过氧化物酶体增殖物激活受体γ的小泛素样修饰在糖尿病易化动脉粥样硬化中的作用

The effect of SUMOylated peroxisome proliferator-activated receptorγin the regulation of diabetes mellitus accelerated atherosclerosis

目的探讨内皮细胞和血管组织过氧化物酶体增殖物激活受体γ(PPARγ)的小泛素样修饰在糖尿病加速动脉粥样硬化中的作用。方法2014年9月至2017年1月选取14周龄雄性健康Sprague-Dawley大鼠(SD大鼠)32只,按随机数字表法分为对照假手术组、对照动脉损伤组、糖尿病动脉损伤组和泛素结合酶9(UBC9)转染组,每组8只。制备1型糖尿病大鼠模型,各组均有1只大鼠造模失败被排除。采用超声仪检测损伤侧颈总侧动脉和健侧颈总动脉收缩期和舒张期内径、动脉内膜厚度,计算标化颈总动脉舒张期直径(sCADD);采用HE染色和免疫组织化学染色检测内膜增殖情况,在中膜面积相当的情况下计算内膜面积与中膜面积比值。人脐静脉内皮细胞(HUVEC)在高糖中培养24 h后采用实时反转录-聚合酶链反应(RT-PCR)检测白细胞介素(IL)-8和IL-1βmRNA表达水平,蛋白质印迹法检测动脉组织UBC9的表达水平,小泛素样修饰试剂盒检测PPARγ小泛素样修饰水平。体外培养HUVEC,检测在不同浓度和不同时间的高糖刺激下PPARγ小泛素样修饰水平。在体内和体外利用慢病毒过表达UBC9,再检测PPARγ小泛素样修饰水平。结果对照假手术组、对照动脉损伤组和糖尿病动脉损伤组颈总动脉损伤8周后动脉内膜厚度、内膜增殖面积和内膜面积与中膜面积比值逐渐增加[(0.026±0.018)、(0.084±0.007)和(0.264±0.022)mm,(0.18±0.09)×10^(6)、(0.32±0.06)×10^(6)和(1.64±0.22)×10^(6)μm2,0.345±0.073、0.570±0.080和2.710±0.220],sCADD逐渐降低(0.903±0.084、0.800±0.071和0.330±0.036),差异有统计学意义(F=10.40、9.40、8.20和8.60,P<0.05)。高糖培养HUVEC 24 h后,糖浓度10、20和40 mmol/L时IL-8 mRNA分别为1.00±0.11、3.57±0.22和4.07±0.40,IL-1βmRNA分别为1.00±0.07、3.32±0.29和5.13±0.19,差异有统计学意义(F=73.05和205.80,P<0.05)。糖尿病动脉损伤组动脉组织PPARγ小泛素样修饰水平和UBC9表达水平明显低于对照动脉损伤组(0.46±0.25比1.00±0.21和0.45±0.02比1.00±0.07),差异有统计学意义(P<0.05);两组PPARγ表达水平比较差异无统计学意义(0.94±0.07比1.00±0.04,P>0.05)。糖浓度10、20和40 mmol/L时UBC9表达水平和PPARγ小泛素样修饰水平逐渐降低(0.99±0.05、0.80±0.06和0.62±0.05,1.00±0.05、0.57±0.13和0.55±0.08),差异有统计学意义(F=21.02和14.31,P<0.05),PPARγ表达水平比较差异无统计学意义(1.00±0.03、0.90±0.04和0.91±0.05,F=3.11,P>0.05)。对HUVEC进行0、6、12、24、48 h的20 mmol/L高糖刺激后,UBC9表达水平和PPARγ小泛素样修饰水平逐渐下调(1.00±0.09、0.75±0.05、0.70±0.08、0.38±0.04和0.35±0.03,1.00±0.03、0.86±0.01、0.59±0.01、0.51±0.11和0.35±0.08),差异有统计学意义(F=36.06和33.13,P<0.05),但PPARγ表达水平比较差异无统计学意义(1.00±0.03、1.14±0.02、1.18±0.17、0.98±0.01和1.04±0.05,F=1.90,P>0.05)。在糖尿病大鼠动脉中过表达UBC9后,组织学分析结果显示,对照动脉损伤组、糖尿病动脉损伤组和UBC9转染组UBC9相对表达水平分别为1.53±0.18、1.00±0.22和3.62±0.35,三组比较差异有统计学意义(F=5.64,P<0.05)。超声检测结果显示,对照动脉损伤组、糖尿病动脉损伤组和UBC9转染组颈动脉内膜厚度逐渐增厚[(0.077±0.015)、(0.216±0.007)和(0.125±0.014)mm],差异有统计学意义(F=27.18,P<0.05)。组织学分析结果显示,对照动脉损伤组、糖尿病动脉损伤组和UBC9转染组颈动脉损伤后内膜增殖面积分别为(0.335±0.066)×10^(6)、(1.053±0.103)×10^(6)和(0.544±0.040)×10^(6)μm2,内膜面积与中膜面积比值分别为0.630±0.063、2.030±0.052和0.930±0.100,三组比较差异均有统计学意义(F=13.58和53.96,P<0.05)。结论高血糖下调HUVEC和大鼠动脉组织UBC9表达水平及抑制PPARγ小泛素样修饰的作用,揭示高血糖下调的UBC9和PPARγ小泛素样修饰水平是糖尿病加速动脉粥样硬化的重要机制。

Objective To explore the role of SUMOylaiton of peroxisome proliferator-activated receptorγ(PPARγ)in diabetes mellitus prompted inflammation and atherosclerosis in vascular and endothelial cells.Methods From September 2014 to January 2017,32 Sprague-Dawley rats in 14 weeks-old were divided into sham operated group,artery injured without diabetes group,artery injured with diabetes group and ubiquitin-conjugating enzyme 9(UBC9)transfection group(Group D)by random digits table method with 8 rats each.Model of type 1 diabetes mellitus(T1DM)and rat carotid artery balloon injury was made in the assigned group.One rat was excluded because of model failure in each group.Systolic and diastolic common carotid artery diameter and intimal thickness of injured and healthy common carotid artery were evaluated by vascular ultrasound,and the standardized common carotid artery diastolic diameter(sCADD)was calculated.Histological tests and immunohistochemical staining were performed to evaluate intimal hyperplasia,and the ratio of intimal area to media area was calculated when the media area was equal.Human umbilical vein endothelial cells(HUVEC)were cultured 24 h in high glucose medium with different duration and concentration,and the expression levels of interleukin(IL)-8 and IL-1βmRNA were determined by real time reverse transcription polymerase chain reaction(RT-PCR),the expression level of UBC9 was determined by Western blot method,SUMOylation assay kit was used to evaluate SUMOylation of PPARγ.HUVEC was cultured in vitro and PPAR was stimulated by high glucose at different concentrations and different times PPARγSUMOylation level.UBC9 was overexpressed by lentivirus in vivo and in vitro,and the PPARγSUMOylation level was detected.Results The intimal thickness,intimal area and ratio of intimal area to media area 8 weeks after carotid artery injuring in sham operated group,artery injured without diabetes group and artery injured with diabetes group were increased respectively:(0.026±0.018),(0.084±0.007)and(0.264±0.022)mm;(0.18±0.09)×10^(6),(0.32±0.06)×10^(6)and(1.64±0.22)×10^(6)μm2;0.345±0.073,0.570±0.080 and 2.710±0.220,the sCADD was decreased respectively:0.903±0.084,0.800±0.071 and 0.330±0.036,and there were statistical differences(F=10.40,9.40,8.20 and 8.60;P<0.05).After HUVEC was cultured in high glucose for 24 h,the IL-8 mRNA at sugar concentrations of 10,20 and 40 mmol/L was 1.00±0.11,3.57±0.22 and 4.07±0.40,the IL-1βmRNA was 1.00±0.07,3.32±0.29 and 5.13±0.19,and there were statistical differences(F=73.05 and 205.80,P<0.05).The level of PPARγSUMOylation and UBC9 in artery injured with diabetes group were significantly lower than those in artery injured without diabetes group(0.46±0.25 vs.1.00±0.21 and 0.45±0.02 vs.1.00±0.07),and there were statistical differences(P<0.05);there was no statistical difference in PPARγbetween 2 groups(0.94±0.07 vs.1.00±0.04,P>0.05).The UBC9 and PPARγSUMOylation at sugar concentrations of 0,10,20 and 40 mmol/L were decreased respectively(0.99±0.05,0.80±0.06 and 0.62±0.05;1.00±0.05,0.57±0.13 and 0.55±0.08),and there were statistical differences(F=21.02 and 14.31,P<0.05);there was no statistical difference in PPARγ(1.00±0.03,0.90±0.04 and 0.91±0.05;F=3.11,P>0.05).In HUVEC cultured in high glucose medium(20 mmol/L)for 6,12,24 and 48 h,the UBC9 and PPARγSUMOylation were downregulated progressively(1.00±0.09,0.75±0.05,0.70±0.08,0.38±0.04 and 0.35±0.03;1.00±0.03,0.86±0.01,0.59±0.01,0.51±0.11 and 0.35±0.08),and there were statistical differences(F=36.06 and 33.13,P<0.05);but there was no statistical difference in PPARγ(1.00±0.03,1.14±0.02,1.18±0.17,0.98±0.01 and 1.04±0.05;F=1.90,P>0.05).After overexpression of UBC9 in rats with diabetes,histological analysis showed that UBC9 in artery injured without diabetes group,artery injured with diabetes group and UBC9 transfection group was 1.53±0.18,1.00±0.22 and 3.62±0.35,there was statistical difference(F=5.64,P<0.05).Ultrasonic test results show that in artery injured without diabetes group,artery injured with diabetes group and UBC9 transfection group intimal thickness was increased respectively:(0.077±0.015),(0.216±0.007)and(0.125±0.014)mm,and there was statistical difference(F=27.18,P<0.05).Histological analysis showed that intimal area in artery injured without diabetes group,artery injured with diabetes group and UBC9 transfection group was(0.335±0.066)×10^(6),(1.053±0.103)×10^(6)and(0.544±0.040)×10^(6)μm2,the ratio of intimal area to media area was 0.63±0.063,2.03±0.052 and 0.93±0.100,there were statistical differences(F=13.58 and 53.96,P<0.05).Conclusions Diabetes mellitus could inhibit the PPARγSUMOylaiton and prompt inflammation and atherosclerosis in vascular and endothelial cells.Upregulation of PPARγSUMOylaiton though UBC9 overexpressioncould play a protecting role in diabetes mellitus prompted atherosclerosis.