Cytoprotective propensity of green tea polyphenols against citrinin-induced skeletal-myotube damage in C2C12 cells

Abstract

The mycotoxin citrinin, is produced by several species of Penicillium, Aspergillus and Monascus, and is capable of inducing cytotoxicity, oxidative stress and apoptosis. The aim of the present study was to investigate the effect of citrinin in mouse skeletal muscle cells (C2C12) and to overcome the cellular adverse effects by supplementing green tea extract (GTE) rich in polyphenols. C2C12 myoblasts were differentiated to myotubes and were exposed to citrinin in a dose dependent manner (0-100 µM) for 24 h and IC₅₀ value was found to be 100 µM that resulted in decreased cell viability, increased LDH leakage and compromised membrane integrity. Mitochondrial membrane potential loss, increased accumulation of intracellular ROS and sub G1 phase of cell cycle was observed. To ameliorate the cytotoxic effects of CTN, C2C12 cells were pretreated with GTE (20, 40, 80 µg/ml) for 2 h followed by citrinin (100 µM) treatment for 24 h. GTE pretreatment combated citrinin-induced cytotoxicity and oxidative stress. GTE at 40 and 80 µg/ml significantly promoted cell survival and upregulated antioxidant enzyme activities (CAT, SOD, GPx) and endogenous antioxidant GSH, while the gene and protein expression levels were significantly restored through its effective antioxidant mechanism. Present study results suggested the antioxidant properties of GTE as a herbal source in ameliorating the citrinin-induced oxidative stress.

Keywords: C2C12 cells; Citrinin; Cytotoxicity; Green tea polyphenols; Lactate dehydrogenase; Oxidative stress.

Fig. 1

HPLC chromatogram of phenolic compounds present in green tea extract

Fig. 2

Identification of phenolic compounds in green tea extract using UPLC-MS

Fig. 3

Cell viability was measured using MTT assay a CTN treatment with 0–100 μM CTN concentration for 24 h, b cells were pretreated with GTE (20, 40, 80 µg/ml) for 2 h followed by with or without CTN (100 µM) treatment for 24 h. Data are expressed as the mean ± standard deviation from three independent experiments, each performed in triplicate. *p < 0.05 versus control cells, ^# p < 0.05 versus CTN treated cells

Fig. 4

Effect of CTN on LDH leakage in C2C12 cells a CTN treatment with 0–100 μM CTN concentration for 24 h, b cells were pretreated with GTE (20, 40, 80 µg/ml) for 2 h followed by with or without CTN (100 µM) treatment for 24 h. Data are expressed as the mean ± standard deviation from three independent experiments, each performed in triplicate. *p < 0.05 versus control cells, ^# p < 0.05 versus CTN treated cells

Fig. 5

Morphological features of C2C12 cells under phase contrast microscope, a control, b CTN 100 µM, c GTE 20 µg/ml + CTN, d GTE 40 µg/ml + CTN, e GTE 80 µg/ml + CTN

Fig. 6

Effect of CTN on loss of MMP in C2C12 cells a C2C12 cells were treated with 0–100 µM CTN for 24 h, b cells were pre-treated with GTE (20, 40, 80 µg/ml) for 2 h followed by with or without CTN (100 µM) treatment for 24 h. Data are expressed as the mean ± standard deviation from three independent experiments, each performed in triplicate. *p < 0.05 versus control cells, ^# p < 0.05 versus CTN treated cells

Fig. 7

Effect of CTN on ROS generation a C2C12 cells were treated with 0–100 µM CTN for 24 h, b ameliorative effect of GTE on CTN-induced ROS generation in C2C12 cells. Data are expressed as the mean ± standard deviation from three independent experiments, each performed in triplicate. *p < 0.05 versus control cells, ^# p < 0.05 versus CTN treated cells

Fig. 8

Flow cytometry analysis of propidium iodide stained C2C12 myotubes after 24 h treatment indicating the percentage of cells at sub-G1 phase. a Control, b CTN 100 µM, c GTE 20 µg/ml + CTN, d GTE 40 µg/ml + CTN, e GTE 80 µg/ml + CTN

Fig. 9

a The protective effect of GTE on CTN-induced expression of oxidative stress marker genes GPx, catalase and SOD by semi-quantitative RT-PCR analysis, b densitometry analysis of GPx, catalase and SOD gene expression (% of control). Data are expressed as the mean ± standard deviation from three independent experiments. *p < 0.05 versus control cells, ^# p < 0.05 versus CTN treated cells

Fig. 10

a Western blot analysis of oxidative stress marker proteins GPx, catalase and SOD, b densitometry analysis of GPx, catalase and SOD protein expression (% of control). Data are expressed as the mean ± standard deviation from three independent experiments. *p < 0.05 versus control cells, ^# p < 0.05 versus CTN treated cells