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Objective To investigate the effects of tanshinone II A ( Tan II A) on the intermittent high glucose (IHG)- induced oxidative stress and apoptosis of Schwann cells (SCs) in suckling rat. Methods Cultured SCs of newborn suckling rats were randomized into several groups including control (con, 5.6 mmol/L glucose), stable high glucose (HG, 50 mmol/L glucose), IHG (5.6 and 50.0 mmol/L glucoseper 8 h alternative), osmotic control, ALA (500 µmol/L) and IHG in the presence of 0.1, 1.0, 10.0 µmol/L Tan II A. Apoptosis rate, oxidative stress level, expression of apoptosis related proteins and the mRNA in the cells were detected. Results (1) The percentages of apoptotic cells exposed to HG ((34.57± 2.45)%) and IHG ((40.17±2.33)%) were significantly higher than that in control group ((4.74±0.26)%). Furthermore, apoptosis rate of IHG was much higher than that of HG (t=3.70,P<0.01). (2) ROS and 8-OHdG levels increased significantly in the IHG group compared with those in control and HG groups (F=73.44, 194.21, P<0.01). IHG exposure up-regulated expression of bax protein and mRNA (F=66.78, 126.69, both P<0.01), while down-regulating expression of bcl-2 protein and mRNA (F=151.88,54.99,both P<0.01). In addition, IHG increased activities of caspase-3 and (poly(ADP-ribose) polymerase, PARP) (129% ,18% , 139% ,56% ,all P<0.01). (3) Treatment with 1.0 µmol/L and 10.0 µmol/L of Tan IIA inhibited IHG-enhanced ROS production and 8-OHdG levels (t=3.50-6.47,P<0.05). Bax protein and mRNA (t=3.59-5.39,P<0.01), PARP and caspase-3 activation (42%,61%,13%,34%,all P<0.01), and SC apoptosis (t= 12.39, 21.26, P<0.01)were decreased with Tan µ A, too. Conclusions Tan µ A decreases IHG-induced apoptosis via reducing oxidative stressin SCs of newborn suckling rats. [ABSTRACT FROM AUTHOR]

Objective To investigate the molecular mechanism of sterol regulatory element binding protein-1c (SREBP- 1c) suppressing insulin receptor substrate-1(IRS-1) in skeletal muscle cells. Methods Luciferase plasmid for rat IRS- 1 promoter, expression plasmid of SREBP-1c and pRL-TK renilla plasmid were cotransfected into L6 cells using Lipofectamine 2000 (Invitrogen). After transfection for 36 h, luciferase activity was measured using the dual-luciferase reporter assay system following the manufacturer' s instructions. L6 myotubes were infected with adenoviral vectors expressing SREBP-1c. Adenovirus expressing green fluorescent protein (GFP) was used as control. The cells were harvested for nucleus protein after infection for 48 h. Electrophoretic mobility shift assay (EMSA) was performed using a Light Shift Chemiluminescent EMSA Kit. Chromatin immunoprecipitation assay was performed by CHIP Kit. The interaction between the transcription factor SREBP-1c and the promoter region of IRS-1 was assessed by above experiments. Differences among the groups were determined using two-way ANOVA. Results The luciferase deletion studies suggested the region from -450 to -210 bp on the IRS-1 promoter was a potential target region for SREBP-1c. Sequence analysis showed that the target region of the rat IRS-1 promoter gene contained a potential binding site (GCCTCCCGAG), which was located between -302 and -292 bp. PCR site- directed mutagenesis (TGTTAAATTA) was generated and analyzed using a luciferase assay. The transcriptional activity of wild-type IRS-1 promoter was dramatically down-regulated by SREBP-1c, whereas SREBP-1c had no effect on the IRS-1 promoter bearing mutation of SREBP-1c binding site(7.03±1.28 vs 19.09±2.45,3.55±1.68 vs 3.96±1.09,F=114.437,0.251,all P>0.05). The EMSA results showed that the labeled wild- type probe successfully formed a complex with nuclear proteins. But the unlabelled mutant probe did not interfere the complex. The results from CHIP assay showed that SREBP-1c could bind directly to the IRS- 1 promoter region. The quantity of SREBP- 1c protein binding to the IRS- 1 promoter was two times of control under PA conditions in L6 cells or five times of control when SREBP-1c was over-expressed (2.15±0.03 vs 1.07±0.24,5.48±1.28 vs 0.86±0.19,t=6.877,5.495,all P<0.05). Conclusion SREBP- 1c could directly bind to the atypical SRE sequence in the promoter region of IRS- 1, suppressing IRS- 1 expression and the subsequent insulin signaling pathway. [ABSTRACT FROM AUTHOR]