镇心省睡益智方抗Aβ25-35诱导人脑微血管内皮细胞损伤的作用

Effect of Zhenxin Shengshui Yizhi Fang on Damage of Human Brain Microvascular Endothelial Cells Induced by Aβ25-35

目的:观察镇心省睡益智方(省睡方)水提液对抗β淀粉样蛋白25-35(Aβ25-35)诱导的人脑微血管内皮细胞(HBMEC)损伤的神经保护作用和潜在机制。方法:采用Aβ25-35诱导的HBMEC细胞损伤作为阿尔茨海默病(AD)细胞模型。本研究分为空白组,Aβ25-35组,省睡方水提液低、中、高剂量组(125,250,500 mg·L^-1)。对其进行相关治疗后,采用噻唑蓝(MTT)比色法确定不同浓度药物及Aβ25-35的细胞毒性,采用Hoechst-33258染色观察细胞凋亡情况,采用比色法检测半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)活性,采用蛋白免疫印迹法(Western blot)检测晚期糖基化终产物受体(RAGE),低密度脂蛋白受体相关蛋白(LRP1),葡萄糖转运蛋白1(GLUT1)及葡萄糖转运蛋白3(GLUT3)的表达。结果:与空白组比较,Aβ25-35组细胞活力显著下降(P<0.01),Hoechst-33258染色观察到明亮的蓝色荧光、染色质固缩、呈致密浓染或碎块状致密浓染,颜色有些发白,凋亡细胞百分比显著增加(P<0.01),Caspase-3活性显著增加(P<0.01),RAGE蛋白表达显著增加(P<0.01),LRP1,GLUT1和GLUT3蛋白表达显著下降(P<0.01);与Aβ25-35组比较,省睡方水提液组细胞存活率以剂量依赖性方式显著增加,500 mg·L^-1省睡方水提液组保护作用比其他组更明显(P<0.05),500 mg·L^-1省睡方水提液组及显著抑制凋亡细胞的数量(P<0.01),且显著降低Caspase-3活性(P<0.01),125 mg·L^-1省睡方水提液组RAGE蛋白表达未显著降低,250,500 mg·L^-1省睡方水提液组RAGE蛋白表达显著下降(P<0.01),而各剂量省睡方水提液组LRP1,GLUT1和GLUT3蛋白表达明显增加(P<0.05,P<0.01)。结论:省睡方水提液能够减弱Aβ25-35寡聚体诱导的HBMEC细胞毒性,抑制细胞凋亡,降低Caspase-3活性,降低RAGE蛋白的表达,升高LRP1,GLUT1和GLUT3蛋白的表达,从而降低Aβ在脑内异常聚集和沉积可能是其防治AD的机制。

Objective:To observe the neuroprotective effect and potential mechanism of Zhenxin Shengshui Yizhi Fang(XSF)aqueous extract on human brain microvascular endothelial cells(HBMEC)injury induced by amyloid-βprotein(Aβ)25-35.Method:HBMEC cells damage induced by Aβ25-35was used as Alzheimer’s disease(AD)cell model.The study included control group,Aβ25-35group,and low,medium and high-dose XSF aqueous extract groups(125,250,500 mg·L^-1).After treatment,the cytotoxicity of different concentrations of drugs and Aβ25-35was determined by methyl thiazolyl tetrazolium(MTT)colorimetry.Apoptosis was observed by Hoechst-33258 staining.The activity of Caspase-3 was detected by colorimetry.Western blot was used to detect the expression levels of the receptor of advanced glycation end products(RAGE)and low-density lipoprotein receptorrelated protein(LRP1).Result:Compared with the control group,the cell viability of Aβ25-35group was significantly decreased(P<0.01).Hoechst-33258 staining showed bright blue fluorescence,chromatin condensation,dense staining or fragmentation dense staining,whitening in color,and significant increase of the percentage of apoptotic cells(P<0.01).Caspase-3 activity increased significantly(P<0.01).Western blot showed that RAGE protein expression increased significantly(P<0.01),while low-density lipoprotein receptorrelated protein(LRP1),glucose transporter 1(GLUT1)and GLUT3 protein expressions decreased significantly(P<0.01).Compared with the Aβ25-35group,the cell viability of XSF aqueous extract groups was significantly increased in a dose-dependent manner.The XSF aqueous extract had a more significant protective effect of than the other groups(P<0.05).The XSF aqueous extract group(500 mg·L^-1)significantly inhibited the number of apoptotic cells(P<0.01),but significantly reduced the Caspase-3 activity(P<0.01).RAGE protein expression was not significantly decreased in XSF aqueous extract group(125 mg·L^-1),but significantly decreased in XSF aqueous extract group(250,500 mg·L^-1,P<0.01),while LRP1,GLUT1 and GLUT3 protein expression significantly increased(P<0.05,P<0.01)in a dose-dependent manner.Conclusion:XSF aqueous extract can attenuate the cytotoxicity of HBMEC induced by Aβ25-35oligomer,inhibit apoptosis,decrease caspase-3 activity and RAGE protein expression,increase LRP1,GLUT1 and GLUT3 protein expressions,and reduce the abnormal accumulation and deposition of Aβin the brain,which may be its mechanisms for prevention and treatment of AD.