Comparison Study Of TGF-?1 And D-galactose In Inducing Spiral Ligament Fibrocytes Mimetic Aging

Objectives: To compare the effect of TGF-?1 and D-galactose in inducing spiral ligament fibrocytes mimetic aging and to investigate the underlying mechanisms.Methods : The cochlear lateral wall was isolated from 3 days old SD rats and digested with type II collagenase. The spiral ligament fibrocytes were collected and cultured with DMEM/F12 medium(10% fetal bovine serum). To determine the optimal treatment concentration and duration of TGF-?1, the spiral ligament fibrocytes were incubated with different concentration of TGF-?1/ D-galactose for different time and the cell viability was tested with CCK8 methods. After confirming the optimal concentration and duration of TGF-?1/ D-galactose, the spiral ligament fibrocytes were divided into three groups: TGF-?1 treatment group(8ng / ml), D- galactose treatment group(8mg / ml), the control group and were cultured for 48 h. The flow cytometry method was conducted to detect the influence of TGF-?1/ D-galactose on cell cycle. ?- galactosidase staining method was conducted to detect the degree of cell aging. RT-PCR methods were separately conducted to detect the expression of BMI1, p16INK4 a in three groups. Western Blot methods were separately conducted to detect the expression of BMI1, p16INK4 A and PCNA in three groups.Results: The CCK-8 test showed that the cell activity of TGF-?1/ D- galactose treatment groups were lower compared with control group. The minimum concentration and duration with statistically significance were: 8ng/ml, 48 h for TGF-?1 and 8mg/ml, 48 h for Dgalactose. In the TGF-?1 and D- galactose treatment group, the spiral ligament fibrocytes senescence occurred both. In TGF-?1 treatment group and D- galactose trentment group G0/ G1 phase ratio increased and cells stranded mainly in the G1 phase of the cell cycle. The expression of PCNA decreased in both TGF-?1 treatment group and D- galactose trentment groups. Real Time PCR showed that in TGF-?1 treatment group the m RNA level of P16INK4 a increased significantly compared with the control group(p<0.05). In the Dgalactose treatment group the m RNA level of BMI1 decreased and that of p16INK4 a increased compared with the control group(p<0.05).Results of Western Blot revealed that compared with the control group the protein expression of P16INK4 a increased(p<0.05) and that of bmi1 increased but not reached statistical significant difference in TGF-?1 treatment group; in D- galactose treatment group the protein expression of P16INK4 a increased and that of bmi1 decreased compared with the control group(p<0.05)Conclusion: TGF-?1 induces the spiral ligament fibrocytes senescence by promoting the expression of p16INK4 a and this effect is independent of BMI1. D- galactose induced the spiral ligament fibrocytes senescence through downregulating the expression of BMI1 which could suppressive the expression of p16INK4 a.