2.2　Cells culture and treatments

Rattus pancreaticβ-cell line(Rin-m5F)was purchased from tumor cells bank of Chinese Academy of Medical Sciences.Rin-m5F cells were cultured at 37℃，5% CO2 atmosphere in RPMI-1640 medium(Hyclone)containing 12% fetal calf serum(Hyclone).On attaining 75%～80%confluency，cells(after passages 4)were seeded into 6-well plate at a density of 1×106 per well and grown for 24h.Then the cells were treated as different groups：control group(10mM glucose)，model group(25mM glucose，1000μg/mL BSA(Bovine serum albumin))，100μg/mL TGSO group(25mM glucose，100μg/mL TGSO，1000μg/mL BSA)and cultured for 48h.The concentration of glucose，time for glucose exposure of RIN cells and TGSO concentration were obtained from our preliminary experiment.Rin-m5F cell was exposed to the glucose of 5，15，25mmol/L for 12，24，36 and 48hours.The treatment of exposure to 25mmol/L glucose for 48h was observed with amount of apoptotic cell and high NO content that it was appropriate for constructing high glucose-induced apoptosis model［38］.TGSO with the concentration of 50，100，200μg/mL were set for cell culture.The results showed that 100μg/mL TGSO played the best anti-apoptotic role by measuring apoptotic rate and insulin release.All cell cultures were set up in triplicates for following assay.(https://www.daowen.com)