二甲双胍通过AMPK/PPAR-γ通路诱导自噬抑制高糖对血管平滑肌细胞的增殖作用

张紫微; 郑甲林; 许晓宇; 王红

doi:10.12259/j.issn.2095-610X.S20231026

二甲双胍通过AMPK/PPAR-γ通路诱导自噬抑制高糖对血管平滑肌细胞的增殖作用

doi: 10.12259/j.issn.2095-610X.S20231026

张紫微¹,
郑甲林¹,
许晓宇^{1, 2},
王红^1, ,

1.
云南省阜外心血管病医院冠心病中心，云南昆明　650032
2.
昆明医科大学研究生院，云南昆明　650500

基金项目: 云南省科技厅-昆明医科大学应用基础研究联合专项基金资助项目（81170250）；云南省医学创新团队基金资助项目（202005AE160020）

详细信息

作者简介:
张紫微（1985～），男，江西九江人，医学博士，副主任医师，主要从事冠心病基础研究与介入诊疗工作

通讯作者:
王红，E-mail：wangh43@126.com

中图分类号: R54
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出版历程
- 收稿日期: 2023-08-23
- 网络出版日期: 2023-09-12
- 刊出日期: 2023-10-25

Metformin Inhibits high Glucose-induced Vascular Smooth Muscle Cells Proliferation by Promoting Autophagy via AMPK/PPAR-γ

Ziwei ZHANG¹,
Jialin ZHENG¹,
Xiaoyu XU^{1, 2},
Hong WANG^{1
, ,}

1.
Center of Coronary Heart Disease，Fuwai Yunnan Cardiovascular Hospital， Kunming Yunnan 650032，China
2.
Graduate School of Kunming Medical University， Kunming Yunnan 650500，China

摘要

摘要: 目的探讨二甲双胍（Metformin）对高糖培养的小鼠主动脉血管平滑肌细胞（vascular smooth muscle cell，VSMCs）自噬、细胞增殖的影响及其调控机制。方法用Western blot检测不同处理组微管相关蛋白轻链-3-Ⅱ（LC3-Ⅱ），Becline-1蛋白水平变化，透射电镜观察各处理组VSMCs自噬小体水平；用Western blot检测不同处理组p-AMPK/AMPK、PPAR-γ蛋白表达水平；用EDU法检测不同处理组VSMCs增殖情况。结果二甲双胍、雷帕霉素逆转高糖抑制VSMCs自噬的作用（P < 0.001）；Compound C、GW9662增加高糖抑制VSMCs自噬的作用（P < 0.05）；二甲双胍逆转高糖对AMPK/PPAR-γ通路的负调控（P < 0.01）；二甲双胍、雷帕霉素抑制高糖促进的VSMCs增殖（P < 0.001），Compound C、GW9662逆转二甲双胍抑制高糖促VSMCs增殖的作用（P < 0.05）。结论高糖促进VSMCs增殖可能是自噬调节相关的，二甲双胍可通过激活AMPK/PPAR-γ通路上调VSMCs自噬水平，抑制高糖促进的VSMCs异常增殖。
- 二甲双胍 /
- 高糖 /
- 自噬 /
- 血管平滑肌细胞 /
- 增殖
Abstract: Objective To investigate the effect and regulatory mechanism of metformin on autophagy and cell proliferation of vascular smooth muscle cells （VSMCs） cultured in high glucose in mice aortic. Methods Western blot was used to detect the changes in microtubule-associated protein light chain 3-II （LC3-II） and Beclin-1 protein levels in different treatment groups. Transmission electron microscope was used to observe the the level of autophagosomes in VSMCs of each treatment group. Western blot was used to detect the expression levels of p-AMPK/AMPK and PPAR-γ proteins in different treatment groups. The EDU method was used to detect the proliferation of VSMCs in different treatment groups. Results Metformin and rapamycin reversed the inhibitory effect of high glucose on VSMC autophagy （P < 0.001）. Compound C and GW9662 enhanced the inhibitory effect of high glucose on VSMC autophagy （P < 0.05）. Metformin reversed the negative regulation of high glucose on the AMPK/PPAR-γ pathway （P < 0.01）. Metformin and rapamycin inhibited high glucose-induced VSMC proliferation （P < 0.001）, and Compound C and GW9662 reversed the inhibitory effect of metformin on high glucose-induced VSMC proliferation （P < 0.05）. Conclusion High glucose promotes the proliferation of VSMCs, which may be regulated by autophagy. Metformin can upregulate autophagy levels in VSMCs by activating the AMPK/PPAR-γ pathway, thereby inhibiting the abnormal proliferation of VSMCs induced by high sugar.
- Metformin /
- High glucose /
- Autophagy /
- Vascular smooth muscle cell /
- Proliferation

HTML全文

图 1 二甲双胍对高糖培养下的VSMCs自噬水平的影响

A：Western blot检测对照组、HG组、HG+MET组及HG+RAP组LC3-Ⅱ/LC3-Ⅰ、Becline-1的表达情况；B：定量分析不同处理组LC3-Ⅱ/LC3-Ⅰ的表达水平；C：定量分析不同处理组Becline-1蛋白的表达水平；D：电镜检测对照组、HG组、MET+HG组自噬小体（12000×）（黑色箭头示自噬小体）。^*P < 0.05，^**P < 0.01，^***P < 0.001 。

Figure 1. Effect of metforin on autophagy in HG-cultured VSMCs

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图 2 AMPK、PPAR-γ抑制剂对VSMCs自噬的影响

A：Western blot检测对照组、HG组、HG+CC组及HG+GW组LC3-Ⅱ/LC3-Ⅰ、Becline-1的表达情况；B：定量分析不同处理组LC3-Ⅱ/LC3-Ⅰ的表达水平；C：定量分析不同处理组Becline-1的表达水平。^*P < 0.05，^**P < 0.01，^***P < 0.001。

Figure 2. Effect of AMPK or PPAR-γ inhibitor on autophagy in VSMCs

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图 3 二甲双胍对高糖培养下VSMCs p-AMPK/AMPK、PPAR-γ表达的影响

A：Western blot检测对照组、HG组、HG+MET组及HG+CC组p-AMPK/AMPK的表达；B：定量分析不同处理组p-AMPK/AMPK的表达水平；C：Western blot检测对照组、HG组、HG+MET组及HG+GW组PPAR-γ的表达；D：定量分析不同处理组PPAR-γ的表达水平。^*P < 0.05，^**P < 0.01，^***P < 0.001。

Figure 3. Effect of metformin and high glucose on p-AMPK/AMPK、PPAR-γexpression of VSMCs

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图 4 二甲双胍抑制高糖诱导的VSMCs增殖

A：EDV法检测不同处理组VSMCs增殖情况；B：EDV阳性细胞统计比较。^* P < 0.05，^** P < 0.01，^***P < 0.001 VS CTRL；^#P < 0.05，^###P < 0.001 VS HG，^&P < 0.05，^&&P < 0.01，^&&&P < 0.001 VS HG+MET。

Figure 4. Effect of metformin on high glucose-induced proliferation of VSMCs

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参考文献(24)

[1]	Chiong M,Morales P,Torres G,et al. Influence of glucose metabolism on vascular smooth muscle cell proliferation[J]. Vasa,2013,42(1):8-16. doi: 10.1024/0301-1526/a000243
[2]	Yang M,Fang J,Liu Q,et al. Role of ROS-TRPM7-ERK1/2 axis in high concentration glucose-mediated proliferation and phenotype switching of rat aortic vascular smooth muscle cells[J]. Biochemical and Biophysical Research Communications,2017,494(3-4):526-533. doi: 10.1016/j.bbrc.2017.10.122
[3]	Marino G,Niso-Santano M,Baehrecke E H,et al. Self-consumption: The interplay of autophagy and apoptosis[J]. Nat Rev Mol Cell Biol,2014,15(2):81-94. doi: 10.1038/nrm3735
[4]	Wu H,Song A,Hu W,et al. The anti-atherosclerotic effect of paeonol against vascular smooth muscle cell proliferation by up-regulation of autophagy via the AMPK/mTOR signaling pathway[J]. Frontiers in Pharmacology,2018,8:948. doi: 10.3389/fphar.2017.00948
[5]	张紫微,杨丽霞,吕晋琳,等. 血管紧张素Ⅱ诱发自噬对血管平滑肌细胞表型转换的调控作用[J]. 中国动脉硬化杂志,2017,25(5):452-456.
[6]	Phadwal K,Feng D,Zhu D,et al. Autophagy as a novel therapeutic target in vascular calcification[J]. Pharmacology & Therapeutics,2020,206:107430.
[7]	Peng S,Xu L,Che X,et al. Atorvastatin inhibits inflammatory response,attenuates lipid deposition,and improves the stability of vulnerable atherosclerotic plaques by modulating autophagy[J]. Frontiers in Ppharmacology,2018,9:438. doi: 10.3389/fphar.2018.00438
[8]	Poznyak A V,Litvinova L,Poggio P,et al. From diabetes to atherosclerosis: Potential of metformin for management of cardiovascular disease[J]. International Journal of Molecular Sciences,2022,23(17):9738. doi: 10.3390/ijms23179738
[9]	Yang Q,Yuan H,Chen M,et al. Metformin ameliorates the progression of atherosclerosis via suppressing macrophage infiltration and inflammatory responses in rabbits[J]. Life Sciences,2018,198:56-64. doi: 10.1016/j.lfs.2018.02.017
[10]	Wu H,Feng K,Zhang C,et al. Metformin attenuates atherosclerosis and plaque vulnerability by upregulating KLF2-mediated autophagy in apoE−/-mice[J]. Biochemical and Biophysical Research Communications,2021,557:334-341. doi: 10.1016/j.bbrc.2021.04.029
[11]	Phadwal K,Koo E,Jones R A,et al. Metformin protects against vascular calcification through the selective degradation of Runx2 by the p62 autophagy receptor[J]. Journal of Cellular Physiology,2022,237(11):4303-4316. doi: 10.1002/jcp.30887
[12]	Yang B,Gao X,Sun Y,et al. Dihydroartemisinin alleviates high glucose-induced vascular smooth muscle cells proliferation and inflammation by depressing the miR-376b-3p/KLF15 pathway[J]. Biochemical and Biophysical Research Communications,2020,530(3):574-580. doi: 10.1016/j.bbrc.2020.07.095
[13]	Yuan T,Yang T,Chen H,et al. New insights into oxidative stress and inflammation during diabetes mellitus-accelerated atherosclerosis[J]. Redox Biology,2019,20:247-260. doi: 10.1016/j.redox.2018.09.025
[14]	Tiong Y L,Ng K Y,Koh R Y,et al. Melatonin inhibits high glucose-induced ox-LDL/LDL expression and apoptosis in human umbilical endothelial cells[J]. Hormone Molecular Biology and Clinical Investigation,2020,41(4):20200009. doi: 10.1515/hmbci-2020-0009
[15]	Zhao Y,Lu N,Zhang Y,et al. High glucose induced rat aorta vascular smooth muscle cell oxidative injury: Involvement of protein tyrosine nitration[J]. Journal of Physiology and Biochemistry,2011,67(4):539-549. doi: 10.1007/s13105-011-0099-x
[16]	Liu J,Wu J,Sun A,et al. Hydrogen sulfide decreases high glucose/palmitate-induced autophagy in endothelial cells by the Nrf2-ROS-AMPK signaling pathway[J]. Cell & Bioscience,2016,6(1):1-17.
[17]	Xu K,Liu X,Ke Z,et al. Resveratrol modulates apoptosis and autophagy induced by high glucose and palmitate in cardiac cells[J]. Cellular Physiology and Biochemistry,2018,46(5):2031-2040. doi: 10.1159/000489442
[18]	De Jager J,Kooy A,Schalkwijk C,et al. Long‐term effects of metformin on endothelial function in type 2 diabetes: A randomized controlled trial[J]. Journal of Internal Medicine,2014,275(1):59-70. doi: 10.1111/joim.12128
[19]	Van der Aa M P,Elst M,Van De Garde E,et al. Long-term treatment with metformin in obese,insulin-resistant adolescents: Results of a randomized double-blinded placebo-controlled trial[J]. Nutrition & Diabetes,2016,6(8):e228.
[20]	He X,Chen X,Wang L,et al. Metformin ameliorates Ox-LDL-induced foam cell formation in raw264.7 cells by promoting ABCG-1 mediated cholesterol efflux[J]. Life Sciences,2019,216:67-74. doi: 10.1016/j.lfs.2018.09.024
[21]	Gopoju R,Panangipalli S,Kotamraju S. Metformin treatment prevents SREBP2-mediated cholesterol uptake and improves lipid homeostasis during oxidative stress-induced atherosclerosis[J]. Free Radical Biology and Medicine,2018,118:85-97. doi: 10.1016/j.freeradbiomed.2018.02.031
[22]	You G,Long X,Song F,et al. Metformin activates the AMPK-mTOR pathway by modulating lncRNA TUG1 to induce autophagy and inhibit atherosclerosis[J]. Drug Design,Development and Therapy,2020,14:457. doi: 10.2147/DDDT.S233932
[23]	Faghfouri A H,Khajebishak Y,Payahoo L,et al. PPAR-gamma agonists: Potential modulators of autophagy in obesity[J]. European Journal of Pharmacology,2021,912:174562. doi: 10.1016/j.ejphar.2021.174562
[24]	Wang H,Wang A,Wang X,et al. AMPK/PPAR-γ/NF-κB axis participates in ROS-mediated apoptosis and autophagy caused by cadmium in pig liver[J]. Environmental Pollution,2022,294:118659. doi: 10.1016/j.envpol.2021.118659

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