Norcantharidin alleviates cyclophosphamide-induced immunosuppression via circBCL2L1/miR-30c-3-3p/TRAF6 axis

Cyclophosphamide is a widely used antitumor drug, with induced adverse effects, such as intestinal mucosal injury and immunosuppression. Norcantharidin possesses anticancer activity through enhancement of antitumor immunity. We investigated the role of norcantharidin in cyclophosphamide-induced immunosuppression. Mice were treated with cyclophosphamide, and exposed to norcantharidin. Enzyme-linked-immunosorbent serologic assay was performed to assess the levels of immunoglobulin and cytokines in serum, and the splenic T lymphocytes were analyzed by immunohistochemistry. Incubation with norcantharidin increased the serum levels of immunoglobulin G (IgG), interleukin (IL)-12, interferon-gamma (IFN-γ), and IL-6, and enhanced the percentage of CD4+ and CD8+ T lymphocytes in cyclophosphamide-induced mice. Expression of circBCL2L1 was down-regulated in the spleen of cyclophosphamide-induced mice, while up-regulated by norcantharidin incuba-tion. Norcantharidin attenuated cyclophosphamide-induced up-regulation of miR-30c-3-3p and down-regulation of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) in mice. Over-expression of circBCL2L1 increased serum levels of immunoglobulin and cytokines, and enhanced the percentage of splenic CD4+ and CD8+ T lymphocytes in cyclophosphamide-induced mice. Moreover, over-expression of circBCL2L1 increased TRAF6 in cyclophosphamide-induced mice through down-regulation of miR-30c-3-3p. Knockdown of TRAF6 attenuated norcantharidin-induced increase of serum levels of IgG, IL-12, IFN-γ, and IL-6, and up-regulation of CD4+ and CD8+ T lymphocytes in cyclophosphamide-induced mice. Norcantharidin exhibited protective effect against cyclophosphamide-induced immunosuppression in mice through regulation of circBCL2L1/miR-30c-3-3p/TRAF6 axis.