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摘要:背景
氯菊酯是一种常用的拟除虫菊酯类杀虫剂,研究发现其具有潜在神经系统毒性。小胶质细胞是中枢神经系统中的天然免疫细胞,参与一系列神经退行性疾病的发生。
目的本研究观察氯菊酯在体外对人小胶质细胞HMC3的毒性效应,并探讨其机制。
方法使用0、10、25、55 μmol·L−1的氯菊酯染毒HMC3 72 h后,使用流式细胞仪检测细胞周期和细胞凋亡,实时荧光定量PCR(qPCR)检测细胞周期蛋白依赖性激酶1基因(
CDK1 )、细胞周期蛋白依赖性激酶抑制因子1A基因(CDKN1A )、细胞周期蛋白B2基因(CCNB2 )、肿瘤蛋白p53基因(p53 )、凋亡相关因子基因(FAS )、胱天蛋白酶3基因(CASP3 )和H2A变体组蛋白基因(H2AX )的表达。转录组测序(RNA-seq)检测0、25 μmol·L−1氯菊酯染毒后HMC3的差异基因和富集通路。再次使用0、10、25、55 μmol·L−1的氯菊酯染毒HMC3 72 h后,使用格里斯试剂法检测上清中一氧化氮(NO)含量,酶联免疫吸附法检测白细胞介素(IL)-6的分泌水平,qPCR检测丝裂原活化蛋白激酶(MAPK)通路(包括MAPK1 、MAPK8 、MAPK14 )、IL-1 β 、IL-6 和基质金属蛋白酶(MMP)家族(包括MMP1 、MMP2 、MMP3 和MMP9 )的mRNA表达情况,蛋白质印记法(Western blot)检测磷酸化p38(p-p38)、磷酸化细胞外信号调节激酶(p-ERK)、IL-1β、IL-6和MMP1蛋白表达情况。结果0、10、25、55 μmol·L−1氯菊酯染毒细胞后,HMC3在G2/M期阻滞,其中55 μmol·L−1氯菊酯染毒组与对照组差异有统计学意义(
P <0.01),qPCR结果显示CDKN1A mRNA表达较对照组上调(P <0.05)。各组细胞凋亡比例差异无统计学意义(P >0.05)。RNA-seq结果提示差异基因富集于MAPK通路。qPCR结果提示55 μmol·L−1染毒组MAPK1 、MAPK8 和MAPK14 的mRNA表达较对照组上调(P <0.05)。Western blot发现,与对照组相比,10 μmol·L−1氯菊酯染毒组的p-p38和p-ERK水平均升高(P <0.05),25 μmol·L−1氯菊酯染毒组的p-ERK水平升高(P <0.05),55 μmol·L−1氯菊酯染毒组的p-p38水平升高(P <0.05)。与对照组相比,染毒后的HMC3上清液中NO分泌量增加(P <0.05),IL-6的mRNA、蛋白表达和分泌量呈上升趋势,IL-1β的mRNA和蛋白表达上调(P <0.05),25、55 μmol·L−1组MMP1的mRNA和蛋白表达上调(P <0.05)。结论氯菊酯在体外抑制HMC3细胞增殖,诱导细胞周期阻滞,可激活MAPK通路,促进炎症因子IL-1β以及MMP1表达,可能是氯菊酯致人神经毒性的机制之一。
Abstract:BackgroundPermethrin is a commonly used pyrethroid insecticide and has been found to be potentially neurotoxic. Microglia are innate immune cells in the central nervous system and are involved in the development of a range of neurodegenerative diseases.
ObjectiveTo observe possible toxic effects of permethrin on human microglia clone 3 (HMC3)
in vitro and explore associated mechanism.MethodsHMC3 were treated with 0, 10, 25, and 55 μmol·L−1 permethrin for 72 h. Cell cycle and apoptosis were measured using flow cytometry. Cyclin-dependent kinase 1 (
CDK1 ), cyclin-dependent kinase inhibitor 1A (CDKN1A ), cyclin B2 (CCNB2 ), cellular tumor antigen p53 (p53 ), factor-related apoptosis (FAS ), caspase 3 (CASP3 ), and H2A histone family member X (H2AX ) were detected by quantitative real-time PCR (qPCR). The differential genes and enrichment pathways of HMC3 after 0 and 25 μmol·L−1 permethrin treatment was analyzed by RNA sequencing. HMC3 was treated by 0, 10, 25, and 55 μmol· L−1 permethrin for 72 h. The content of nitric oxide (NO) in the supernatant was detected using Griess reagent. The secretion level of interleukin-6 (IL-6) was detected by enzyme linked immunosorbent assay (ELISA). The mRNA expression levels of mitogen-activated protein kinase (MAPK) pathway (includingMAPK1 ,MAPK8 , andMAPK14 ), interleukin-1β (IL-1 β ),IL-6 , and matrix metalloproteinase (MMP) families (includingMMP1 ,MMP2 ,MMP3 , andMMP9 ) were detected by qPCR. The protein expressions of phosphorylated p38 mitogen-activated protein kinase (p-p38), phosphorylated extracellular signal-regulated kinase (p-ERK), IL-1β, IL-6, and MMP1 were detected by Western blot.ResultsHMC3 was arrested in G2/M phase after 0, 10, 25, and 55 μmol·L−1 permethrin treatment for 72 h, of which there was a statistically significant difference between the 55 μmol·L−1 permethrin treatment group and the control group (
P <0.01), and the mRNA expression ofCDKN1A was up-regulated according to the qPCR (P <0.05). There was no statistically significant difference in the proportions of apoptosis between the groups (P >0.05). The RNA sequencing showed that the differential genes were enriched in the MAPK pathway, and the mRNA expressions ofMAPK1 ,MAPK8 , andMAPK14 were up-regulated after the permethrin treatment at 55 μmol·L−1 compared to the control group by qPCR (P <0.05). The Western blot revealed that, compared to the control group, the levels of p-p38 and p-ERK were increased after the 10 μmol·L−1 permetrin treatment (P <0.05), the p-ERK level was increased after the 25 μmol·L−1 permetrin treatment (P <0.05), and the p-p38 level was up-regulated after the 55 μmol·L−1 permetrin treatment (P <0.05). The secretion of NO in the supernatant of HMC3 increased after permetrin treatment compared to the control group (P <0.05), the mRNA and protein expressions and the secretion of IL-6 showed an upward trend, the mRNA and protein expressions of IL-1β were up-regulated (P <0.05), and the mRNA and protein expressions of MMP1 were up-regulated in the 25 and 55 μmol·L−1 permethrin groups (P <0.05).ConclusionPermethrin inhibits HMC3 cell proliferation
in vitro , induces cell cycle arrest, activates MAPK pathway, and promotes the expression of inflammatory factors IL-1β and MMP1, which may be one of the mechanism of neurotoxicity induced by permethrin. -
氟咯草酮(flurochloridone, FLC)作为新型除草剂,自上市以来在欧盟和北美国家中广泛应用。欧洲食品安全局(European Food Safety Authority, EFSA)在对FLC危险度评估报告中指出FLC的靶器官为睾丸和附睾[1]。前期研究结果显示,FLC能够导致精子畸形率增加,支持细胞空泡化,并可诱导支持细胞凋亡或自噬发生[2-3],损伤血睾屏障完整性[4]。FLC可诱导支持细胞产生大量活性氧,损伤内质网结构,破坏内质网钙离子(Ca2+)稳态[3],提示FLC可能会诱导支持细胞发生内质网应激,进而诱导细胞凋亡发生。
内质网在细胞内承担着重要的生命活动,主要包括蛋白质的正确折叠与合成运输、钙离子的储存与调节等[5]。当内质网中出现未折叠及错误折叠蛋白质累积,会引起内质网应激[6],同时启动未折叠蛋白反应[7-8],通过降低蛋白质转录和翻译、上调内质网伴侣蛋白葡萄糖调节蛋白78(glucose regulated protein 78, GRP78)水平、加强错误折叠蛋白降解等方式,使细胞恢复正常[9-10]。未折叠蛋白反应的激活涉及三条信号通路的参与,分别由肌醇需酶1α(inosital-requiring enzyme 1α, IRE1α)、蛋白激酶R样内质网激酶(protein kinase R like endoplasmic reticulum kinase, PERK)和活化转录因子6(activating transcription factor 6, ATF6)介导[11-12],它们与内质网伴侣蛋白GRP78解离并通过自身二聚体或磷酸化激活下一级信号通路,引发不同的细胞反应[13]。
越来越多的研究证实,内质网应激与细胞凋亡之间的关系十分密切。未折叠蛋白反应的下游蛋白,如c-Jun氨基末端激酶(c-Jun N-terminal kinase, JNK)[14],可通过调节B淋巴细胞瘤-2蛋白(B-cell lymphoma-2, Bcl-2)家族蛋白,如Bcl-2、Bcl-2相互作用的死亡介质(Bcl-2 interacting mediator of cell death, Bim)和Bcl-2相关X蛋白(Bcl-2 associated X protein, Bax)等的表达水平来促进细胞凋亡的发生[15-16]。
在FLC诱导睾丸组织及支持细胞损伤的过程中,是否启动了内质网应激和未折叠蛋白反应,以及未折叠蛋白反应的哪些信号通路在细胞凋亡的过程发挥作用还有待进一步研究证实。本研究通过对FLC染毒的C57BL/6小鼠睾丸组织中细胞凋亡发生情况的研究,利用小鼠睾丸支持细胞系TM4细胞模型,围绕内质网应激及未折叠蛋白反应IRE1α-JNK信号通路,观察其在不同染毒浓度下的改变情况,并且通过相应的干预实验探索其在FLC诱导支持细胞凋亡中的作用,为FLC诱导睾丸组织细胞凋亡的机制研究提供新思路。
1. 材料与方法
1.1 实验设计
本研究利用了实验室先前完成的6~8周龄雄性C57BL/6小鼠28 d经口染毒FLC的标本。FLC染毒剂量分别为0(0.5%羧甲基纤维素钠)、3、15、75和375 mg·(kg·d)−1[2]。观察睾丸组织中细胞凋亡发生情况,检测睾丸组织中的凋亡蛋白表达水平。在体外实验中,使用小鼠睾丸支持细胞系TM4细胞进行FLC(40、80和160 μmol·L−1)染毒6 h,检测细胞凋亡情况及内质网应激相关蛋白水平。干预实验中使用IRE1α磷酸化抑制剂4μ8C(上海Selleck)、JNK磷酸化抑制剂SP600125(上海碧云天)预处理TM4细胞6 h后再进行160 μmol·L−1 FLC染毒6 h,检测细胞活力、细胞凋亡情况及内质网应激相关蛋白水平。根据文献报道,选择4μ8C的处理浓度为25、50 μmol·L−1[17],SP600125的处理浓度为10、20 μmol·L−1[18-19]。
1.2 TM4细胞培养与FLC染毒液配制
小鼠睾丸支持细胞系TM4细胞(购自国家细胞资源平台,编号:1101MOU-PUMC000298)的培养条件为5%二氧化碳、37 ℃恒温,完全培养基为含有10%胎牛血清(美国Gibco)、1%青霉素-链霉素(美国Sigma)的高糖培养基(美国Gibco)。FLC(上海笃玛)固体粉末溶解于二甲基亚砜中制成不同浓度的染毒液用于后续实验。
1.3 TUNEL染色检测睾丸组织凋亡水平
小鼠睾丸组织石蜡切片置于二甲苯中浸洗进行脱蜡处理,再依次置于100%、95%、85%、75%乙醇中浸洗,根据TUNEL凋亡检测试剂盒(上海碧云天)的说明书配制并在切片上滴加适量TUNEL检测液,最后滴加抗荧光淬灭封片液[含4',6-二脒基-2-苯基吲哚(DAPI)]进行封片处理,尽快在荧光显微镜下观察,每张切片随机选择3个独立视野进行观察。
1.4 流式细胞术检测TM4细胞凋亡率
将细胞接种至6孔板中,待细胞贴壁后进行染毒处理,处理结束后,收集培养液,每孔中加入500 μL胰酶消化液消化细胞,收集细胞悬液,1000×g离心5 min,弃上清,随后加入195 μL膜联蛋白V(Annexin V)-异硫氰酸荧光素(FITC)结合液重悬细胞,再分别加入5 μL Annexin V-FITC和10 μL碘化丙啶(PI),混匀后室温避光孵育20 min,立即上机检测。使用FlowJo软件(版本号v10.0.7r2)进行凋亡细胞分析。
1.5 CCK-8法检测TM4细胞活力
将细胞接种至96孔板中,待细胞贴壁后进行染毒处理,处理结束后,每孔中加入10 μL CCK-8溶液,继续置于培养箱中培养1.5 h,取出96孔板于酶标仪中在波长为450 nm处测定光密度,将处理组与对照组光密度的比值作为相对细胞活力值。
1.6 Western blotting检测蛋白水平
采用Western blotting检测睾丸组织匀浆及TM4细胞内抗凋亡蛋白BCL-2、促凋亡蛋白Bim和Bax的蛋白水平,进一步检测TM4细胞中内质网应激标记蛋白GRP78、未折叠蛋白反应信号通路蛋白磷酸化PERK(p-PERK)、ATF6和磷酸化IREIα(p-IRE1α)以及内质网应激诱导凋亡关键蛋白p-JNK的表达水平。具体条件如下:蛋白电泳条件为80 V,1 h,待蛋白样本进入分离胶后转至100 V,1 h。转膜条件为100 V,1.5 h。将膜置于5%脱脂奶粉溶液中,室温封闭2 h。随后与含0.05% Tween20的三羟甲基氨基甲烷缓冲液(TBST)稀释的一抗(Bcl-2,1∶500稀释;Bim,1∶1000稀释;Bax,1∶500稀释;GRP78,1∶1000稀释;p-PERK,1∶500稀释;ATF6,1∶1000稀释;p-IRE1α,1∶1000稀释;p-JNK,1∶1000稀释)在4 °C孵育过夜。一抗孵育结束后,将蛋白条带与辣根过氧化物酶结合的二抗置于室温中孵育1 h,滴加适量增强化学发光法(ECL)超敏检测化学发光液,在ECL化学发光检测系统(美国Syngene)中检测目的蛋白条带的发光情况。使用ImageJ软件(版本号1.52v)分析蛋白条带灰度值,以β-actin或甘油醛-3-磷酸脱氢酶(GAPDH)作为内参蛋白,计算目的蛋白灰度值/内参灰度值的数值。
1.7 统计学分析
使用GraphPad Prism软件(版本号8.3.0)进行统计分析,不同处理组数据间的统计分析采用单因素方差分析,随后使用LSD进行两两组间比较。Western blotting及CCK-8实验每组包含6个平行数据,流式细胞术实验每组包含3个平行数据。检验水准α=0.05。
2. 结果
2.1 FLC诱导小鼠睾丸组织中细胞凋亡发生
雄性小鼠经口染毒FLC 28 d后,睾丸组织TUNEL染色结果(图1A)显示,375 mg·(kg·d)−1染毒组发生明显的细胞凋亡,主要集中在睾丸间质及生精小管基底部,提示有间质细胞及支持细胞凋亡发生。对凋亡蛋白水平的检测发现(图1B、图1C),Bcl-2蛋白水平呈现随染毒剂量增加而降低的趋势,Bim蛋白、Bax蛋白水平分别在75、375 mg·(kg·d)−1组中较对照组增加(P<0.05)。
图 1 FLC诱导小鼠睾丸组织损伤及凋亡发生A:睾丸组织TUNEL染色,蓝色荧光为细胞核,绿色荧光为凋亡细胞,白色箭头表示FLC引起的支持细胞凋亡;B:Western blotting检测凋亡蛋白的特征性条带图;C:Western blotting条带量化图(n=6)。与对照组相比,*:P<0.05,**:P<0.01,***:P<0.001。Figure 1. FLC-induced testis injury and apoptosis in miceA: TUNEL staining of mice testis tissue, cell nuclei are stained blue and apoptotic cells are stained green, apoptotic cells induced by FLC are pointed by white arrows; B: The representative blots of apoptotic proteins determinated by Western blotting; C: The quantitative data of Western blotting (n=6). Compared with the control group, *: P<0.05, **: P<0.01, ***: P<0.001.2.2 FLC诱导TM4细胞凋亡发生
细胞凋亡检测发现(图2A、图2B),40、80和160 μmol·L−1 FLC染毒组的细胞凋亡率分别为4.8%±1.3%、9.4%±0.3%、13.2%±0.2%,均高于对照组(2.7%±0.2%)(P<0.05)。检测Bcl-2家族凋亡蛋白的表达水平(图2C、图2D),与对照组相比,抗凋亡蛋白Bcl-2水平在160 μmol·L−1下降(P<0.05),而促凋亡蛋白Bim和Bax的水平在80和160 μmol·L−1时均出现上调(P<0.05)。
2 FLC诱导TM4凋亡发生A:流式细胞术检测细胞凋亡特征图;B:细胞凋亡率(n=3);C:Western blotting检测凋亡蛋白水平特征性条带图;D:Western blotting条带量化图(n=6)。与对照组相比,*:P<0.05,**:P<0.01,***:P<0.001。2. FLC-induced apoptosis in TM4 cellsA: Apoptotic cells detected by flow cytometry; B: Apoptotic rate (n=3); C: The representative blots of apoptotic proteins determinaed by Western blotting; D: The quantitative data of Western blotting (n=6). Compared with the control group, *: P<0.05, **: P<0.01, ***: P<0.001.2.3 FLC诱导TM4细胞内质网应激及未折叠蛋白反应发生
对内质网应激相关蛋白水平的检测结果显示(图3),与对照组相比,GRP78蛋白表达水平在80、160 μmol·L−1染毒组中上升,p-PERK蛋白表达水平在40 μmol·L−1时即出现上调(P<0.05),ATF6和p-IRE1α蛋白表达水平在160 μmol·L−1时上升(P<0.05)。同时,p-JNK蛋白表达水平也在40和160 μmol·L−1时上升(P<0.05),在80 μmol·L−1时上升不明显,呈“U型”效应曲线。
图 3 FLC诱导TM4细胞内质网应激及未折叠蛋白反应发生A:Western blotting检测内质网应激及未折叠蛋白反应蛋白特征性条带图;B1~B5:Western blotting条带量化图(n=6)。与对照组相比,*:P<0.05,**:P<0.01,***:P<0.001。Figure 3. FLC-activated endoplasmic reticulum stress and unfolded protein response in TM4 cellsA: The representative blots of endoplasmic reticulum stress- and unfolded protein response-related proteins determinated by Western blotting; B1-B5: The quantitative data of Western blotting (n=6). Compared with the control group, *: P<0.05, **: P<0.01, ***: P<0.001.2.4 IRE1α在FLC诱导细胞凋亡中的作用
单纯的4μ8C能够降低p-IRE1α的蛋白表达水平,同时降低GRP78及促凋亡蛋白Bax的表达水平(图4A、图4B、图4C、图4D),但是在一定程度上可抑制细胞活力(图4E)。在经过FLC染毒的细胞中,4μ8C能够降低GRP78、p-IRE1α、p-JNK以及Bax的表达水平,同时可以改善FLC诱导的细胞活力损伤。
图 4 IRE1α在FLC诱导细胞凋亡中的作用A:Western blotting检测内质网应激蛋白水平特征性条带图;B:Western blotting条带量化图(n=6);C:Western blotting检测凋亡蛋白水平特征性条带图;D:Western blotting条带量化图(n=6);E:CCK-8检测细胞活力(n=6)。与对照组相比,*:P<0.05,**:P<0.01,***:P<0.001。与160 μmol·L−1 FLC且0 μmol·L−1 4μ8C组相比,#:P<0.05,##:P<0.01,###:P<0.001。Figure 4. The role of IRE1α in cell apoptosis induced by FLCA: The representative blots of endoplasmic reticulum stress-related proteins determinated by Western blotting; B: The quantitative data of Western blotting (n=6); C: The representative blots of apoptotic proteins determinated by Western blotting; D: The quantitative data of Western blotting (n=6); E: Cell viability detected by CCK-8 assay. Compared with the control group, *: P<0.05, **: P<0.01, ***: P<0.001. Compared with the 160 μmol·L−1 FLC and 0 μmol·L−1 4μ8C group, #: P<0.05, ##: P<0.01, ###: P<0.001.2.5 JNK在FLC诱导细胞凋亡中的作用
SP600125预处理细胞后的内质网应激相关蛋白水平的检测结果显示,SP600125能够降低JNK的磷酸化水平,并呈浓度依赖性下调(图5A、图5B)。同时SP600125在高浓度时能够下调p-IRE1α、GRP78的蛋白表达水平,浓度依赖性抑制Bax的蛋白表达水平(图5C、图5D),并且可以缓解FLC诱导的细胞活力损伤(图5E)。
图 5 JNK在FLC诱导细胞凋亡中的作用A:Western blotting检测内质网应激蛋白水平特征性条带图;B:Western blotting条带量化图(n=6);C:Western blotting检测凋亡蛋白水平特征性条带图;D:Western blotting条带量化图(n=6);E:CCK-8检测细胞活力(n=6)。与对照组相比,*:P<0.05,**:P<0.01,***:P<0.001。与160 μmol·L−1 FLC且0 μmol·L−1 SP600125组相比,#:P<0.05,##:P<0.01,###:P<0.001。Figure 5. The role of JNK in cell apoptosis induced by FLCA: The representative blots of endoplasmic reticulum stress-related proteins determinated by Western blotting; B: The quantitative data of Western blotting (n=6); C: The representative blots of apoptotic proteins determinated by Western blotting; D: The quantitative data of Western blotting (n=6); E: Cell viability detected by CCK-8 assay. Compared with the control group, *: P<0.05, **: P<0.01, ***: P<0.001. Compared with the 160 μmol·L−1 FLC and 0 μmol·L−1 SP600125 group, #: P<0.05, ##: P<0.01, ###: P<0.001.3. 讨论
FLC可诱导小鼠睾丸组织中发生细胞凋亡,改变Bcl-2家族凋亡蛋白(Bcl-2、Bim、Bax)的表达水平,并且诱导TM4细胞活力损伤及细胞凋亡发生,在该过程中存在内质网应激发生,并且激活未折叠蛋白反应信号通路之一IRE1α通路,进一步针对IRE1α及其下游JNK通路进行抑制剂干预实验,证实了FLC通过激活IRE1α-JNK信号通路介导TM4细胞凋亡发生。
关于FLC的毒性研究主要集中在其对雄性动物生殖系统的损伤,其可损伤血睾屏障完整性[4],通过细胞外调节蛋白激酶1/2(extracellular regulated protein kinases 1/2, ERK1/2)信号通路导致支持细胞内质网Ca2+稳态失调[3],并通过线粒体途径诱导支持细胞凋亡[4],激活磷脂酰肌醇3-激酶(phosphoinositide 3-kinase, PI3K)/丝氨酸/苏氨酸激酶(serine/threonine kinases, Akt)/雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)信号通路引起自噬发生[2],还可激活促分裂素原活化蛋白激酶(mitogen-activated protein kinases, MAPK)信号通路[20],同时FLC还可激活内质网应激,通过PERK通路诱导细胞凋亡发生[21]。本研究补充了内质网应激与细胞凋亡发生之间可能存在其他的信号通路,进一步完善了FLC诱导凋亡发生的机制网络。
FLC的靶细胞为支持细胞,鉴于TM4细胞系在众多研究中作为小鼠睾丸支持细胞体外模型[22-24],本研究选择TM4细胞系进行体外实验,因此在睾丸组织中观察到凋亡发生后,进一步检测了TM4支持细胞凋亡发生情况,其凋亡率随染毒浓度增加而增加,凋亡蛋白水平也发生改变,提示FLC诱导TM4细胞凋亡发生。
细胞凋亡可由死亡受体依赖途径、线粒体途径及内质网途径介导[25],其中线粒体途径和内质网途径均依赖Bcl-2家族蛋白介导凋亡发生[26],通过促凋亡蛋白Bim与线粒体膜上表达的蛋白Bax结合,导致线粒体外膜通透性增加,促进半胱天冬酶和细胞色素c释放到胞浆中,引发细胞凋亡[27]。相比于线粒体途径诱导的凋亡,内质网应激诱导的凋亡还涉及未折叠蛋白反应三条信号通路的活化,分别由PERK、ATF6和IRE1α介导。前期研究已经探究了FLC可通过PERK通路激活CCAAT增强子结合蛋白同源蛋白(CCAAT enhancer binding protein homologous protein, CHOP),诱导凋亡发生[21],而许多研究表明,IRE1α信号通路也可以诱导细胞凋亡的发生[28]。
过量或持续的内质网应激可激活IRE1α的磷酸激酶活性,使下游凋亡信号调节激酶1(apoptosis signal regulating kinase-1, ASK1)活化,激活的ASK1可增加下游蛋白JNK的表达[29]。IRE1α还可与肿瘤坏死因子受体相关因子-2(tumor necrosis factor receptor associated factor-2, TRAF2)相互作用激活JNK。JNK激活后可与凋亡相关蛋白相互作用,如促进Bax的表达诱导线粒体凋亡途径[30]。因此本研究通过抑制剂体外干预TM4细胞着重探究了IRE1α和JNK在FLC诱导细胞凋亡中的作用。
4μ8C通过抑制IRE1α的RNase结构域限制x盒结合蛋白1(X-box binding protein 1, XBP1)的剪切,从而阻断未折叠蛋白反应的激活[31]。在本研究中,使用不同浓度的4μ8C预处理TM4细胞6 h后,可以看到4μ8C可降低磷酸化IRE1α的水平,其下游蛋白JNK的磷酸化水平也降低,并且促凋亡蛋白Bax在高浓度4μ8C的预处理组中发生下调,提示IRE1α参与了FLC诱导的内质网应激以及JNK的活化,能够通过抑制IRE1α的磷酸化缓解FLC诱导的细胞凋亡,证实FLC可能通过IRE1α-JNK-Bax信号通路诱导TM4细胞凋亡发生。为了进一步验证JNK在连接IRE1α和凋亡之间的作用,使用了JNK磷酸化抑制剂SP600125进行干预实验[32],SP600125能够浓度依赖性地抑制JNK的磷酸化,进而导致Bax蛋白表达水平下降,提示FLC诱导的TM4细胞凋亡受到JNK活化的影响,p-JNK参与了IRE1α介导细胞凋亡的过程,进一步证实了IRE1α-JNK-Bax信号通路在FLC诱导TM4细胞凋亡中的作用。同时两种抑制剂能够显著缓解FLC诱导的细胞活力受损,进一步证实了IRE1α/JNK通路参与了FLC诱导的TM4细胞活力受损。
本研究结果进一步证实了FLC能够诱导小鼠睾丸组织发生细胞凋亡,同时诱导TM4细胞凋亡发生,并且诱导内质网应激发生,激活未折叠蛋白反应IRE1α通路,通过抑制剂干预实验发现,FLC通过IRE1α-JNK信号通路介导凋亡发生。本研究为FLC诱导凋亡发生的机制网络补充了新的信号通路,但是该通路与其他通路之间是否存在关联尚需进一步研究,如内质网应激与氧化应激之间的关系、内质网途径诱导凋亡与线粒体途径诱导凋亡之间的关系等,还需再进一步研究中进行探究。
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图 2 氯菊酯对HMC3细胞周期及相关基因表达的影响
A:流式细胞仪检测氯菊酯染毒72 h后的HMC3细胞周期;B:各周期细胞比例定量;C:细胞周期相关基因表达情况。*:P<0.05;**:P<0.01; ****:P<0.0001。
Figure 2. Cell cycle and related-gene expressions of HMC3 after 72 h permethrin treatment
A: HMC3 cell cycle after 72 h permethrin treatment by flow cytometry. B: Quantification of cell proportions in each cycle. C: Cell cycle-related gene expression. *: P<0.05; **: P<0.01; ****: P<0.0001.
图 4 氯菊酯对HMC3 MAPK通路的影响
A:qPCR检测MAPK通路基因表达情况;B:Western blot检测MAPK通路蛋白表达条带及定量。**: P<0.01;***: P<0.001;****: P<0.0001。
Figure 4. Effects of permethrin on MAPK pathway of HMC3
A: Expression of genes related to MAPK pathway by qPCR. B: Protein expression bands of MAPK pathway and quantification by Western blot. **: P<0.01; ***: P<0.001; ****: P<0.0001.
图 5 氯菊酯对HMC3炎症因子表达的影响
A:格里斯试剂法检测氯菊酯染毒后的HMC3上清中NO含量;B:qPCR检测氯菊酯染毒后HMC3炎症因子mRNA表达情况;C:Western blot检测氯菊酯染毒后HMC3细胞IL-6和IL-1β的蛋白表达情况及相对定量;D:qPCR检测氯菊酯染毒后HMC3 MMP家族mRNA表达情况;E:Western blot检测氯菊酯染毒后HMC3细胞MMP1的蛋白表达情况及相对定量。*: P<0.05;**: P<0.01;***: P<0.001;****: P<0.0001。
Figure 5. Effects of permethrin on the expression of inflammatory cytokines in HMC3
A: NO content in the supernatant of HMC3 after permethrin treatment by Greiss method. B: mRNA expressions of HMC3 inflammatory factors after permethrin treatment by qPCR. C: Protein expressions of IL-6 and IL-1β in HMC3 after permethrin treatment and their relative quantification by Western blot; D: Relative mRNA expressions of HMC3 MMP family by qPCR. D: Protein expressions of MMP1 in HMC3 after permethrin treatment and their relative quantification by Western blot. *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001.
表 1 引物序列
Table 1 Primer sequences
基因名称
(Genes)正向(Forward)
5′→ 3′反向(Reverse)
5′→ 3′GAPDH GGAGCGAGATCCCTCCAAAAT GGCTGTTGTCATACTTCTCATGG CDK1 AAACTACAGGTCAAGTGGTAGCC TCCTGCATAAGCACATCCTGA CDKN1A TGTCCGTCAGAACCCATGC AAAGTCGAAGTTCCATCGCTC CCNB2 CCGACGGTGTCCAGTGATTT TGTTGTTTTGGTGGGTTGAACT p53 GGCGTAAACGCTTCGAGATG CTTCAGGTAGCTGGAGTGAGC FAS AGATTGTGTGATGAAGGACATGG TGTTGCTGGTGAGTGTGCATT CASP3 CATGGAAGCGAATCAATGGACT CTGTACCAGACCGAGATGTCA H2AX CTGTACCAGACCGAGATGTCA TGTGCCTGTTACCAAGTGCT MAPK1 TACACCAACCTCTCGTACATCG CATGTCTGAAGCGCAGTAAGATT MAPK8 TACAGAGCACCCGAGGTCAT TCTCCCATGATGCACCCAAC MAPK14 TCAGTCCATCATTCATGCGAAA AACGTCCAACAGACCAATCAC IL-1α AGATGCCTGAGATACCCAAAACC CCAAGCACACCCAGTAGTCT IL-1β ATGATGGCTTATTACAGTGGCAA GTCGGAGATTCGTAGCTGGA IL-4 CGGCAACTTTGTCCACGGA TCTGTTACGGTCAACTCGGTG IL-6 ACTCACCTCTTCAGAACGAATTG CCATCTTTGGAAGGTTCAGGTTG MMP1 AAAATTACACGCCAGATTTGCC GGTGTGACATTACTCCAGAGTTG MMP2 CGCTACGATGGAGGCGCTAA AGAAGGTGTTCAGGTATTGCACTG MMP3 CTGGACTCCGACACTCTGGA CAGGAAAGGTTCTGAAGTGACC MMP9 GGCAGCTGGCAGAGGAATAC GGCCCCAGAGATTTCGACTC -
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