内脂素抑制游离胆固醇过载血管平滑肌细胞的自噬并促进脂质沉积

Visfatin inhibits autophagy and promotes lipid accumulation in free cholesterol-overloading vascular smooth muscle cells

目的探讨内脂素对游离胆固醇(FC)过载血管平滑肌细胞(VSMCs)自噬及脂质代谢的影响。方法体外培养人主动脉VSMCs(CRL-1999),通过与甲基环糊精包被的胆固醇(Chol∶MβCD)共孵育构建VSMCs源性泡沫细胞模型,再结合酰基辅酶A:胆固醇酰基转移酶(ACAT)特异性阻断剂Sandoz58035,建立FC过载VSMCs模型。油红O染色及胞内胆固醇检测确定造模成功后,蛋白质印迹法(Western blot)检测自噬体膜蛋白微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)的相对表达水平,激光共聚焦显微镜下观察绿色荧光蛋白(GFP)-LC3的表达评估FC过载VSMCs模型的自噬水平。以不同浓度的内脂素(0、60、120、240μg/L)作用48 h及240μg/L的内脂素不同时间(0、12、24、48 h)作用于FC过载VSMCs,Western blot检测LC3-Ⅱ蛋白的表达,转染GFP-LC3后,激光共聚焦显微镜下观察GFP-LC3的表达,计算绿色荧光点数,胆固醇检测评估胞内脂质代谢情况。多组间差异采用单因素方差分析,组间进一步两两比较采用LSD-t检验。结果FC过载VSMCs的LC3-Ⅱ蛋白的相对表达水平明显高于VSMCs源性泡沫细胞(0.32±0.06比0.21±0.03,t=3.875,P<0.05)和正常VSMCs(0.32±0.06比0.10±0.04,t=7.169,P<0.05),差异均有统计学意义;转染GFP-LC3的FC过载VSMCs胞浆中的绿色荧光点数明显高于VSMCs源性泡沫细胞[(71.34±7.79)个/细胞比(49.67±7.12)个/细胞,t=5.031,P<0.05]和正常VSMCs[(71.34±7.79)个/细胞比(12.10±3.41)个/细胞,t=17.100,P<0.05],差异均有统计学意义,FC过载VSMCs的自噬现象更为明显。此外,内脂素可呈浓度和时间依赖性抑制FC过载VSMCs内LC3-Ⅱ蛋白的相对表达水平,转染GFP-LC3后,内脂素可使FC过载VSMCs内的绿色荧光点数呈浓度和时间依赖性减少,而胞内的脂质沉积程度随着内脂素作用浓度及时间的增加而明显加重。结论内脂素可呈浓度和时间依赖性抑制FC过载VSMCs的自噬水平,并促进脂质沉积。

Objective To explore the effect of visfatin on autophagy and lipid metabolism in free cholesterol(FC)-overloading vascular smooth muscle cells(VSMCs).Methods Human primary VSMCs(CRL-1999)were cultured in vitro and incubated with cholesterol:methyl-β-cyclodextrin(Chol∶MβCD)complexes to establish the VSMCs-derived foam cells model.The FC-overloading VSMCs model were then established with Chol∶MβCD accompanied with the acyl-coenzyme A:cholesterol acyltransferase(ACAT)inhibitor Sandoz58035.After confirming the success of modeling by oil red O staining and cholesterol detection,Western blottiing was used to detect the protein expression of autophagy membrane protein microtubule-associated protein 1 light chain 3-Ⅱ(LC3-Ⅱ),and the expression of green fluorescent protein(GFP)-LC3 was observed by confocal laser microscopy to evaluate the level of autophagy in FC-overloading VSMCs.Furthermore,the visfatin with different dose(0,60,120,240μg/L for 48 h)and different time(0,12,24,48 hof 240μg/L)were used to intervene FC-overloading VSMCs,and the expression of LC3-Ⅱin each group was then detected by Western blotting.After transfection of GFP-LC3,the expression of GFP-LC3 was observed by confocal laser microscopy in FC-overloading VSMCs and the number of green fluorescence spots was calculated.The intracellular lipid metabolism was evaluated by cholesterol detection.Results Western blotting showed that the protein expression of LC3-Ⅱin FC-overloading VSMCs was significantly higher than that in VSMCs-derived foam cells(0.32±0.06 vs.0.21±0.03,t=3.875,P<0.05)and normal VSMCs(0.32±0.06 vs.0.10±0.04,t=7.169,P<0.05).The number of green fluorescence spots in FC-overloading VSMCs was significantly higher than that in VSMCs-derived foam cells[(71.34±7.79)/cell vs.(49.67±7.12)/cell,t=5.031,P<0.05]and normal VSMCs[(71.34±7.79)/cell vs.(12.10±3.41)/cell,t=17.100,P<0.05],suggesting that FC-overloading VSMCs had more obvious autophagy.Further research showed that visfatin could inhibit the protein expression of LC3-Ⅱwith some dose-and time-dependent manner in FC-overloading VSMCs.After transfection of GFP-LC3,visfatin could also decrease green fluorescence spots with some dose-and time-dependent manner in FC-overloading VSMCs,whereas the intracellular lipid accumulation was more serious with the increased dose and time of visfatin intervention.Conclusion Visfatin can inhibit the autophagy and promote lipid accumulation in a time-and dose-dependent manner in FC-overloading VSMCs.