邻苯二甲酸二-2-乙基己酯通过ROS/PTEN/PI3K/AKT轴诱导HD11细胞凋亡和程序性坏死

doi:10.11843/j.issn.0366-6964.2023.12.033

摘要/Abstract

摘要： 旨在探究邻苯二甲酸二-2-乙基己酯[di (2-ethylhexyl) phthalate, (DEHP)]暴露通过ROS/PTEN/PI3K/AKT通路诱导细胞凋亡和程序性坏死的作用机制。本试验以HD11细胞为研究对象,设置对照组(C组)、30 μmol·L^-1 DEHP组(L组)、60 μmol·L^-1 DEHP组(M组)和90 μmol·L^-1 DEHP组(H组)。用不同浓度的DEHP处理HD11细胞24 h后,采用CCK-8检测细胞存活情况,生化试剂盒检测氧化应激指标,包括活性氧(ROS)水平,丙二醛(MDA)含量与总抗氧化能力(T-AOC)水平,以及总超氧化物歧化酶(T-SOD)和谷胱甘肽过氧化物酶(GSH-PX)活性。采用AO/EB染色和流式细胞术检测凋亡率和坏死率,荧光定量RT-PCR和Western blot法检测PTEN/PI3K/AKT通路基因及凋亡与坏死相关基因的mRNA和蛋白表达,结果表明,与对照组相比,DEHP暴露可显著抑制HD11细胞的活力,且半数抑制浓度(IC₅₀)为180.644 μmol·L^-1。与对照组相比,L、M、H DEHP组AO/EB染色和流式细胞术结果显示DEHP暴露导致HD11细胞具有典型的凋亡和坏死特征。与对照组相比,L、M、H DEHP组ROS水平呈现上升趋势(P<0.01),L、M、H DEHP组MDA含量显著升高(P<0.01),而T-AOC水平显著降低(P<0.01),T-SOD和GSH-PX活性显著降低(P<0.01),提示DEHP暴露诱导HD11细胞发生氧化应激损伤。此外,与对照组相比,DEHP暴露上调了PTEN/Bax/Caspase-3/Caspase-9/RIPK1/RIPK3/MLKL的mRNA和蛋白表达(P<0.01),而 PI3K/AKT/BCL-2的mRNA和蛋白表达显著降低(P<0.01),提示DEHP暴露通过PTEN/PI3K/AKT信号通路诱导HD11细胞凋亡和程序性坏死。综上所述,DEHP可以通过调控ROS/PTEN/PI3K/AKT轴诱导HD11细胞凋亡和程序性坏死,并存在明显的剂量-效应关系。

关键词: DEHP, 氧化应激, 细胞凋亡, 程序性坏死

Abstract: To explore the mechanism of apoptosis and necroptosis induced by di(2-ethylhexyl)phthalate (DEHP) exposure through ROS/PTEN/PI3K/AKT pathway. HD11 cells were used in this study, and control group(C group), 30 μmol·L^-1 DEHP group(L group), 60 μmol·L^-1 DEHP group(M group), 90 μmol·L^-1 DEHP group(H group)were set. After cells were treated with different concentrations of DEHP for 24 h, cell survival was detected by CCK-8, oxidative stress was detected by biochemical kit, included reactive oxygen species (ROS)levels, malondialdehyde (MDA)content, total antioxidant capacity (T-AOC)levels, and total superoxide dismutase (T-SOD)and glutathione peroxidase (GSH-PX)activities. AO/EB staining and flow cytometry were used to detect the rate of apoptosis and necrosis, mRNA and protein expressions of PTEN/PI3K/AKT pathway genes and apoptosis and necroptosis related genes were detected by qRT-PCR and Western blot. The results showed that, compared with the control group, DEHP considerably reduced the viability of HD11 cells, with the median inhibitory concentration (IC₅₀)being 180.644 μmol·L^-1. Besides, compared with the control group, AO/EB staining and flow cytometry validated the L, M and H DEHP group with typical characteristics of apoptosis and necroptosis. Additionally, compared with the control group, ROS expression levels in groups L, M and H showed an increasing trend (P<0.01), and the content of MDA were considerably increased (P<0.01), the T-AOC level was significantly decreased (P<0.01), and the activities of T-SOD and GSH-PX were significantly decreased (P<0.01). It was suggested that DEHP exposure induced oxidative stress injury of HD11 cells. In addition, compared with the control group, DEHP exposure up-regulated the mRNA and protein expression levels of PTEN/Bax/Caspase-9/Caspase-3/RIPK1/RIPK3/MLKL and down-regulated the mRNA and protein expression levels of PI3K/AKT/BCL-2(P<0.01), these results suggested that DEHP exposure induced apoptosis and necroptosis of HD11 cells through the PTEN/PI3K/AKT signaling pathway. Overall, DEHP can induce apoptosis and necroptosis in HD11 cells by modulating the ROS/PTEN/PI3K/AKT axis, and there is a dose-effect manner.

Key words: DEHP, oxidative stress, apoptosis, necroptosis

中图分类号:

S859.8

李广兴, 陈阳, 陈凯婷, 武梦林, 张迪, 黄小丹. 邻苯二甲酸二-2-乙基己酯通过ROS/PTEN/PI3K/AKT轴诱导HD11细胞凋亡和程序性坏死[J]. 畜牧兽医学报, 2023, 54(12): 5240-5251.

LI Guangxing, CHEN Yang, CHEN Kaiting, WU Menglin, ZHANG Di, HUANG Xiaodan. Di (2-ethylhexyl) Phthalate Induces the Apoptosis and Necroptosis of HD11 Cells through the ROS/PTEN/PI3K/AKT Axis[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5240-5251.

参考文献

[1] ROWDHWAL S S S, CHEN J X. Toxic effects of Di-2-ethylhexyl phthalate:an overview[J]. Biomed Res Int, 2018, 2018:1750368.
[2] ERYTHROPEL H C, MARIC M, NICELL J A, et al. Leaching of the plasticizer di(2-ethylhexyl)phthalate (DEHP) from plastic containers and the question of human exposure[J]. Appl Microbiol Biotechnol, 2014, 98(24):9967-9981.
[3] LI X T, LV Z M, CHEN J, et al. Bacillus amyloliquefaciens B10 can alleviate liver apoptosis and oxidative stress induced by aflatoxin B1[J]. Food Chem Toxicol, 2021, 151:112124.
[4] WANG Y, ZHU H K, KANNAN K. A review of biomonitoring of phthalate exposures[J]. Toxics, 2019, 7(2):21.
[5] WANG S T, CAO Y R, WANG S C, et al. DEHP induces immunosuppression through disturbing inflammatory factors and CYPs system homeostasis in common carp neutrophils[J]. Fish Shellfish Immunol, 2020, 96:26-31.
[6] YI W E I, TANG X L, ZHOU Y, et al. DEHP exposure destroys blood-testis barrier (BTB) integrity of immature testes through excessive ROS-mediated autophagy[J]. Genes Dis, 2018, 5(3):263-274.
[7] SUN Y Y, YANG F, LIU Y, et al. Di-2-ethylhexyl phthalate induces heart looping disorders during zebrafish development[J]. Toxicol Ind Health, 2021, 37(7):391-397.
[8] FENG W W, LIU Y C, DING Y Y, et al. Typical neurobehavioral methods and transcriptome analysis reveal the neurotoxicity and mechanisms of di(2-ethylhexyl) phthalate on pubertal male ICR mice with type 2 diabetes mellitus[J]. Arch Toxicol, 2020, 94(4):1279-1302.
[9] YOU H H, LI R, WEI C X, et al. Thymic stromal lymphopoietin neutralization inhibits the immune adjuvant effect of Di-(2-ethylhexyl) phthalate in balb/c mouse asthma model[J]. PLoS One, 2016, 11(7):e0159479.
[10] SERRANO K, LEVIN E, CHEN D, et al. An investigation of red blood cell concentrate quality during storage in paediatric-sized polyvinylchloride bags plasticized with alternatives to di-2-ethylhexyl phthalate (DEHP)[J]. Vox Sang, 2016, 110(3):227-235.
[11] LEE J W, PARK S, HAN H K, et al. Di-(2-ethylhexyl) phthalate enhances melanoma tumor growth via differential effect on M1-and M2-polarized macrophages in mouse model[J]. Environ Pollut, 2018, 233:833-843.
[12] YU L, LI H X, GUO J Y, et al. Di (2-ethyl hexyl) phthalate (DEHP)-induced spleen toxicity in quail (Coturnix japonica) via disturbing Nrf2-mediated defense response[J]. Environ Pollut, 2019, 251:984-989.
[13] YAHYAVY S, VALIZADEH A, SAKI G, et al. Taurine ameliorates cytotoxic effects of Di(2-ethylhexyl) phthalate on Leydig cells[J]. Andrologia, 2021, 53(8):e14146.
[14] HONG Y F, ZHOU Y, SHEN L J, et al. Exposure to DEHP induces testis toxicity and injury through the ROS/mTOR/NLRP3 signaling pathway in immature rats[J]. Ecotoxicol Environ Saf, 2021, 227:112889.
[15] TRIPATHI A, PANDEY V, SAHU A N, et al. Di-(2-ethylhexyl) phthalate (DEHP) inhibits steroidogenesis and induces mitochondria-ROS mediated apoptosis in rat ovarian granulosa cells[J]. Toxicol Res, 2019, 8(3):381-394.
[16] HAN Q, ZHANG J Y, SUN Q, et al. Oxidative stress and mitochondrial dysfunction involved in ammonia-induced nephrocyte necroptosis in chickens[J]. Ecotoxicol Environ Saf, 2020, 203:110974.
[17] TANG Y S, ZHAO Y H, ZHONG Y, et al. Neferine inhibits LPS-ATP-induced endothelial cell pyroptosis via regulation of ROS/NLRP3/Caspase-1 signaling pathway[J]. Inflamm Res, 2019, 68(9):727-738.
[18] LI Z L, MI J, LU L, et al. The main anthocyanin monomer of Lycium ruthenicum Murray induces apoptosis through the ROS/PTEN/PI3K/Akt/caspase 3 signaling pathway in prostate cancer DU-145 cells[J]. Food Funct, 2021, 12(4):1818-1828.
[19] WANG L Q, WANG L X, SHI X, et al. Chlorpyrifos induces the apoptosis and necroptosis of L8824 cells through the ROS/PTEN/PI3K/AKT axis[J]. J Hazard Mater, 2020, 398:122905.
[20] LIU H Y, ZHANG Y Y, ZHU B L, et al. miR-21 regulates the proliferation and apoptosis of ovarian cancer cells through PTEN/PI3K/AKT[J]. Eur Rev Med Pharmacol Sci, 2019, 23(10):4149-4155.
[21] HU S P, CHANG X X, ZHU H B, et al. PI3K mediates tumor necrosis factor induced-necroptosis through initiating RIP1-RIP3-MLKL signaling pathway activation[J]. Cytokine, 2020, 129:155046.
[22] CHEN H J, ZHANG Y, ZOU M M, et al. Bisphenol A aggravates renal apoptosis and necroptosis in selenium-deficient chickens via oxidative stress and PI3K/AKT pathway[J]. J Cell Physiol, 2022, 237(8):3292-3304.
[23] ZHANG J Q, ZHENG S F, WANG S C, et al. Cadmium-induced oxidative stress promotes apoptosis and necrosis through the regulation of the miR-216a-PI3K/AKT axis in common carp lymphocytes and antagonized by selenium[J]. Chemosphere, 2020, 258:127341.
[24] FU Y Y, DONG J, WANG J N, et al. Developmental exposure to Di-(2-ethylhexyl) phthalate induces cerebellar granule cell apoptosis via the PI3K/AKT signaling pathway[J]. Exp Neurobiol, 2018, 27(6):472-488.
[25] FU G Q, DAI J, LI Z, et al. The role of STAT3/p53 and PI3K-Akt-mTOR signaling pathway on DEHP-induced reproductive toxicity in pubertal male rat[J]. Toxicol Appl Pharmacol, 2020, 404:115151.
[26] KITAOKA M, HIRAI S, TERAYAMA H, et al. Effects on the local immunity in the testis by exposure to di-(2-ethylhexyl) phthalate (DEHP) in mice[J]. J Reprod Dev, 2013, 59(5):485-490.
[27] HOSSEINZADEH A, MEHRZADI S, SIAHPOOSH A, et al. The ameliorative effect of ellagic acid on di-(2-ethylhexyl) phthalate-induced testicular structural alterations, oxidative stress, inflammation and sperm damages in adult mice[J]. Reprod Biol Endocrinol, 2021, 19(1):146.
[28] FORRESTER S J, KIKUCHI D S, HERNANDES M S, et al. Reactive oxygen species in metabolic and inflammatory signaling[J]. Circ Res, 2018, 122(6):877-902.
[29] JAKUBCZYK K, DEC K, KAȽDUŃSKA J, et al. Reactive oxygen species-sources, functions, oxidative damage[J]. Pol Merkur Lekarski, 2020, 48(284):124-127.
[30] CHENG C H, MA H L, DENG Y Q, et al. Oxidative stress, cell cycle arrest, DNA damage and apoptosis in the mud crab (Scylla paramamosain) induced by cadmium exposure[J]. Chemosphere, 2021, 263:128277.
[31] HUANG M Q, LIU S, FU L, et al. Bisphenol A and its analogues bisphenol S, bisphenol F and bisphenol AF induce oxidative stress and biomacromolecular damage in human granulosa KGN cells[J]. Chemosphere, 2020, 253:126707.
[32] GENG L J, XIA Z H, YUAN L, et al. Effects of β-HgS on cell viability and intracellular oxidative stress in PC-12 cells[J]. Metallomics, 2020, 12(9):1389-1399.
[33] WU Z M, WANG H G, FANG S Y, et al. Roles of endoplasmic reticulum stress and autophagy on H₂O₂-induced oxidative stress injury in HepG2 cells[J]. Mol Med Rep, 2018, 18(5):4163-4174.
[34] WANG X L, YAN X, YANG Y Y, et al. Dibutyl phthalate-mediated oxidative stress induces splenic injury in mice and the attenuating effects of vitamin E and curcumin[J]. Food Chem Toxicol, 2020, 136:110955.
[35] PARK C G, SUNG B, RYU C S, et al. Mono-(2-ethylhexyl) phthalate induces oxidative stress and lipid accumulation in zebrafish liver cells[J]. Comp Biochem Physiol Part C:Toxicol Pharmacol, 2020, 230:108704.
[36] BALLY M B, OPHEIM D J, SHERTZER H G. Di-(2-ethylhexyl) phthalate enhances the release of lysosomal enzymes from alveolar macrophages during phagocytosis[J]. Toxicology, 1980, 18(1):49-60.
[37] CHO Y J, PARK S B, HAN M. Di-(2-ethylhexyl)-phthalate induces oxidative stress in human endometrial stromal cells in vitro[J]. Mol Cell Endocrinol, 2015, 407:9-17.
[38] LIAO H P, LIU S L, JUNAID M, et al. Di-(2-ethylhexyl) phthalate exacerbated the toxicity of polystyrene nanoplastics through histological damage and intestinal microbiota dysbiosis in freshwater Micropterus salmoides[J]. Water Res, 2022, 219:118608.
[39] FANG H Q, FANG W T, CAO H W, et al. Di-(2-ethylhexyl)-phthalate induces apoptosis via the PPARγ/PTEN/AKT pathway in differentiated human embryonic stem cells[J]. Food Chem Toxicol, 2019, 131:110552.
[40] YU D M, ZHU D W, WANG X F, et al. Prenatal di-(2-ethylhexyl) phthalate exposure induced myocardial cytotoxicity via the regulation of the NRG1-dependent ErbB2/ErbB4-PI3K/AKT signaling pathway in fetal mice[J]. Ecotoxicol Environ Saf, 2022, 241:113771.
[41] LIU X J, ZHANG Y L, SUN X Y, et al. Di-(2-ethyl hexyl) phthalate induced oxidative stress promotes microplastics mediated apoptosis and necroptosis in mice skeletal muscle by inhibiting PI3K/AKT/mTOR pathway[J]. Toxicology, 2022, 474:153226.
[42] GONG Y, LUO S, FAN P, et al. Growth hormone activates PI3K/Akt signaling and inhibits ROS accumulation and apoptosis in granulosa cells of patients with polycystic ovary syndrome[J]. Reprod Biol Endocrinol, 2020, 18(1):121.
[43] LIU L L, LIU X L, ZHAO L Y, et al. 1, 8-cineole alleviates bisphenol A-induced apoptosis and necroptosis in bursa of Fabricius in chicken through regulating oxidative stress and PI3K/AKT pathway[J]. Ecotoxicol Environ Saf, 2021, 226:112877.
[44] ZHU B B, ZHANG Z C, LI J, et al. Di-(2-ethylhexyl) phthalate induces testicular endoplasmic reticulum stress and germ cell apoptosis in adolescent mice[J]. Environ Sci Pollut Res Int, 2021, 28(17):21696-21705.
[45] ZHENG Y Y, SHI G L, CAI J Z, et al. Di-(2-ethyl hexyl) phthalate induces necroptosis in chicken cardiomyocytes by triggering calcium overload[J]. J Hazard Mater, 2020, 387:121696.