Elsevier

Toxicology in Vitro

Volume 26, Issue 1, February 2012, Pages 1-6
Toxicology in Vitro

Anti-inflammatory effects of sophocarpine in LPS-induced RAW 264.7 cells via NF-κB and MAPKs signaling pathways

https://doi.org/10.1016/j.tiv.2011.09.019Get rights and content

Abstract

Sophocarpine, a tetracyclic quinolizidine alkaloid, is one of the most abundant active ingredients in Sophora alopecuroides L. Our previous studies have showed that sophocarpine exerts anti-inflammatory activity in animal models. In the present study, anti-inflammatory mechanisms of sophocarpine were investigated in lipopolysaccharide (LPS)-induced responses in RAW 264.7 cells. Furthermore, the cytotoxicity of sophocarpine was tested. The results indicated that sophocarpine could increase the LDH level and inhibit cell viability up to 800 μg/ml, and which was far higher than that of the plasma concentration of sophocarpine in clinical effective dosage. The results also demonstrated that sophocarpine (50 and 100 μg/ml) suppressed LPS-stimulated NO production and pro-inflammatory cytokines secretion, including tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). These were associated with the decrease of the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Furthermore, sophocarpine inhibited LPS-mediated nuclear factor-κB (NF-κB) activation via the prevention of inhibitor κB (IκB) phosphorylation. Sophocarpine had no effect on the LPS-induced phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2), whereas it attenuated the phosphorylation of p38 mitogen-activated protein (MAP) kinase and c-Jun NH2-terminal kinase (JNK). Our data suggested that sophocarpine exerted anti-inflammatory activity in vitro, and it might attribute to the inhibition of iNOS and COX-2 expressions via down-regulation of the JNK and p38 MAP kinase signal pathways and inhibition of NF-κB activation.

Highlights

► Sophocarpine inhibits the production of TNF-α and IL-6 in RAW 264.7 cells. ► Sophocarpine inhibited the production of iNOS and COX-2 expression in RAW 264.7 cells. ► Anti-inflammatory mechanisms of sophocarpine via NF-κB and MAPKs inactivation.

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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