颅内动脉瘤组织中Septin4的表达及其通过调节自噬对人脑血管平滑肌细胞的作用研究-东南大学学报医学版

颅内动脉瘤组织中Septin4的表达及其通过调节自噬对人脑血管平滑肌细胞的作用研究

单位：1. 十堰市太和医院(湖北医药学院附属医院), 湖北十堰 442000;
2. 珠海市中西医结合医院(南方医科大学附属珠海医院) 脑外科，广东珠海 519000

分类号：R739.41;Q245

出版年·卷·期（页码）：2021·40·第六期（795-802）

摘要：

目的: 探究Septin4在颅内动脉瘤中的表达及其对人脑血管平滑肌细胞(HBVSMCs)的作用及可能的作用机制。方法: 收集颅内动脉瘤组织和正常颅动脉组织各32份, 通过免疫组化(IHC)、逆转录-聚合酶链反应(RT-PCR)和蛋白质印迹法检测组织中Septin4的表达; 免疫荧光染色(IF)检测平滑肌22α(SM22α)和平滑肌α肌动蛋白(αSMA)表达; 分别将Septin4过表达载体(Septin4)、Septin4干扰序列(si-Septin4)及其阴性对照(NC和si-NC)转染HBVSMCs, RT-PCR和蛋白质印迹法检测HBVSMCs中Septin4的表达; 蛋白质印迹法检测αSMA、SM22α、基质金属蛋白酶(MMP)2、MMP9、微管相关蛋白1轻链3(LC3)、sequestosome 1(p62)、磷酸化腺苷酸活化蛋白激酶(p-AMPK)、腺苷酸活化蛋白激酶(AMPK)、磷酸化哺乳动物类雷帕霉素靶蛋白(p-mTOR)和哺乳动物类雷帕霉素靶蛋白(mTOR)蛋白表达。结果: 与正常颅动脉组织相比, 颅内动脉瘤组织中Septin4阳性细胞数、mRNA表达、蛋白表达及SM22α和αSMA荧光强度明显降低(P < 0.05)。与NC组相比, Septin4组细胞中Septin4 mRNA表达及Septin4、αSMA、SM22α、p62、p-mTOR/mTOR蛋白表达明显升高(P < 0.05), MMP2、MMP9、LC3Ⅱ/LC3Ⅰ和p-AMPK/AMPK蛋白表达明显降低(P < 0.05);与si-NC组相比, si-Septin4组细胞中Septin4 mRNA表达及Septin4、αSMA、SM22α、p62、p-mTOR/mTOR蛋白表达明显降低(P < 0.05), MMP2、MMP9、LC3Ⅱ/LC3Ⅰ和p-AMPK/AMPK蛋白表达明显升高(P < 0.05)。结论: Septin4可能通过调节AMPK/mTOR信号通路抑制自噬, 促进HBVSMCs向收缩型表型转化, 在颅内动脉瘤中发挥保护作用。

Objective: To explore the expression of Septin4 in intracranial aneurysms and its effect on human brain vascular smooth muscle cells (HBVSMCs) and its possible mechanism.Methods: 32 samples of intracranial aneurysm tissue and normal cranial artery tissue were collected, and the expression of Septin4 in the tissue was detected by immunohistochemistry(IHC), reverse transcription-polymerase chain reaction (RT-PCR) and Western blot; immunofluorescence staining were used to detect smooth muscle 22α (SM22α) and smooth muscle α actin (ΑSMA) expression. Septin4 overexpression vector (Septin4), interference sequence (si-Septin4) and negative control (NC and si-NC) were transfected into HBVSMCs, respectively. RT-PCR and Western blot experiments were used to detect the expression of Septin4 in cells; Western blot was used to detect the expression of αSMA, SM22α, matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), Microtubule-associated protein 1 light chain 3 (LC3), sequestosome 1 (p62), Phosphorylated adenylate activated protein kinase (p-AMPK), adenosine monophosphate activated protein kinase (AMPK), Phosphorylated mammalian target of rapamycin (p-mTOR) and mammalian target of rapamycin (mTOR) protein.Results: Compared with the normal cranial artery tissue, the number of Septin4 positive cells, mRNA expression, protein expression and the fluorescence intensity of SM22α and αSMA in the intracranial aneurysm tissue were significantly reduced (P < 0.05). Compared with the NC group, Septin4 mRNA expression, Septin4, αSMA, SM22α, p62 and p-mTOR/mTOR protein expression were significantly increased in Septin4 cells (P < 0.05), MMP2, MMP9, LC3Ⅱ/LC3Ⅰ and p-AMPK/AMPK protein expression was significantly reduced (P < 0.05); compared with si-NC group, Septin4 mRNA expression, Septin4, αSMA, SM22α, p62 and p-mTOR/mTOR protein expression were significantly reduced in si-Septin4 group cells (P < 0.05), MMP2, MMP9, LC3Ⅱ/LC3Ⅰ and p-AMPK/AMPK protein expression were significantly increased (P < 0.05).Conclusion: Septin4 may inhibit autophagy by regulating AMPK/mTOR signaling pathway, promote the transformation of HBVSMCs to contractile phenotype, and play a protective role in intracranial aneurysms.

参考文献：

[1] KLEINLOOG R, DE MUL N, VERWEIJ B H, et al. Risk factors for intracranial aneurysm rupture: a systematic review[J]. Neurosurgery, 2018, 82(4): 431-440.
[2] CAN A, CASTRO V M, OZDEMIR Y H, et al. Association of intracranial aneurysm rupture with smoking duration, intensity, and cessation[J]. Neurology, 2017, 89(13): 1408-1415.
[3] SIGNORELLI F, SELA S, GESUALDOL L, et al. Hemodynamic stress, inflammation, and intracranial aneurysm development and rupture: a systematic review[J]. World Neurosurg, 2018, 115: 234-244.
[4] FRÖSEN J, JOUTEL A. Smooth muscle cells of intracranial vessels: from development to disease[J]. Cardiovasc Res, 2018, 114: 501-512.
[5] TAO L, ZHAO S, TAO Z, et al. Septin4 regulates endoplasmic reticulum stress and apoptosis in melatonin induced osteoblasts[J]. Mol Med Rep, 2020, 22: 1179-1186.
[6] ZHANG N, ZHANG Y, YOU S, et al. Septin4 prevents PDGF-BB-induced HAVSMC phenotypic transformation, proliferation and migration by promoting SIRT1-STAT3 deacetylation and dephosphorylation[J]. Int J Biol Sci, 2020, 16: 708-718.
[7] TOTH G, CEREJO R. Intracranial aneurysms: Review of current science and management[J]. Vasc Med, 2018, 23(3): 276-288.
[8] OKA M, SHIMO S, OHNO N, et al. Dedifferentiation of smooth muscle cells in intracranial aneurysms and its potential contribution to the pathogenesis[J]. Sci Rep, 2020, 10: 8330-8338.
[9] BENNETT M R, SINHA S, OWENS G K. Vascular smooth muscle cells in atherosclerosis[J]. Circ Res, 2016, 118(4): 692-702.
[10] WANG G, JACQUET L, KARAMARITI E, et al. Origin and differentiation of vascular smooth muscle cells[J]. J Physiol, 2015, 593(14): 3013-3030.
[11] SUN L, ZHAO M, ZHANG J, et al. MiR-29b downregulation induces phenotypic modulation of vascular smooth muscle cells: implication for intracranial aneurysm formation and progression to rupture[J]. Cell Physiol Biochem, 2017, 41: 510-518.
[12] ABBEY M, GAESTEL M, MENON M B, et al. Septins: Active GTPases or just GTP‐binding proteins? [J]. Cytoskeleton, 2019, 76(1): 55-62.
[13] WANG Z, GUO J, HAN X, et al. Metformin represses the pathophysiology of AAA by suppressing the activation of PI3K/AKT/mTOR/autophagy pathway in ApoE mice[J]. Cell Biosci, 2019, 9: 68-81.
[14] 袁广胜, 吴海东, 陈胜利, 等. 细胞自噬调控颅内动脉瘤血管平滑肌细胞表型转化的体外研究[J]. 中国免疫学杂志, 2019, 35(11): 1337-1340, 1353.
[15] GAO G, ZHANG Y, CHAO Y, et al. miR-4735-3p regulates phenotypic modulation of vascular smooth muscle cells by targeting HIF-1-mediated autophagy in intracranial aneurysm[J]. J Cell Biochem, 2019, 120: 19432-19441.
[16] WANG N, XU F, LU S, et al. Septin4 as an autophagy modulator regulates Angiotensin-Ⅱ mediated VSMCs proliferation and migration[J]. Biochem Biophys Res Commun, 2020, 525: 272-279.
[17] JIANG P, JIANG B, LI Y, et al. Autophagy promotes angiogenesis via AMPK/Akt/mTOR signaling during the recovery of heat-denatured endothelial cells[J]. Cell Death Dis, 2018, 9(12): 115-124.
[18] SUN L, ZHAO M, LIU A, et al. Shear stress induces phenotypic modulation of vascular smooth muscle cells via AMPK/mTOR/ULK1-mediated autophagy[J]. Cell Mol Neurobiol, 2018, 38: 541-548.

服务与反馈：

【文章下载】【发表评论】【查看评论】【加入收藏】

提示：您还未登录，请登录！点此登录