Objective: To optimize the hydrolytic process of antioxidant silk fibroin (SF) by pancreatin

and investigate the effects and mechanism of the resulting hydrolysates on H2O2-induced HepG2 cell injuries. Method: With the superoxide radical scavenging rate as index

the effects of reaction time

pH

temperature

substrate concentration and enzyme concentration on silk fibroin hydrolytic process were investigated by orthogonal tests. The molecular weight distribution of resulting hydrolysates

the scavenging rate of hydroxyl radicals and the inhibition rate of H2O2-induced erythrocyte hemolysis were determined. Cells were treated with different concentrations of H2O2 for 24 h

and then the optimal H2O2 concentration was selected to determine cell activity. Then cells were divided into blank group (without drug treatment)

H2O2-induced group

H2O2 pretreatment+5 mmol·L-1 N-acetyl cysteine(NAC) treatment as positive control group

H2O2 pretreatment+10

20

30

50 g·L-1 SF treatment groups. Changes in cells activity

malondialdehyde (MDA) content

tumor necrosis factor-α (TNF-α) content

superoxide dismutase (SOD) activity

catalase (CAT) activity and total antioxidant capacity (T-AOC) were observed. Result: Optimal conditions for SF hydrolysis in the study were as follows:enzyme concentration of 6%

reaction time of 160 min

substrate concentration of 20 g·L-1

pH of 8

and temperature 38℃. Under this hydrolysis process

the hydrolyzate's superoxide radical scavenging rate was 72.73%

and the molecular weight of hydrolyzate was below 10 kDa

with greatly increased hydroxyl radical scavenging rate and inhibition rate of H2O2-induced erythrocyte hemolysis. Cell activity was decreased and MDA content was increased after H2O2 induced cells injury (P<0.05). Cell activity was increased and MDA content was decreased in SF treatment group as compared with the H2O2-induced injury group (P<0.05). The TNF-α content was increased and the levels of SOD

CAT

and T-AOC capacity were decreased after H2O2 induced injury (P<0.05). SF treatment could reduce TNF-α content

increase SOD

CAT secretion and the T-AOC capacity(P<0.05). Conclusion: Study demonstrated that SF could improve H2O2-induced injury of HepG2 cells

and its mechanism may be associated with lowering TNF-α content and increasing autioxidant enzyme activities in cells.