Ivermectin induces mitophagy in H9c2 cells via activation of the PINK1/Parkin pathway

Yanmin Jia,Baoxue Han,Rene Wang

doi:10.1590/fst.65621

Abstract

Abstract Ivermectin (IVM), a remarkable broad-spectrum anthelmintic and insecticides, which also contributes to clinical application. However, IVM induction of cytotoxicity through mitophagy has not been consistently proven in vitro. Here we investigate the cytotoxic effects of ivermectin in mammalian nontarget cells. We have used cck-8 assay to evaluate the cell viability. The Fluorescence labeling was detected the occurrence of autophagy and mitophagy. The ATP bioluminescence detection kit was used to detect changes in intracellular ATP levels. In addition, the western blot assay was applied to reflect the expression of autophagy-related and mitophagy- related proteins. Cellular calcium concentration analysis was captured with a flow cytometer. We also used western blot to reveal expression of the lysosomal membrane protein Lamp2. The expression of lysosomal cathepsin mRNA was evaluated by RT-PCR. The cell viability was significantly diminished in H9c2 cells and the number of autophagosomes increases in a dose-dependent way, at the same time, LC3-II / I ratio was increased. We also found that the H9c2 cellular ATP level was decreased and observed the mPTP opening. The co-localization of mitochondria and lysosomes was detected, also, we found the concentration of Ca2+, the expression of Lamp 2 and mRNA expression levels of Cathepsin B and Cathepsin L significantly increased in a dose-dependent way in H9c2 cells. Finally, the results of expression of PINK1 and Parkin protein increased simultaneously in H9c2 cells. IVM induced mitophagy in H9c2 cells via PINK1/Parkin signaling.

Highlights

Ivermectin (IVM), a mixture of more than 80% 22,23-dihydroavermectin B1a and approximately 20% 22,23-dihydroavermectin B1b, was well-known anti-helminthic agent from the late-1970s (Yang et al, 2020)
The results showed that IVM has potential cytotoxicity, which will have a risk of endanger mammalian health
The cell viability of H9c2 cells treated with IVM for 6 h was assessed by CCK-8 assay

Summary

Introduction

Ivermectin (IVM), a mixture of more than 80% 22,23-dihydroavermectin B1a and approximately 20% 22,23-dihydroavermectin B1b, was well-known anti-helminthic agent from the late-1970s (Yang et al, 2020). The IVM have extraordinary efficacy against parasitic diseases such as river blindness, elephantiasis and scabies (Ashour, 2019; Vos et al, 2012). IVM has the ability to re-treat new diseases and will continue to offer new clinical applications (Formiga et al, 2021). The report confirmed that IVM has great antiviral activity against West Nile virus ( flavivirus) even in the low dosage. The mechanism of IVM was caused accumulation of gamma amino butyric acid (GABA)-gated-Cl− channels, contributing a long-lasting hyperpolarization and paralysis of the infesting organism (Santos et al, 2009)

Methods

Results

Conclusion