Diosbulbin B-Induced Mitochondria-Dependent Apoptosis in L-02 Hepatocytes is Regulated by Reactive Oxygen Species-Mediated Autophagy.

Jing Ye,Sirui Zhu,Yamin Liu,Yu Li,Jia Rui,Xiaoli Liu,Mei Xue,Liang Zhang,Danhong Cai

doi:10.3389/fphar.2019.00676

Jing Ye, Sirui Zhu + Show 7 more

Open Access

https://doi.org/10.3389/fphar.2019.00676

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Abstract

Aim: Diosbulbin B (DB) is a major diterpenoid compound found in Dioscorea bulbifera L, a traditional medicinal herb in China. Clinical reports have confirmed that Dioscorea bulbifera L. can induce significant hepatotoxicity. In this study, we showed that DB can induce mitochondria-dependent apoptosis and investigated the role of autophagy in DB-induced hepatotoxicity in L-02 hepatocytes. Methods: L-02 hepatocytes were treated with different concentrations of DB for 48 h, after which indicators of autophagy and apoptosis were measured. 3-Methyladenine (3-MA) and rapamycin (Rapa) were used as inhibitor and agonist of autophagy, respectively. Furthermore, the reactive oxygen species (ROS) scavenger N-acetyl-l-cysteine (NAC) was used in combination with DB to evaluate the relationship between ROS and autophagy. Results: L-02 cell viability was significantly decreased after treatment with DB for 48 h. Additionally, DB induced concentration-dependent apoptosis and autophagy and increased the activities of caspase-3, caspase-9, alanine aminotransferase (ALT), and aspartate transaminase (AST), and induced excessive leakage of lactate dehydrogenase (LDH). Inhibition of autophagy by 3-MA increased DB-induced apoptosis, resulting in aggravation of hepatotoxicity. Conversely, treatment with Rapa increased malondialdehyde (MDA) content and reduced superoxide dismutase (SOD) activity. Moreover, we found that DB treatment increased the level of intracellular ROS, decreased the mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) production, and caused abnormal opening of the mitochondrial permeability transition pore (mPTP), which were finally restored by the ROS scavenger NAC. Conclusions: Accumulation of ROS can induce mitochondria-dependent apoptosis and likely to play a key role in DB-induced hepatocellular injury. Activation of autophagy may inhibit apoptosis, but also reduces antioxidant capacity.

Highlights

Diosbulbin B (DB) is a diterpene lactone compound in Dioscorea bulbifera L., which is well known for its unique therapeutic effect on treating thyroid disease in China (Wang et al, 2012)
MDC staining results demonstrated enhanced fluorescence signals in the DB treatment groups (Supplementary Figure S2). These results suggested the activation of autophagy during DB-induced hepatocyte injury
Enhanced protein expression of Bax, up-regulated activities of caspase-3 and caspase-9, and excessive leakage of lactate dehydrogenase (LDH) revealed that DB can induce cell apoptosis in L-02 hepatocytes (Figure 2C-E)

Summary

Introduction

Diosbulbin B (DB) is a diterpene lactone compound in Dioscorea bulbifera L., which is well known for its unique therapeutic effect on treating thyroid disease in China (Wang et al, 2012). A previous report showed that DB induced oxidative stress and cholestasis in animal experiments (Ma et al, 2014). Mitochondrial dysfunction can inhibit the chain of energy supplementation and cause oxidative stress, as well as a shift in metabolic pathways (Halbrook et al, 2018). The breakdown of inner mitochondrial transmembrane potential (MMP) is an early stage of mitochondria-dependent apoptosis (Wang et al, 2015), whereby excessive production of reactive oxygen species (ROS) from the mitochondrial electron transport chain (ETC) attacks lipids and proteins on the membrane. The mutation, deletion, or insertion of mitochondrial DNA (mtDNA) caused by ROS creates an irreversible cycle that triggers mitochondrial dysfunction (Vakifahmetoglunorberg et al, 2017)

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