Hainan Provincial Key Laboratory for Tropical Hydrobiology and Biotechnology, College of Marine Science, Hainan University, Haikou, 570228, People's Republic of China.
School of Life Sciences, Hainan University, Haikou, 570228, People's Republic of China.
Parasit Vectors. 2023 Aug 16;16(1):287. doi: 10.1186/s13071-023-05910-1.
Cryptocaryon irritans, a common parasite in tropical and subtropical marine teleost fish, has caused serious harm to the marine aquaculture industry. Honokiol was proven to induce C. irritans tomont cytoplasm shrinkage and death in our previous study, but the mechanism by which it works remains unknown.
In this study, the changes of apoptotic morphology and apoptotic ratio were detected by microscopic observation and AnnexinV-FITC/PI staining. The effects of honokiol on intracellular calcium ([Ca]) concentration, mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS), quantity of DNA fragmentations (QDF) and caspase activities were detected by Fluo-3 staining, JC-1 staining, DCFH-DA staining, Tunel method and caspase activity assay kit. The effects of honokiol on mRNA expression levels of 61 apoptosis-related genes in tomonts of C. irritans were detected by real-time PCR.
The results of the study on the effects of honokiol concentration on C. irritans tomont apoptosis-like death showed that the highest levels of prophase apoptosis-like death rate (PADR), [Ca] concentration, ROS, the activities of caspase-3/9 and the lowest necrosis ratio (NER) were obtained at a concentration of 1 μg/ml, which was considered the most suitable for inducing C. irritans tomont apoptosis-like death. When C. irritans tomonts were treated with 1 μg/ml honokiol, the [Ca] concentration began to increase significantly at 1 h. Following this, the ROS, QDF and activities of caspase-3/9 began to increase significantly, and the ΔΨm began to decrease significantly at 2 h; the highest PADR was obtained at 4 h. The mRNA expression of 14 genes was significantly upregulated during honokiol treatment. Of these genes, itpr2, capn1, mc, actg1, actb, parp2, traf2 and fos were enriched in the pathway related to apoptosis induced by endoplasmic reticulum (ER) stress.
This article shows that honokiol can induce C. irritans tomont apoptosis-like death. These results suggest that honokiol may disrupt [Ca] homeostasis in ER and then induce C. irritans tomont apoptosis-like death by caspase cascade or mitochondrial pathway, which might represent a novel therapeutic intervention for C. irritans infection.
刺激隐核虫是热带和亚热带海洋硬骨鱼类的一种常见寄生虫，已对海水养殖业造成严重危害。我们之前的研究表明，厚朴酚能诱导刺激隐核虫的脱囊幼体细胞质收缩和死亡，但作用机制尚不清楚。
在本研究中，通过显微镜观察和 AnnexinV-FITC/PI 染色检测凋亡形态和凋亡比例的变化。通过 Fluo-3 染色、JC-1 染色、DCFH-DA 染色、Tunel 法和 caspase 活性测定试剂盒检测厚朴酚对细胞内钙离子([Ca])浓度、线粒体膜电位（ΔΨm）、活性氧（ROS）、DNA 片段化数量（QDF）和 caspase 活性的影响。通过实时 PCR 检测厚朴酚对刺激隐核虫脱囊幼体中 61 个凋亡相关基因的 mRNA 表达水平的影响。
研究厚朴酚浓度对刺激隐核虫脱囊幼体凋亡样死亡的影响结果表明，在 1μg/ml 浓度下获得最高的前期凋亡样死亡率（PADR）、[Ca]浓度、ROS、caspase-3/9 活性和最低的坏死率（NER），这被认为是最适合诱导刺激隐核虫脱囊幼体凋亡样死亡的浓度。当刺激隐核虫脱囊幼体用 1μg/ml 厚朴酚处理时，[Ca]浓度在 1 小时时开始显著增加。随后，ROS、QDF 和 caspase-3/9 的活性开始显著增加，ΔΨm 在 2 小时时开始显著降低；在 4 小时时获得最高的 PADR。厚朴酚处理时，14 个基因的 mRNA 表达显著上调。其中，itpr2、capn1、mc、actg1、actb、parp2、traf2 和 fos 在与内质网（ER）应激诱导的细胞凋亡相关的途径中被富集。
本文表明厚朴酚能诱导刺激隐核虫脱囊幼体凋亡样死亡。这些结果表明，厚朴酚可能通过钙蛋白酶级联或线粒体途径破坏 ER 中的[Ca]稳态，然后诱导刺激隐核虫脱囊幼体凋亡样死亡，这可能代表了一种治疗刺激隐核虫感染的新方法。
