7, 8-Dihydroxycoumarin (daphnetin) protects INS-1 pancreatic β-cells against streptozotocin-induced apoptosis.
OBJECTIVE
Daphnetin (7, 8-dihydroxycoumarin), a natural coumarin compound, is known to exhibit antioxidant and anti-inflammatrory effects. However, the underlying mechanisms of its anti-apoptotic and antidiabetic effects yet not been examined. Therefore, the present work studied the anti-apoptotic and anti-diabetic effects of daphnetin by in vitro experiments.


METHODS
The rat insulinoma (INS-1) cells were pre-treated with daphnetin at different concentrations (1, 10, 20 and 40µM) for 24h followed by exposition to streptozotocin (STZ) (3mM) for 12h. Effects of daphnetin and STZ on INS-1 cells were determined by MTT assay, glucose stimulated insulin secretion (GSIS) assay, lipid peroxidation, antioxidant status (SOD, CAT, GPx, and GST) Apoptosis staining (DAPI, Hoechst 33342, AO/EB and ROS) was performed by fluorescence microscopy, and Bcl-2, Bax and NF-κB protein expression was detected by Western blotting.


RESULTS
MTT assay indicated that the viability of INS-1 cells was significantly reduced with exposure to STZ for 12h as compared to control cells, while pre-treated with daphnetin for 24h resulted in a significant improvement of cell viability. The effects daphnetin treatment in INS-1 cells on insulin secretion was tested and results showed that the pre-treatment of daphnetin could improve GSIS. Further, daphnetin pre-treatment significantly reduced the levels of lipid peroxidation markers and also improved antioxidant enzymes' activities in STZ-induced INS-1 cells. Western blotting assay revealed that daphnetin could suppress apoptosis through up-regulation of anti-apoptotic Bcl-2 protein expression and the down-regulation of pro-apoptotic Bax and nuclear factor NF-κB protein levels.


CONCLUSIONS
The results showed that daphnetin might be used in treating diabetes due to its insulin stimulating property and subsequent regulation of apoptotic pathway.
Food Science and Technology Program, Beijing Normal University-Hong Kong Baptist University United International College, Zhuhai, Guangdong 519085, China.
Ramachandran
R
Vinayagam
Food Science and Technology Program, Beijing Normal University-Hong Kong Baptist University United International College, Zhuhai, Guangdong 519085, China. Electronic address: baojunxu@uic.edu.hk.
Baojun
B
Xu
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology / Q000627:therapeutic use*; D019661:Apiaceae / Q000737:chemistry; D017209:Apoptosis / Q000187:drug effects*; D003920:Diabetes Mellitus / Q000188:drug therapy*; D007004:Hypoglycemic Agents / Q000627:therapeutic use*; D050417:Insulin-Secreting Cells / Q000187:drug effects*; D018384:Oxidative Stress / Q000187:drug effects; D010190:Pancreatic Neoplasms / Q000188:drug therapy*; D008517:Phytotherapy; D010936:Plant Extracts / Q000494:pharmacology; D020011:Protective Agents / Q000494:pharmacology; D051381:Rats; D013311:Streptozocin / Q000009:adverse effects; D014468:Umbelliferones / Q000627:therapeutic use*
D000975:Antioxidants; D007004:Hypoglycemic Agents; D010936:Plant Extracts; D020011:Protective Agents; D014468:Umbelliferones; D013311:Streptozocin
Apoptosis; Daphnetin; INS-1 cells; Oxidative stress; Streptozotocin; β-cell destruction