Trichloroethylene (TCE) is a commonly used organic solvent in industry. Our previous studies have found that TCE can cause liver injury accompanied by macrophage polarization, but the specific mechanism is unclear. The epigenetic regulation of macrophage polarization is mainly focused on histone modification. Histone lysine demethylase 4A (KDM4A) is involved in the activation of macrophages. In this study, we used a mouse model we investigated the role of KDM4A in the livers of TCE-drinking mice and found that the expression of KDM4A, M1-type polarization indicators, and related inflammatory factors in the livers of TCE-drinking mice. In the study, BALB/c mice were randomly divided into four groups: 2.5 mg/mL TCE dose group and 5.0 mg/mL TCE dose group, the vehicle control group, and the blank control group. We found that TCE triggered M1 polarization of mouse macrophages, characterized by the expression of CD11c and robust production of inflammatory cytokines. Notably, exposure to TCE resulted in markedly increased expression of KDM4A in macrophages. Functionally, the increased expression of KDM4A significantly impaired the expression of H3K9me3 and H3K9me2 and increased the expression of H3K9me1. In addition, KDM4A potentially represents a novel epigenetic modulator, with its upregulation connected to β-catenin activation, a signal critical for the pro-inflammatory activation of macrophages. Furthermore, KDM4A inhibitor JIB-04 treatment resulted in a decrease in β-catenin expression and prevented TCE-induced M1 polarization and the expression of the pro-inflammatory cytokines TNF-α and IL-1β. These results suggest that the association of KDM4A and Wnt/β-catenin cooperatively establishes the activation and polarization of macrophages and global changes in H3K9me3/me2/me1. Our findings identify KDM4A as an essential regulator of the polarization of macrophages and the expression of inflammatory cytokines, which might serve as a potential target for preventing and treating liver injury caused by TCE.

Abstract Background & Aims: Trichloroethylene (TCE) is a commonly used organic solvent in industry. Our previous studies have found that TCE can cause liver injury accompanied by macrophage polarization, but the specific mechanism is unclear. The epigenetic regulation of macrophage polarization is mainly focused on histone modification. Histone lysine demethylase 4A (KDM4A) is involved in the activation of macrophages. In this study, we used a mouse model we investigated the role of KDM4A in the liversof TCE-drinking mice and found that the expression of KDM4A, M1-type polarization indicators, and related inflammatory factors in the livers of TCE-drinking mice. Methods: In the study, BALB/c mice were treated with TCE by drinking water. The mice were randomly divided into 4 groups: 2.5 milligrams per milliliter (mg/mL) TCE dose group (n=24) and 5.0 mg/mL TCE dose group (n=24), TCE used 1% DMSO to aid solubilization, the vehicle control group (drinking water containing 1% DMSO) (n=24), and the blank control group (fed with normal drinking water) (n=24). Drinking water was replaced every 24 h to ensure the stability of the TCE concentration. The mice were sacrificed at four time points of 2, 4, 8 and 12 weeks, and biological sample material were taken aseptically. Results: TCE triggered M1 polarization of mouse macrophages, characterized by the expression of CD11c and robust production of inflammatory cytokines (e.g., TNF-α andIL-1β). Notably, exposure to TCE resulted in markedly increased expression of KDM4A in macrophages. Functionally, the increased expression of KDM4A significantly impaired the expression of H3K9me3 and H3K9me2 andincreased the expression of H3K9me1. In addition, KDM4A potentially represents a novel epigenetic modulator, with its upregulation connected to β-catenin activation, a signal critical for the proinflammatoryactivation of macrophages. Furthermore, KDM4A inhibitor JIB-04 treatment resulted in a decrease in β-catenin expression andprevented TCE-induced M1 polarization and the expression of the proinflammatory cytokines TNF-α and IL-1β. These results suggest that the association of KDM4A and Wnt/β-catenin cooperatively establishes the activation and polarization of macrophages and global changes in H3K9me3/me2/me1. Conclusion: Our findings identify KDM4A as an essential regulator of the polarization of macrophages and the expression of inflammatory cytokines, which might serve as a potential target for preventing and treating liver injury caused by TCE.

Trichloroethylene (TCE) induces occupational medicamentosa-like dermatitis due to TCE (OMDT) with immune liver injury, and TNF-α plays an important role in macrophage polarization and liver injury. However, TNF-α regulating macrophage polarization in liver injury induced by TCE is still unknown. Thus, on the basis of our previous research, we established the TCE-sensitized BALB/c mouse model with R7050, a specific inhibitor of TNFR1. Then, we observed significant decreases in autophagy related protein and gene levels in M1 macrophage in TCE positive group, and R7050 can relieve M1 macrophage autophagy. We also found the phosphorylated form of mammalian target of Rapamycin (mTOR) was activated and the expression of p-mTOR protein increased induce by TCE. In vitro, we found TNFR1 and CD11c were increased in RAW264.7 cell line with TNF-α. And then we use Zafirlukast (Zaf), an TNFR1 antagonist, CD11c and TNFR1 reduced significantly, we also found p-mTOR expression increased after TNF-α treatment, but decreased in TNF-α + Zaf group. Further, we used Rapamycin (RAP), a mTOR-specific inhibitor, to establish a TCE-sensitized mice model and found the expression levels of p62 and p-mTOR proteins increased and LC3B decreased in the TCE positive group, while RAP treatment reversed the trends of all of these proteins. Rapamycin prevented the TNF-α-induced p-mTOR increase and dramatically downregulated IL-1β expression in the RAW264.7 cell line with TNF-α treatment. The results uncover a novel role for TNF-α/TNFR1, which promotes M1 polarization of macrophage and suppresses macrophage autophagy via the mTOR pathway.

目的 观察三氯乙烯(TCE)致敏小鼠肝脏组织中Wnt家族成员5A(WNT5A)的表达情况,探讨肿瘤坏死因子(TNF)-α/TNF受体1(TNFR1)介导WNT5A的表达参与TCE致敏小鼠免疫性肝损伤的机制.方法 无特定病原体级雌性BALB/c小鼠随机分为4组:空白对照组小鼠5只,不予任何处理;溶剂对照组小鼠5只,予丙酮与橄榄油混合溶液处理;TCE处理组小鼠18只,予TCE、丙酮和橄榄油混合物进行经皮激发与致敏;联合处理组小鼠17只,在首次激发前腹腔注射剂量为12 mg/kg体质量的TNFR1抑制剂R7050,再按照TCE处理组进行处理.在末次激发24 h后,根据皮肤评分将TCE处理组和联合处理组小鼠分为未致敏亚组和致敏亚组.末次激发72 h后处死小鼠,取肝脏组织进行组织病理学检查,以蛋白质印迹法检测TNF-α、TNFR1、白细胞介素(IL)-6和WNT5A的表达,以免疫荧光法观察WNT5A分布,以实时荧光定量聚合酶链式反应法检测Wnt5a mRNA表达.结果 TCE处理组、联合组小鼠致敏率分别为38.9％(7/18)和35.3％(6/17),差异无统计学意义(P＞0.05).组织病理学检查结果显示,TCE处理组致敏亚组小鼠出现肝损伤,表现为细胞空泡样变性和形态改变,联合处理组致敏亚组小鼠肝损伤较TCE处理组致敏亚组减轻;其余4组小鼠无出现肝损伤.免疫荧光法检查结果示,TCE处理组致敏亚组小鼠肝脏组织中WNT5A蛋白荧光信号表达增多,且高于联合处理组致敏亚组;其余4组小鼠肝脏组织中WNT5A蛋白几乎不表达.TCE处理组致敏亚组小鼠肝脏组织中TNF-α、TNFR1、IL-6、WNT5A的蛋白相对表达水平和Wnt5α mRNA相对表达水平分别高于空白对照组、溶剂对照组、TCE处理组未致敏亚组、联合处理组未致敏亚组和联合处理组未致敏亚组(P值均＜0.05).结论 TCE可通过介导TNF-α/TNFR1信号通路,上调WNT5A的表达,启动促炎反应,参与TCE致敏小鼠的免疫性肝损伤.TNFR1抑制剂可通过TNF-α/TNFR1信号通路抑制WNT5A的表达,减轻炎症反应,改善TCE致敏小鼠的免疫性肝损伤.

Objective: To detect the expression levels of M1-type polarization and autophagy-related indicators in the liver of trichloroethylene (TCE) -sensitized mice, and to explore the role of liver tumor necrosis factor-α (TNF-α) and tumor necrosis factor receptor 1 (TNFR1) in regulating M1-type Kupffer cells autophagy in liver injury in TCE-sensitized mice. Methods: In November 2019, according to simple random grouping, 45 SPF grade BALB/c female mice (6-8 weeks old) were divided into 4 groups: blank control group (n=5) , solvent control group (n=5) , TCE treatment group (n=18) , TCE+R7050 (inhibitor) treatment group (n=17) . Transdermally sensitized mice, 24 h after the last challenge, the mice were divided into TCE sensitized group and TCE non-sensitized group according to the skin reaction score. The livers of mice were harvested, and the pathological changes of the livers were observed under light and electron microscopes. Western blotting was used to detect the expressions of TNF-α, TNFR1 and autophagy-related indexes. The expression of inducible nitric oxide synthase (iNOS) , a marker of M1-type Kupffer cells, was detected by immunohistochemistry, and the occurrence of autophagy in M1-type Kupffer cells was detected by immunofluorescence double-labeling method. Results: The sensitization rate of TCE treatment group was 38.9% (7/18) , and TCE+R7050 treatment group was 35.3% (6/17) , with no significant difference between the two groups (P=1.000) . Compared with the blank control group, mice in the TCE sensitized group had abnormal liver ocytes, obvious liver injury, reduced mitochondria and broken endoplasmic reticulum. Western blotting results showed that the expressions of TNF-α and TNFR1 protein in the liver of the mice in the TCE sensitized group increased, the expression of iNOS protein in M1-type Kupffer cells increased, and the expressions of autophagic microtubule-associated protein 1 light-chain 3 (LC3B) and Beclin1 protein were decreased (P<0.05) . The results of immunohistochemistry showed that iNOS was not significantly expressed in the blank control group and solvent control group, and a small amount of expression was found in the TCE non-sensitized group, the positive staining area was obvious in TCE sensitized group, and the expression of iNOS was significantly increased (P<0.05) . Immunofluorescence results showed that the iNOS protein levels in the blank control group, solvent control group and TCE non-sensitized group were lower, and only partially colocalized with P62; the colocalization of iNOS with P62 in the TCE sensitized group was significantly increased. Conclusion: TNF-α/TNFR1 signaling pathway may promote liver injury in TCE-sensitized mice by inhibiting autophagy of M1-type Kupffer cells.

We have previously shown trichloroethylene (TCE) induced immune liver injury, and TNF-α/TNFR1 pathway as a probably mechanism underlying the immune damage, but the pathogenic mechanism is still unclear. The study aims to investigate whether TNF-α and its receptors regulate Kupffer cell polarization and downstream inflammation signaling pathways during TCE sensitization, to clarify the mechanism of TCE-mediated immune liver injury. 6–8 weeks old SPF BALB/c female mice were used to establish a TCE sensitization model. We found that in the TCE sensitization positive group, liver injury was aggravated, Kupffer cells activated and polarized to M1 type. The expression of M1 Kupffer cell marker proteins CD11c and CD16/32 increased in the TCE positive group, so did TNF-α and TNFR1 in liver. The expression of P-IKK protein, PP65 protein and P-STAT3 protein increased in the TCE sensitization positive group, and the downstream inflammatory factors IL-1β and IL-6 also increased in the TCE sensitization positive group. After using the TNFR1 inhibitor R7050, we found that M1 Kupffer cell polarization, TNF-α expression, signal pathway expression and inflammatory factors IL-1β and IL-6 expression declined, and the liver damage relieved. Briefly, the use of R7050 to inhibit TNF-α/TNFR1 changing the polarization of liver M1 Kupffer cell, thereby inhibiting the activation of related downstream signaling pathways and reducing the secretion of inflammatory factors. TNF-α/TNFR1 regulates the polarization of M1 Kupffer cells inflammatory play an important role in liver immune damage.

[背景]三氯乙烯(TCE)可通过受污染的水或空气经生物蓄积作用进入人体,从而产生健康危害,受累脏器以肝脏最为多见.[目的]观察TCE经口暴露小鼠肝脏中M1型库普弗细胞(KCs)极化的情况,探讨组蛋白赖氨酸去甲基化酶JMJD3与KCs M1型极化之间的关系.[方法]选取72只SPF级雌性BALB/c小鼠,6～8周龄,将其随机分为4组:空白对照组(18只)、溶剂对照组(18只)、2.5 mg·mL–1 TCE组(18只)和5.0 mg·mL–1 TCE组(18只),适应性喂养1周.按照课题组前期研究建立小鼠TCE经口暴露模型,染毒8周.分别在第2、4、8周时处死小鼠取肝脏组织,采用Western blotting法检测小鼠肝脏组织中组蛋白赖氨酸去甲基化酶JMJD3的蛋白表达水平,采用免疫荧光法对巨噬细胞标志F4/80和M1型巨噬细胞表面标志CD11c进行共定位,采用免疫组织化学法检测巨噬细胞M1型标志CD16/32和小鼠肝脏M1型巨噬细胞相关炎性因子肿瘤坏死因子-α(TNF-α)的表达情况.[结果]在第2、4、8周时,各组小鼠状态良好,没有出现因TCE染毒而死亡的个体,各组间小鼠耗水量差异无统计学意义,各时间点小鼠体重增长及肝脏系数的组间差异没有统计学意义(P>0.05).Western blotting法检测结果显示:在各时间点空白对照组和溶剂对照组JMJD3蛋白表达水平差异均无统计学意义,2.5 mg·mL–1 TCE组和5.0 mg·mL–1 TCE组JMJD3蛋白表达水平相较于对照组均升高,且第4、8周5.0 mg·mL–1 TCE组JMJD3蛋白表达水平高于2.5 mg·mL–1 TCE组(P<0.05).免疫荧光共定位结果显示:在各时间点空白对照组和溶剂对照组F4/80与CD11c表达较少,2.5 mg·mL–1 TCE组和5.0 mg·mL–1 TCE组F4/80与CD11c表达较高.免疫组织化学检测结果显示:CD16/32和TNF-α在空白对照组和溶剂对照组表达较低,而在2.5 mg·mL–1 TCE组和5.0 mg·mL?1 TCE组出现大量沉积.[结论]KCs M1型极化及促炎因子的表达可能与经口暴露TCE所致的JMJD3表达水平增加有关.

目的 观察三氯乙烯(TCE)致敏小鼠肝脏内TNFR2表达情况,探讨TNFR2与免疫性肝脏损伤之间的关系.方法 选取21只SPF级雌性BALB/c小鼠,6～8周龄,随机将其分为空白对照组(5只),溶剂对照组(5只),TCE处理组(11只),建立TCE致敏小鼠模型,进行小鼠背部皮肤致敏反应评分,评分大于1分为TCE致敏组,反之为未致敏组.模型完成后处死小鼠,摘取眼球取血,测定血清肝功能活性指标ALT和AST;取出小鼠肝脏制成石蜡切片,HE染色观察小鼠肝脏病理表现,免疫组织化学(IHC)法和Western blot法观察小鼠肝脏肿瘤坏死因子(TNF)-α与TNFR2的表达情况.实时荧光定量PCR法检测TNFR2的mRNA水平.结果 TCE组致敏率为45.45％(5/11);致敏组小鼠的肝功能指标较高,与其他组比较差异有统计学意义(P＜0.05).HE染色结果显示TCE致敏组可见细胞发生空泡样变性,细胞呈蜂窝状且胞质疏松,其余组小鼠的肝细胞形态正常,染色均匀.免疫组化实验显示,TCE致敏组TNF-α表达较高,且与其他组相比TCE致敏组可以观察到大量TNFR2阳性表达,评分结果差异有统计学意义(P＜0.05).实时荧光PCR结果显示TCE致敏组TNFR2的mRNA表达也比其他组表达升高(P＜0.05).Western blot实验表明TCE致敏组比其他组TNFR2蛋白表达水平增高,差异有统计学意义(P＜0.05).结论 TNFR2在TCE致敏组小鼠肝脏中表达升高,其表达的改变可能参与了 TCE致敏小鼠免疫性肝脏损伤过程.

纳米二氧化硅是目前生产量最大的3种纳米颗粒之一,广泛应用于光电学、催化、材料改性和药物载体等方面,职业暴露与环境暴露是纳米二氧化硅致病的重要接触途径,长期接触易对人体健康产生危害.该文综合了国内外最新研究成果,探讨纳米二氧化硅的致病分子机制,分别从纳米二氧化硅引起氧化应激、自噬、凋亡及炎症反应4个方面进行综述,阐明纳米二氧化硅可能引发的致病机制,为发病机制研究及疾病防治工作提供参考.