KLF4通过促进自噬减轻高糖浓度下巨噬细胞胆固醇沉积

doi:10.11958/20240283

天津医药 ›› 2024, Vol. 52 ›› Issue (10): 1014-1019.doi: 10.11958/20240283

KLF4通过促进自噬减轻高糖浓度下巨噬细胞胆固醇沉积

张睿(), 陈思思, 王彤丹, 于珮^△()

天津医科大学朱宪彝纪念医院血液净化中心、天津市内分泌研究所、国家卫健委激素与发育重点实验室、天津市代谢性疾病重点实验室（邮编300134）

收稿日期:2024-03-08 修回日期:2024-04-21 出版日期:2024-10-15 发布日期:2024-10-14
通讯作者: △ E-mail：peiyu@tmu.edu.cn
作者简介:张睿（1986），女，主治医师，主要从事糖尿病及其并发症、肾脏病、血液净化相关方面研究。E-mail：zhangruiyes@163.com
基金资助:
天津市教委科研计划项目(2019KJ193)

Krüppel-like factor 4 alleviated cholesterol deposition in macrophages by promoting autophagy at high glucose concentration

ZHANG Rui(), CHEN Sisi, WANG Tongdan, YU Pei^△()

Department of Blood Purifying Center, Chu Hsien-I Memorial Hospital and Tianjin Institute of Endocrinology, Tianjin Medical University, NHC Key Laboratory of Hormones and Development, Tianjin Key Laboratory of Metabolic Diseases, Tianjin 300134, China

Received:2024-03-08 Revised:2024-04-21 Published:2024-10-15 Online:2024-10-14
Contact: △ E-mail: peiyu@tmu.edu.cn

张睿, 陈思思, 王彤丹, 于珮. KLF4通过促进自噬减轻高糖浓度下巨噬细胞胆固醇沉积[J]. 天津医药, 2024, 52(10): 1014-1019.

ZHANG Rui, CHEN Sisi, WANG Tongdan, YU Pei. Krüppel-like factor 4 alleviated cholesterol deposition in macrophages by promoting autophagy at high glucose concentration[J]. Tianjin Medical Journal, 2024, 52(10): 1014-1019.

摘要/Abstract

摘要：

目的观察Krüppel样因子4（KLF4）对高糖处理的巨噬细胞胆固醇沉积的影响，并探讨其作用机制是否与巨噬细胞自噬相关。方法 10只Balb/c小鼠随机分为正常对照（NC）组和糖尿病（DM）组，每组5只。建立DM小鼠模型，给予高脂饮食后检测小鼠主动脉KLF4蛋白表达水平。人白血病单核细胞THP-1诱导为巨噬细胞后分为LV-NC组、LV-KLF4组、si-NC组、si-KLF4组。观察过表达或敲低KLF4表达后，THP-1巨噬细胞胆固醇含量、细胞凋亡、自噬相关蛋白及蛋白激酶B/雷帕霉素靶蛋白（AKT/mTOR）信号通路相关蛋白的表达变化。结果 DM组小鼠主动脉KLF4蛋白表达水平低于NC组（P<0.05）。KLF4过表达可显著降低高糖条件下的THP-1巨噬细胞胆固醇积累，增加苄氯素1（Beclin1）表达，降低P62表达，提高微管相关蛋白1轻链3（LC3）荧光强度，减少细胞凋亡（P<0.05），抑制AKT/mTOR信号通路相关蛋白表达（P<0.05）。敲低KLF4表达后，结果反之。结论 KLF4通过促进自噬减轻高糖浓度下巨噬细胞胆固醇沉积。

关键词: 自噬, 巨噬细胞, Krüppel样因子4, 高糖, 胆固醇沉积

Abstract:

Objective To observe the effect of Krüppel-like factor 4 (KLF4) on cholesterol deposition in macrophages treated with high glucose, and to explore the mechanism related to macrophage autophagy. Methods Ten Balb/c mice were randomly divided into the normal control (NC, n=5) group and the DM group (n=5). A diabetic mouse model was established, and the expression level of KLF4 protein in aorta was detected after high fat diet. After induction of THP-1 monocytes into macrophages, they were divided into the LV-NC group, the LV-KLF4 group, the si-NC group and the si-KLF4 group. Changes of cholesterol content, cell apoptosis and the expression level of autophagy related proteins and AKT/mTOR signaling pathway related proteins in THP-1 macrophages were observed after overexpression or knockout of KLF4. Results The expression level of KLF4 protein in aorta of diabetic mice was lower in the DM group than that of the NC group (P<0.05). Meanwhile, under high glucose concentration, overexpression of KLF4 in THP-1 macrophages significantly decreased cholesterol deposition, cell apoptosis and P62 expression, increased Beclin1 expression, LC3 fluorescence intensity and also inhibited AKT/mTOR signaling pathway related protein expression (P<0.05). After knocking down KLF4 expression, the results were reversed. Conclusion KLF4 alleviates cholesterol deposition in THP-1 macrophages by promoting autophagy under high glucose concentration.

Key words: autophagy, macrophages, Krüppel-like factor 4, high glucose, cholesterol deposition

中图分类号:

R587.1

图/表 9

表1 细胞转染的引物序列

Tab.1 Primer sequence for cell transfection

基因	引物序列（5'→3'）
KLF4-siRNA	上游：GGAGAAGACACUGCGUCAATT
	下游：UUGACGCAGUGUCUUCUCCTT
KLF4-siRNA NC	上游：UUCUCCGAACGUGUCACGUTT
	下游：ACGUGACACGUUCGGAGAATT

表2 造模4周时2组小鼠血糖、血脂比较 ($\bar{x}±s$)

Tab.2 Comparison of blood glucose and blood lipid after 4 weeks of modeling between the two groups

组别	n	血糖/（mmol/L）	TG/（mmol/L）	TC/（mmol/L）
NC组	5	5.40±0.35	1.26±0.11	2.25±0.10
DM组	5	17.17±1.47	2.25±0.07	7.13±0.84
t		15.600^＊＊	15.030^＊＊	11.550^＊＊

图1 Western blot检测DM小鼠主动脉KLF4蛋白表达

Fig.1 Expression of KLF4 protein in aorta of DM mice detected by Western blot assay

图2 Western blot验证KLF4转染效果

Fig.2 The transfection effect of KLF4 verified by Western blot assay

图3 各组细胞中脂滴比较（油红O染色）

Fig.3 Comparison of lipid deposition between the four groups (oil red O staining)

图4 TUNEL染色检测各组细胞凋亡

Fig.4 Cell apoptosis in each group detected by TUNEL staining

图5 免疫荧光检测各组细胞LC3自噬流

Fig.5 LC3 autophagy flow in each group detected by immunofluorescence assay

图6 Western blot检测各组自噬相关蛋白表达 1：LV-NC组；2：LV-KLF4组；3：si-NC组；4：si-KLF4组。

Fig.6 The expression of autophagy related proteins in each group detected by Western blot assay

表3 各组细胞自噬相关蛋白表达水平比较（n=3，$\bar{x}±s$）

Tab.3 Comparison of autophagy related protein expression level between the four groups

组别	Beclin1/ β-actin	LC3/ β-actin	P62/ β-actin	p-AKT/ AKT	p-mTOR/ mTOR
LV-NC组	1.00±0.20	0.99±0.16	1.01±0.07	1.01±0.17	1.00±0.17
LV-KLF4组	1.40±0.28	1.42±0.23	0.83±0.10	0.68±0.22	0.76±0.15
t	7.822^＊	9.811^＊	5.678^＊	8.837^＊	19.970^＊＊
si-NC组	1.00±0.16	0.96±0.13	1.13±0.09	0.95±0.23	1.07±0.12
si-KLF4组	0.78±0.10	0.54±0.08	1.47±0.17	1.15±0.17	1.40±0.10
t	5.949^＊	13.500^＊＊	7.921^＊	5.415^＊	25.840^＊＊

参考文献 24

[1]	POZNYAK A, GRECHKO A V, POGGIO P, et al. The diabetes mellitus-atherosclerosis connection: the role of lipid and glucose metabolism and chronic inflammation[J]. Int J Mol Sci, 2020, 21(5):1835. doi:10.3390/ijms21051835.
[2]	TABAS I, GARCIA C G, OWENS G K. Recent insights into the cellular biology of atherosclerosis[J]. J Cell Biol, 2015, 209:13-22. doi:10.1083/jcb.201412052.
[3]	TESSA J B. Macrophages in atherosclerosis regression[J]. Arterioscler Thromb Vasc Biol, 2020, 40(1):20-33. doi:10.1161/ATVBAHA.119.312802.
[4]	PEET C, IVETIC A, BROMAGE D I, et al. Cardiac monocytes and macrophages after myocardial infarction[J]. Cardiovasc Res, 2020, 116(6):1101-1112. doi:10.1093/cvr/cvz336.
[5]	MADHAVI J, YUGUANG Z, LU C. Krϋppel-like factors (KLFs) in renal physiology and disease[J]. E Bio Medicine, 2019, 40:743-750. doi:10.1016/j.ebiom.2019.01.021.
[6]	SWEET D R, FAN L, HSIEH PN, et al. Krϋppel-like factors in vascular inflammation:mechanistic insights and therapeutic potential[J]. Front Cardiovasc Med, 2018, 5:6. doi:10.3389/fcvm.2018.00006.
[7]	ZHANG R, ZHOU S J, LI C J, et al. C-reactive protein/oxidised low-density lipoprotein/β2-glycoprotein I complex promotes atherosclerosis in diabetic BALB/c mice via p38mitogen-activated protein kinase signal pathway[J]. Lipids Health Dis, 2013, 12:42. doi:10.1186/1476-511X-12-42.
[8]	NYANDWI J B, KO Y S, JIN H, et al. Rosmarinic acid inhibits oxLDL-induced inflammasome activation under high-glucose conditions through downregulating the p38-FOXO1-TXNIP pathway[J]. Biochem Pharmacol, 2020, 182:114246. doi:10.1016/j.bcp.2020.114246.
[9]	XU X, ZHANG A, LI N, et al. Concentration-dependent diversifcation effects of free cholesterol loading on macrophage viability and polarization[J]. Cell Physiol Biochem, 2015, 37(2):419-431. doi: 10.1159/000430365.
[10]	MANGANELLI V, LONGO A, MATTEI V, et al. Role of ERLINs in the control of cell fate through lipid rafts[J]. Cells, 2021, 10(9):2408. doi:10.3390/cells10092408.
[11]	WANG Y, XIAO S M, ZHOU S J, et al. High glucose aggravates cholesterol accumulation in glomerular endothelial cells through the LXRs/LncRNAOR13C9/ABCA1 regulatory network[J]. Front Physiol, 2020, 19(11):552483. doi:10.3389/fphys.2020.552483.
[12]	KHAWAR M B, GAO H, LI W. Autophagy and lipid metabolism[J]. Adv Exp Med Biol, 2019, 1206:359-374. doi:10.1007/978-981-15-0602-4_17.
[13]	SERGIN I, BHATTACHARYA S, EMANUEL R, et al. Inclusion bodies enriched for p62 and polyubiquitinated proteins in macrophages protect against atherosclerosis[J]. Sci Sign, 2016, 9(409):ra2. doi:10.1126/scisignal.aad5614.
[14]	SWAMINATHAN B, GOIKURIA H, VEGA R, et al. Autophagic marker MAP1LC3B expression levels are associated with carotid atherosclerosis symptomatology[J]. PLoS One, 2014, 9:e115176. doi:10.1371/journal.pone.0115176.
[15]	NAHAPETYAN H, MOULIS M, GROUSSET E, et al. Altered mitochondrial quality control in Atg7-deficient VSMCs promotes enhanced apoptosis and is linked to unstable atherosclerotic plaque phenotype[J]. Cell Death Dis, 2019, 10(2):119. doi:10.1038/s41419-019-1400-0.
[16]	DUBNER A M, LU S, JOLLY A J, et al. Smoothmuscle-derived adventitial progenitor cells direct atherosclerotic plaque composition complexity in a Klf4-dependent manner[J]. JCI Insight, 2023, 8(22):e174639. doi:10.1172/jci.insight.174639.
[17]	WU Q, WANG H, HE F, et al. Depletion of microRNA-92a enhances the role of sevoflurane treatment in reducing myocardial ischemia-reperfusion injury by upregulating KLF4[J]. Cardiovasc Drugs Ther, 2023, 37(6):1053-1064. doi:10.1007/s10557-021-07303-x.
[18]	SALMON M, SPINOSA M, ZEHNER Z E, et al. Klf4,Klf2,and Zfp148 activate autophagy-related genes in smooth muscle cells during aortic aneurysm formation[J]. Phys Rep, 2019, 7:e14058. doi:10.14814/phy2.14058.
[19]	饶超峰, 薛笑楠, 朱明英, 等. 巨噬细胞外泌体通过抑制自噬诱导高血糖对肾小球足细胞的损伤作用[J]. 天津医药, 2021, 49(2):119-125.
	RAO C F, XUE X N, ZHU M Y, et al. Effects of exosomes from high glucose-treated macrophage on the injury of glomerular podocytes via inhibiting autophage[J]. Tianjin Med J, 2021, 49(2):119-125. doi:10.11958/20201991.
[20]	WANG Y, ZHAO B L, ZHANG Y, et al. Krüppel-like factor 4 is induced by rapamycin and mediates the anti-proliferative effect of rapamycin in rat carotid arteries after balloon injury[J]. Br J Pharmacol, 2012, 165(7):2378-2388. doi:10.1111/j.1476-5381.2011.01734.x.
[21]	SETO B. Rapamycin and mTOR:a serendipitous discovery and implications for breast cancer[J]. Clin Transl Med, 2012, 1:29. doi:10.1186/2001-1326-1-29.
[22]	POZNYAK A V, SUKHORUKOV V N, ZHURAVLEV A, et al. Modulating mTOR signaling as a promising therapeutic strategy for atherosclerosis[J]. Int J Mol Sci, 2022, 23(3):1153. doi:10.3390/ijms23031153.
[23]	ZHANG G, HE C, WU Q, et al. Impaired autophagy induced by oxLDL/β2GPI/anti- β2GPI complex through PI3K/AKT/mTOR and eNOS signaling pathways contributes to endothelial cell dysfunction[J]. Oxid Med Cell Longev, 2021, 2021:6662225. doi: 10.1155/2021/6662225.
[24]	ZHAI C, CHENG J, MUJAHID H, et al. Selective inhibition of PI3K/Akt/mTOR signaling pathway regulates autophagy of macrophage and vulnerability of atherosclerotic plaque[J]. PLos One, 2014, 9:e90563. doi:10.1371/journal.pone.0090563.

KLF4通过促进自噬减轻高糖浓度下巨噬细胞胆固醇沉积

Krüppel-like factor 4 alleviated cholesterol deposition in macrophages by promoting autophagy at high glucose concentration

引用本文

RichHTML

PDF (PC)

可视化

摘要/Abstract

使用本文

图/表 9

参考文献 24

相关文章 15

编辑推荐

Metrics

本文评价

[1]	贾维宁, 鲍亚玲, 雷慧, 殷晓宁. 夏枯草提取物对脓毒症小鼠炎症反应和腹腔巨噬细胞的影响[J]. 天津医药, 2024, 52(9): 930-935.
[2]	方杰, 黄芮, 郑红慧, 贾倩倩, 鲍静. miR-9-5p靶向TIMP2诱导多发性骨髓瘤细胞自噬和凋亡的机制[J]. 天津医药, 2024, 52(8): 785-790.
[3]	李大强, 李坚, 陆哲明, 曹阳. 毛蕊异黄酮对脊髓损伤大鼠神经元自噬及凋亡的影响[J]. 天津医药, 2024, 52(8): 798-803.
[4]	钟敏, 施震, 周劲松, 李晋杰. GABA信号通路对脓毒症大鼠急性肺损伤内质网应激和线粒体自噬的影响[J]. 天津医药, 2024, 52(7): 733-737.
[5]	王柯, 叶寒露. 隐丹参酮调节HIF-1α/BNIP3信号通路对兔膝骨关节炎模型软骨细胞自噬和凋亡的影响[J]. 天津医药, 2024, 52(4): 372-378.
[6]	何颖, 张广华, 田立东, 于泳浩. 富氢液通过增加自噬治疗大鼠神经病理性疼痛[J]. 天津医药, 2024, 52(3): 261-265.
[7]	汪爱华, 张飞忠, 王红英. 麝香酮调节SHH介导的自噬对卵巢癌细胞恶性进展的影响[J]. 天津医药, 2024, 52(2): 142-147.
[8]	赵元元, 吴小华. 基于PI3K/Akt/mTOR信号通路探讨LINC00173对多囊卵巢综合征颗粒细胞自噬的影响[J]. 天津医药, 2024, 52(11): 1121-1126.
[9]	乔娜, 田英, 陈杨, 郝静. LncRNA MALAT1对PCOS颗粒细胞凋亡、自噬和PI3K/Akt/mTOR通路的影响[J]. 天津医药, 2024, 52(10): 1020-1024.
[10]	廖忠, 廖伟健, 赖国利, 文茵, 苏志威, 曾举浩, 丁洪光. 漆黄素抑制小胶质细胞NLRP3炎症小体活化缓解脓毒症后认知功能损害的研究[J]. 天津医药, 2024, 52(10): 1025-1030.
[11]	侯永波, 余骏马, 朱海娟. 外泌体在心肌梗死治疗中的研究新进展[J]. 天津医药, 2023, 51(9): 1016-1019.
[12]	李宏军, 钱亮, 邓新超, 余庭, 吴岩, 吴红丽. 舒筋活血胶囊通过JAK2/STAT3通路缓解大鼠膝骨关节炎的机制研究[J]. 天津医药, 2023, 51(9): 961-967.
[13]	杨渊, 宋爽, 陈容, 刘永莲, 刘春燕. 人参皂苷Rg1拮抗亚砷酸钠诱导C57BL/6小鼠肾毒性研究[J]. 天津医药, 2023, 51(8): 820-824.
[14]	黄承军, 徐宇, 秘乐, 王秀军, 刘振峰, 王红嫚. 细胞自噬在急性呼吸窘迫综合征中的研究进展[J]. 天津医药, 2023, 51(6): 668-672.
[15]	王飞, 张小蕾, 李含章, 李亚楠, 胡梦妮, 马骏. 抑制PI3K/Akt/mTOR信号通路对MPP⁺处理的SH-SY5Y细胞自噬、凋亡及PD特征蛋白表达的影响[J]. 天津医药, 2023, 51(5): 449-453.