactivated fetal calf serum, penicillin, and streptomycin (Biological Industries, Israel) at 37 °C in a humidified atmosphere in the presence of 5% CO2 (Chiou et al., 2003a, Chiou et al., 2003b).
Natural products
Cryptotanshinone and tanshinone IIA were isolated by our laboratory (Sun et al., 2006). The dried roots
Effects of cryptotanshinone and tanshinone IIA on C5a- and MIP-1α-induced chemotactic migration
The stimulation dose of 1 μg/ml of C5a was selected according to our previous findings (Chiou et al., 2003a, Chiou et al., 2004). Non-stimulated control macrophages displayed spontaneous migration with a total cell number of 72 ± 16. The concentration gradient generated by 1 μg/ml of C5a induced an 8-fold increase (cell number: 615 ± 49) in cell migration as compared with non-stimulated control and was represented as 100% (Fig. 2). Cryptotanshinone (1–30 μM) alone did not influence the spontaneous
Discussion
We report here that cryptotanshinone displayed inhibitory effect on chemotactic migration and was more potent than tanshinone IIA, suggesting that cryptotanshinone might be one of the active component existing in S. miltiorrhiza Bunge (Danshen). Indeed, our results indicated that cryptotanshinone not only inhibited C5a-induced migration, but also inhibited cell migration in response to MIP-1α. These observations indicated that cryptotanshinone may act as an inhibitor to block a variety of
Acknowledgment
This work was supported by National Research Institute of Chinese Medicine (NRICM95-DBCMR-01), Taipei, Taiwan, ROC.
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