颗粒蛋白前体通过调控调节性T细胞影响急性肺损伤白细胞介素-10表达和肺巨噬细胞极化的实验研究

Effect of progranulin on expression of interleukin-10 and polarization of pulmonary macrophages through the regulation of regulatory T cells

摘要:

目的：研究颗粒蛋白前体（progranulin，PGRN）在小鼠急性肺损伤（acute lung injury，ALI）模型中对调节性T细胞（regulatory T cells，Tregs）的调控及其对白细胞介素-10（interleukin-10，IL-10）表达和肺巨噬细胞极化的影响。方法：将 C57BL/6小鼠随机分为 Control 组、脂多糖（lipopolysaccharides，LPS）组、LPS+PGRN 治疗组。用 LPS 气道滴注诱导小鼠 ALI 模型，LPS+PGRN 治疗组在 LPS 处理半小时后采用 0.1 mg/kg 的 PGRN 气道滴注。给药 24 h 后用水合氯醒麻醉小鼠，取小鼠肺组织及肝素抗凝血。肺组织病理切片HE染色评估肺损伤；流式细胞术检测外周血 Tregs；酶联免疫吸附法检测血浆 IL-6、肿瘤坏死因子（tumor necrosis factor，TNF）-α、IL-10 表达；免疫荧光检测肺巨噬细胞表型。结果：不同处理组间外周血 Tregs，血浆中 IL-6、TNF-α、IL-10，肺组织中 M1、M2 型肺巨噬细胞存在统计学差异（F=69.340，P=0.000；F=73.100，P=0.000；F=17.800，P=0.000；F=11.780，P=0.003；F=67.560，P=0.000；F=197.100，P=0.000）。并且 PGRN 治疗后，ALI 小鼠肺损伤明显减轻，外周血 Tregs 明显增高[LPS：（4.714±0.265）%，LPS+PGRN：（8.930±0.255）%，P=0.000]，血浆中促炎因子 IL-6[LPS：（3 021.000±294.900） pg/mL，LPS+PGRN：（276.700±148.000） pg/mL，P=0.000]和TNF-α表达[LPS：（90.980±10.240） pg/mL，LPS+PGRN：（20.410±9.190） pg/mL，P=0.002]明显降低,血浆抑炎因子 IL-10 表达明显上升[LPS：（296.400±44.280） pg/mL，LPS+PGRN：（2 551.000±0 655.100） pg/mL，P=0.014]，M1 型肺巨噬细胞减少[LPS：（17.870±1.890）%，LPS+PGRN：（4.436±0.248）%，P=0.000]，M2 型肺巨噬细胞增多[LPS:（2.284±0.444）%，LPS+PGRN：（11.920±0.458）%，P=0.000]。结论：在小鼠ALI模型中，PGRN能够通过调控Tregs改变IL-10的表达，影响肺巨噬细胞极化。

Abstract:

Objective：To study the effect of progranulin（PGRN） on the expression of interleukin（IL）-10 and the polarization of pulmonary macrophages through the regulation of regulatory T cells（Tregs） in mouse model with acute lung injury（ALI）. Methods：The C57BL/6 mice were randomly divided into the normal control group（Control group），the lipopolysaccharides（LPS） group and the LPS+PGRN treatment group（LPS+PGRN group）. The ALI mouse model was induced by LPS（2.5 mg/kg） via intratracheal instillation. The LPS+PGRN group was treated with 0.1 mg/kg PGRN after half an hour of LPS instillation. The mice were anesthetized with 3% chloral hydrate at 24h after LPS instillation，lung tissues and heparin anticoagulant blood were taken. The severity of lung injury was evaluated by histopathology with hematoxylin and eosin staining. The Tregs in peripheral blood was detected by flow cytometry. The expression of IL-6，tumor necrosis factor（TNF）-α and IL-10 in plasma were tested by enzyme linked immunosorbent assay. The phenotype of macrophages in lung tissue was detected by immunofluorescence. Results：The ratio of Tregs in peripheral blood，the expression of IL-6，TNF-α，and IL-10 in plasma，and the proportion of M1 and M2 macrophages in the lung tissue between different groups had statistically significant differences（F=69.340，P=0.000；F=73.100，P=0.000；F=17.800，P=0.000；F=11.780，P=0.003；F=67.560，P=0.000；F=197.100，P=0.000）. After PGRN treatment，the lung injury of ALI mouse model was significantly reduced and the proportion of Tregs in peripheral blood was higher[LPS：（4.714±0.265）%，LPS+PGRN：（8.930±0.255）%，P=0.000]. The expression of proinflammatory cytokines IL-6[LPS：（3 021.000±294.900） pg/mL，LPS+PGRN：（276.700±148.000） pg/mL，P=0.000] and TNF-α[LPS：（90.980±10.240） pg/mL，LPS+PGRN：（20.410±9.190） pg/mL，P=0.002] was significantly decreased. The expression of anti-inflammatory cytokine IL-10 in plasma significantly was increased[LPS：（296.400±44.280） pg/mL，LPS+PGRN：（2551.000±655.100） pg/mL，P=0.014]，and M1 lung macrophages was decreased[LPS：（17.870±1.890）%，LPS+PGRN：（4.436±0.248）%，P=0.000]，while M2 lung macrophages was increased[LPS：（2.284±0.444）%，LPS+PGRN：(11.920±0.458）%，P=0.000]. Conclusion：PGRN can increase the expression of IL-10 and M2 lung macrophages through the regulation of Tregs in ALI mouse model，so as to influence the polarization of lung macrophages.