Anti-inflammatory effects of sophocarpine in LPS-induced RAW 264.7 cells via NF-κB and MAPKs signaling pathways
Introduction
Lipopolysaccharide (LPS)-induced gene products are pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), adhesion enzymes such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) (Ahn and Aggrawal, 2005). COX-2 is induced by several stimuli, and is responsible for the production of large amounts of pro-inflammatory at the inflammatory site (Lee et al., 1992). High-output NO by iNOS can provoke deleterious consequences such as septic shock and inflammatory diseases (Titheradge, 1999, Zamora et al., 2000). Based on these observations, it has been hypothesized that inhibiting high-output NO, IL-6 and TNF-α production in macrophages, by blocking iNOS and COX-2 expressions or their activities, can serve as the basis for the potential development of anti-inflammatory drugs. Furthermore, nuclear factor-κB (NF-κB)-dependent gene expression plays an important role in inflammatory responses and increases the expression of genes encoding cytokines and receptors involved in pro-inflammatory enzyme pathways such as iNOS and COX-2 (Park et al., 2007a, Park et al., 2007b). Recently, many studies have demonstrated the role of phytochemicals in anti-inflammatory activity is through downregulation of the NF-κB pathway (Kundu and Surh, 2005).
The mitogen-activated protein kinases (MAPKs) family, including extracellular signal-regulated kinase 1/2 (Erk1/2), p38 MAPK and c-Jun NH2-terminal kinase (JNK), has been shown to play a significant role in the mediation of signals triggered by cytokines, growth factors, and environmental stress and is known to be involved in various cellular functions (Reibman et al., 2000). Increased activity of MAPKs, in particular p38 MAPK, and their involvement in the regulation of the synthesis of inflammation mediators at the level of transcription and translation, make them potential targets for anti-inflammatory therapeutics (Kaminska, 2005). Several studies have shown that the activation of MAPKs are involved in LPS-induced iNOS expression and NF-κB activation (Carter et al., 1999, Nakano et al., 1998).
Sophora alopecuroides L. (the clinical usefulness of compound Kudouzi injection) has been used mainly for the treatment of fever, inflammation, edema and pain (Xiao, 1993). The active ingredients of compound Kudouzi injection include matrine, sophoridine and sophocarpine (Fig. 1). Our previous reports have found that sophocarpine exerts anti-inflammatory activity on carrageenan-induced rat paw edema, xylene-induced mouse ear edema and acetic acid-induced vascular permeability in mice (Gao et al., 2009). Zhang et al. (2008) have found that sophocarpine inhibits the production of TNF-α and IL-6 in murine macrophages and prevents cachexia-related symptoms induced by colon 26 adenocarcinoma in mice. However, up to now, few studies on its mechanisms have been carried out. In the present study, anti-inflammatory mechanisms of sophocarpine were investigated in LPS-induced RAW 264.7 macrophage cells.
Section snippets
Plant materials
Sophora alopecuroides L. was purchased from Ning-Xia province, China and was identified by Prof. B. Xu, School of Pharmacy, Yantai University. A voucher specimen was deposited in the Herbarium of the School of Pharmacy, Yantai University. Sophocarpine was isolated as described previously (Gao et al., 2009).
Cell culture
RAW 264.7 mouse monocyte-macrophage (ATCC TIB-71) was maintained in RPMI 1640 medium supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml) and 10% heat inactivated fetal bovine
Sophocarpine did not exhibit cytotoxicity against RAW 264.7 cells
RAW 264.7 cells were treated with various concentrations of sophocarpine for 24 h, and the viability and cytotoxicity were determined by metabolic reduction of a tetrazolium salt to a formazan dye (MTT assay) and by release of LDH from membrane damaged (LDH release assay) cells, respectively. As shown in Fig. 2, sophocarpine did not exhibit cytotoxicity to RAW 264.7 cells at the dose of 25, 50 and 100 μg/ml, and these doses were used for treatment of sophocarpine in the following experiments.
Discussion
MTT assay is a sensitive and accurate test, with color development strongly correlating with cell numbers. It is widely used as a measure of cytotoxicity (Keiser et al., 2000). In the present study, RAW 264.7 cells were treated with various concentrations of sophocarpine for 24 h, and the viability was tested by MTT assay. The results indicated that sophocarpine did not exhibit cytotoxicity in the range of 10–100 μg/ml against RAW 264.7 cells. To further investigate the cytotoxicity of
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgments
This work was supported by the Natural Science Foundation of Shandong Province (No. ZR2010HQ024) and the National Natural Science Foundation of China (No. 81001661). The authors would like to thank Mr. C.Yao for valuable technical assistance.
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