摘要
目的观察d7亚基烟碱乙酰胆碱受体（α7nAChR）在右美托咪定（DEX）防治内毒素血症谵妄发生中的作用，并探讨其作用机制。方法将100只雄性C57BL／6小鼠按随机数字表法分为生理盐水对照组（Ns组）、DEX对照组、脂多糖（LPS）诱导内毒素血症模型组（LPS组）、DEX保护组（DEX＋LPS组）、α-银环蛇毒素拮抗组（α-BGT＋DEX＋LPS组），每组20只。采用腹腔注射LPS20mg/kg制备内毒素血症模型，Ns组和DEX对照组给予等量生理盐水。DEX对照组、DEX＋LPS组和α—BGT＋DEX＋LPS组小鼠于制模前15min腹腔注射DEX40ug／kg，α—BGT＋DEX＋LPS组于注射DEX前15min腹腔注射d7nAChR抑制剂α—BGT1ug／kg；Ns组给予等量生理盐水。制模前及制模后24h每组取10只小鼠进行旷场实验，实验结束后处死小鼠留取标本，采用酶联免疫吸附试验（ELlSA）检测血清肿瘤坏死因子-α（TNF-α）和神经元特异性烯醇化酶（NSE）水平，蛋白质免疫印迹试验（Western Blot）检测海马组织胆碱乙酰转移酶（ChAT）及乙酰胆碱酯酶（AChE）表达量。另取10只小鼠行新物体识别实验，观察训练阶段探究总时间及制模后3h和24h的偏好指数。结果Ns组和DEX对照组各项指标比较差异均无统计学意义。①旷场实验结果显示，制模前各组旷场实验观察指标均无差异。制模后24h，与NS组比较，LPS组小鼠表现为对新环境的认知、学习和记忆能力均下降，紧张度增加；DEX可明显改善小鼠的上述变化；而α-BGT可部分拮抗DEX对谵妄小鼠的保护作用。②新物体识别实验结果显示，与Ns组比较，LPS组小鼠探究新事物的能力下降；DEX可改善小鼠的探究能力；而α-BGT可拮抗DEX的保护作用。⑧ELISA结果显示，LPS组血清TNF-α和NSE水平较Ns组明显升高，DEX可抑制LPS诱导的血清TNF-α和NSE水平升高，而α-BGT可拮抗DEX的保护作用（Ns组、LPS组、DEX＋LPS组、α-BGT＋DEX＋LPS组TNF-α（ng／L）分别为23．72±3．13、808．78±87．86、192．96±31．47、829．99±80．98。NSE（-L）分别为8．70±0．74、25．90±3．03、18．10±2．14、23．12±2．21，均P〈0．01]。④WesternBlot结果显示，与Ns组比较，LPS组海马组织ChAT表达明显下降，AChE表达明显升高，DEX可逆转ChAT和AChE的表达变化，而α-BGT可拮抗DEX的保护作用[Ns组、LPS组、DEX＋LPS组、α-BGT＋DEX＋LPS组ChAT（灰度值）分别为1．536±0．150、0．381±0．138、0．914±0．173、0．628±0．088，AChE（灰度值）分别为0．382±0．201、1．843±0．325、0．898±0．155、1．470±0．220，P〈0．05或P〈0．01]。结论内毒素血症小鼠可发生谵妄等认知功能障碍；DEX可通过调节脑内神经递质AChE和ChAT的表达量而改善谵妄症状，从而起到脑保护作用，其机制可能与仅7nAChR有关。
Objective To observe the role of α7 nicotinic acetylcholine receptor （α7nAChR） in the protection against deliriμm by the use of dexmedetomidine （DEX） in endotoxin derived deliriμm and its mechanism. Methods 100 male adult C57BL/6 mice were randomly divided into normal saline control group （NS group）, DEX control group, lipopolysaccharide （LPS） induced endotoxemia model group （LPS group）, DEX protection group （DEX＋LPS group）, and a-bungarotoxin antagonism group （α-BGT＋DEX＋LPS group）, with 20 naice in each group. A model of endotoxemia was reproduced by intraperitoneal injection of LPS 20 mg/kg, and the mice in NS group and DEX control group were given equivalent sterile normal saline. The mice in DEX control group, DEX＋LPS group,and α-BGT＋DEX＋LPS group were intraperitoneally injected with DEX 40 ug/kg 15 minutes before LPS injection. The mice in α-BGT＋DEX＋LPS group were intraperitoneally injected with ot 7nAChR inhibitor α-BGT 1 p-g/kg 15 minutes before DEX injection. The mice in NS group were given equivalent sterile normal saline. Ten mice in each group were assigned for open field test before and 24 hours after model reproduction, and the mice were then sacrificed to obtain the specimens. The levels of tμmor necrosis factor- α （TNF- α） and neuron-specific enolase （NSE） in serμm were determined by enzyme-linked immune sorbent assay （ELISA）. Western Blot method was used to determine the expression of choline acetyhransferase （CHAT） and acetylcholinesterase （ACHE） in hippocampus. Another 10 mice were subjected to new object recognition test to observe the total exploration time during training period and preference index at 3 hours and 24 hours after LPS challenge. Results There were no significant differences in all parameters between NS group and DEX control group. （1） It was shown by the open field test results that there were no significant differences in all parameters of open field test before model reproduction among all the groups. Twenty-four hours after model reproduction, when compared with NS group, the mice in LPS group showed that they had the ability of cognition of new environment, but learning and memory abilities were lowered, and tension was increased. DEX could significantly attenuate the degree of deliriμm, however, the protection of DEX from the delirious syndrome was antagonized partly by cL-BGT. （3） The new object recognition test results showed that compared with NS group, the ability of exploring new object was decreased in LPS group. DEX could significantly improve the exploration ability. However, DEX failed to control the delirious syndrome in α-BGT＋DEX＋LPS group. （2） The results of ELISA showed that the levels of TNF-α and NSE in serμm were significantly increased in LPS groups as compared with that in NS group, and the levels of TNF-α and NSE were significantly decreased in DEX＋LPS group. However, α -BGT could antagonise the protective effect of DEX [TNF-α （ng/L） in NS, LPS, DEX＋LPS and α-BGT＋DEX＋LPS groups was 23.72±3.13, 808.78±87.86, 192.96±31.47, 829.99±80.98, respectively, and NSE （p.g/L） was 8.70±0.74, 25.90±3.03, 18.10 ± 2.14, and 23.12 ± 2.21, respectively, all P 〈 0.01]. （1） The results of Western Blot showed that compared with NS group, the protein expression of ChAT in LPS group was significantly declined, and the protein expression of AChE was significantly increased. DEX could reverse the expressions of ChAT and AChT, however, α -BGT could reverse the protective effect of DEX [CHAT （gray value） in NS, LPS, DEX＋LPS and oL-BGT＋DEX＋LPS groups was 1.536 ±0.150, 0.381 ±0.138, 0.914±0.173, 0.628 ±0.088, respectively, and AChE （gray value） was 0.382±0.201, 1.843 ±0.325, 0.898 ±0.155, and 1.470±0.220, respectively, P 〈 0.05 or P〈0.01]. Conclusions Deliriμm syndrome may occur in mice with endotoxemia. DEX could attenuate endotoxemia-associated deliriμm syndrome through transforming central neurotransmitter, and its mechanism maybe related with α- 7nAChR.
出处
《中华危重病急救医学》
CAS
CSCD
北大核心
2016年第2期127-133,共7页
Chinese Critical Care Medicine
基金
国家自然科学基金（30972852）
