从心论治方改善ApoE^(-/-)小鼠动脉粥样硬化及上调ACE2蛋白表达

Congxin Lunzhi Fang improves aortic atherosclerosis and regulates ACE2 expression in ApoE^(-/-)mice

目的探讨从心论治方(CXLZF)对ApoE^(-/-)小鼠动脉粥样硬化(AS)损伤的作用机制。方法采用高脂饮食喂养ApoE^(-/-)小鼠12周建立AS模型,随机分为模型组,CXLZF低(4.5 g·kg^(-1)·d^(-1))、中(9 g·kg^(-1)·d^(-1))、高(18 g·kg^(-1)·d^(-1))剂量组,辛伐他汀(5 mg·kg^(-1)·d^(-1))组,同时以普通饮食喂养C57BL/6小鼠作为对照组,6只/组。各组小鼠给予相应药物灌胃,对照组、模型组灌胃等体积生理盐水,干预12周。分别采用油红O、Masson、HE染色法观察斑块及肝脏病变情况,检测血脂、血糖及肝功指标;Westernblotting法检测血管紧张素转换酶2(ACE2)、E-选择素(E-selectin)蛋白表达,免疫荧光法对ACE2蛋白表达进行定位。在体外,以CXLZF灌胃SD大鼠制作含药血清、生理盐水灌胃大鼠制作对照血清后,采用100μg/m Lox-LDL干预人脐静脉内皮细胞(HUVECs)建立细胞损伤模型,CXLZF含药血清(5%、10%、20%)干预人脐静脉内皮细胞,对照组、模型组细胞给予10%对照血清。检测细胞ACE2表达。结果CXLZF低、中、高剂量干预均有效缩小AS小鼠主动脉斑块面积,增加斑块稳定性;同时,CXLZF干预显著降低AS小鼠血清甘油三酯、总胆固醇、低密度脂蛋白胆固醇水平(P<0.05),高剂量组降脂效果最佳;CXLZF干预还可降低AS小鼠血清天门冬氨酸转氨酶和丙氨酸转氨酶含量(P<0.05)、改善肝脏脂肪变性。CXLZF干预显著上调AS小鼠主动脉ACE2、下调E-selectin表达(P<0.05)。细胞实验中,CXLZF含药血清(5%、10%、20%)显著上调氧化低密度脂蛋白干预人脐静脉内皮细胞的ACE2表达(P<0.05)。结论CXLZF可有效减轻小鼠AS损伤,可能与调控ACE2蛋白有关。

Objective To investigate the effect of Congxin Lunzhi Fang(CXLZF)for improving atherosclerosis(AS)and explore its therapeutic mechanism.Methods Thirty ApoE^(-/-)mice were fed on high-fat diet(HFD)to establish AS models,which were randomized equally into model group,low-,medium-and high-dose CXLZF groups(daily dose 4.5,9,and 18 g/kg,respectively),and simvastatin(5 mg/kg daily)group,with 6 male C57BL/6 mice fed a normal diet as the control group.The mice in the control and model groups were gavaged with normal saline,and those in the treatment groups were gavaged with the indicated drugs for a treatment course of 12 weeks.The changes of atherosclerotic plaque and liver pathologies were observed using oil-red O,HE and Masson staining,and blood lipid profiles and serum levels of glucose,Alanine aminotransferase(ALT)and Aspartate aminotransferase(AST)of the mice were examined.The protein expressions of ACE2 and E-selectin were determined with Western blotting.Drug-containing serum was collected from SD rats gavaged with CXLZF for treatment of human umbilical vein endothelial cells(HUVECs)with oxidized low-density lipoprotein(ox-LDL)-induced injury,and the protein expression of ACE2 in the cells was determined.Results In ApoE^(-/-)mice,CXLZF at all the 3 doses significantly reduced the plaque area and increased the collagen content in atherosclerotic plaque,indicating improved plaque stability.CXLZF treatment,especially at the high dose,also significantly reduced the levels of serum lipids(P<0.05).CXLZF significantly lowered serum ALT and AST levels,mitigated hepatocyte steatosis(P<0.05),increased the protein expression of ACE2 and down-regulated E-selectin expression(P<0.05)in the mice.In HUVEC cultures,CXLZFcontaining serum(5%,10%,and 20%)significantly up-regulated the cellular expression of ACE2 protein(P<0.05).Conclusion CXLZF relieves aortic atherosclerosis in ApoE^(-/-)mice possibly by regulating ACE2 protein.