The Effects Of Sirt1/FOXO1 On High Glucose-induced Apoptosis In Mouse Mesangial Cells

Objectives: Diabetic nephropathy(DN) is one of the most common diabetes microvascular complications. The patients of DN are increasing. In our country, the patients of DN cost a lot of money every year. But the mechanism of DN is unknown, a large number of studies have shown that oxidative stress played an important role in the pathogenesis of DN. FOXO1 is a impotent transcription factors and able to regulate PI3K/AKT signals pathway, resist oxidative stress, activate transcription factors that promote the growth and proliferation of cells, and inhibit cell apoptosis. FOXO1 plays a role in mediating oxidative stress, resisting cell proliferation and inducing apoptosis. However, the role of FOXO1 in high glucose(HG)-induced apoptosis in MMC is unknown. In this study, we examined the effects of FOXO1 by using a small interference RNA on high glucose-induced apoptosis,expression of cleaved caspase-3 and Bax/Bcl-2 ratio in mouse mesangial cells.Methods: FOXO1 si RNA plasmids were constructed. Using Lipofectamine 2000, the stable transfect MMCs of FOXO1 si RNA plasmids were obtained through selection by G418. MMCs were stimulated with HG and cultured with normal glucose(NG). The number of apoptotic cells was detected using TUNEL method. The proteins of SIRT1、FOXO1、Bax、Bcl-2、Cleaved caspase-3、SOD1、SOD2、P47, NOX4 and β-actin were detected by western blot. The m RNA levels of FOXO1、Bax and Bcl-2were detected by RT-PCR.Results:1. The expression of FOXO1 protein was efficiently induced by transfection with FOXO1 si RNA plasmid.Compared with control groups, the expression of FOXO1 protein was efficiently induced by transfection with FOXO1 si RNA plasmid in highglucose(HG) codition.2. Effect of FOXO1 in HG induced apoptosis in MMC.The result of TUNEL indicated that compared with NG group, HG significantly promoted cell apoptosis. The number of apoptosis cells was decreased in transfection with FOXO1 si RNA group.3. Effect of FOXO1 in HG induced-ratio of Bax/Bcl-2.The results of Western blot and RT-PCR indicated that compared with NG group, the m RNA and protein ratio of Bax/Bcl-2 was increased in high glucose group, and decreased in high glucose with transfection of FOXO1 si RNA group.4. Effects of FOXO1 on expression of cleaved Caspase-3.The results of Western blot indicated that compared with NG group, the protein ratio of cleaved Caspase-3/ Caspase-3 was increased in high glucose group, and decreased in high glucose with transfection of FOXO1 si RNA group.5. Effect of FOXO1 on HG-induced e NOS productionThe results of Western blot indicated that compared with NG group, the proteins of SOD1 and SOD2 were decreased in high glucose group, and increased in high glucose with transfection of FOXO1 si RNA group.Compared with NG group, the proteins of P47 and NOX4 were increased in high glucose group, and decreased in high glucose with transfection of FOXO1 si RNA group.6. Effect of FOXO1 when silencing on SIRT1.Compared with those of the NG groups, the FOXO1 level significantly decreased in HG group and increased in high glucose with transfection of SIRT1 si RNA group.Conclusion:1. FOXO1 was involved in high glucose induced mouse mesangial cell(MMC)apoptosis and reduced HG-mediated expression of cleaved caspase-3,Bax/Bcl-2 ratio.2. FOXO1 was involved in high glucose induced expression of SOD1, SOD2,P47 and NOX4.3. SIRT1 can decrease(HG)-induced apoptosis in MMC by inhibited the expression of FOXO1.