| Background:Hyperglycemia-induced oxidative stress is known to play an important role in the development of several diabetic complications, such as atherosclerosis. Although a number of antioxidants are available, none have been found to be suitable for regulating the oxidative stress response and enhancing antioxidative defense mechanisms. Magnesium lithospermate B (LAB) is an active compinent isolated from Danshen with known to have antioxidative effects. It has been reported that LAB exerts a protective effects against the diabetes-accelerated atherosclerotic processes by inhibiting multiple pathways associated with hyperglycemia-induced vascular damages, and LAB may become a new therapeutic agent for the treatment of diabetic and its severalcomplations.Objective:This study was to further confirm the effect of LAB on intracellular reactive oxygen species (ROS) production induced by high glucose or H2O2. We also observe the effect of LAB on HO-1 expression in HEK293T cells cultured under high glucose and preliminarily study its modulated mechasim.Method:1. Determination of ROS generation. The cells were resuspended in culture medium containing 10μmol/L of DCFH-DA and incubated for 30 min. DCFH-DA is a nonfluorescent compound, it can enter the cells and is trapped by removal of the diacetate group. Upon interaction with intracellular ROS, DCFH-DA is converted into DCF,which is a fluorescent product. After staining, DCF-DA was removed by washing the cells with the washing buffer, which were then immediately subjected to flow cytometric analysis using a FACScan at excitation 488 nm and emission 525 nm.2. We determined the expressive level of HO-1 mRNA and HO-1 protein under the treatment of high glucose and LAB by use of quantative reverse transcription polymerase chain reaction (QRT-PCR) and western blotting.3. Detection of nuclear translocation of Nrf2. HEK293T cells were treated with high glucose or pretreated with LAB. Total nuclear proteins were extracted by the use of cytoplasmic protein extraction Kit and Nrf2 was detected by Western blotting.4. Construction of RNA interfering expression vector directed against Nrf2 (pRNAT-U6.1/Neo-siNrf2). We transiently transfected Non-specific control shRNA and pRNAT-U6.1/Neo-siNrf2 into HEK293T cells. After transfection for 48h, the cells were treated with high glucose or pretreated with 50μmol/L LAB, the expression levels of HO-1 and Nrf2 protein were detected by Western blotting.Results:1. Compared with control (30.72±0.47%),30 mmol/L glucose and 100μmol/L H2O2 stimulated intracellular ROS generation (both P<0.05) in HEK293T incubation cells. Furthermore, ROS generation was significantly inhibited in HEK293T cells pretreated with LAB plus 30 mmol/L glucose (27.41±4.13%) compared with high glucose (79.00±5.8%) (P<0.05). However, ROS generation was also significantly inhibited in HEK293T cells pretreated with LAB plus 100μmol/L H2O2 (43.66±5.27%) compared with 100μmol/L H2O2 (92.83±8.9%) (P<0.01).2. The expression level of HO-1 mRNA increased in HEK293T cells treated with high glucose, reached the peak value at 24h and reduced at 48h. The relative quantation of HO-1 mRNA in cells at 2,6,24,48h after the treatment of high glucose were 3.96±0.71,9.57±1.39,12.29±2.54,7.80±0.40, respectively (P=0.022, P=0.003, P=0.000). The change of HO-1 protein was similar to HO-1 mRNA.The ratios of absorbency (HO-1/β-actin) in normal HEK293T cell and high glucose treatment cells for 6,12,24,48h were 0.087±0.01,0.326±0.092,0.718±0.206,0.889±0.128,0.274±0.036, respectively. The protein levels of HO-1 in cells treated with high glucose for 24,48h were higher than control (both P<0.01), whereas there was no difference in 2h cells after high glucose treatment compared with control. The relative quantation of HO-1 mRNA in cells treated with high glucose or 10μmol/L /L,50μmol/L,100μmol/L LAB were 1.37±0.32,1.69±0.31, 2.19±0.26, respectively. The expression of HO-1 mRNA increased in all groups pretreated with LAB compared with high glucose (P=0.045,P=0.015, P=0.000, respectively). The change of HO-1 protein was similar to HO-1 mRNA. The ratios of absorbency in cells treated with high glucose or 10μmol/L/L,50μmol/L,100μmol/L LAB were 0.853±0.107,0.977±0.061, 1.156±0.0182,1.470±0.138, respectively. The cells was pretreated with 50μmol/L,100μmol/L LAB could futher increase the HO-1 protein levels (P= 0.021, P=0.001, respectively).3. Nrf2 was translocated into nucleus and the amount of Nrf2 translocation was enhancced in HEK293T cells were pretreated with 50μmol/L,100μmol/L LAB compare to high glucose (P=0.015, P=0.000, respectively).4. After the Nrf2 gene was silenced, the enhanced expression level of HO-1 protein in cells induced by high glucose plus LAB was vanished.Conclusion:LAB reduced the ROS production in HEK293T cells cultured under oxidative stress. LAB enhanced the expression levels of HO-1 mRNA and HO-1 protein in HEK293T cells treated with high glucose, which is probably modulated via Nrf2 signal pathway. |
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