The Mechanism Of Reduced β2GPⅠ Inhibiting Glomerular Mesangial Cells VEGF-NO Axis Uncoupling Induced By High Glucose

Objective: Diabetic nephropathy(DN) is one of the most common and most serious chronic complications of diabetes, which is a significant cause for end-stage renal failure. DN has already become a leading cause of morbidity and mortality of diabetic patients. The mechanism of DN is very complex. In recent years, it has been proposed that VEGF-NO axis uncoupling may be an important pathogenesis for DN. Our previous studies found that reduced β2-glycoprotein I(r-β2GPI) inhibited the angiogenesis of diabetic retinal neovascularization through regulating the VEGF signaling pathway. Besides, r-β2GPI inhibited the progress of diabetic nephropathy in STZ-induced diabetic mice and reduced the damage of mesangial cells induced by high glucose associated with suppressing the activation of the TGF-β1-p38 MAPK pathway. So rat glomerular mesangial cell line(HBZY-1) was used in this study to investigate: 1. The effect of β2GPI and r-β2GPI to high glucose inducing VEGF-NO axis uncoupling on glomerular mesangial cells. 2. The mechanism of the contribution of β2GPI and r-β2GPI to high glucose inducing VEGF-NO axis uncoupling on glomerular mesangial cells.Method: 1. HBZY-1 cells were cultivated and divided into six groups, ① normal control(NC group): 5.5m M glucose. ② high isotonic group(MC group): 5.5m M glucose+19.5m M mannitol. ③ high glucose group(HG group): 25 m M glucose. ④ HSA group: 25 m M glucose + 100 μg/ml HSA. ⑤ β2GPI group: 25 m M glucose + 100 μg/ml β2GPI. ⑥ r-β2GPI group: 25 m M glucose + 100 μg/ml r-β2GPI. 2. NO assay kit was used to detect the role of β2GPI and r-β2GPI on the production of NO by glomerular mesangial cells under high glucose condition.and DCFH-DA fluorescent probe was used to explore the content of ROS in each group detected by fluorescence microscope and flow cytometer. 3. Real time PCR was used to investigate the contribution of β2GPI and r-β2GPI to the m RNA expression of VEGF、VEGFR-2、e NOS、GCH-1 in VEGF-NO axis of glomerular mesangial cells intervened by high glucose. 4. Western blot was used to determine the role of β2GPI and r-β2GPI to the activity of e NOS in VEGF-NO axis and the phosphorylation of Akt in VEGF intracellular signal transduction pathway on glomerular mesangial cells induced by high glucose.Results: 1. High glucose markedly inhibited the production of NO in glomerular mesangial cells by 33%(P<0.05) which was promoted by β2GPI and r-β2GPI(P<0.05). The effect of r-β2GPI was stronger than β2GPI(P<0.05), respectively 2.10 times and 1.88 times higher than HG group. 2. Compared to NC group, the production of ROS was conspicuously increased under high glucose or high osmotic pressure condition in glomerular mesangial cell(P<0.05), which was restrained by β2GPI and r-β2GPI(P<0.05), of which r-β2GPI displayed the stronger inhibition(P<0.05). 3. Under high glucose condition, the m RNA expression of VEGF and VEGFR-2 in glomerular mesangial cell was observably increased(P<0.05), while the m RNA expression of GCH-1 was inhibited, and the effect on the m RNA expression of e NOS was not obvious(P>0.05). Both β2GPI and r-β2GPI decreased high glucose induced the m RNA expression of VEGFR-2(P < 0.05) in glomerular mesangial cell, promoted the m RNA expression of GCH-1 and e NOS(P<0.05), and had no effect on the m RNA expression of VEGF(P>0.05). The effect of β2GPI and r-β2GPI on VEGF、VEGFR2、GCH-1、e NOS expression was not statistically different(P>0.05). 4. High glucose markedly inhibited the phosphorylation of e NOS in glomerular mesangial cell(P<0.05), which was improved by β2GPI and r-β2GPI(P<0.05). The effect of β2GPI and r-β2GPI was not statistically different(P>0.05). 5. High glucose markedly inhibited the phosphorylation of Akt in glomerular mesangial cell(P<0.05), which was improved by β2GPI and r-β2GPI(P<0.05). The effect of β2GPI and r-β2GPI was not statistically different(P>0.05).Conclusion: 1. High glucose inhibited NO production and increased ROS level in glomerular mesangial cell, which might be via increasing VEGF and VEGFR-2 expression, decreasing GCH-1 expression, lowering e NOS activity but not its expression. 2. Both β2GPI and r-β2GPI antagonized high glucose induced effect on NO production and ROS level in glomerular mesangial cell, which might via decreasing VEGFR-2 expression, increasing GCH-1 expression, lowering Akt phosphorylation and improving e NOS activity, thus leading to the reduced level of oxidative stress and the protective role on glomerular mesangial cell.3. The effect of r-β2GPI on promoting NO production and decreasing ROS level was stronger than β2GPI, which could be ascribed to the free thiol contained by r-β2GPI, leading to the change of its space structure and the amount of charge on r-β2GPI. Though r-β2GPI had stronger antioxidant capacity, both r-β2GPI and β2GPI could regulate VEGF signal pathway.