乙酰胆碱受体对急性呼吸窘迫综合征小鼠T细胞亚群和炎症因子的影响

Effects of acetylcholine receptor on T cell subsets and inflammatory cytokines in ARDS mice

目的探讨乙酰胆碱受体对急性呼吸窘迫综合征(ARDS)小鼠T细胞亚群CD3^(+)、CD25^(+)Foxp3^(+)T细胞、CD11b和炎症因子IL-1β、TNF-α的影响。方法健康清洁级雄性balb/C小鼠50只(6周),完全数字随机分为正常组(N组)、生理盐水对照组(NS组)、ARDS组(A组)、ARDS^(+)断离颈部两侧迷走神经组(D组)、ARDS^(+)断离颈部两侧迷走神经给予乙酰胆碱受体激动剂组(A^(+)J组),每组各10只正常饲养。HE染色观察各组小鼠右肺结构,采用流式细胞仪检测小鼠支气管肺泡灌洗液中CD11b、CD3^(+)、CD25^(+)Foxp3^(+)T细胞占T淋巴细胞的百分比。采用实时荧光定量聚合酶链反应(RT-qPCR)法检测左肺组织IL-1βmRNA和TNF-αmRNA的表达水平。结果N组和NS组小鼠右肺HE染色显示结构正常,而A组和D组小鼠肺间质有大量的炎性细胞浸润,肺泡壁增厚、肺泡结构破坏明显,肺泡腔融合。A^(+)J组小鼠肺泡结构较完整、有少许破损、肺泡腔存在。A组和D组小鼠支气管肺泡灌洗液CD11b、CD3^(+)、CD25^(+)Foxp3^(+)T细胞百分比高于其他3组,A^(+)J组CD11b(10.77±2.41)、CD3^(+)(5.94±0.27)、CD25^(+)Foxp3^(+)T(5.00±1.15)细胞百分比分别与D组(15.32±1.56,9.78±0.88,9.84±1.11)、A组(13.49±2.06,8.55±1.07,8.98±0.91)比较,均差异有统计学意义(t=3.04,2.61,2.87,3.87,2.54,2.53;均P<0.05)。A^(+)J组小鼠左肺IL-1βmRNA(198.98±54.05)和TNF-αmRNA(52.41±12.26)的表达分别与A组(384.26±69.03,96.84±20.02)和D组(397.26±64.31,99.45±18.34)比较,均差异有统计学意义(t=94.42,99.54,91.32,98.57,P<0.05)。结论激活胆碱能抗炎通路的乙酰胆碱受体不仅抑制肺组织T淋巴细胞的释放,还可以抑制肺组织IL-1βmRNA和TNF-αmRNA的表达以及CD11b的释放,从而抑制ARDS的炎症反应,减轻ARDS病理变化。

Objective To investigate the effects of acetylcholine receptor(AChR)on T-cell subpopulations CD3^(+),CD25^(+)Foxp3^(+)T cells,CD11b,and inflammatory factors IL-1β,TNF-αin acute respiratory distress syndrome(ARDS)mice.Methods Fifty(6 weeks)healthy and clean male balb/C mice were randomly divided into normal group(N group),normal saline control group(NS group),ARDS group(A group),ARDS^(+)transection of the vagus nerve on both sides of the neck(D group),ARDS^(+)group given AChR after transection of the vagus nerve on both sides of the neck(A^(+)J group),10 mice in each group were fed normally.HE staining was used to observe the right lung structure of mice in each group,and flow cytometry was used to detect the percentages of CD3^(+),CD11b,CD25^(+)Foxp3^(+)T cells in the bronchoalveolar lavage fluid of mice.Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression of IL-1βmRNA and TNF-αmRNA in the left lung tissue.Results The HE staining of the right lung in the N group and NS group of mice showed normal structure,but that of the A and D groups of mice showed a large amount of inflammatory cell infiltration in the interstitium,significant thickening of the alveolar walls,and obvious destruction of the alveolar structure with alveolar fusion.The alveolar structure of mice in A^(+)J group was relatively complete with a little damage and alveolar cavities.The percentages of CD11b,CD3^(+),and CD25^(+)Foxp3^(+)T cells in the bronchoalveolar lavage fluid of mice in groups A and D were higher than those in the other 3 groups.In the A^(+)J group,the percentages of CD11b(10.77±2.41),CD3^(+)(5.94±0.27),and CD25^(+)Foxp3^(+)T cells(5.00±1.15)were statistically significantly different from the D group(15.32±1.56,9.78±0.88,9.84±1.11)and the A group(13.49±2.06,8.55±1.07,8.98±0.91)(t=3.04,2.61,2.87,3.87,2.54,2.53,all P<0.05).In the A^(+)J group of mice,the expressions of IL-1βmRNA(198.98±54.05)and TNF-αmRNA(52.41±12.26)in the left lung were significantly different from the A group(384.26±69.03,96.84±20.02)and the D group(397.26±64.31,99.45±18.34)(t=94.42,99.54,91.32,98.57,P<0.05).Conclusion The activation of the cholinergic anti-inflammatory pathway by acetylcholine receptors not only inhibits the release of T lymphocytes in lung tissue,but also suppresses the expressions of IL-1βmRNA and TNF-αmRNA in lung tissue,as well as the release of CD11b,thus inhibiting the inflammatory response of ARDS and alleviating the pathological changes in ARDS.