Özlem Çalışkan Serçin, Özen Hüsne, Kaya İlayda, Ilıkçı Sağkan Rahşan, Ertabaklar Hatice
Uşak University Faculty of Medicine, Department of Biophysics, Uşak, Türkiye.
Student at Uşak University Faculty of Medicine, Uşak, Türkiye.
Mikrobiyol Bul. 2022 Oct;56(4):706-721. doi: 10.5578/mb.20229608.
Leishmaniasis is an infectious disease that is transmitted by Phlebotomus, 400 thousand new cases appearing every year, and approximately 350 million people are at risk, and accepted by the World Health Organization as one of the six important tropical diseases. Cutaneous leishmaniasis is a disease that occurs on exposed areas of the body and is characterized by long-term non-healing skin lesions. Although the treatment methods applied today vary according to the clinical picture of the patient, the immune system of the person and the causative agent Leishmania species, there is still no standard treatment scheme that has few side effects and can be used in the treatment of leishmaniasis. Therefore, alternative treatment methods with less side effects are being tried. Sonodynamic therapy (SDT) has also emerged as an active antimicrobial research area in recent years. SDT, a new modality for antibacterial therapy, aims to increase antibacterial effects with the simultaneous combination of low-intensity ultrasound and sonosensitizer. There is no information in the literature about the effect of SDT on parasites. In this study, it was aimed to demontrate the anti-leishmanial effect and possible mechanisms of curcumin mediated SDT on L.tropica promastigotes in vitro. Parasites were incubated with 0.25, 1.0, 4.0 and 15.6 micromolar (μM) of curcumin for one hour and subjected to 1 MHz frequency, 50% duty cycle and 3 W/cm2 intensity ultrasound irradiation. XTT assay was used to evaluate the viability of the cells and morphological changes were analyzed by Giemsa staining. Flow cytometry was used to quantify the fluorescence emitted by intracellular reactive oxygen species (ROS) signal, JC-1, cell cycle, Annexin V/PI staining reagents. With the combination of curcumin (15.6 μM) and ultrasound (3 W/cm2 intensity, seven minutes), L.tropica promastigote viability was found to be significantly decreased compared to the control group. Giemsa staining results showed that 15.6 μM curcumin mediated SDT induced several morphological alterations in L.tropica promastigotes typical for apoptosis. Late apoptosis was observed in 15.6 μM curcumin combined SDT treated parasites according to Annexin/PI staining. Besides, curcumin mediated SDT caused mitochondrial membrane potential (∆ᴪm) loss. Cell cycle analysis data indicated that curcumin based SDT caused an subG1 arrest in the cell cycle of L.tropica promastigotes. The generation of intracellular ROS detected by flow cytometry was increased in L.tropica promastigotes treated with curcumin mediated SDT. This study provided new data elucidating the molecular mechanism underlying the anti-leishmanial effect of curcumin mediated SDT. Curcumin mediated SDT has the potential to inactivate L.tropica promastigotes. However, further testing with amastigote or animal models is needed.
利什曼病是一种由白蛉传播的传染病,每年出现40万新病例,约3.5亿人面临风险,被世界卫生组织列为六种重要的热带疾病之一。皮肤利什曼病是一种发生在身体暴露部位的疾病,其特征是皮肤损伤长期不愈合。尽管目前应用的治疗方法根据患者的临床症状、人的免疫系统和致病原利什曼原虫种类而有所不同,但仍然没有一种副作用少且可用于治疗利什曼病的标准治疗方案。因此,正在尝试副作用较小的替代治疗方法。近年来,声动力疗法(SDT)也已成为一个活跃的抗菌研究领域。SDT是一种抗菌治疗的新模式,旨在通过低强度超声和超声敏化剂的同时联合来增强抗菌效果。文献中没有关于SDT对寄生虫作用的信息。在本研究中,旨在证明姜黄素介导的SDT对热带利什曼原虫前鞭毛体的体外抗利什曼作用及可能机制。将寄生虫与0.25、1.0、4.0和15.6微摩尔(μM)的姜黄素孵育1小时,然后进行1兆赫频率、50%占空比和3瓦/平方厘米强度的超声照射。采用XTT法评估细胞活力,通过吉姆萨染色分析形态变化。流式细胞术用于量化细胞内活性氧(ROS)信号、JC-1、细胞周期、膜联蛋白V/碘化丙啶(PI)染色试剂发出的荧光。与对照组相比,姜黄素(15.6 μM)与超声(3瓦/平方厘米强度,7分钟)联合使用时,热带利什曼原虫前鞭毛体的活力显著降低。吉姆萨染色结果显示,15.6 μM姜黄素介导的SDT诱导热带利什曼原虫前鞭毛体出现几种典型的凋亡形态改变。根据膜联蛋白/PI染色,在15.6 μM姜黄素联合SDT处理的寄生虫中观察到晚期凋亡。此外,姜黄素介导的SDT导致线粒体膜电位(∆ᴪm)丧失。细胞周期分析数据表明,基于姜黄素的SDT导致热带利什曼原虫前鞭毛体细胞周期出现亚G1期停滞。流式细胞术检测到,用姜黄素介导的SDT处理的热带利什曼原虫前鞭毛体细胞内ROS的生成增加。本研究提供了新的数据,阐明了姜黄素介导的SDT抗利什曼作用的分子机制。姜黄素介导的SDT有可能使热带利什曼原虫前鞭毛体失活。然而,需要用无鞭毛体或动物模型进行进一步测试。
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