中华危重病急救医学
中華危重病急救醫學
중화위중병급구의학
Chinese Critical Care Medicine
2015年
10期
811-815
,共5页
刘芬%赵宁%李东海%曾振国%邵强%彭菲菲%王燕%钱克俭
劉芬%趙寧%李東海%曾振國%邵彊%彭菲菲%王燕%錢剋儉
류분%조저%리동해%증진국%소강%팽비비%왕연%전극검
乙酰胆碱%脂多糖%肺泡巨噬细胞%炎症反应
乙酰膽堿%脂多糖%肺泡巨噬細胞%炎癥反應
을선담감%지다당%폐포거서세포%염증반응
Acetylcholine%Lipopolysaccharide%Alveolar macrophage%Inflammatory response
目的：观察乙酰胆碱（ACh）对脂多糖（LPS）诱导肺泡巨噬细胞炎症反应模型大鼠胆碱能抗炎通路的作用，以及胆碱酯酶抑制剂毒扁豆碱（Phy）对ACh抗炎作用的影响。方法体外培养NR8383大鼠肺泡巨噬细胞株并分为空白对照组、LPS组（1 mg/L LPS刺激12 h）、LPS+ ACh组（LPS刺激前加入终浓度0.01、0.1、1、10、100μmol/L的ACh处理5 min）、 LPS+Phy组（LPS刺激前加入1 mmol/L的Phy处理5 min）、 LPS+ACh+Phy组（LPS刺激前加入1 mmol/L Phy及10μmol/L Ach分别处理5 min）5组。收集各组细胞上清液，采用酶联免疫吸附试验（ELISA）检测肿瘤坏死因子-α（TNF-α）、白细胞介素（IL-1β、IL-6）的含量；同时测定乙酰胆碱酯酶（AChE）活性。结果① LPS组细胞上清液中TNF-α（ng/L：605.09±57.13比34.07±8.62）、IL-1β（ng/L：377.09±28.55比32.33±10.62）、IL-6（ng/L：558.04±77.45比42.62±11.21）含量均较空白对照组显著升高（均P＜0.05），证实肺泡巨噬细胞炎症反应模型构建成功。②与LPS组相比，终浓度0.01、0.1和1μmol/L的ACh对 LPS刺激的肺泡巨噬细胞上清液中TNF-α、IL-1β、IL-6含量影响较小（均P＞0.05）；而终浓度10μmol/L及100μmol/L的ACh可显著降低细胞上清液中TNF-α（ng/L：451.19±30.67、332.19±32.19比604.96±22.56）、IL-1β（ng/L：261.08±24.78、143.98±28.39比367.06±10.44）、IL-6（ng/L：342.75±54.60、235.48±29.75比562.69±63.34）的含量（均P＜0.05）。③ LPS组细胞上清液中AChE活性较空白对照组显著增加（kU/L：5.21±0.63比3.09±0.10，P＜0.05）；与LPS组相比，给予1 mmol/L胆碱酯酶抑制剂Phy预处理可成功抑制AChE的活性（1.51±0.12比5.21±0.63，P＜0.05）。④与LPS+ ACh组相比，LPS+ ACh+ Phy组可显著降低细胞上清液中TNF-α（ng/L：183.17±35.44比451.19±30.67）、IL-1β（ng/L：91.49±12.27比261.08±24.78）、IL-6（ng/L：108.17±22.82比342.75±54.60）的含量（均P＜0.05）。结论终浓度10μmol/L及100μmol/L的ACh可抑制LPS诱导的大鼠肺泡巨噬细胞炎症反应；胆碱酯酶抑制剂Phy可增强ACh对肺泡巨噬细胞炎症反应模型的抗炎作用。
目的：觀察乙酰膽堿（ACh）對脂多糖（LPS）誘導肺泡巨噬細胞炎癥反應模型大鼠膽堿能抗炎通路的作用，以及膽堿酯酶抑製劑毒扁豆堿（Phy）對ACh抗炎作用的影響。方法體外培養NR8383大鼠肺泡巨噬細胞株併分為空白對照組、LPS組（1 mg/L LPS刺激12 h）、LPS+ ACh組（LPS刺激前加入終濃度0.01、0.1、1、10、100μmol/L的ACh處理5 min）、 LPS+Phy組（LPS刺激前加入1 mmol/L的Phy處理5 min）、 LPS+ACh+Phy組（LPS刺激前加入1 mmol/L Phy及10μmol/L Ach分彆處理5 min）5組。收集各組細胞上清液，採用酶聯免疫吸附試驗（ELISA）檢測腫瘤壞死因子-α（TNF-α）、白細胞介素（IL-1β、IL-6）的含量；同時測定乙酰膽堿酯酶（AChE）活性。結果① LPS組細胞上清液中TNF-α（ng/L：605.09±57.13比34.07±8.62）、IL-1β（ng/L：377.09±28.55比32.33±10.62）、IL-6（ng/L：558.04±77.45比42.62±11.21）含量均較空白對照組顯著升高（均P＜0.05），證實肺泡巨噬細胞炎癥反應模型構建成功。②與LPS組相比，終濃度0.01、0.1和1μmol/L的ACh對 LPS刺激的肺泡巨噬細胞上清液中TNF-α、IL-1β、IL-6含量影響較小（均P＞0.05）；而終濃度10μmol/L及100μmol/L的ACh可顯著降低細胞上清液中TNF-α（ng/L：451.19±30.67、332.19±32.19比604.96±22.56）、IL-1β（ng/L：261.08±24.78、143.98±28.39比367.06±10.44）、IL-6（ng/L：342.75±54.60、235.48±29.75比562.69±63.34）的含量（均P＜0.05）。③ LPS組細胞上清液中AChE活性較空白對照組顯著增加（kU/L：5.21±0.63比3.09±0.10，P＜0.05）；與LPS組相比，給予1 mmol/L膽堿酯酶抑製劑Phy預處理可成功抑製AChE的活性（1.51±0.12比5.21±0.63，P＜0.05）。④與LPS+ ACh組相比，LPS+ ACh+ Phy組可顯著降低細胞上清液中TNF-α（ng/L：183.17±35.44比451.19±30.67）、IL-1β（ng/L：91.49±12.27比261.08±24.78）、IL-6（ng/L：108.17±22.82比342.75±54.60）的含量（均P＜0.05）。結論終濃度10μmol/L及100μmol/L的ACh可抑製LPS誘導的大鼠肺泡巨噬細胞炎癥反應；膽堿酯酶抑製劑Phy可增彊ACh對肺泡巨噬細胞炎癥反應模型的抗炎作用。
목적：관찰을선담감（ACh）대지다당（LPS）유도폐포거서세포염증반응모형대서담감능항염통로적작용，이급담감지매억제제독편두감（Phy）대ACh항염작용적영향。방법체외배양NR8383대서폐포거서세포주병분위공백대조조、LPS조（1 mg/L LPS자격12 h）、LPS+ ACh조（LPS자격전가입종농도0.01、0.1、1、10、100μmol/L적ACh처리5 min）、 LPS+Phy조（LPS자격전가입1 mmol/L적Phy처리5 min）、 LPS+ACh+Phy조（LPS자격전가입1 mmol/L Phy급10μmol/L Ach분별처리5 min）5조。수집각조세포상청액，채용매련면역흡부시험（ELISA）검측종류배사인자-α（TNF-α）、백세포개소（IL-1β、IL-6）적함량；동시측정을선담감지매（AChE）활성。결과① LPS조세포상청액중TNF-α（ng/L：605.09±57.13비34.07±8.62）、IL-1β（ng/L：377.09±28.55비32.33±10.62）、IL-6（ng/L：558.04±77.45비42.62±11.21）함량균교공백대조조현저승고（균P＜0.05），증실폐포거서세포염증반응모형구건성공。②여LPS조상비，종농도0.01、0.1화1μmol/L적ACh대 LPS자격적폐포거서세포상청액중TNF-α、IL-1β、IL-6함량영향교소（균P＞0.05）；이종농도10μmol/L급100μmol/L적ACh가현저강저세포상청액중TNF-α（ng/L：451.19±30.67、332.19±32.19비604.96±22.56）、IL-1β（ng/L：261.08±24.78、143.98±28.39비367.06±10.44）、IL-6（ng/L：342.75±54.60、235.48±29.75비562.69±63.34）적함량（균P＜0.05）。③ LPS조세포상청액중AChE활성교공백대조조현저증가（kU/L：5.21±0.63비3.09±0.10，P＜0.05）；여LPS조상비，급여1 mmol/L담감지매억제제Phy예처리가성공억제AChE적활성（1.51±0.12비5.21±0.63，P＜0.05）。④여LPS+ ACh조상비，LPS+ ACh+ Phy조가현저강저세포상청액중TNF-α（ng/L：183.17±35.44비451.19±30.67）、IL-1β（ng/L：91.49±12.27비261.08±24.78）、IL-6（ng/L：108.17±22.82비342.75±54.60）적함량（균P＜0.05）。결론종농도10μmol/L급100μmol/L적ACh가억제LPS유도적대서폐포거서세포염증반응；담감지매억제제Phy가증강ACh대폐포거서세포염증반응모형적항염작용。
ObjectiveTo observe the effect of acetylcholine (ACh) on lipopolysaccharide (LPS) induced inflammatory model of rat alveolar macrophages, and to observe the effect of the acetylcholinesterase inhibitor physostigmine (Phy) on the anti-inflammatory effect of ACh.Methods The rat alveolar macrophages NR8383 were cultured in vitro, which were divided into five groups: blank control group, LPS group (stimulated with 1 mg/L LPS for 12 hours), LPS+ ACh group (0.01, 0.1, 1, 10, 100μmol/L of ACh were added for 5 minutes before LPS stimulation), LPS+ Phy group (1 mmol/L Phy was added for 5 minutes before LPS stimulation), and LPS+ ACh+ Phy group (1 mmol/L Phy and 10μmol/L ACh were added for 5 minutes before LPS stimulation). The supernatants were collected in each group, the enzyme-linked immunosorbent assay (ELISA) was used to assay the contents of tumor necrosis factor-α (TNF-α), interleukins (IL-1β, and IL-6). The activity of acetylcholine esterase (AChE ) in the supernatant was also determined.Results① The contents of TNF-α (ng/L: 605.09±57.13 vs. 34.07±8.62), IL-1β (ng/L: 377.09±28.55 vs. 32.33±10.62) and IL-6 (ng/L: 558.04±77.45 vs. 42.62±11.21) in the LPS group were significantly higher than those in the blank control group (allP< 0.05). These results indicated that the inflammatory model of rat alveolar macrophages was constructed successfully.② ACh with the final concentrations of 0.01, 0.1, and 1μmol/L had less influence on the production of TNF-α, IL-1β and IL-6 in the culture supernatants of alveolar macrophages stimulated with LPS compared with LPS group (allP> 0.05). Nevertheless, 10μmol/L and 100μmol/L ACh notably reduced the production of TNF-α (ng/L: 451.19±30.67, 332.19±32.19 vs. 604.96±22.56), IL-1β(ng/L: 261.08±24.78, 143.98±28.39 vs. 367.06±10.44) and IL-6 (ng/L: 342.75±54.60, 235.48±29.75 vs. 562.69±63.34) in the culture supernatants compared with the LPS group (allP< 0.05).③ The activity of AChE in the LPS group was significantly higher than that in the blank control group (kU/L: 5.21±0.63 vs. 3.09±0.10,P< 0.05). The activity of AChE was successfully inhibited by 1 mmol/L acetylcholinesterase inhibitor Phy pretreatment compared with that in the LPS group (1.51±0.12 vs. 5.21±0.63,P< 0.05).④ The level of TNF-α (ng/L: 183.17±35.44 vs. 451.19±30.67), IL-1β (ng/L: 91.49±12.27 vs. 261.08±24.78) and IL-6 (ng/L: 108.17±22.82 vs. 342.75±54.60) in the culture supernatants of LPS+ ACh+ Phy group was significantly decreased as compared with LPS+ ACh group (allP< 0.05).Conclusions ACh with the final concentrations of 10μmol/L and 100μmol/L can inhibit the LPS induced inflammatory reaction in alveolar macrophages. The acetylcholinesterase inhibitor Phy can reinforce the ACh-mediated anti-inflammatory effect on alveolar macrophages inflammatory model.
