Citral, A Monoterpene Protect Against High Glucose Induced Oxidative Injury in HepG2 Cell In Vitro-An Experimental Study

Abstract

Introduction: Diabetes mellitus, a major metabolic disorder associated with hyperglycaemia is one of the leading cause of death in many developed countries. However, use of natural phytochemicals have been proved to have a protective effect against oxidative damage.

Aim: To investigate the effect of citral, a monoterpene on high glucose induced cytotoxicity and oxidative stress in human hepatocellular liver carcinoma (Hep G2) cell line.

Materials and methods: Cells were treated with 50 mM concentration of glucose for 24 hours incubation following citral (30 μM) was added to confluent HepG2 cells. Cell viability, Reactive Oxygen Species (ROS) generation, DNA damage, lipid peroxidation, antioxidants and Mitogen Activated Protein Kinases (MAPKs) signaling were assessed in citral and/or high glucose induced HepG2 cells.

Results: Cells treated with glucose (50 mM), resulted in increased cytotoxicity, ROS generation, DNA damage, lipid peroxidation and depletion of enzymatic and non enzymatic antioxidants. In contrast, treatment with citral (30 μM) significantly decreased cell cytotoxicity, ROS generation, DNA damage, lipid peroxidation and increased antioxidants enzymes in high glucose induced HepG2 cells. In addition, the present study highlighted that high glucose treated cells showed increased expression of Extracellular Signal Regulated Protein Kinase-1 (ERK-1), c-Jun N-terminal Kinase (JNK) and p38 in HepG2 cells. On the other hand treatment with citral significantly suppressed the expression of ERK-1, JNK and p38 in high glucose induced HepG2 cells.

Conclusion: Citral protects against high glucose induced oxidative stress through inhibiting ROS activated MAPK signaling pathway in HepG2 cells.

Keywords: Antioxidants; Hyperglycaemia; Oxidative stress.

Figures

[Table/Fig-1]:

Effect of citral on high glucose induced cytotoxicity in HepG2 cells. HepG2 (1X106) cells were incubated at 37°C with citral (0–60 μM) for 24 hours, followed by exposure to 50 mM glucose for 24 hours. (a) Cytotoxic effect of citral on HepG2 cells without exposure of high glucose. (b) Cytotoxic effect citral on HepG2 cells with exposure to high glucose. Data are given as means ± SD of six experiments in each group. Values not sharing a common marking (a, b, c,..) differ significantly at p

[Table/Fig-2]:

Effect of citral on high glucose induced ROS generation in HepG2 cells by DCFH-DA staining. (a) Photomicrographs showed (40X) enhanced green fluorescence in high glucose induced HepG2 cells, citral treatment decreased high glucose induced ROS generation. (b) Bar diagram represents the fluroscence intensity for accumulation DCFH-DA. Values are given as means ± SD of six experiments in each group. Values not sharing a common marking (a, b, c,..) differ significantly at p

[Table/Fig-3]:

Effect of citral on high glucose induced lipid peroxidation in HepG2 cells. Lipid Peroxidation (LPO) values were expressed as nmol per mg protein. Values are given as means ± SD of six experiments in each group. Values not sharing a common marking (a, b, c,..) differ significantly at p

[Table/Fig-4]:

Effect of citral on high glucose induced depletion of antioxidants status in HepG2 cells. Values are given as means±SD of six experiments in each group. Values not sharing a common marking (a, b, c,..) differ significantly at p

[Table/Fig-5]:

Effect of citral on high glucose induced DNA damage in HepG2 cells. (a) Fluorescence photomicrographs showed enhanced tail DNA and tail movement in high glucose induced HepG2 cells, citral treatment decreased high glucose induced comet formation. (b) Bar diagram represents % of DNA damage were assessed by CASP online software. Values are given as means±SD of six experiments in each group. Values not sharing a common marking (a, b, c,..) differ significantly at p

[Table/Fig-6]:

Effect of citral on high glucose induced p-ERK1, p-JNK and p-p38 expressions in HepG2 cells.