卵泡抑素样蛋白1对氧化低密度脂蛋白诱导血管平滑肌细胞增殖的影响

doi:10.16138/j.1673-6087.2021.03.007

摘要/Abstract

摘要：

目的：探讨在氧化低密度脂蛋白（oxidized low-density lipoprotein,Ox-LDL）刺激下,卵泡抑素样蛋白1（follistatin like protein 1,FSTL1）在小鼠血管平滑肌细胞（vascular smooth muscle cell,VSMC）表型转化过程中的作用。方法：检测小鼠血管沉积Ox-LDL的病变区域中FSTL1的表达水平,再通过体外实验进一步验证Ox-LDL对FSTL1表达量的影响。运用蛋白质印迹法检测VSMC不同状态下收缩型标志物α-平滑肌肌动蛋白（α-smooth muscle actin,α-SMA）、沉默信息调节蛋白1（silencing information regulatory protein 1,SIRT1）和分泌型标志物骨桥蛋白（osteopontin,OPN）的表达情况。通过增殖实验验证Ox-LDL和FSTL1作用下VSMC的增殖情况。结果：与对照组相比,小鼠Ox-LDL沉积的病变血管区FSTL1表达减少（0.223±0.010比0.097±0.019,t=27.381,P<0.01）。采用Ox-LDL 0、12.5、25、50 mg/L分别刺激 VSMC 24 h, FSTL1表达水平分别为1.330±0.055、0.905±0.027、0.753±0.037和0.243±0.016,Ox-LDL浓度为50 mg/L时,FSTL1表达水平最低（F=260.600,P<0.000 1）。在此基础上用50 mg/L Ox-LDL分别刺激 VSMC 0、6、12、24 h,FSTL1的表达分别为1.383±0.033、0.782±0.047、0.381±0.022和0.230±0.017,Ox-LDL诱导时间为24 h时,FSTL1表达水平最低（F=151.000,P<0.000 1）。Ox-LDL诱导下,VSMC分泌的α-SMA（1.303±0.030比0.493±0.069,P<0.01）和SIRT1（0.993±0.044比0.613±0.030,P<0.01）表达减少,但OPN表达增多（1.001±0.031比2.698±0.001,P<0.01）。在Ox-LDL刺激的基础上加入FSTL1共刺激,结果与Ox-LDL刺激组相反[OPN 2.698±0.002比1.590±0.001（P<0.05）,α-SMA 0.493±0.062比0.653±0.015（P<0.05）,SIRT1 0.613±0.030比1.231±0.011（P<0.05）]。加入SIRT1抑制剂,Ox-LDL+FSTL1的条件下出现VSMC中α-SMA（0.530±0.033比0.283±0.032,P<0.01）和SIRT1（1.056±0.020比0.207±0.021,P<0.01）表达减少,但OPN表达增多(1.643±0.047比3.533±0.100,P<0.01)。Ox-LDL刺激组细胞增殖能力上升（0.870±0.010比1.890±0.020,P<0.01）,但Ox-LDL联合FSTL1组的增殖能力比Ox-LDL刺激组弱（1.890±0.021比1.200±0.023,P<0.05）。通过阻断SIRT1的作用,FSTL1失去抵抗Ox-LDL诱导的平滑肌细胞增殖的能力（1.280±0.033 比 2.030±0.092,P<0.01）。结论：Ox-LDL的刺激下调FSTL1的表达。通过回补FSTL1,能有效抑制Ox-LDL刺激导致的VSMC过度增殖。

关键词: 卵泡抑素样蛋白1, 氧化低密度脂蛋白, 增殖, 表型转化, α-平滑肌肌动蛋白, 沉默信息调节蛋白1, 骨桥蛋白

Abstract:

Objective To investigate the effect of follistatin like protein 1(FSTL1) on oxidized low-density lipoprotein (Ox-LDL)-stimulated the phenotypic transformation of mouse vascular smooth muscle cell (VSMC). Methods Firstly, under Ox-LDL-stimulating, the expression of FSTL1 in VSMC was detected. Secondly, Western blotting was used to detect the signature proteins, α-smooth muscle actin(α-SMA) and osteopontin(OPN). Finally, the proliferation of VSMC was verified by proliferation experiments under the influence of Ox-LDL and FSTL1. Results Comparing to the normal group, the expression of FSTL1 was lower in the abnormal vessel regions, which was filled with Ox-LDL（0.223±0.010 vs. 0.097±0.019, P<0.01）. VSMC was stimulated by Ox-LDL at 0, 12.5, 25 and 50 mg/L for 24 h respectively and the expression level of FSTL1 detected by Western blotting was 1.330±0.055, 0.905±0.027, 0.753±0.037 and 0.243±0.016 accordingly. It showed that the lowest expression level of FSTL1 was observed when Ox-LDL concentration increased to 50 mg/L (F=260.600, P<0.000 1). The expression of FSTL1 was 1.383±0.033, 0.782±0.047, 0.381±0.022 and 0.230±0.017 after 50 mg/L Ox-LDL stimulating VSMC for 0, 6, 12 and 24 h respectively, in which the expression of FSTL1 was the lowest when Ox-LDL induced 24 h(F=151.000, P<0.000 1). In addition, Ox-LDL stimulation reduced the expression of α-SMA(1.303±0.030 vs. 0.493±0.069, P<0.01) and SIRT1(0.993±0.044 vs. 0.613±0.030, P<0.01), while increasing the expression of OPN(1.001±0.031 vs. 2.698±0.001, P<0.01). However, the expression of OPN, α-SMA and SIRT1 showed opposite trend when VSMC was stimulated with both FSTL1 and Ox-LDL（OPN 2.698±0.002 vs. 1.590±0.001, P<0.05; α-SMA 0.493±0.062 vs. 0.653±0.015, P<0.05; SIRT1 0.613±0.030 vs. 1.231±0.011, P<0.05）. Compared with Ox-LDL+FSTL1+DMSO group, VSMC in Ox-LDL+FSTL1+SIRT1 inhibitor group showed reduced α-SMA(0.530±0.033 vs. 0.283±0.032, P<0.01) and SIRT1(1.056±0.020 vs. 0.207±0.021, P<0.01), and increased OPN(1.643±0.047 vs. 3.533±0.100, P<0.01). The proliferation of VSMC was enhanced by Ox-LDL stimulation（0.870±0.010 vs. 1.890±0.020, P<0.01）, while FSTL1 reduced this proliferation（1.890±0.021 vs. 1.200±0.023, P<0.05） via SIRT1（1.280±0.033 vs. 2.030±0.092, P<0.01）. Conclusions Ox-LDL reduced the expression of FSTL1. FSTL1 could mitigate Ox-LDL-stimulated proliferation via SIRT1 effectively.

Key words: Follistatin like protein 1, Oxidized low-density lipoprotein, Proliferation, Phenotypic transformation, α-Smooth muscle actin, Silencing information regulatory protein 1, Osteopontin

中图分类号:

R392

肖凡, 查晴, 刘亚, 杨玲, 叶佳雯, 刘艳. 卵泡抑素样蛋白1对氧化低密度脂蛋白诱导血管平滑肌细胞增殖的影响[J]. 内科理论与实践, 2021, 16(03): 172-177.

XIAO Fan, ZHA Qing, LIU Ya, YANG Ling, YE Jiawen, LIU Yan. Follistatin like protein 1 mitigates oxidized low-density lipoprotein-stimulated phenotypic transformation of mouse vascular smooth muscle cell[J]. Journal of Internal Medicine Concepts & Practice, 2021, 16(03): 172-177.

图/表 7

图1

图2

图3

表1

图4

表2

图5

参考文献 16

[1]	Silva MC, Magalhães TA, Meira ZM, et al. Myocardial fibrosis progression in duchenne and becker muscular dystrophy[J]. JAMA Cardiol, 2017, 2(2): 190-199. doi: 10.1001/jamacardio.2016.4801 URL
[2]	Alshehry ZH, Mundra PA, Barlow CK, et al. Plasma lipidomic profiles improve on traditional risk factors for the prediction of cardiovascular events in type 2 diabetes mellitus[J]. Circulation, 2016, 134(21): 1637-1650. pmid: 27756783
[3]	Hwang JS, Ham SA, Yoo T, et al. Sirtuin 1 mediates the actions of peroxisome proliferator-activated receptor δ on the oxidized low-density lipoprotein-triggered migration and proliferation of vascular smooth muscle cells[J]. Mol Pharmacol, 2016, 90(5): 522-529. pmid: 27573670
[4]	Boucherat O, Peterlini T, Bourgeois A, et al. Mitochondrial HSP90 accumulation promotes vascular remodeling in pulmonary arterial hypertensionn[J]. Am J Respir Crit Care Med, 2018, 198(1): 90-103. doi: 10.1164/rccm.201708-1751OC URL
[5]	Thenappan T, Ormiston ML, Ryan JJ, et al. Pulmonary arterial hypertension: pathogenesis and clinical management[J]. BMJ, 2018, 360: j5492.
[6]	Hu S, Liu H, Hu Z, et al. Follistatin-like 1: a dual regulator that promotes cardiomyocyte proliferation and fibrosis[J]. J Cell Physiol, 2020, 235(9): 5893-5902. doi: 10.1002/jcp.29588 URL
[7]	Ni X, Cao X, Wu Y, et al. FSTL1 suppresses tumor cell proliferation, invasion and survival in non-small cell lung cancer[J]. Oncol Rep, 2018, 39(1): 13-20.
[8]	Shi N, Mei X, Chen SY. Smooth muscle cells in vascular remodeling[J]. Arterioscler Thromb Vasc Biol, 2019, 39(12): e247-e252.
[9]	He D, Xu L, Wu Y, et al. Rac3, but not Rac1, promotes ox-LDL induced endothelial dysfunction by downregulating autophagy[J]. J Cell Physiol, 2020, 235(2): 1531-1542. doi: 10.1002/jcp.29072 URL
[10]	Grebe A, Hoss F, Latz E. NLRP3 inflammasome and the IL-1 pathway in atherosclerosis[J]. Circ Res, 2018, 122(12): 1722-1740. doi: 10.1161/CIRCRESAHA.118.311362 URL
[11]	Yuan T, Yang T, Chen H, et al. New insights into oxidative stress and inflammation during diabetes mellitus-accelerated atherosclerosis[J]. Redox Biol, 2019, 20: 247-260. doi: 10.1016/j.redox.2018.09.025 URL
[12]	Chellan B, Rojas E, Zhang C, et al. Enzyme-modified non-oxidized LDL (ELDL) induces human coronary artery smooth muscle cell transformation to a migratory and osteoblast-like phenotype[J]. Sci Rep, 2018, 8(1): 11954. doi: 10.1038/s41598-018-30073-w pmid: 30097618
[13]	Gliozzi M, Scicchitano M, Bosco F, et al. Modulation of nitric oxide synthases by oxidized LDLs: role in vascular inflammation and atherosclerosis development[J]. Int J Mol Sci, 2019, 20(13): 3294. doi: 10.3390/ijms20133294 URL
[14]	Zhang Y, Xu X, Yang Y, et al. Deficiency of follistatin-like protein 1 accelerates the growth of breast cancer cells at lung metastatic sites[J]. J Breast Cancer, 2018, 21(3): 267-276. doi: 10.4048/jbc.2018.21.e43 pmid: 30275855
[15]	Liu Q, Yu S, Zhao W, et al. EGFR-TKIs resistance via EGFR-independent signaling pathways[J]. Mol Cancer, 2018, 17(1): 53. doi: 10.1186/s12943-018-0793-1 URL
[16]	Walsh K. Adipokines, myokines and cardiovascular disease[J]. Circ J, 2009, 73(1): 13-18. doi: 10.1253/circj.CJ-08-0961 URL

项目	对照组	Ox-LDL刺激组	Ox-LDL+FSTL1组	F	P
A_{450 nm}	0.870±0.010	1.890±0.020¹⁾	1.200±0.023²⁾	314.800	<0.000 1
OPN	1.001±0.031	2.698±0.001¹⁾	1.590±0.001²⁾	236.200	<0.000 1
α-SMA	1.303±0.030	0.493±0.069¹⁾	0.653±0.015²⁾	407.700	<0.000 1
SIRT1	0.993±0.044	0.613±0.030¹⁾	1.231±0.011²⁾	383.500	<0.000 1