Inflammatory cytokines are important factors in chronic inflammation, allergy, asthma, atherogenesis, and autoimmune diseases. Human mast cells play an integral role in the inflammatory response by accumulating at sites of inflammation and mediating the production of inflammatory cytokines . In spite of advances in the pharmacological management of above mentioned diseases and symptoms, to discover effective, alternative anti-inflammatory reagents is still in need. Several Chinese herbal medicines have anti-bacterial and viral properties and been used for treatment of chronic inflammation. Previously, we have screened several Chinese herbal medicines and found that the compound Baicalein (BAI, Fig 1) isolated from Huangqin (Scutellaria baicalensis Georgi) has a great inhibitory effect on the production of IL-6 from IL-1β-activated HMC-1 in a dose dependent fashion . The purpose of this study is to further investigate inhibitory effects and mechanisms of BAI on inflammatory cytokine expression from IL-1β- and TNF-α-activated human mast cells. Ultimately it is hoped that BAI will be a possible candidate for future development of novel anti-inflammatory therapies.
In this study, we examined effects of BAI on the production of important inflammatory cytokines, IL-6, IL-8, and MCP-1, from IL-1β- or TNF-α-activated HMC-1. We observed that BAI (1.8 to 30 μM) significantly inhibited production of IL-6, IL-8, and MCP-1 in a dose-dependent manner in IL-1β-activated HMC-1 (Fig. 2). Since BAI 30 μM was the most effective concentration, we only used this dose to treat TNF-α-activated HMC-1 cells and found it also significantly inhibited production of IL-6, IL-8, and MCP-1 in TNF-α-activated HMC-1 (Fig. 3). The results show that BAI significantly inhibit the production of inflammatory cytokines from human mast cells. The cell viabilities of the drug groups in this study were ranging from 93 to 95%, while that of medium control cultures was 93% (Data not shown). Thus, this inhibitory effect appears not due to the toxic effect of BAI on HMC-1 cells. Moreover, the gene expression, analyzed by RT-PCR, of these inflammatory cytokines was mildly decreased in IL-1β-activated HMC-1 (Fig. 4A) and markedly decreased in TNF-α-activated HMC-1 (Fig. 4B) when BAI was presented. These suggest that inhibitory effect of BAI on cytokine productions is through the decrease of cytokine mRNA transcription.
BAI is a flavonoid extracted from the root of Scutellaria baicalensis Georgi, which has been used as anti-inflammatory medicine in China for years. In recent studies, an important flavonoid, quercetin, has been reported to exert a strong inhibitory effect on the production of IL-6, MCP-1, and histidine decarboxylase (HDC) mRNA transcription from mast cells [36–38]. Our results confirmed that BAI, as a flavonoid, could also strongly inhibit production of inflammatory cytokines of IL-6, IL-8, and MCP-1 from activated mast cells through the decrease of mRNA transcription. On the other hand, in our study, the cytokine gene expression was mildly decreased in IL-1β-activated HMC-1 (Fig. 4A), but markedly decreased in TNF-α-activated HMC-1 (Fig. 4B) by addition of BAI. It appears that BAI had a differential effect on the cytokine gene expression in mast cells activated by different stimulants. It has been shown that acute phase response cytokines, IL-1β and TNF-α, activate human mast cells by IL-1 receptor (IL-1R) and TNF-α receptor (TNFR) signaling pathways, respectively, involving MyD88 dependent and/or independent protein kinases [39, 40]. This differential effect of BAI on activated mast cells warrants further studies.
The expression of various inflammatory cytokines is regulated by transcription factors. The activation of the NF-κB transcription plays an important role in inflammation through its ability to induce the transcription of proinflammatory genes . Previously, glucocorticoids that have frequently been used for the treatment of inflammatory diseases, allergy, and autoimmune diseases were suggested to suppress NF-κB activation. Glucocorticoids are thought to induce the transcription of IκBα, resulting in an enlarged IκBα pool, and therefore reduced active NF-κB in the nucleus . Additionally, 12-lipoxygenase (12-LOX) has been implicated as a mediator of inflammation, atherosclerosis, and cancer [43–45]. Several in vitro studies have suggested 12/15-LOX products to be co-activators of peroxisomal proliferator activating-receptors (PPAR), regulators of cytokine generation, and modulators of gene expression related to inflammation resolution. The dampening effect of PPAR on inflammation is via their inhibitory activity on expression of NF-κB [46–48]. As BAI is known as a 12-LOX inhibitor, we speculated the mechanism by which BAI inhibited inflammatory cytokines was through the NF-κB/IκBα pathway. Therefore, we analyzed NF-κB activation and examined the cytoplasmic levels of IκBα in HMC-1 after treatment with IL-1β or TNF-α in the presence or absence of BAI. Our data showed BAI decreased NF-κB binding activity (Fig. 5) and increased IκBα proteins in cytoplasm in IL-1β- and TNF-α-activated mast cells (Fig. 6). The results suggest BAI inhibits the NF-κB activation via inhibition of IκBα phosphorylation and degradation.