| Summary: | 博士 === 高雄醫學大學 === 毒理學博士學位學程 === 106 === Neolitsea (N.) genus, belonging to the family Lauraceae, consists of about 100 species distributed in the tropics, especially in Brazil and Southern Eastern Asia. There are 12 Neolitsea species and four of them, including N. acuminatissima, N. daibuensis, N. hiiranensis, and N. parvigemma, are endemic in Taiwan. Neolitsea species have found to be rich in sesquiterpenoids, triterpenoids, alkaloids, and steroids, and Neolitsea species plants which have been reported to have various bioactivities, such as antibacterial, antibacterial, and anti-inflammatory activities. Due to the complexity of compositions within Neolitsea species, it is still unclear how Neolitsea species modulate the functionality of immune cells. The objective of this study aimed to examine the immunomodulatory effects of Taiwanese Neolitsea species on T helper cell immunity.
T helper (Th) cells play a pivotal role in our immune system against environmental stimulations. They participated in a wide range of immune responses via cell-cell interaction with other cells through a complicated cytokine network. Th1 cells, producing interferon-gamma (IFN-γ), interleukin-2 (IL-2) in the regulation of cellular immunity. On the other side, Th2 cells promoted humoral immunity via secreting IL-4, IL-5, and IL-13. Most of the 10 Neolitsea plants were not toxic to splenocytes, except for the root extracts of N. buisanensis. Neolitsea species possess potential immunomodulation in adaptive immunity, the leaf extracts of N. aciculata and N. villosa possessed differential immunomodulatory effects in ConA-stimulated splenocytes in vitro. The leaf extracts of N. aciculata var. variabillima and N. hiiranensis suppressed both Th1 and Th2 cytokines while N. konishii dramatically inhibited interferon-γ (IFN-γ) production through the downregulation of upstream IL-12 production. As N. aciculata var. variabillima and N. konishii leaf extracts significantly attenuated Th1 functionality, we also further evaluated their effects on CD4 cells under CD3/CD28 stimulation. N. aciculata var. variabillima significantly suppressed IFN-γ, interleukin-10 (IL-10), and IL-13; while N. konishii mainly significantly suppressed IFN-γ and IL-10 production.
The secondary metabolites from Taiwanese endemic N. hiiranensis leaf extracts exhibited inhibitory activity against N-formyl-methionyl-leucyl-phenylalanine fMLP-induced superoxide production by human neutrophils, we therefore further investigated its influences on antigen-specific T cells immunity in vivo. N. hiiranensis significantly inhibited IL- 12, IFN-γ, and IL-2 cytokine productions as well as the serum levels of antigen-specific IgM and IgG2a in vivo. Fourteen secondary metabolites with none/less risk of hepatotoxicity and genotoxicity from leaves of N. hiiranensis based on the in silico prediction were selected for the further studies. Two terpenoids, β-caryophyllene oxide (CRYO) and hiiranlactone D, and one diterpenoid (trans-phytol) suppressed IFN-γ in vitro. CRYO displayed the immunomodulatory potential to inhibit several aspects of adaptive immune responses, including suppression of IFN-γ production and the expression of Th1-assocaited genes, including IFN-γ, T-box transcription factor (T-bet), and IL-12Rβ2 in a dose-dependent manner.
Abnormal immunostimulation and the imbalance of Th1/Th2 responses may lead to a variety of immune diseases. The immunosuppressive drugs were developed to treat the over-reactive immune responses or T-cell mediated immune disorders. However, severe adverse effects have been associated with the long-term use of these immunosuppressants. To discover new botanicals with differential immunomodulatory effects on T-cell function may provide more therapeutics for different T-cell-mediated immune disorders, e.g., multiple sclerosis, rheumatoid arthritis, and delayed-type hypersensitivity (DTH). DTH is a class of Th1-mediated immune response triggered by Th1-driven inflammation. Th1-related cytokines released by Th1 cells will activate macrophage to release lysosomal enzymes, toxic reactive oxygen species (ROS), nitric oxide (NO), and pro-inflammatory cytokines. Activated antigen-specific Th1 cells and macrophages produced chemotactic and inflammatory mediators including interferon IFN-γ and tumor necrosis factor-alpha (TNF-α) to induce vascular permeability, fibrin deposition, and local inflammation. Our results demonstrated the N. hiiranensis leaf extracts and CRYO locally attenuated inflammatory reactions associated with DTH, including the footpad swelling and the infiltration of Th1 cells and macrophages.
The Notch signaling cascade is highly conserved in organisms, which is a single-pass transmembrane receptor protein that regulates a broad range of cell fate decisions and plays a key role in a broad range of cellular processes including activation, proliferation, differentiation, and apoptosis. When a Notch receptor engages a Notch ligand expressed on antigen-presenting cells will regulate the down-stream genes, such as T-bet and IFN-γ, and drive naive CD4+ T cells to differentiate into Th1 cells. In our data, CRYO significantly suppressed the expression of T-bet, Notch, and STAT-1. In addition, CRYO decreased the proportion of Notch+/CD4+ cells suggesting the therapeutic potential of CRYO on Th1 immune disorders. The histopathological examination of the kidney and liver tissues obtained from the treated mice did not show significant toxic effects. In addition, CRYO did not significantly induce micronucleus formation in CHO-K1 cells.
Taken together, these data demonstrated that leaf extracts of Taiwanese Neolitsea species and its-derived secondary metabolites, CRYO, could modulate antigen-specific Th1 cell responses by inhibiting IFN-γ production and the expression of Th1-associated genes. In addition, CRYO possesses therapeutic potential for Th1-mediated immune disorders without undesired hepatotoxicity, nephrotoxicity, and genotoxicity.
|
|---|
