经口暴露三氯乙烯对小鼠肝脏JMJD3表达和M1型库普弗细胞极化的影响

黄华; 丁百旺; 左绪磊; 杨呓; 彭家乐; 王燚灿; 张家祥; 朱启星

doi:10.11836/JEOM21242

经口暴露三氯乙烯对小鼠肝脏JMJD3表达和M1型库普弗细胞极化的影响

Effect of oral exposure to trichloroethylene on JMJD3 expression and polarization of M1 Kupffer cells

摘要

摘要:
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。

Abstract:
Background Trichloroethylene (TCE) can enter human body through biological accumulation of polluted water or air, resulting in health hazards. The most commonly involved organs are the liver.

Objective To observe potential polarization of M1 Kupffer cells (KCs) in mice liver exposed to TCE orally, and to investigate the relationship between histones lysin demethylase JMJD3 and M1 KCs polarization.

Methods A total of 72 SPF BALB/c mice aged 6 to 8 weeks were randomly divided into a blank control group (n=18), a vehicle control group (n=18), a 2.5 mg·mL⁻¹ TCE group (n=18), and a 5.0 mg·mL⁻¹ TCE group (n=18) after adaptive feed for one week. A TCE transoral exposure model was established after eight weeks of administration according to previous research of the research group. In the 2nd, 4th, and 8th weeks, the mice were sacrificed and liver tissue samples were collected. Western blotting was used to detect the expression level of JMJD3 in the liver tissue samples. Immunofluorescence was used to co-locate the macrophage marker F4/80 and the surface marker CD11c of M1 macrophages. Immunohistochemistry was used to detect the expressions of CD16/32, a marker of M1 macrophages, and TNF-α, an inflammatory factor of M1 macrophages in mouse liver.

Results In the 2nd, 4th, and 8th weeks, the mice in each group were generally in good condition, and no individual died due to TCE. There was no statistically significant difference in the amount of water consumed by each group, nor in the body weight gain and the liver coefficient of mice at each time point (P>0.05). The results of Western blotting analysis showed that there was no statistically significant difference in JMJD3 protein expression level between the blank control group and the vehicle control group at each time point, the expression levels of JMJD3 protein in the 2.5 mg·mL⁻¹ TCE group and the 5.0 mg·mL⁻¹ TCE group were higher than that in the control group , and the expression level of JMJD3 protein in the 5.0 mg·mL⁻¹ TCE group was higher than that in the 2.5 mg·mL⁻¹ TCE group (P<0.05). The results of immunofluorescence co-localization showed that the expressions of F4/80 and CD11c were low in the blank control group and the vehicle control group, while the expressions of F4/80 and CD11c were increased in the 2.5 mg·mL⁻¹ and the 5.0 mg·mL⁻¹ TCE groups. The results of immunohistochemistry showed that the expressions of CD16/32 and TNF-α in the blank control group and the vehicle control group were low, and there were large deposits in the 2.5 mg·mL⁻¹ TCE group and the 5.0 mg·mL⁻¹ TCE group.

Conclusion The polarization of M1 KCs and the expression of proinflammatory factors may be related to an increased expression level of JMJD3 induced by oral TCE exposure.

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