5-羟色胺通过上调2B受体抑制放线菌素D诱导肝细胞凋亡初步实验研究

肝脏 ›› 2019, Vol. 24 ›› Issue (12): 1387-1392.

5-羟色胺通过上调2B受体抑制放线菌素D诱导肝细胞凋亡初步实验研究

马丽霞, 高玉娟, 刘晓慧, 张晶

100069 首都医科大学附属北京佑安医院丙型肝炎与中毒性肝病科(马丽霞,刘晓慧,张晶);南京大学附属南京鼓楼医院呼吸与危重症医学科(高玉娟)

收稿日期:2019-09-18 发布日期:2020-03-28
通讯作者: 张晶,Email:drzhangjing@163.com

5-HT inhibits actinomycin D induced hepatocyte apoptosis by upregulating 2B receptor

MA Li-xia, GAO Yu-juan, LIU Xiao-hui, ZHANG Jing

Department of Hepatitis C and Toxic Hepatology, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China

Received:2019-09-18 Published:2020-03-28
Contact: ZHANG Jing, Email: drzhangjing@163.com

摘要/Abstract

摘要： 目的探讨5-羟色胺(5-HT)在肝细胞凋亡中的作用及机制。方法以不同浓度梯度放线菌素D(AcD)分别诱导HepG2和7702细胞凋亡,采用AnnexinV-FITC/PI流式细胞术和TUNEL方法检测细胞凋亡,MTT法检测细胞存活率。AcD预处理HepG2细胞后分别加入5-HT、5-HT 2A受体激动剂DOI、5-HT 2B受体激动剂M110、5-HT 2B受体拮抗剂SB204+5-HT,AnnexinV-FITC/PI流式细胞术和MTT法分别检测细胞凋亡率和存活率,蛋白质印迹法检测蛋白水解酶3(caspase3)、丝氨酸/苏氨酸蛋白激酶(AKT)及磷酸化丝氨酸/苏氨酸蛋白激酶(p-AKT)的表达。结果 AcD以浓度依赖性方式分别诱导HepG2细胞和7702细胞的凋亡,50 ng/mL AcD处理两种细胞凋亡率均高于无血清对照组(HePG2细胞t=1.71,7702细胞t=1.98,均P<0.05),与单用AcD组相比,5-HT使HepG2细胞凋亡率从31.96%±2.13%降至10.24%±2.59%(t=1.62,P<0.05),5-HT 2B受体激动剂使其降至8.41%±0.96%(t=1.75,P<0.05),反之,5-HT 2B受体拮抗剂能拮抗5-HT的抗凋亡作用,其凋亡率为25.92%±1.33%(t=1.45,P>0.05)。5-HT及5-HT 2B受体激动剂均能够减少caspase3活化,增加AKT的磷酸化,使细胞凋亡减少。结论 5-HT通过上调5-HT 2B受体促进AKT磷酸化,从而抑制AcD诱导的肝细胞凋亡。

关键词: 放线菌素D, 5-HT, 肝细胞凋亡, AKT磷酸化, 5-HT 2B受体

Abstract: Objective To investigate the role and mechanism of 5-hydroxytryptamine (5-HT) in hepatocyte apoptosis, and to provide evidence for the application of 5-HT in liver diseases.Methods Apoptosis of HepG2 and 7702 cells was induced by different concentrations of actinomycin-D (AcD). After AcD pretreatment of HepG2 cells, 5-HT, 5-HT 2A receptor agonist DOI, 5-HT 2B receptor agonist M110, and 5-HT 2B receptor antagonist SB204 with 5-HT were added respectively. Then, apoptotic rates and survival rates were detected by Annexin V-FITC/PI flow cytometry and MTT assay, expressions of aspartate specific protease 3 (caspase-3), serine/threonine protein kinase (AKT) and phosphorylated serine/threonine protein kinase (p-AKT) were detected by Western blot method. Results AcD induced apoptosis of HepG2 cells and 7702 cells in a concentration-dependent manner. The apoptotic rates of cells treated with 50 ng/ml AcD were higher than those of serum-free control group (HepG2 cells t=1.71, 7702 cells t=1.98, P<0.05). Compared with the AcD group, 5-HT decreased the apoptosis rate of HepG2 cells from 31.96% ± 2.13% to 10.24% ± 2.59% (t=1.62, P<0.05), and the 5-HT 2B receptor agonist decreased the apoptosis rate to 8.41% ± 0.96% (t=1.75, P<0.05). On the contrary, the 5-HT 2B receptor antagonist antagonize the anti-apoptotic effect of 5-HT, making the apoptotic rate 25.92% ± 1.33% (t=1.45, P>0.05). Both 5-HT and 5-HT 2B receptor agonists reduced the activation of caspase3, increased the phosphorylation of AKT, and decreased the apoptosis.Conclusion 5-HT promotes AKT phosphorylation by up-regulating 5-HT 2B receptor, thereby inhibiting AcD-induced hepatocyte apoptosis.

Key words: Actinomycin-D, 5-hydroxtryptamine, Hepatocyte apoptosis, AKT phosphorylation, 5-HT 2B receptor

马丽霞, 高玉娟, 刘晓慧, 张晶. 5-羟色胺通过上调2B受体抑制放线菌素D诱导肝细胞凋亡初步实验研究[J]. 肝脏, 2019, 24(12): 1387-1392.

MA Li-xia, GAO Yu-juan, LIU Xiao-hui, ZHANG Jing. 5-HT inhibits actinomycin D induced hepatocyte apoptosis by upregulating 2B receptor[J]. Chinese Hepatolgy, 2019, 24(12): 1387-1392.

参考文献

[1] Wang K, Lin B. Pathophysiological significance of hepatic apoptosis. ISRN Hepatol, 2013, 2013:740149.
[2] Chakraborty JB, Oakley F, Walsh MJ. Mechanisms and biomarkers of apoptosis in liver disease and fibrosis. Int J Hepatol, 2012, 2012: 648915.
[3] Zhang G, Yan C, Chen D, et al. Up-regulation of miR-155 contributes to TNF-mediated hepatocyte apoptosis in acute liver failure. Turk J Gastroenterol, 2019, 30: 475-484.
[4] Chen P, Hu M, Liu F, et al. S-allyl-l-cysteine (SAC) protects hepatocytes from alcohol-inducedapoptosis. FEBS Open Bio, 2019,9:1327-1336.
[5] Liu T, Yang T, Xu Z, et al. MicroRNA-193b-3p regulates hepatocyte apoptosis in selenium-defici- ent broilers by targeting MAML1. J Inorg Biochem, 2018, 186: 235-245.
[6] Niture S, Gyamfi MA, Kedir H, et al. Serotonin induced hepatic steatosis is associated with modu-lation of autophagy and notch signaling pathway. Cell Commun Signal,2018 , 16:78.
[7] Chang CF, Yang J, Zhao WM, et al. Gene expression profiling analysis of 5-hydroxytryptamine s-ignaling pathway in rat regenerating liver and different types of liver cells. Genet Mol Res, 2015,14:3409-3420.
[8] Zhang J, Song S, Pang Q, et al. Corrigendum: Serotonin deficiency exacerbates acetaminophen- induced liver toxicity in mice. Sci Rep, 2015, 5: 12184.
[9] Wang K, Wang M, Gannon M, et al. Growth Hormone Mediates Its Protective Effect in Hepatic Apoptosis through Hnf6. PLoS One, 2016 ,11:e0167085.
[10] Jiang J, Yu S, Jiang Z, et al. N-acetyl-serotonin protects HepG2 cells from oxidative stress injury induced by hydrogen peroxide. Oxid Med Cell Longev, 2014, 2014: 310504.
[11] Woodhead JL, Howell BA, Yang Y, et al. An analysis of N-acetylcysteine treatment for acetaminophen overdose using a systems model of drug-induced liver injury. J Pharmacol Exp Ther, 2012, 342: 529-540.
[12] Soll C, Jang JH, Riener MO, et al. Serotonin promotes tumor growth in human hepatocellular can- cer. Hepatology, 2010, 51: 1244-1254.
[13] Soll C, Riener MO, Oberkofler CE, et al. Expression of serotonin receptors in human hepatocellular cancer. Clin Cancer Res, 2012, 18:5902-5910.
[14] Tzirogiannis KN, Demonakou MD, Papadimas GK, et al. Effect of 5-HT(2) receptor blockade on cadmium-induced acute toxicity. Dig Dis Sci, 2007, 52: 2351-2358.
[15] 韦新焕,马丽霞,张晶.肝细胞凋亡与肝纤维化.肝脏,2012,17:887-890.
[16] Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol, 2007, 35: 495-516.
[17] Zhang X, Jiang W, Zhou AL, et al. Inhibitory effect of oxymatrine on hepatocyte apoptosis via TLR4/PI3K/Akt/GSK-3beta signaling pathway. World J Gastroenterol, 2017, 23: 3839-3849.
[18] Sun YY, Zhao YX, Li XF, et al. β-Arrestin 2 promotes hepatocyte apoptosis by inhibiting akt pathway in alcoholic liver disease. Front Pharmacol, 2018, 9: 1031.
[19] Li W, Cai S, Cai L, et al. Anti-apoptotic effect of hepatocyte growth factor from actinomycin D in hepatocyte-derived HL7702 cells is associated with activation of PI3K/Akt signaling. Toxicol Lett, 2006, 165: 142-148.