荠菜提取物抑制2型糖尿病小鼠血管损伤的作用研究

Inhibitory effect of capsella bursa pastoris extract on vascular injury in Type 2 diabetes mellitus mice

目的构建2型糖尿病(T2DM)小鼠及高糖诱导人脐静脉内皮细胞(HUVECs)损伤模型,观察荠菜提取物(CBP)对T2DM血管损伤的作用。方法雄性昆明小鼠60只,随机取10只作为正常对照组,其余小鼠均以高糖高脂饲料结合小剂量链脲佐菌素(STZ)制备T2DM模型小鼠。选取建模成功小鼠30只,随机分为T2DM组、阳性对照(二甲双胍)组和CBP低、中、高剂量组(0.75、1.5、3 g·kg^(-1)),每组6只,灌胃8周后,检测各组小鼠空腹血糖(FBG)、低密度脂蛋白(LDL)、甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白(HDL)及NO含量,ELISA法检测血清中胰岛素、超氧化物歧化酶(SOD)、丙二醛(MDA)的含量,Real-time PCR法检测小鼠胸主动脉Caspase-3 mRNA的表达。建立高糖诱导HUVECs损伤模型,分为正常对照组、高糖组、溶剂对照组、高糖+CBP(0.25、0.5、1 mg·mL-1)组,MTT法检测HUVECs的活性,荧光探针DCFH-DA测定细胞内活性氧(ROS)水平、Griess Reagent法检测细胞上清液中NO的含量、Hoechst染色法检测细胞凋亡。结果动物实验中,与对照组比较,T2DM组FBG、胰岛素抵抗指数(HOMA-IR)、LDL、TC、TG、MDA含量及血管组织中Caspase-3 mRNA的表达均升高(P<0.05),胰岛素敏感指数(HOMA-IS)、HDL、SOD、NO含量下降(P<0.05);各剂量CBP组对T2DM小鼠的FBG、HOMA-IR、HOMA-IS无明显改善作用;与T2DM组比较,各剂量CBP组TC、TG、MDA含量下降(P<0.05),SOD含量上升(P<0.05),CBP中、高剂量组LDL含量下降、HDL及NO含量升高(P<0.05),CBP高剂量组Caspase-3 mRNA的表达下降(P<0.05)。细胞实验中,与对照组比较,高糖组可导致HUVECs活性及NO含量下降(P<0.01),细胞凋亡率及ROS水平上升(P<0.01);与高糖组比较,CBP组能改善高糖导致的细胞活性及ROS水平下降的现象,减少细胞凋亡率(P<0.05),NO含量升高(P<0.05)。结论CBP可抑制T2DM小鼠的血管损伤,其机制可能与其能改善血脂、减轻氧化应激、抑制血管内皮细胞凋亡有关。

Objective To establish an injury model of human umbilical vein endothelial cells(HUVECs)induced by high glucose in type 2 diabetes mellitus(T2DM)mice and to observe the effect of capsella bursa pastoris extract(CBP)on vascular injury in T2DM mice.Methods 60 male Kunming mice were randomly selected as normal control group.The rest of the mice were fed with high glucose and high fat diet combined with low-dose streptozotocin(STZ)to prepare type 2 diabetes mellitus(T2DM)model mice.After successful modeling,30 mice were randomly divided into model group(T2DM),positive control group(metformin)and CBP low-dose,medium-dose and high-dose groups(0.75、1.5、3 g·kg^(-1)),with 6 mice in each group.After 8 weeks of intragastric administration,the contents of fasting blood glucose,low density lipoprotein(LDL),triglyceride(TG),total cholesterol(TC),high density lipoprotein(HDL)and NO were detected.The levels of insulin,SOD and MDA in serum were detected by ELISA,and the expression of Caspase-3 mRNA in thoracic aorta was detected by Real-time PCR.HUVECs injury model induced by high glucose was established and divided into normal control group,high glucose injury group,solvent control group and high glucose+CBP(0.25,0.5,1 mg·mL-1)group,The activity of HUVECs was detected by MTT assay,intracellular reactive oxygen species(ROS)level was determined by fluorescent probe DCFH-DA,the content of NO in cell supernatant was detected by Griess Reagent method,and cell apoptosis was detected by Hoechst staining method.Results In the animal experiment,compared with the control group,the contents of FBG,insulin resistance index(HOMA-IR),LDL,TC,TG,MDA and the expression of Caspase-3 mRNA in vascular tissue in T2DM group were significantly increased(P<0.05),while the contents of insulin sensitivity index(HOMA-IS),HDL,SOD and NO were significantly decreased(P<0.05);All mice in CBP groups showed no significant improvement of FBG,HOMA-IR and HOMA-IS.Compared with T2DM group,the contents of TC,TG and MDA in CBP groups were decreased(P<0.05),SOD content was increased(P<0.05),LDL content was decreased,HDL and NO content were increased in CBP medium-dose and high-dose groups(P<0.05),and the expression of Caspase-3 mRNA was decreased in CBP high-dose group(P<0.05).In cell experiment,compared with the control group,the activity of HUVECs and the content of NO were decreased in high glucose group(P<0.01),and the apoptosis rate and ROS level were increased in high glucose group(P<0.01).Compared with high glucose group,CBP groups could improve the decrease of cell activity caused by high glucose.The apoptosis rate and ROS level were decreased(P<0.05),and the content of NO was increased(P<0.05).Conclusion CBP can inhibit vascular injury in T2DM mice,and the mechanism may be related to the improvement of blood lipid,reduction of oxidative stress,and inhibition of vascular endothelial cell apoptosis.