柚皮苷调控PI3K/Akt/eNOS通路控制2型糖尿病大鼠脑血管内皮氧化损伤的作用探讨

Naringin regulates PI3K/Akt/eNOS pathway to control cerebral vascular endothelial oxida⁃tive damage in type 2 diabetic rats

目的探讨柚皮苷对2型糖尿病大鼠脑血管内皮氧化损伤的治疗效果,以及柚皮苷对3-磷酸肌醇激酶(PI3K)/蛋白激酶B(AKT)/内皮型一氧化氮(eNOS)信号通路的调控作用。方法 2019年3月15日至2020年5月20日,将SD大鼠(南京医科大学实验动物中心)按随机字母表法分为4组,对照组大鼠给予标准饲料,其他组给予高糖高脂饲料。通过腹腔注射60 mg/kg的链脲佐菌素(STZ)建立2型糖尿病模型,柚皮苷组大鼠灌胃100 mg/kg的柚皮苷溶液,柚皮苷+LY294002组大鼠在灌胃100mg/kg的柚皮苷溶液的基础上腹腔注射100 mg/kg的PI3K抑制剂LY294002。治疗8周后,分别检测各组大鼠空腹血糖、一氧化氮(NO)、血脂代谢指标[总胆固醇(TC)、三酰甘油(TG)、高密度脂蛋白胆固醇(HDL-C)和低密度脂蛋白胆固醇(LDL-C)]、脑组织中丙二醛(MDA)和超氧化物歧化酶(SOD)水平。苏木精-伊红(HE)染色评价脑组织病理改变。蛋白质印迹法(Western blotting)检测脑组织中PI3K/Akt/eNOS通路的活化情况。结果柚皮苷组的空腹血糖(13.46±1.09)mmol/L、TC(1.65±0.13)mmol/L、TG(2.89±0.23)mmol/L、LDL-C(1.59±0.13)mmol/L、MDA(55.17±4.24)U/mgprot水平均低于模型组[空腹血糖(25.32±2.04)mmol/L、TC(2.81±0.23)mmol/L、TG(4.36±0.35)mmol/L、LDL-C(2.97±0.24)mmol/L、MDA(9.37±0.72)nmol/mgprot],而HDL-C(1.08±0.08)mmol/L、血清NO(28.76±2.21)μmol/L、SOD(55.17±4.24)U/mgprot水平均高于模型组[HDL-C(0.65±0.05)mmol/L、血清NO(19.11±1.47)μmol/L、SOD(41.22±3.17)U/mgprot](P<0.05)。柚皮苷组的PI3K蛋白表达水平(0.81±0.06)及Akt(0.91±0.07)和eNOS(0.97±0.07)的磷酸化水平均高于模型组[PI3K蛋白表达水平(0.31±0.02)、(0.21±0.02)和eNOS(0.18±0.01)](P<0.05)。此外,LY294002处理可显著减弱柚皮苷对上述指标的影响(P<0.05)。结论柚皮苷对2型糖尿病大鼠的脑保护作用主要由PI3K/AKT/eNOS信号通路介导。

Objective To explore the therapeutic effect of naringin on cerebral vascular endothelial oxidative damage in type 2 diabetic rats,and the regulating effect of naringin on 3-phosphoinositide kinase(PI3K)/protein kinase B(AKT)/endothelial nitric oxide(eNOS)signaling pathway.Methods From March 15,2019 to May 20,2020,SD rats(Experimental Animal Center of Nanjing Medical University)were assigned into 4 groups according to the random alphabet method.Normal control rats were fed with standard feed,and other groups were fed with high-sugar and high-fat feed.A model of type 2 diabetes was established by intraperitoneal injection of 60 mg/kg streptozotocin(STZ).Rats in the naringin group were administrated with a 100 mg/kg naringin solution.Rats in the naringin+LY294002 group were intraperitoneally injected with a 100 mg/kg PI3K inhibitor LY294002 on the basis of a 100 mg/kg naringin solution.After 8 weeks of treatment,fasting blood glucose,nitric oxide(NO),lipid metabolism indicators[total cholesterol(TC),triglyceride(TG),high density lipoprotein cholesterol(HDL-C)and low density lipoprotein cholesterol(LDL-C)],malondialdehyde(MDA),and superoxide dismutase(SOD)levels in brain tissues were measured in each group.Hematoxylin-eosin(HE)staining was used to evaluate the pathological changes of brain tissues.Western blotting was used to detect the activation of PI3K/Akt/eNOS pathway in brain tissues.Results Fasting blood glucose(13.46±1.09)mmol/L,TC(1.65±0.13)mmol/L,TG(2.89±0.23)mmol/L,LDL-C(1.59±0.13)mmol/L,MDA(55.17±4.24)U/mgprot levels in naringin group were lower than those in the model group[fasting blood glucose,(25.32±2.04)mmol/L;TC,(2.81±0.23)mmol/L;TG,(4.36±0.35)mmol/L;LDL-C,(2.97±0.24)mmol/L;MDA,(9.37±0.72)nmol/mgprot],while HDLC(1.08±0.08)mmol/L,serum NO(28.76±2.21)μmol/L,SOD(55.17±4.24)U/mgprot levels were higher in the naringin group than in the model group[HDL-C,(0.65±0.05)mmol/L;serum NO,(19.11±1.47)μmol/L;SOD,(41.22±3.17)U/mgprot](P<0.05).The PI3K protein expression level(0.81±0.06)and the phosphorylation levels of Akt(0.91±0.07)and eNOS(0.97±0.07)in the naringin group were higher than those in the model group[PI3K,(0.31±0.02);Akt,(0.21±0.02);eNOS,(0.18±0.01)](P<0.05).Additionally,LY294002 treatment significantly reduced the effect of naringin on the above indicators(P<0.05).Conclusion The brain protective effect of naringin on type 2 diabetic rats is mainly mediated by the PI3K/AKT/eNOS signaling pathway.