血糖波动加剧1型糖尿病大鼠主动脉纤维化的作用机制

Impact and related mechanisms of glucose fluctuations on aortic fibrosis in type 1 diabetic rats

目的探讨血糖波动对糖尿病大鼠主动脉纤维化的影响及其机制。方法雄性SD大鼠24只(8~12周龄)通过腹腔注射链脲霉素建立1型糖尿病大鼠模型,按照随机数字表法分为3组:血糖控制组、血糖未控制组和血糖波动组,每组8只。3周后取大鼠主动脉,Masson染色后光镜观察其改变;用免疫荧光法测定Ⅰ型胶原蛋白(ColⅠ)的表达;用免疫组化法测定Runt相关转录因子2(Runx2)的表达;用实时荧光定量聚合酶链反应法(qRT-PCR)分别测定ColⅠ和Runx2的mRNA表达水平;用Western blot法分别测定ColⅠ、Runx2和核因子κB(NF-κB)的蛋白表达。体外培养原代大鼠主动脉平滑肌细胞,分别以含有5.5和25 mmol/L葡萄糖的培养液培养细胞,即为正常葡萄糖组和高浓度葡萄糖组;通过交替给予含有5.5和25 mmol/L葡萄糖的培养液,每12 h更换1次,培养72 h构建血糖波动细胞模型。3组细胞分别加入0.5μmol/L NF-κB阻滞剂5-(4-氟苯基)-2-脲基噻吩-3-甲酰胺(TPCA-1),用免疫荧光法测定各组细胞ColⅠ的表达;用Western blot法分别测定ColⅠ、Runx2和NF-κB的蛋白表达。结果(1)血糖控制组、血糖未控制组和血糖波动组大鼠主动脉中纤维化占总面积百分比分别为(8.42±0.10)%、(21.30±0.74)%和(44.39±1.09)%(P<0.05);ColⅠ的平均吸光度值分别为11.92±0.88、50.04±3.56和77.52±2.69(P<0.05),其mRNA表达水平为1.00±0.10、2.02±0.28和2.83±0.33(P<0.05),蛋白表达水平为1.05±0.03、2.06±0.32和4.93±0.25(P<0.05)。(2)血糖控制组、血糖未控制组和血糖波动组大鼠主动脉中,Runx2的平均吸光度值分别为150.00±7.35、204.84±2.32和391.48±7.13(P<0.05),其mRNA水平分别为1.02±0.02、1.27±0.04和2.18±0.12(P<0.05),蛋白表达水平分别为1.03±0.01、2.34±0.36和4.52±0.75(P<0.05)。(3)血糖控制组、血糖未控制组和血糖波动组大鼠主动脉NF-κB的蛋白表达水平分别为1.02±0.01、1.96±0.13和2.64±0.21(P<0.05)。(4)加入TPCA-1后,高浓度葡萄糖组和波动葡萄糖组ColⅠ和Runx2的蛋白表达均较未加TPCA-1明显减少(P<0.05)。结论血糖波动可能通过激活NF-κB上调Runx2的表达,从而加剧糖尿病大鼠主动脉纤维化进程。

Objective To investigate the impact and related mechanisms of glucose fluctuations on aortic fibrosis in rats with type 1 diabetes mellitus.Methods After injection of streptozotocin(STZ),male Sprague Dawley(SD)(8-12 weeks)rats(n=24)were randomly divided into three groups in accordance with the random number table:controlled STZ-induced diabetes(C-STZ)group(n=8);uncontrolled STZ-induced diabetes(U-STZ)group(n=8);STZ-induced diabetes with glucose fluctuations(STZ-GF)group(n=8).After three weeks,rats were sacrificed and aorta was obtained,aortic fibrosis was detected by Masson trichrome staining.The expression of collagen type 1(collagenⅠ)was tested by immunofluorescence.The expression of runt-related transcription factor 2(Runx2)was tested by immunohistochemistry.The mRNA levels of collagenⅠand Runx2 were detected by quantitative real-time PCR(qRT-PCR).The protein expressions of collagenⅠ,Runx2 and nuclear factor(NF)-κB were determined by Western blot.Primary rat aortic smooth muscle cells(VSMCs)were cultured in three conditions:normal glucose(NG),high glucose(HG)and glucose fluctuations(GF).Cells in GF group were incubated for 72 hours with glucose alternating between 5.5 and 25 mmol/L every 12 hours.TPCA-1,the inhibitor of NF-κB,the expression of collagenⅠin different groups of cells was tested by immunofluorescence.The protein expressions of collagenⅠ,Runx2 and NF-κB were also determined by Western blot.Results(1)The quantitative ratios of the area of fibrosis in the C-STZ group,U-STZ group,STZ-GF group were(8.42±0.10)%,(21.30±0.74)%and(44.39±1.09)%(P<0.05),respectively.The means of integral optical density(IOD)of collagenⅠin the three groups were 11.92±0.88,50.04±3.56 and 77.52±2.69,respectively(P<0.05).The mRNA levels of collagenⅠin the three groups were 1.00±0.10,2.02±0.28 and 2.83±0.33,respectively(P<0.05).The protein expressions of collagenⅠin the three groups were 1.05±0.03,2.06±0.32 and 4.93±0.25,respectively(P<0.05).(2)The average IOD of Runx2 in the three groups were 150.00±7.35,204.84±2.32 and 391.48±7.13,respectively(P<0.05).The mRNA levels of Runx2 in the three groups were 1.02±0.02,1.27±0.04 and 2.18±0.12,respectively(P<0.05).The protein expressions of Runx2 in the three groups were 1.03±0.01,2.34±0.36 and 4.52±0.75,respectively(P<0.05).(3)The protein expressions of NF-κB in the three groups were 1.02±0.01,1.96±0.13 and 2.64±0.21,respectively(P<0.05).(4)In vitro,application of inhibitor of NF-κB reversed glucose fluctuations-induced upregulation of protein levels of ColⅠand Runx2(P<0.05).Conclusion Glucose fluctuations could aggravate aortic fibrosis through activating Runx2 via NF-κB signaling pathways.