腺相关病毒感染GDF11对2型糖尿病大鼠血管损伤的保护作用

Protective effect of adeno-associated virus sense transfection of GDF11 on vascular injury in type 2 diabetic rats

目的探讨腺相关病毒感染上调体内生长和分化因子11(GDF11)表达水平对2型糖尿病大鼠(T2DM)主动脉损伤的影响。方法随机选取9周龄雄性SD大鼠,采用高糖高脂饲料加小剂量链脲佐菌素(STZ)联合诱导,建立T2DM模型。正常大鼠和糖尿病模型大鼠随机分为5组:空白对照组(Control组)、阴性病毒对照组(NC组)、GDF11腺相关病毒组(GDF11组)、糖尿病组(DM组)、糖尿病+GDF11腺相关病毒组(DM+GDF11组)。喂养8周后,检测大鼠血清中胰岛素(INS)、晚期糖基化终末产物(AGES)、重组生长转化因子11(GDF11)、总胆固醇(T-CHO)、三酰甘油(TG)、低密度脂蛋白(LDL-C)、高密度脂蛋白(HDL-C)、不对称二甲基精氨酸(ADMA)、丙二醛(MDA)浓度;采用过碘酸雪夫氏染色(PAS染色)观察糖原沉积部位,苏木精-伊红染色(HE)观察血管损伤情况,扫描电镜观察血管内皮细胞和血管弹性纤维损伤情况,蛋白印迹和免疫组化检测血管损伤相关蛋白的表达水平。结果在动物实验中与Control组相比,生化指标提示:DM组大鼠血清中AGES、T-CHO、TG、LDL-C和MDA浓度升高(P<0.05),INS、GDF11、HDL-C、ADMA显著低于Control组(P<0.05),DM+GDF11组AGES和HDL-C与DM组无明显差异,T-CHO、TG、LDL-C和MDA相比较DM组降低(P<0.05),INS、GDF11和ADMA相比较DM组升高(P<0.05)。病理染色提示:DM组PAS染色提示糖原颗粒在主动脉内皮及内皮下沉积,HE染色观察到主动脉中膜增厚,内皮细胞及弹性纤维断裂走形不规则,电镜观察到DM组血管内皮损伤,弹性纤维断裂,DM+GDF11组这些变化有所减轻。蛋白印迹和免疫组化表示内皮细胞相关蛋白在DM组中表达下降(P<0.05),间充质标志物在DM组中表达升高(P<0.05),DM+GDF11组中这些蛋白有所回调,且差异有统计学意义(P<0.05)。结论提高体内GDF11表达水平可以改善糖尿病导致的主动脉血管损伤,推断可能与其抑制内皮间充质转化保护血管内皮细胞的功能从而改善血管损伤有关。

Objective To explore the effect of adeno-associated virus sense transfection up-regulating the expression level of the growth and differential factor 11(GDF11)in vivo on aortic injury in type 2 diabetic mellitus rats(T2DM).Methods Nine-week-old male SD rats were randomly selected to establish a T2DM model by using high-sugar and high-fat chow plus small-dose streptozotocin(STZ)combined induction.Both normal rats and diabetic model rats were randomly divided into five groups:blank control group(Control group),negative virus control group(NC group),GDF11 adeno-associated virus group(GDF11 group),diabetic group(DM group),and diabetic+GDF11 adeno-associated virus group(DM+GDF11 group).After 8 weeks of feeding,the serum concentrations of insulin(INS),advanced glycosylation end products(AGES),recombinant growth transforming factor 11(GDF11),total cholesterol(T-CHO),triglycerides(TG),low-density lipoproteins(LDL-C),high-density lipoproteins(HDL-C),asymmetric dimethylarginine(ADMA),and malondialdehyde(MDA)were assayed in the rats;periodic acid-schiff stain(PAS stain)was used to observe the sites of glycogen deposition,and hematoxylin-eosin staining(HE)was used to observe vascular damage.Scanning electron microscopy was used to observe the damage of vascular endothelial cells and vascular elastic fibers,and protein blotting and immunohistochemistry were used to detect the expression levels of vascular injury-related proteins.Protein blotting and immunohistochemistry were used to detect the expression levels of vascular injury-related proteins.Results The biochemical indexes showed that the serum concentrations of AGES,T-CHO,TG,LDL-C and MDA were higher in the DM group than those in the Control group(P<0.05),the concentrations of INS,GDF11,HDL-C and ADMA were significantly lower than those in the Control group(P<0.05),and the concentrations of AGES and HDL-C were not significantly lower in the DM+GDF11 group compared with the DM group(P<0.05).HDL-C was not significantly different from the DM group,and several other data were improved(P<0.05).Pathological staining suggested that PAS staining in the DM group suggested that glycogen particles deposited in the endothelium and subendothelium of the aorta,HE staining observed thickening of the aortic mesentery,endothelial cells and elastic fibers broke off in an irregular alignment,and electron microscopy observed endothelial damage in the vasculature and elastic fibers broke off in the DM group,and these changes attenuated in the DM+GDF11 group.Protein blotting and immunohistochemistry indicated that the expression of endothelial cell-associated proteins decreased in the DM group(P<0.05),and mesenchymal markers elevated in the DM group(P<0.05),these proteins were regressed in the DM+GDF11 group,and the difference was statistically significant(P<0.05).Conclusion Increasing the expression level of GDF11 in vivo can improve aortic vascular injury caused by diabetes mellitus,which is inferred that it may be related to the inhibition of endothelial mesenchymal transition to protect the function of vascular endothelial cells and thus improve vascular injury.