Dan-Zhi-Yin formula against IL-17A/F-induced chemokines via JNK pathway in mouse embryo fibroblasts NIH 3T3 cells

Dan-Zhi-Yin formula (DZY) is a traditional Chinese medicine formula consisting of 8 herbs, which has been used to treat chronic pelvic inflammation and pelvic pain. The aim of the present study was to observe the mechanism of DZY on IL-17A/F-induced chemokines in the mouse embryonic fibroblasts (NIH 3T3). The viability of cells was determined by MTT assay. We evaluated the expression of the chemokines monocyte chemoattractant protein- 1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) by ELISA and real-time RT-PCR. The phosphorylation of JNK was assessed by Cytometric Bead Array. IL-17RA and IL-17RC were inhibited by small interfering RNA (siRNA). Our study demonstrated that IL-17A/F were able to increase the gene levels and protein secretion of MCP-1 and MIP-2. However, the DZY could disrupt the expression of MCP-1 and MIP-2 stimulated by IL-17A/F. We observed that IL-17A/F could induce JNK activation and that pharmacological inhibitors of JNK (SP600125) blocked the IL-17A/F mediated MCP-1 and MIP-2 release. DZY could inhibited JNK expression at each time point, compared with Il-17A/F stimulated group alone. In addition, we also found that NIH 3T3 cells expressed IL-17 receptors. The expression of MCP-1 and MIP-2 stimulated by IL-17A/F could be reduced by inhibiting IL-17RA or IL-17RC expression via siRNA. In conclusion, IL-17A/F could induce the expression of MCP-1 and MIP-2 through JNK pathway in NIH 3T3 cells. However, DZY can inhibit IL-17A/F-induce MCP-1 and MIP-2 expression which might be regulated by JNK pathway. All these above indicate that DZY plays a role in anti-inflammatory process in NIH 3T3 cells.