| Cantharidin (CTD) is an anti-tumor compound extracted from blister beetles and has been used for the treatment of primary liver cancer, breast cancer, gastric cancer and other cancers. But the side effects have become the biggest obstacle for CTD to be further applied in clinical trials. Norcantharidin (NCTD), a demethylated analog of CTD, has much little toxicity to the patients. Now, NCTD has been widely used as an anti-tumor drug in China to inhibit proliferation and metastasis of various kinds of cancers, such as hepatoma, lung cancer, colorectal cancer, breast cancer and oral cancer, with a strong activity of anti-tumor and enhancing white blood cell function. Till now, most of studies in NCTD are focusing on its anti-tumor activity. However, there is little definitive evidence to prove its effects of immune response.Many studies have shown that patients’ immune system always be restricted by most chemotherapy drugs in clinical application of cancer treatment, which is likely to cause infection and other complications. Thus, it is very necessary to enhance the immune response in patients in order to achieve better therapeutic effect.In this study, we chose some compounds that have been proved posses anti-tumor activity to test their immune functions in macrophages. First, to obtain less toxic compounds, we screened compounds by MTS assay to test toxicity in macrophages. Then we detected the effects of compounds on expressions of some important cytokines in innate immunity, including IL-6, TNF-α, MCP-1, iNOS, by RT-PCR. The results showed that NCTD has less cytotoxic and more immune responses than other drugs.To demonstrate whether NCTD promotes cytokine gene expression, LPS-induced cytokines mRNA expression in RAW264.7were tested by realtime PCR. NCTD increased TNF-α, IL-6and IFN-β mRNA expression in a dose-dependent manner, but had no effect on G-CSF, MIF, MCP-1, IFN-γ mRNA level. We also investigated NCTD-promoted cytokines production by means of enzyme-linked immunosorbent assay. ELISA of RAW264.7and bone marrow-derived macrophages show that NCTD was critical for TNF-α and IL-6induction by LPS. These data demonstrated that NCTD promoted some LPS-induced cytokine expression in both primary and immortalized macrophages. Additionally, NCTD facilitates activation of macrophages with respect to the cytokine expression, NO production, MMP-9induction and phagocytosis.To investigate the function of NCTD in regulating innate immunity, we set up acute peritonitis model and GFP-labeled bacteria infection model. Therefore, NCTD probably enhanced the host defense in against pathogens and promoted the induction of cytokines via effecting macrophages.As is known, TLR downstream signaling pathways mainly involve MAPK signaling and NF-κB signaling. And LPS is the agonist of toll-like receptor member4(TLR4), which suggests that the effects of NCTD may be through the TLR-related signaling pathway. Therefore, we tested key components in these two signaling pathways, such as p65, p38, JNK, ERK1/2, AKT, etc. We found that NCTD facilitated LPS-induced phosphorylation of AKT and p65, as well as the translocation of p65. Using EMSA, we also found NCTD stimulates LPS-mediated NF-κB activation in a dose-dependent manner in RAW264.7. Taken together, NCTD regulates immune response of macrophages and protects host from invading microbes.Our results demonstrate that:1. NCTD is a positive regulator of innate immune in macrophage mediated immune responses such as phagocytes and the production of MMPs, cytokines, NO.2. NCTD probably enhanced the host defense in against infectious diseases.3. NCTD up-regulated LPS-induced phosphorylation of AKT and p65, facilitated the translocation of p65and therefore enhances NF-κB transcriptional activity. |
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