In vitro studies on radioprotective efficacy of silymarin against γ-irradiation

Abstract

Purpose: Silymarin has been widely exploited for its hepatoprotective activities. This study aimed to evaluate the protective efficacy of silymarin against γ-radiation.

Materials and methods: The radioprotective properties of silymarin were studied using different assays. Cytotoxicity of silymarin on Human embryonic kidney (HEK) cells was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Protective efficacy against γ-radiation was assessed by studying reduction in micronuclei frequency and free radical generation using 2′,7′-dichlorodihydroflurescin diacetate (H₂DCFDA). Radiation-induced apoptosis was estimated by Annexin V-PI (propidium iodide) analysis and cell cycle analysis. γ-radiation induced changes in mitochondrial membrane potential (MMP) and DNA damage was estimated employing flow-cytometry and comet assay respectively.

Results: MTT assay and Annexin V-PI studies showed that pre-incubation of HEK cells with silymarin protected them from γ-irradiation. Significant reduction in apoptosis (76.36%) was observed. Silymarin also decreased the percentage of radiation-induced micronuclei (> 69%) (p < 0.05 ). Measurement of intracellular reactive oxygen species (ROS) by H₂DCFDA revealed a reduction in ROS (21%) at 0.5 h. Cell cycle analysis revealed G₁ block in the unirradiated control, which declined in the silymarin pretreated irradiated group (0.5 h). Silymarin treatment resulted in a significant increase in MMP (2 h) against the radiation control. Moreover, the presence of silymarin during irradiation significantly decreased the DNA damage (as measured by comet assay).

Conclusions: Protection against radiation-induced cell-death and DNA damage by silymarin could be attributed to a reduction in ROS induced by γ-radiation. In vitro experiments on HEK cells explicitly prove that silymarin is a promising, effective and safe radiation countermeasure agent.

Keywords::

• HEK cells
• micronuclei frequency
• mitochondrial membrane potential
• radiation
• radioprotection
• reactive oxygen species

Acknowledgements

The authors are grateful to Dr W. Selvamurthy, Chief Controller, Research and Development (Life Sciences and International Cooperation), Defence Research and Development Organization, Government of India, Director, Institute of Nuclear Medicine and Allied Sciences, Delhi, India, and Rector, Trakia University, Bulgaria for support in multifarious ways. The authors would like to acknowledge the technical help of Mrs Namita Kalra in flow cytometry. Thanks are also due to Department of Science and Technology (DST), Delhi, India, and Government of the Repubic of Bulgaria for providing resources under the Indo-Bulgarian Joint Collaborative Project.

Declaration of interest:

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper

This work was primarily supported by funds from the Defence Research and Development Organization (DRDO), Ministry of Defence, Government of India, and an Indo-Bulgarian Joint Collaborative Project entitled “Studies on Antioxidants against Oxidation Stress Induced by Drug and Radiation” [INT/Bulgaria/B-57/05]. Manish Adhikari is grateful to DRDO for providing a research fellowship.