Complex molecular mechanism of ammonia-induced apoptosis in chicken peripheral blood lymphocytes: miR-27b-3p, heat shock proteins, immunosuppression, death receptor pathway, and mitochondrial pathway
Complex molecular mechanism of ammonia-induced apoptosis in chicken peripheral blood lymphocytes: miR-27b-3p, heat shock proteins, immunosuppression, death receptor pathway, and mitochondrial pathway
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DOI：
10.1016/j.ecoenv.2022.113471
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发表时间：
2022-04-02
影响因子：
6.8
通讯作者：
Tang, You
中科院分区：
文献类型：
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作者：
Zhang, Jingyang;Cui, Jiawen;Tang, You
氨气是一种有毒的环境污染物，是PM2.5气溶胶的重要组成部分，可以降低人类和动物的免疫力。外周血淋巴细胞是主要的免疫细胞。然而，多氯联苯在氨暴露下的中毒机制尚不清楚。本实验建立鸡pbl氨中毒模型，探讨氨致鸡pbl细胞凋亡的中毒机制。分别采用CCK8法和流式细胞术检测细胞活力和凋亡率。荧光染色法观察线粒体膜电位（MMP）。此外，采用qRT-PCR检测凋亡相关基因(肿瘤坏死因子- α (TNF- α)、肿瘤坏死因子受体1 （TNFR1）、肿瘤坏死因子受体相关死亡结构域（TRADD）、肿瘤坏死因子相关死亡结构域（FADD）、Caspase-8、bh3相互作用结构域死亡激动剂（Bid）、Bcl-2相关X蛋白（Bax）、Bcl-2同源拮抗剂/杀伤剂（Bak）、b细胞淋巴瘤-2 （Bcl-2）、细胞色素-c （Cytc）、凋亡蛋白酶激活因子-1 （APAF1）、Caspase-9、和Caspase-3)、免疫相关基因(干扰素- γ （ifn - γ)、白细胞介素-2 （IL-2）、IL-4、IL-6、IL-1 β、IL- 10、转化生长因子- β 1 （tgf - β 1）、IL-17、IL-21和IL-22）、热休克蛋白（HSP）基因（HSP25、HSP40、HSP60、HSP70、HSP90和HSP110）以及miR-27b-3p。Western blot检测细胞凋亡相关因子（tnf - α、Caspase-8、Bcl-2、Caspase-9、Caspase-3）和热休克蛋白（HSP40、HSP60、HSP70、HSP90）的蛋白水平。结果表明，TRADD、FADD和APAF1是miR-27b-3p的靶基因，miR-27b-3p通过靶向氨处理鸡pbl中TNF α /TNFR1/Caspase-8死亡受体通路触发的Bid/Cytc/Caspase-9线粒体通路参与细胞凋亡的分子机制。此外，我们的研究结果表明，过量的氨通过Th1/Th2失衡和Treg/Th17失衡导致免疫抑制。同时，氨胁迫激活热休克蛋白。总之，我们的数据首次证明了热休克蛋白引发的免疫抑制导致氨暴露下的细胞凋亡。研究结果为氨中毒的分子机制提供了新的认识，为氨中毒环境风险评价提供了重要参考。
Ammonia gas, a toxic environmental pollutant, is a vital component of PM2.5 aerosols, and can decrease human and animal immunity. Peripheral blood lymphocytes (PBLs) are main immune cells. Nevertheless, poisoning mechanism of PBLs under ammonia exposure remains unclear. Here, we established an ammonia poisoning model of chicken PBLs to explore poisoning mechanism of ammonia-caused apoptosis in chicken PBLs. Cell viability and apoptosis rate were detected using CCK8 assay and flow cytometry, respectively. Mitochondrial membrane potential (MMP) was observed using fluorescent staining. In addition, qRT-PCR was performed to measure mRNA levels of apoptosis-related genes (tumor necrosis factor-alpha (TNF-alpha), tumor necrosis factor receptor 1 (TNFR1), TNF receptor-associated death domain (TRADD), Fas-associated death domain (FADD), Caspase-8, BH3-interacting domain death agonist (Bid), Bcl-2-associated X protein (Bax), Bcl-2 homologous antagonist/ killer (Bak), B-cell lymphoma-2 (Bcl-2), Cytochrome-c (Cytc), apoptotic protease activating factor-1 (APAF1), Caspase-9, and Caspase-3), immune-related genes (interferon-gamma (IFN-gamma), interleukin-2 (IL-2), IL-4, IL-6, IL-1 beta, IL 10, transforming growth factor-beta 1 (TGF-beta 1), IL-17, IL-21, and IL-22), heat shock protein (HSP) genes (HSP25, HSP40, HSP60, HSP70, HSP90, and HSP110), as well as miR-27b-3p. Western blot was used to determine protein levels of apoptosis-related factors (TNF-alpha, Caspase-8, Bcl-2, Caspase-9, and Caspase-3), as well as HSPs (HSP40, HSP60, HSP70, and HSP90). The results indicated that TRADD, FADD, and APAF1 were target genes of miR-27b-3p, as well as miR-27b-3p participated in molecular mechanism of apoptosis through targeting TNF alpha/TNFR1/Caspase-8 death receptor pathway-triggered Bid/Cytc/Caspase-9 mitochondrial pathway in ammonia treated chicken PBLs. In addition, our findings demonstrated that excess ammonia led to immunosuppression via Th1/Th2 imbalance and Treg/Th17 imbalance. Simultaneously, ammonia stress activated HSPs. In summary, for the first time, our data demonstrated that HSPs-triggered immunosuppression led to apoptosis under ammonia exposure. Our findings provided a new insight into molecular mechanism of ammonia poisoning and an important reference for environmental risk assessment related to ammonia.