【作者】 张晓艳；

【导师】 姜丽华；

【作者基本信息】 郑州大学 ， 麻醉学， 2023， 硕士

【摘要】 急性肺损伤(Acute lung injury,ALI)是由脓毒症、严重肺炎、急性重症胰腺炎及其他不同的肺内外致病因素累及肺实质,造成的轻度急性呼吸衰竭,病死率高,而致病因素中又以脓毒症最常见。脂多糖(LPS)作为一种内毒素,在基础实验中常用于制作脓毒症急性肺损伤模型,且目前研究发现尚缺乏有效防治ALI的抗内毒素药物。近二十年来的研究证实,胆碱能抗炎通路(CAP)作为一种内源性神经免疫通路具有反馈性调控局部和全身抗炎作用,现已受到业界广泛关注并逐渐成为研究治疗ALI的热点。α7烟碱型乙酰胆碱受体(α7 Nicotinic acetylcholine receptor,α7nAChR)是激活CAP的关键受体,通过负反馈机制调控炎症反应,在减轻LPS致急性肺损伤中发挥重要作用。本实验拟采取腹腔注射脂多糖的方法制造小鼠急性肺损伤模型,并分析盐酸戊乙奎醚(Penehyclidine hydrochloride,PHC)预处理对相关因子表达的影响,探讨α7nAChR在盐酸戊乙奎醚减轻脓毒症小鼠急性肺损伤的作用及机制,为PHC在防治ALI中的应用增加新的药理学依据。目的:探讨α7nAChR是否参与了盐酸戊乙奎醚减轻LPS致小鼠急性肺损伤,并探寻其相关机制,为PHC在防治ALI中的应用提供新的理论依据。方法:SPF级健康雄性C57BL/6小鼠40只,6-8周,体重18-25 g,采用随机数字表法分为4组:对照组(C组),急性肺损伤组(ALI组),盐酸戊乙奎醚组(PHC组),α7nAChR抑制剂MLA组(MLA组)。腹腔注射LPS 15 mg/kg制备小鼠脓毒症ALI模型。PHC组于制备模型前30min腹腔注射PHC 2 mg/kg,α7nAChR抑制剂MLA组于应用PHC前10min腹腔注射MLA10 mg/kg,对照组腹腔注射等量生理盐水。注射LPS后6h时处死小鼠,取肺组织,HE染色显微镜下观察肺组织病理学变化,通过计算肺组织湿/干重(W/D)比值来测定肺含水量,ELISA法测定肺组织中TNF-α、IL-1β、IL-10含量;Western blot检测肺组织中α7nAChR、NF-κB p65、IκB-α蛋白的表达。结果:1.HE染色观察结果:C组可见正常肺组织结构。ALI组肺组织结构明显破坏,肺泡壁增厚,部分肺泡融合或萎缩,组织内可见大量炎性细胞浸润和肺间质水肿。PHC组肺泡结构较完整,肺泡壁稍增厚,少部分肺泡萎缩,炎性细胞浸润程度和肺损伤程度较ALI组减轻。而MLA组的肺损伤程度较PHC组明显加重。2.肺组织湿干重比(W/D):与C组相比,ALI组肺组织W/D比值增加(P<0.05);与ALI组相比较,PHC预处理显著降低了肺组织W/D比值(P<0.01);与PHC组相比,α7nAChR抑制剂MLA使肺组织W/D比值显著增加(P<0.01)。3.肺组织中TNF-α、IL-1β含量:与C组相比,ALI组肺组织TNF-α、IL-1β含量升高(P<0.05);与ALI组相比,PHC组肺组织TNF-α、IL-1β含量降低(P<0.01);与PHC组比较,α7nAChR拮抗剂MLA组肺组织TNF-α、IL-1β含量升高(P<0.01)。4.肺组织中IL-10含量:与C组相比,ALI组肺组织IL-10含量降低(P<0.05);与ALI组相比,PHC组肺组织IL-10含量升高(P<0.01);与PHC组比较,α7nAChR拮抗剂MLA组肺组织IL-10含量降低(P<0.01)。5.肺组织中α7nAChR蛋白表达:与C组相比,ALI组肺组织中α7nAChR蛋白表达增加(P<0.05);PHC组与ALI组相比,肺组织中α7nAChR蛋白表达进一步增加(P<0.01);MLA组与PHC组相比,肺组织中α7nAChR蛋白表达显著降低(P<0.05)。6.肺组织中NF-κB p65、IκB-α蛋白表达:与C组比较,ALI组的NF-κB p65蛋白水平表达升高,而IκB-α蛋白含量降低(P<0.05);与ALI组比较,PHC降低了NF-κB p65蛋白的表达,而升高了IκB-α蛋白的表达(P<0.01);与PHC组比较,α7nAChR拮抗剂MLA组NF-κB p65蛋白表达水平升高而IκB-α蛋白表达水平降低(P<0.01)。结论:盐酸戊乙奎醚预处理可以抑制LPS诱导的炎症反应并减轻急性肺损伤,其机制可能与增加α7nAChR表达及抑制其介导的NF-κB信号通路有关。更多还原

【Abstract】 Acute lung injury(ALI)is a mild acute progressive respiratory failure caused by sepsis,severe pneumonia,acute severe pancreatitis,and other different internal and external pathogenic factors of the lung implicating pulmonary parenchyma,and accompanied by a high case fatality rate.Sepsis is the most common precipitating factor of acute lung injury.Lipopolysaccharide(LPS)is a kind of endotoxin,which is often used in basic experiments to induce acute lung injury models of sepsis.At present,there is no specific anti-endotoxin therapeutic drug.In the past two decades,we have identified that the cholinergic anti-inflammatory pathway(CAP),as an endogenous neuroimmune pathway,has received widespread attention and gradually become the focus of research and treatment of ALI,which has local and systemic anti-inflammatory effects.α7 Nicotinic acetylcholine receptor(α7 Nicotinic acetylcholine receptor,α7n ACh R)is the key receptor of CAP,and plays an important role in mitigating LPSinduced acute lung injury by regulating inflammatory response through a negative feedback mechanism.In this experiment,the animal model of acute lung injury in mice was induced by intraperitoneal injection of LPS,and analyzed the effect of penehyclidine hydrochloride pretreatment was on the expression of related mediators.The aim is to explore the role and mechanism of α7n ACh R in penehyclidine hydrochloride alleviating LPS-induced acute lung injury in mice,to add new pharmacological evidence for the application of PHC in prophylaxis and treatment of ALI.Objective:To investigate whether α7nAChR is involved in penehyclidine hydrochloride alleviating LPS-induced acute lung injury in mice.Furthermore,to explore its related mechanisms,providing new theoretical basis for the application of PHC in prophylaxis and treatment of ALI.Methods:Forty healthy male C57BL/6 mice,aged 6-8 weeks old,weighing 18-25 g,were divided into four groups by using the table of random numbers(n =10 each): control group(group C),acute lung injury group(ALI group),penehyclidine hydrochloride group(PHC group),α7nAChR inhibitor MLA group(MLA group).The ALI model was induced by using LPS(15 mg/kg)through intraperitoneal injection.In PHC group,penehyclidine hydrochloride(2 mg/kg)was intraperitoneally injected at 30 min before establishing the model.MLA(10 mg/kg)was intraperitoneally injected at 10 min before the administration of penehyclidine hydrochloride while in control group,mice were given an equal volume of saline.Mice were sacrificed at 6h after LPS administration and lung tissues were collected.The pulmonary histopathological results were observed by HE staining,and the water content of the lung was measured by reckoning the W/D weight ratio of the lung tissue.The content of TNF?α,IL?1β,and IL-10 was detected by ELISA,and the expression of α7nAChR,NF-κB p65,and IκB-α in lung tissues was tested by Western blot.Results:1.HE staining observation: Normal lung tissue structure can be seen in group C.In ALI group,the lung tissue structure was significantly damaged,the alveolar wall was thickened,and some alveoli fused or atrophied.There was a large number of inflammatory cell infiltration and pulmonary interstitial edema.The alveolar structure of PHC group was relatively integrated,the alveolar wall was slightly thickened,and some of the alveoli were atrophic.Compared with ALI group,the degree of inflammatory cell infiltration and lung tissue injury in PHC group was reduced.However,the degree of lung injury in MLA group was significantly worse than that in PHC group.2.Wet-to-dry weight ratio(W/D)of lung tissues: Compared with group C,the W/D ratio of lung tissues in ALI group was increased(P<0.05).Compared with ALI group,PHC pretreatment significantly reduced the W/D ratio of lung tissues(P<0.01).Compared with PHC group,α7nAChR inhibitor MLA significantly increased the W/D ratio of lung tissues(P<0.01).3.The content of TNF-α and IL-1β in lung tissues: Compared with group C,the content of TNF-α and IL-1β in lung tissues of the group ALI were increased obviously(P<0.05).Compared with group ALI,the content of TNF-α and IL-1β in lung tissues in the group PHC was decreased(P<0.01).Compared with PHC group,the content of TNF-α and IL-1β of lung tissue in group α7nAChR antagonist MLA was increased(P<0.01).4.The content of IL-10 in lung tissues: Compared with group C,the content of IL-10 in lung tissues of the group ALI decreased obviously(P<0.05).Compared with group ALI,the content of IL-10 in lung tissues in the group PHC was increased(P<0.01).Compared with PHC group,the content of IL-10 of lung tissue in groupα7nAChR antagonist MLA was decreased(P<0.01).5.The protein levels of α7nAChR in lung tissues: The protein levels of α7nAChR in lung tissues of the group ALI were higher than that in group C(P<0.05).Compared to group ALI,the protein expression of α7nAChR in lung tissues of group PHC was further raised(P<0.01).There was a significant decrease in α7nAChR expression levels in the lung tissues of group MLA compared to group PHC(P<0.05).6.The protein expression of NF-κB p65 and IκB-α in lung tissues: Compared with group C,the protein expression of NF-κB p65 in lung tissues of the group ALI was increased whereas IκB-α expression levels were decreased(P<0.05).Compared with group ALI,the protein expression of NF-κB p65 in lung tissues of group PHC decreased while the protein expression of IκB-α was increased(P<0.01).Compared with group PHC,NF-κB p65 expression levels in lung tissues of group MLA were significantly increased,whereas IκB-α expression was decreased(P<0.01).Conclusions:Pretreatment with penehyclidine hydrochloride can restrain LPS-induced inflammatory reaction and mitigate acute lung injury,and the mechanism of which may be relevant to the increasing of α7nAChR expression and inhibition of the NF-κB signaling pathway.更多还原