达格列净对动脉粥样硬化的影响以及与钠氢交换体1相关机制  

Effect of dapagliflozin on atherosclerosis and the mechanism related to sodium hydrogen exchanger 1

作　　者：张书萍 莫显刚[1,2] 张诗悦 陈举海 王兰 杨卉 刘大男 ZHANG Shuping;MO Xiangang;ZHANG Shiyue;CHEN Juhai;WANG Lan;YANG Hui;LIU Danan(Guizhou Medical University,Guiyang 550004;China.2.Comprehensive Ward,the Affiliated Hospital of Guizhou Medical University,Guiyang 550004;.3.Department of Cardiovascular Medicine,the Affiliated Hospital of Guizhou Medical University,Guiyang 550004)

机构地区：[1]贵州医科大学,贵阳550004 [2]贵州医科大学附属医院综合病房,贵阳550004 [3]贵州医科大学附属医院心血管内科,贵阳550004

出　　处：《中国比较医学杂志》2022年第9期10-18,共9页Chinese Journal of Comparative Medicine

基　　金：国家自然科学基金(82160097,31660288)。

摘　　要：目的 探讨达格列净对载脂蛋白E敲除(ApoE^(-/-))小鼠动脉粥样硬化(AS)形成的影响以及与钠氢交换体1(NHE1)相关的机制。方法 6周龄雄性ApoE^(-/-)小鼠24只,随机分为普通饮食组、高脂饮食组、高脂饮食+达格列净组(10 mg/(kg·d))以及高脂饮食+格列美脲组(0.5 mg/(kg·d))。8周后,HE染色检测小鼠主动脉的AS斑块情况,定量PCR及免疫印迹法检测主动脉钠葡萄糖协同转运蛋白2(SGTL2)表达,免疫组化法检测主动脉NHE1表达。进而给予达格列净(10μmol/L)、阿米洛利(20μmol/L)以及脂多糖(100 ng/mL)干预小鼠巨噬细胞系RAW 264.7细胞处理24 h,免疫印迹法检测NHE1蛋白表达以及SNARF-1/AM荧光法检测NH_(4)Cl诱导pH值回复率(NHE1活性);酶联免疫吸附法检测TNF-α、IL-1β、IL-6、IL-10分泌情况。结果 高脂饮食+达格列净组主动脉的AS斑块面积显著低于高脂饮食组(P<0.05);各组主动脉斑块内均极低表达SGLT2(P<0.05);高脂饮食+达格列净组内斑块NHE1表达较高脂饮食组下降(P<0.05)。LPS+达格列净组RAW.7细胞的NHE1蛋白水平明显低于LPS组(P<0.05);SNARF-1荧光法提示达格列净处理后细胞内pH值降低(P<0.05),细胞NHE1活性显著降低(P<0.05);与LPS组相比,LPS+达格列净组细胞上清TNF-α、IL-1β、IL-6含量显著减少(P<0.05),IL-10含量显著增加(P<0.05)。结论 达格列净通过抑制NHE1表达及其活性抑制AS斑块发展及细胞因子释放。Objective To investigate the effect of dapagliflozin on the formation of atherosclerosis(AS) in apolipoprotein E knockout(ApoE^(-/-)) mice and the mechanism associated with sodium-hydrogen exchanger 1(NHE1).Methods 24 6-week-old male ApoE^(-/-)mice were randomly divided into ordinary diet group,high-fat diet group,high-fat diet+dapagliflozin group(10 mg/(kg·d) gavage) and high-fat diet+glimepiride group(0.5 mg/(kg·d) gavage).AS plaques in mouse aorta was observed by using HE staining after 8 weeks.The expression of sodium-glucose cotransporter 2(SGTL2) in the aorta was measured by quantitative PCR and Western blot.NHE1 expression was detected by immunohistochemistry.Then,mouse macrophage cell line RAW 264.7 cells were treated with dapagliflozin(10 μmol/L),amiloride(20 μmol/L) and lipopolysaccharide(100 ng/mL) for 24 h.The expression of NHE1 protein was analyzed by Western blot.The recovery rate(NHE1 activity) from the NH_(4)Cl-induced acid load was assayed by SNARF-1/AM fluorescence method.TNF-α,IL-1β,IL-6,IL-10 secretion were determined by enzyme-linked immunosorbent assay.Results The AS plaque area in the aorta of the high-fat diet+dapagliflozin group was significantly lower than that of the high-fat diet group(P<0.05).The expression of SGLT2 in the aortic plaques of each group was significantly decreased(P<0.05).The plaque NHE1 expression in the high-fat diet+dapagliflozin group was lower than that in the high-fat diet group(P<0.05).Compared with the LPS group,the LPS+dapagliflozin group had a significantly lower NHE1 protein level in the RAW 264.7 cells(P<0.05).SNARF-1 fluorescence assay indicated dapagliflozin inhibited NHE1 activity with decreasing intracellular pH(P<0.05).ELISA showed that the contents of TNF-α,IL-1β and IL-6 in the cell supernatant were significantly decreased,whereas the content of IL-10 was significantly increased(P<0.05).Conclusions Dapagliflozin inhibits AS plaque development and cytokine release by inhibiting NHE1 expression and its activity.

关 键 词：动脉粥样硬化 钠-葡萄糖协同转运蛋白2 达格列净 钠氢交换体1 脂多糖 ApoE^(-/-)小鼠 

分 类 号：R-33[医药卫生]