Laboratory of Cellular Immunology, Department of Microbiology, Hellenic Pasteur Institute, Athens, Greece.
Division of Pharmacognosy and Natural Product Chemistry, Department of Pharmacy, National and Kapodistrian University of Athens, Athens, Greece.
PLoS Negl Trop Dis. 2021 Jan 11;15(1):e0008968. doi: 10.1371/journal.pntd.0008968. eCollection 2021 Jan.
Leishmaniasis is a serious multifactorial parasitic disease with limited treatment options. Current chemotherapy is mainly consisted of drugs with serious drawbacks such as toxicity, variable efficacy and resistance. Alternative bioactive phytocompounds may provide a promising source for discovering new anti-leishmanial drugs. Extra Virgin Olive Oil (EVOO), a key-product in the Mediterranean diet, is rich in phenols which are associated with anti-inflammatory, anti-cancer and anti-microbial effects. In this study, we investigate the anti-leishmanial effect of Total Phenolic Fraction (TPF) derived from EVOO in both in vitro and in vivo systems by investigating the contributing mechanism of action.
METHODOLOGY/PRINCIPAL FINDINGS: We tested the ability of TPF to cause apoptotic-like programmed cell death in L. infantum and L. major exponential-phase promastigotes by evaluating several apoptotic indices, such as reduction of proliferation rate, sub-G0/G1 phase cell cycle arrest, phosphatidylserine externalization, mitochondrial transmembrane potential disruption and increased ROS production, by using flow cytometry and microscopy techniques. Moreover, we assessed the therapeutic effect of TPF in L. major-infected BALB/c mice by determining skin lesions, parasite burden in popliteal lymph nodes, Leishmania-specific antibodies and biomarkers of tissue site cellular immune response, five weeks post-treatment termination. Our results show that TPF triggers cell-cycle arrest and apoptotic-like changes in Leishmania spp. promastigotes. Moreover, TPF treatment induces significant reduction of parasite burden in draining lymph nodes together with an antibody profile indicative of the polarization of Th1/Th2 immune balance towards the protective Th1-type response, characterized by the presence of IFN-γ-producing CD4+ T-cells and increased Tbx21/GATA-3 gene expression ratio in splenocytes.
CONCLUSIONS/SIGNIFICANCE: TPF exhibits chemotherapeutic anti-leishmanial activity by inducing programmed cell death on cell-free promastigotes and immunomodulatory properties that induce in vivo T cell-mediated responses towards the protective Th1 response in experimental cutaneous leishmaniasis. These findings enable deeper understanding of TPF's dual mode of action that encourages further studies.
利什曼病是一种严重的多因素寄生虫病，治疗选择有限。目前的化疗主要由毒性大、疗效不一和耐药性等严重缺陷的药物组成。替代生物活性植物化合物可能为发现新的抗利什曼病药物提供有希望的来源。特级初榨橄榄油（EVOO）是地中海饮食的主要产品，富含酚类物质，具有抗炎、抗癌和抗菌作用。在这项研究中，我们通过研究作用机制，在体外和体内系统中研究了源自 EVOO 的总酚类（TPF）的抗利什曼作用。
方法/主要发现：我们通过使用流式细胞术和显微镜技术评估了几种凋亡指数，如增殖率降低、亚 G0/G1 期细胞周期停滞、磷脂酰丝氨酸外翻、线粒体跨膜电位破坏和增加的 ROS 产生，来测试 TPF 引起 L. infantum 和 L. major 指数期前鞭毛体凋亡样程序性细胞死亡的能力。此外，我们通过确定治疗后 5 周时皮肤病变、后肢淋巴结中的寄生虫负荷、利什曼原虫特异性抗体和组织部位细胞免疫反应的生物标志物，评估了 TPF 在 L. major 感染的 BALB/c 小鼠中的治疗效果。我们的结果表明，TPF 可引发利什曼原虫属前鞭毛体的细胞周期停滞和凋亡样变化。此外，TPF 治疗可显著降低引流淋巴结中的寄生虫负荷，并诱导抗体谱发生变化，表明 Th1/Th2 免疫平衡向保护性 Th1 型反应倾斜，其特征是存在 IFN-γ 产生的 CD4+ T 细胞和脾细胞中 Tbx21/GATA-3 基因表达比值增加。
结论/意义：TPF 通过诱导无细胞前鞭毛体程序性细胞死亡和免疫调节特性发挥抗利什曼病的化疗活性，从而诱导体内 T 细胞介导的反应，在实验性皮肤利什曼病中向保护性 Th1 反应倾斜。这些发现使我们更深入地了解 TPF 的双重作用模式，鼓励进一步研究。
