Effects of YAP2 gene on rat H9c2 cardiomyocyte apoptosis and TNF-α and IL-6 levels

doi:10.3760/cma.j.issn.1007-1245.2024.14.003

Abstract

Abstract:

Objective　To investigate the effect of Yes-associated protein 2 (YAP2) gene silencing on rat H9c2 cardiomyocyte viability, apoptosis rate, and inflammatory factors induced by H₂O₂. Methods　The experiment was conducted from May to December 2023. The experiment cells were divided into four groups: a normal control group, a H₂O₂ group, a si-YAP2+H₂O₂ group, and a NC+H₂O₂ group. MTT, flow cytometry, and Western blotting were used to detect the cell viability, apoptosis rate, and the expressions of YAP2, Bcl-2, Bax, and p-AKT protein in the four groups, and the contents of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the cell culture medium of each group were detected by enzyme-linked immunosorbent assay (ELISA) kit. One-way analysis of variance and SNK-q test were used. Results　The expression levels of YAP2 in the H₂O₂ group and the si-YAP2+H₂O₂ group were (0.716±0.056) and (0.204±0.018), respectively, with a statistically significant difference (P<0.05). The viability of H9c2 cardiomyocytes in the H₂O₂ group was significantly lower than that in the si-YAP2+H₂O₂ group and the normal control group, while the apoptosis rate was significantly higher, with statistically significant differences (all P<0.05). There were no statistically significant differences in the viability or apoptosis rate of H9c2 cardiomyocytes between the H₂O₂ group and the NC+H₂O₂ group (both P>0.05). The expression levels of Bcl-2 and p-AKT in the H₂O₂ group were significantly lower than those in the si-YAP2+H₂O₂ group and the normal control group, while the expression level of Bax was significantly higher, with statistically significant differences (all P<0.05). There were no statistically significant differences in the expression levels of Bcl-2, p-AKT, and Bax between the H₂O₂ group and the NC+H₂O₂ group (all P>0.05). The contents of TNF-α and IL-6 in the H₂O₂ group were significantly higher than those in the si-YAP2+H₂O₂ group and the normal control group (all P<0.05). There were no statistically significant differences in the contents of TNF-α and IL-6 between the H₂O₂ group and the NC+H₂O₂ group (both P>0.05). Conclusion　Silencing the expression of YAP2 gene may reduce the occurrence of cardiovascular diseases by reducing the apoptosis of cardiomyocytes and the contents of inflammatory factors TNF-α and IL-6.

Key words:

Cardiomyocytes, YAP2 gene, Apoptosis, Immunity, PI3K/AKT signaling pathway

摘要：

目的　探讨沉默Yes相关蛋白2（YAP2）基因表达对H₂O₂诱导的大鼠H9c2心肌细胞活力、凋亡率及炎症因子的影响及机制。方法　本实验于2023年5月至12月实施。实验细胞分为4个处理组，即正常对照组（Control组）、H₂O₂组、si-YAP2+H₂O₂组、NC+H₂O₂组。通过MTT、流式细胞术及Western blotting分别检测4组细胞活力、凋亡率及YAP2、Bcl-2、Bax、p-AKT蛋白表达；酶联免疫吸附试验（ELISA）试剂盒检测各组细胞培养液中肿瘤坏死因子（TNF）-α和白细胞介素（IL）-6含量。采用单因素方差分析、SNK-q检验。结果　H₂O₂组、si-YAP2+H₂O₂组YAP2表达量分别为（0.716±0.056）、（0.204±0.018），差异有统计学意义（P<0.05）。H₂O₂组H9c2心肌细胞活力低于si-YAP2+H₂O₂组和Control组，细胞凋亡率高于si-YAP2+H₂O₂组和Control组，差异均有统计学意义（均P<0.05）。H₂O₂组和NC+H₂O₂组H9c2心肌细胞活力、细胞凋亡率差异均无统计学意义（均P>0.05）。H₂O₂组Bcl-2、p-AKT表达量低于si-YAP2+H₂O₂组和Control组，Bax表达量高于si-YAP2+H₂O₂组和Control组，差异均有统计学意义（均P<0.05）。H₂O₂组和NC+H₂O₂组Bcl-2、p-AKT、Bax表达量差异均无统计学意义（均P>0.05）。H₂O₂组TNF-α和IL-6含量高于si-YAP2+H₂O₂组和Control组（均P<0.05）。H₂O₂组和NC+H₂O₂组TNF-α和IL-6含量差异均无统计学意义（均P>0.05）。结论　沉默YAP2基因表达可能通过降低心肌细胞凋亡及炎症因子TNF-α和IL-6含量，从而降低心血管疾病发生。

关键词:

心肌细胞, YAP2基因, 凋亡, 免疫, PI3K/AKT信号通路

Huang Yi, Wang Conghan.

Effects of YAP2 gene on rat H9c2 cardiomyocyte apoptosis and TNF-α and IL-6 levels [J]. International Medicine and Health Guidance News, 2024, 30(14): 2297-2302.

黄毅王琮翰.

YAP2基因对大鼠H9c2心肌细胞凋亡及TNF-α和IL-6水平的影响 [J]. 国际医药卫生导报, 2024, 30(14): 2297-2302.

References

[1] 韦颖,莫昌干,韦利元,等.新型心脏康复训练对急性ST段抬高型心肌梗死患者PCI术后心功能及血管内皮功能的影响[J].国际医药卫生导报,2023,29(4):537-541.DOI:10.3760/cma.j.issn.1007-1245.2023.04.022.
[2] Talukder MA, Johnson WM, Varadharaj S, et al. Chronic cigarette smoking causes hypertension, increased oxidative stress, impaired NO bioavailability, endothelial dysfunction, and cardiac remodeling in mice[J]. Am J Physiol Heart Circ Physiol, 2011, 300(1):H388-H396. DOI: 10.1152/ajpheart.00868.2010.
[3] Koka S, Das A, Salloum FN, et al. Phosphodiesterase-5 inhibitor tadalafil attenuates oxidative stress and protects against myocardial ischemia/reperfusion injury in type 2 diabetic mice[J]. Free Radic Biol Med, 2013, 60:80-88. DOI: 10.1016/j.freeradbiomed.2013.01.031.
[4] Wang YL, Sun GY, Zhang Y, et al. Tormentic acid inhibits H2O2-induced oxidative stress and inflammation in rat vascular smooth muscle cells via inhibition of the NF-κB signaling pathway[J]. Mol Med Rep, 2016, 14(4):3559-3564. DOI: 10.3892/mmr.2016.5690.
[5] Jin J, Zhang L, Li X, et al. Oxidative stress-CBP axis modulates MOB1 acetylation and activates the Hippo signaling pathway[J]. Nucleic Acids Res, 2022, 50(7):3817-3834. DOI: 10.1093/nar/gkac189.
[6] Zhang H, Pasolli HA, Fuchs E. Yes-associated protein (YAP) transcriptional coactivator functions in balancing growth and differentiation in skin[J]. Proc Natl Acad Sci U S A, 2011, 108(6):2270-2275. DOI: 10.1073/pnas. 1019603108.
[7] Yang Y, Del Re DP, Nakano N, et al. miR-206 mediates YAP-induced cardiac hypertrophy and survival[J]. Circ Res, 2015, 117(10):891-904. DOI: 10.1161/CIRCRESAHA. 115.306624.
[8] Song B, Dang H, Dong R. The role and the signal pathways of Yes-associated protein 2 in hypertrophic cardiomyopathy[J]. Gen Physiol Biophys, 2021, 40(5):419-426. DOI: 10.4149/gpb_2021024.
[9] Dhar A, Dhar I, Bhat A, et al. Alagebrium attenuates methylglyoxal induced oxidative stress and AGE formation in H9C2 cardiac myocytes[J]. Life Sci, 2016, 146:8-14. DOI: 10.1016/j.lfs.2016.01.006.
[10] Radak Z, Zhao Z, Koltai E, et al. Oxygen consumption and usage during physical exercise: the balance between oxidative stress and ROS-dependent adaptive signaling[J]. Antioxid Redox Signal, 2013, 18(10):1208-1246. DOI: 10.1089/ars.2011.4498.
[11] Lin X, Wu S, Wang Q, et al. Saikosaponin-D reduces H2O2-induced PC12 cell apoptosis by removing ROS and blocking mapk-dependent oxidative damage[J]. Cell Mol Neurobiol, 2016, 36(8):1365-1375. DOI: 10.1007/s10571-016-0336-5.
[12] Chakraborti S, Pramanick A, Saha S, et al. Atypical G protein β5 promotes cardiac oxidative stress, apoptosis, and fibrotic remodeling in response to multiple cancer chemotherapeutics[J]. Cancer Res, 2018, 78(2):528-541. DOI: 10.1158/0008-5472.CAN-17-1280.
[13] Lin X, Jiang S, Jiang Z, et al. Effects of equol on H2O2-induced oxidative stress in primary chicken intestinal epithelial cells[J]. Poult Sci, 2016, 95(6):1380-1386. DOI: 10.3382/ps/pew034.
[14] Zhao B, Li L, Lu Q, et al. Angiomotin is a novel Hippo pathway component that inhibits YAP oncoprotein[J]. Genes Dev, 2011, 25(1):51-63. DOI: 10.1101/gad.2000111.
[15] Li C, Wang S, Xing Z, et al. A ROR1-HER3-lncRNA signalling axis modulates the Hippo-YAP pathway to regulate bone metastasis[J]. Nat Cell Biol, 2017, 19(2):106-119. DOI: 10.1038/ncb3464.
[16] Huang J, Wu S, Barrera J, et al. The Hippo signaling pathway coordinately regulates cell proliferation and apoptosis by inactivating Yorkie, the Drosophila Homolog of YAP[J]. Cell, 2005, 122(3):421-434. DOI: 10.1016/j.cell.2005.06.007.
[17] Ren F, Zhang L, Jiang J. Hippo signaling regulates Yorkie nuclear localization and activity through 14-3-3 dependent and independent mechanisms[J]. Dev Biol, 2010, 337(2):303-312. DOI: 10.1016/j.ydbio.2009.10.046.
[18] Xin M, Kim Y, Sutherland LB, et al. Regulation of insulin-like growth factor signaling by Yap governs cardiomyocyte proliferation and embryonic heart size[J]. Sci Signal, 2011, 4(196):ra70. DOI: 10.1126/scisignal. 2002278.
[19] 赵倩茹,曹梦菲,孙侠,等.Yes相关蛋白在血管紧张素Ⅱ诱导的心肌纤维化中的作用及机制研究[J].中华老年心脑血管病杂志,2022,24(3):306-310. DOI:10.3969/j.issn.1009- 0126.2022.03.021.
[20] Mia MM, Cibi DM, Ghani SABA, et al. Loss of Yap/Taz in cardiac fibroblasts attenuates adverse remodelling and improves cardiac function[J]. Cardiovasc Res, 2022, 118(7):1785-1804. DOI: 10.1093/cvr/cvab205.
[21] Zheng XH, Wang LL, Zheng MZ, et al. RGFP966 inactivation of the YAP pathway attenuates cardiac dysfunction induced by prolonged hypothermic preservation[J]. J Zhejiang Univ Sci B, 2020, 21(9):703-715. DOI: 10.1631/jzus.B2000026.
[22] Li X, Wu C, Chen N, et al. PI3K/Akt/mTOR signaling pathway and targeted therapy for glioblastoma[J]. Oncotarget, 2016, 7(22):33440-33450. DOI: 10.18632/oncotarget.7961.
[23] Xia H, Li Y, Lv X. MicroRNA-107 inhibits tumor growth and metastasis by targeting the BDNF-mediated PI3K/AKT pathway in human non-small lung cancer[J]. Int J Oncol, 2016, 49(4):1325-1333. DOI: 10.3892/ijo.2016. 3628.
[24] Worou ME, Belmokhtar K, Bonnet P, et al. Hemin decreases cardiac oxidative stress and fibrosis in a rat model of systemic hypertension via PI3K/Akt signalling[J]. Cardiovasc Res, 2011, 91(2):320-329. DOI: 10.1093/cvr/cvr072.
[25] Peng X, Shao J, Shen Y, et al. FAT10 protects cardiac myocytes against apoptosis[J]. J Mol Cell Cardiol, 2013, 59:1-10. DOI: 10.1016/j.yjmcc.2013.01.018.
[26] Cheng XY, Gu XY, Gao Q, et al. Effects of dexmedetomidine postconditioning on myocardial ischemia and the role of the PI3K/Akt-dependent signaling pathway in reperfusion injury[J]. Mol Med Rep, 2016, 14(1):797-803. DOI: 10.3892/mmr.2016.5345.
[27] Cheng SM, Ho TJ, Yang AL, et al. Exercise training enhances cardiac IGFI-R/PI3K/Akt and Bcl-2 family associated pro-survival pathways in streptozotocin-induced diabetic rats[J]. Int J Cardiol, 2013, 167(2):478-485. DOI: 10.1016/j.ijcard.2012. 01.031.
[28] Basu S, Totty NF, Irwin MS, et al. Akt phosphorylates the Yes-associated protein, YAP, to induce interaction with 14-3-3 and attenuation of p73-mediated apoptosis[J]. Mol Cell, 2003, 11(1):11-23. DOI: 10.1016/s1097-2765(02)00776-1.
[29] Jin H, Fujita T, Jin M, et al. Epac activation inhibits IL-6-induced cardiac myocyte dysfunction[J]. J Physiol Sci, 2018, 68(1):77-87. DOI: 10.1007/s12576-016-0509-5.
[30] Wu H, Wang G, Li S, et al. TNF-α- mediated-p38-dependent signaling pathway contributes to myocyte apoptosis in rats subjected to surgical trauma[J]. Cell Physiol Biochem, 2015, 35(4):1454-1466. DOI: 10.1159/000373965.
[31] Brown M, McGuinness M, Wright T, et al. Cardiac-specific blockade of NF-kappaB in cardiac pathophysiology: differences between acute and chronic stimuli in vivo[J]. Am J Physiol Heart Circ Physiol, 2005, 289(1):H466-H476. DOI: 10.1152/ajpheart.00170.2004.