Study On The Protective Effect Of Peroxiredoxin ? Gainst Damage Of ?-cell Induced By Streptozotocin

Background: Peroxiredoxin ??Prx ??is a kind of typical antioxidant reductase protein containing 2-Cys residue,which is widely expressed in the cells of mammal.Prx ? has redox reaction with the mercapto of the other molecule's N-end Cys through the Cys mercapto of C-end and forms disulfide bond and removes H2O2.The results of a paper in 2010 on ANTIOXIDANTS & REDOX SIGNALING shows that: after real time-PCR testing that the mice enterocoelia is injected with STZ,the transcription level of the mRNA of Prx ? in pancreatic tissue is significantly decreases comparing with the control group.The STZ molecular has glucosamine structure which can specifically pass GLUT2 to enter into ?-cells,cause oxidative stress and ?-cells damage,and induce blood glucose increase.Though the researches on effect of Prx ? are widely,the effect of Prx ? on induced ?-cells damage process of STZ is unclear.In order to deeply research the protective effect of Prx ? on ?-cells damage induced by STZ,this experiment utilizes the insulin oncocytes of Prx ? gene knockout mice(Prx ?^-/-)and Min6 cell of Prx ? knockdown to research the protective effect of Prx ? on ?-cells damage induced by STZ.Methods: in vivo test,employ single-time 200 mg/kg/per time STZ and five-time 50mg/kg/ per time STZ?1 per time/d?to conduct intraperitoneal injection toward the Prx ?^+/+and Prx ?^-/-mice of six-eight weeks aging to induce ?-cells damage,adopt H&E staining to observe the mice islet histomorphology changes,glucometer to observe the mice blood glucose level and ELISA to observe the fasting insulin level of mice serum;conduct intraperitoneal injection of 2 g/kg IPGTT and 1 IU/kg IPIPP and respectively test the blood glucose change conditions of mice.In vitro test,employ slow virus to infect Min6 cells and establish empty carrier Min6?Scramble?of stable inheritance and Min6 cells?shPrx?of Prx ? knockdown.Firstly,use MTT test to observe the cytotoxicity effect of STZ on Scramble and shPrx ?.Then,employ flow cytometry?FCM?and in situ fluorescence staining to observe the cell cycle,cell apoptosis and ROS level conditions of 5 mM STZ processed Scramble and shPrx ? under different times.Finally,employ Western blotting to observe the expression level of cell cycle,cell apoptosis and upstreat relative signal transducing protein.Results: in vivo test.After staining,it is observed that after STZ intraperitoneal injection,the ?-cells of both kinds of mice have cell death,and the death scale of Prx ?^-/-mice ?-cells is significantly higher than Prx ?^+/+mice.The ELISA results show that the fasting insulin level of Prx ?^-/-mice is significantly lower than that of Prx ?^+/+mice.The mice blood glucose test finds that the blood glucose levels of both kinds of mice increase,and the blood glucose level of Prx ?^-/-mice is significantly higher than Prx ?^+/+mice under same time.And the IPGTT has the same results.In vitro test.Prx ? knockdown Min6 mice islet oncocyte?shRrx ??of stable inheritance is successfully established.The MTT test results show that: with the increase of STZ tretment concentration and extension of time,the survival ratios of Scramble and shPrx ? cells decrease,and the survival ratio of shPrx ? cells is significantly lower than Scramble cells under same processing time or concentration.FCM test finds that: with the extension of 5 m M STZ processing time,the G2/M phase arrest and ROS level increase trends of Scramble and shPrx ? cells become more possible,and under the same processing time,the G2/M phase arrest of shPrx ? cells is significantly higher than Scramble cells,and at 9 h,18 h,the ROS level of shPrx ? cells is significantly higher than Scramble cells.In situ fluorescence staining results find that with the extension of 5 m M STZ processing time,the cell apoptosis of Scramble and shPrx ? cells increase,and under the same processing time,the shPrx ? cells apoptosis is significantly higher than Scramble cells.Western blotting results find that the expression levels of the relative proteins?P21,P16 and CyclinB1?of cell cycle and relative proteins?Bad,Bax,Cleavaged-caspase3?of apoptosis promotion increase with the extension of5 mM STZ processing time,and under the same processing time,the relative proteins' expression level of shPrx ? cells is significantly higher than Scramble,and the expression level of anti-apoptosis protein Bcl-2 decreases with the extension of 5 mM STZ processing time,and under the same processing time,the expression level of Bcl-2 protein of shPrx ? cells is significantly lower than Scramble cells.The expression levels of P-AKT,P-GSK-3b P-P53 and P53 decrease with the extension of 5 mM STZ's processing time on Scramble and shPrx ? cells,and under the same processing time,the phosphorylation level of relative shPrx ? proteins is significantly lower than Scramble cells.Conclusions: above results show that: when islet cells is damaged by STZ,the deficiency of Prx ? causes large-scale ?-cells death and blood glucose increase,the knockdown of Prx ? can cause the expression levels decrease of P-AKT,P-GSK-3b?11?P53 and P-P53 and ?-cells haveG2/M phase arrest and apoptosis.As a result,Prx ? can protect ?-cells through adjusting the expression levels of P-AKT,P-GSK-3b?11?P53 and P-P53.