| Objective: To establish the animal model of Diabetic Retinopathy in Wistar rat. And to observe the therapeutic effect of Niaspan on DR, also to explore the possible mechanisms.Methods:(1)One hundred and fifty 6-8 week-old male Wistar rats were divided into control(CON) group(50 rats),diabetes(DM) group(50 rats) and Niaspan-treated group(50 rats). Rats were induced with STZ injection for diabetes model. Niaspan(40mg/kg/day) was administrated orally everyday in Niaspan-treated group until sacrificed at month 3. All eyeballs were examined hematoxylin and eosin(HE) staining、total cholesterol and High-Density Lipoprotein(HDL), were evaluated at month 3. The integrity of the blood-retinal barrier(BRB) and the vascular permeability was quantified by analyzing albumin leakage using Evans blue method. Apoptosis was examined by terminal deoxynucleotidyl transferase-mediated d UTP nick end labeling(TUNEL) assay. Claudin-5、Occludin、VEGF/VEGFR、Tie-2、VCAM-1 、 TNF-α 、 CD45 were evaluated through immunohistochemistry. The VEGF/VEGFR、Ang-1/Tie-2、VCAM-1/ICAM-1、NF-κB/TNF-α、IL-6、i NOS、ZO-1 were evaluated through western blotting. The relative expressions of micro RNA-126(mi R-126)、Claudin-5、Occludin、ZO-1、 ICAM-1、NF-κB、i NOS were detected through quantitative real-time reverse transcription PCR assay(q RT-PCR).(2) Cells were treated with ECM containing of the following: 5m M D-glucose(euglycemic control)(CON group); 50 m M D-glucose(hyperglycemia)(HI group); 50 m M D-glucose(hyperglycemia)+Niacin(1m M, Sigma Chemical Co., USA)(NA group); 50 m M D-glucose(hyperglycemia) + Niacin(1m M) +micro RNA126 inhibitor(NI group). HREC cells were treated mi R-126 inhibitor(mi R-126-inhibitor)(Invitrogen, CA, USA) using Lipofectamine 2000. The relative expressions of micro RNA-126(mi R-126) were detected through quantitative real-time reverse transcription PCR assay(q RT-PCR). The VEGF and Ang-1 were evaluated through western blotting.Results: Compared with the rats in control group, significantly higher blood glucose and lower body weight(t=9.45、25.01, P<0.05) were found in model group rats. Diabetes significantly increases blood serum total cholesterol level and decreases HDL level compared to normal control rats(t=4.03、5.81, P<0.05); while Niaspan treatment in diabetic rats significantly decreases serum total cholesterol and increases HDL level compared to non-treatment diabetic rats(t=6.87 、 3.37, P<0.05). Non-diabetic animal showed a normal retina, all cell layers of retina were clear and neatly arranged. In the DR group, cells were disorganized after DM modeling. Obvious vacuolated、hemorrhage、inflammatory cell infiltration in ganglionic layers were observed. The Niaspan-treated retina significantly attenuated the retina edema, hemorrhage and ganglionic layers were neatly arranged. Evans blue was shown to be confined to the retinal blood vessels without any leakage occurring in control rats. The dye was shown to leak from the vessels to the surrounding tissue in diabetic rats(t=24.712, P<0.05). The Niaspan treatment also significantly prevented BRB breakdown in diabetic rats when compared to untreated diabetic animals(t=16.414, P<0.05). There are significant reduced cell number of retinal ganglion cell layer(RGCL) and increased the numbers of TUNEL(+) cells in the GCL of the diabetic retina compared with to CON retina(t=18.28, P<0.05). Treatment with Niaspan was able to significantly prevent the reduction of retinal cells and decrease retinal cell apoptosis compared with that of the diabetic retina(t=6.17, P<0.05). Immunohistochemistry staining shows that there was a significant increase in VEGF/VEGFR、VCAM-1、TNF-α、CD45 in diabetic retina when compared to control rats(t=12.02、12.88、11.24、15.60、7.64, P<0.05);treatment with Niaspan significantly decreases VEGF/VEGFR、VCAM-1、TNF-α、CD45 expressions in retina when compared to non-treatment diabetic rats(t=6.44、11.58、7.49、8.83、3.82,P<0.05). there was a significant decreases in Occludin and Claudin expressions in diabetic retina when compared to control rats(t=4.36、13.05, P<0.05); treatment with Niaspan significantly increases Occludin and Claudin expressions in the vessel of retina when compared to non-treatment diabetic rats(t=3.62、7.30,P<0.05). Treatment with Niaspan significantly increase Tie-2 expression in the retina when compared to non-treatment diabetic rats(t=3.79,P<0.05). Western blot shows that there was a significant increase in VEGF/VEGFR、VCAM-1/ICAM-1、TNF-α/NF-κB、IL-6、i NOS in diabetic retina when compared to control rats(t=5.45、3.51、7.44、19.48、6.58、8.95、11.11、6.12, P<0.05);treatment with Niaspan significantly decreases VEGF/VEGFR、VCAM-1/ICAM-1、TNF-α/NF-κB、IL-6、i NOS expressions in retina when compared to non-treatment diabetic rats(t=2.63、2.05、4.25、16.92、5.02、4.40、10.13、5.45,P<0.05). There was a significant decrease in ZO-1 in diabetic retina when compared to control rats(t=14.10, P<0.05);treatment with Niaspan significantly decreases ZO-1expression in retina when compared to non-treatment diabetic rats(t=5.77,P<0.05). RT-PCR shows there was a significant decrease in Occludin, Claudin-5, ZO-1 and micro RNA-126 in diabetic rats when compared to control rats(t=11.422、12.638、12.060、15.53, P<0.05). Treatment with Niaspan was able to significantly prevent the decrease in Occludin, Claudin-5、ZO-1 and micro RNA-126 when compared to diabetic rats(t=5.278、3.952、8.030、7.18,P<0.05). There was a significant increase in ICAM-1、NF-κB、i NOS m RNA in diabetic retina when compared to control rats(t=11.422、12.638、12.060, P<0.05);treatment with Niaspan significantly decreases ICAM-1、NF-κB、i NOS m RNA expressions in retina when compared to non-treatment diabetic rats(t=5.278 、 3.952 、 8.030, P<0.05).(2) Hyperglycemia significantly decreases mi R-126 expression in the hyperglycemia-induced cells and mi R-126-inhibitor pretreatment cells compared to the normal control cells(t=13.43、7.16,P<0.05). However, Niaspan intervention markedly increases the mi R-126 expression in the HREC(t=4.62, P<0.05). Western blot shows that hyperglycemia-induced cells increases the expression of VEGF compared to the CON(t=8.54,P<0.05). However, treatment with Niaspan decreases VEGF expression and increases Ang-1 expression(t=7.76 、 16.25, P<0.05). Pretreatment with mi R-126-inhibitors bring down the reduction of VEGF and the increase of Ang-1 expression which resulted in by Niaspan in hyperglycemia-induced HREC cells(t=6.95、4.24,P<0.05).Conclusion(1) STZ can induced the DR model successfully.(2)Niaspan can relive DR clinically,alleviate the immunological damage in the retina.(3) Niaspan has the protective effect of blood-retina barrier, the mi R-126 pathway may contribute to Niaspan treatment induced benefit effects.(4) Niaspan treatment ameliorates DR via inhibiting inflammatory effects and the NF-κB pathway may contribute to Niaspan treatment induced benefit effects. |
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