Honokiol induces apoptosis-like death in Cryptocaryon irritans Tomont

Abstract

Background: 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.

Methods: 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 ([Ca²⁺]_i) 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.

Results: 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), [Ca²⁺]_i 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 [Ca²⁺]_i 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.

Conclusions: This article shows that honokiol can induce C. irritans tomont apoptosis-like death. These results suggest that honokiol may disrupt [Ca²⁺]_i 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.

Keywords: Apoptosis-like death; Cryptocaryon irritans; Endoplasmic reticulum stress; Honokiol.