M1型库普弗细胞极化在三氯乙烯致敏小鼠肝脏损伤中的作用
Role of M1 Kupffer cell polarization in liver injury induced by trichloroethylene in mice
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摘要:
背景 三氯乙烯（TCE）可致职业性药疹样皮炎并伴有严重的肝脏损伤，是致死的主要原因之一，但其发生机制尚不清楚。
目的 观察TCE致敏小鼠肝脏中库普弗细胞（KCs）极化的情况，探讨KCs的M1型极化在TCE免疫性肝损伤中的作用。
方法 36只雌性BALB/c小鼠，6~8周龄，随机分为4组：空白对照组（n=5），溶剂对照组（n=5），TCE处理组（TCE组）（n=11），抑制剂处理组（TCE+GdCl3组）（n=15），适应性喂养一周。按照本课题组的建模方法建立TCE致敏模型。在末次激发24 h后对TCE组和TCE+GdCl3组进行皮肤评分，将TCE组和TCE+GdCl3组小鼠分为致敏阳性组和致敏阴性组；末次激发72 h后处死小鼠，取肝脏。全自动生化分析仪检测肝脏损伤指标丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）水平，HE染色法检测小鼠肝脏病理变化，免疫组化法检测KCs的表面标志物F4/80的表达情况，免疫荧光法检测小鼠肝脏M1型巨噬细胞表面标志物CD11c的表达水平，免疫组化法检测小鼠肝脏肿瘤坏死因子（TNF）-α的表达情况。
结果 TCE组致敏率为45.45%（5/11），TCE+GdCl3组致敏率为33.33%（5/15），差异无统计学意义（P>0.05）。血清ALT和AST水平在空白对照组、溶剂对照组之间差异无统计学意义（P>0.05）；与溶剂对照组和TCE阴性组相比，TCE阳性组血清ALT和AST升高有统计学意义（P < 0.05），TCE阳性组ALT及AST水平分别为（115.05±13.74）U/L和（224.68±30.98）U/L；TCE+GdCl3阳性组ALT及AST水平分别为（97.59±9.16）U/L和（124.69±11.26）U/L，低于TCE阳性组（P < 0.05）。HE染色结果显示：对照组及阴性组小鼠肝脏细胞形态正常，染色均匀；TCE阳性组小鼠肝脏细胞形态异常，细胞空泡样变性；TCE+GdCl3阳性组小鼠肝脏部分细胞出现水肿。肝脏F4/80的免疫组化结果显示：空白对照组、溶剂对照组表达较低，TCE阳性组F4/80大量沉积，TCE+GdCl3阳性组表达较TCE阳性组减少（P < 0.05）。肝脏CD11c免疫荧光结果显示：空白对照组、溶剂对照组CD11c表达较少，TCE阳性组CD11c表达水平较高，而TCE+GdCl3阳性组该指标表达水平下降。免疫组化结果显示：空白对照组和溶剂对照组TNF-α表达水平极低，TCE阳性组肝脏TNF-α大量沉积，TCE+GdCl3阳性组肝脏中TNF-α表达水平较TCE阳性组下降（P < 0.05）。
结论 KCs在TCE致敏小鼠免疫性肝损伤中发挥重要作用，其M1型极化会加重小鼠肝脏损伤。
Abstract:Background Trichloroethylene (TCE) can cause occupational drug-like dermatitis with severe liver damage, which is one of the leading causes of death, but its mechanism is still unclear.
Objective This study observes the polarization of Kupffer cells (KCs) in the liver of TCE sensitized mice, and to explore the role of M1 type polarization of KCs in immune liver injury induced by TCE.
Methods Thirty-six female BALB/c mice, 6-8 weeks old, were randomly divided into four groups:blank control group (n=5), vehicle control group (n=5), TCE-treated group (TCE group, n=11), and inhibitor-treated group (TCE+GdCl3 group, n=15), with adaptive feeding for one week. A TCE sensitization model was established according to an protocol developed by our team. Twenty-four hours after the last challenge, the skin of the TCE group and the TCE+GdCl3 group was scored and the mice in the TCE group and the TCE+GdCl3 group were divided into sensitization positive group and sensitization negative group. Seventy-two hours after the last challenge, the mice were sacrificed and the livers were taken. The levels of alanine transaminase (ALT) and aspartate transaminase (AST) as selected liver injury indicators were determined using automatic biochemical analyzer; the pathological changes of liver were observed after HE staining; the expression level of surface marker F4/80 of KCs was detected by immunohistochemistry; the expression level of surface marker CD11c in M1 type macrophages was detected by immunofluorescence; and the expression level of tumor necrosis factor (TNF)-α in mouse liver was detected by immunohistochemistry.
Results The sensitization rate was 45.45% (5/11) in the TCE group and 33.33% in the TCE+GdCl3 group (5/15), with no significant difference (P>0.05). The serum ALT and AST levels were not significantly different between the blank control group and the vehicle control group (P>0.05). Compared with the vehicle control group and the TCE negative group, the serum ALT and AST levels were significantly increased in the TCE positive group (P < 0.05). The ALT level and AST level in the TCE positive group were (115.05±13.74) U/L and (224.68±30.98) U/L, respectively; the levels in the TCE+GdCl3 positive group were (97.59±9.16) U/L and (124.69±11.26) U/L, respectively, lower than those in the TCE positive group (P < 0.05). The results of HE staining showed that the liver cells of the control groups and the negative groups were normal in morphology and color; in the TCE positive group, the cells had abnormal cell morphology and vacuolar degeneration; some cells in the liver of the TCE+GdCl3 positive group showed edema. The immunohistochemical results of liver F4/80 showed that the expression levels in the blank control group and the vehicle control group were low, the TCE positive group had more deposition of F4/80 than the vehicle control group, while the TCE+GdCl3 positive group had less than the TCE positive group (P < 0.05). The results of liver CD11c immunofluorescence showed that the expression levels were low in the blank control group and the vehicle control group, the expression level of CD11c was higher in the TCE positive group than in the vehicle control group, while the expression level of this index was lower in the TCE+GdCl3 positive group than in the TCE positive group. The results of immunohistochemistry showed that the expression levels of TNF-α were very low in the blank control group and the vehicle control group, and higher in the TCE positive group than in the vehicle control group, and lower in the TCE+GdCl3 positive group than in the TCE positive group (P < 0.05).
Conclusion KCs play an important role in immune liver injury induced by TCE in mice, and M1 type polarization may aggravate the injury.
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