研究背景： 气道高反应（Airway hyperresponsiveness, AHR）代表一种支气管对吸入的化学 和物理刺激发生过度收缩的状态，是呼吸系统炎性疾病的特征性症状，且气道重构 与炎症均为 AHR 的重要发生机制。AHR 不仅加剧肺功能的恶化，未得到及时缓解 的严重 AHR 还将导致低氧血症、酸中毒甚至心跳骤停。氧化应激是氧化物质累积 与抗氧化能力削弱或丧失的结果，其不仅与呼吸系统炎性疾病的发病机制和严重程 度密切相关，还是导致患者对 β2-肾上腺素能受体激动剂和皮质类固醇激素治疗反 应不佳的因素之一。氯胺酮是一种传统麻醉药物，近年来因其抗氧化及抗炎作用得 到广泛关注，有研究表明氯胺酮可缓解对传统支气管扩张剂及皮质类固醇治疗不敏 感的 AHR，但是作用机制有待探索。鉴于氧化应激在 AHR、气道炎症与气道重构 发生机制中的重要地位，本课题拟探究抗氧化机制在氯胺酮缓解气道高反应中的关 键作用。

研究目的： 1. 建立小鼠混合粒细胞型哮喘模型并明确氯胺酮在本模型中的最佳治疗浓度。 2. 明确氯胺酮对氧化应激和气道高反应的调控作用及机制。 3. 明确氯胺酮对气道炎症中粒细胞凋亡的调控作用及机制。

研究方法与结果：

第一部分：小鼠混合粒细胞型哮喘模型的建立及氯胺酮治疗浓度的探索 方法： 课 题 第 一 部 分 利 用 卵 清 蛋 白 （ Ovalbumin ， OVA ） 与 脂 多 糖 （Lipopolysaccharide，LPS）建立一种同时具有混合粒细胞性气道炎症、AHR 以及 气道重构特征的小鼠混合粒细胞型哮喘模型，并分别给予小鼠三种不同浓度氯胺酮 （50mg/kg、75mg/kg、100mg/kg）治疗。将 30 只雌性 BALB/c 小鼠随机平分为 6 组 （n=5）：Control 组(给予小鼠生理盐水)、K100 组（给予小鼠 100mg/kg 氯胺酮）、 OVA+LPS 组（给予小鼠 OVA+LPS 建立哮喘模型）、OVA+LPS+K50 组（给予小 鼠 OVA+LPS 以及 50mg/kg 氯胺酮）、OVA+LPS+K75 组（给予小鼠 OVA+LPS 以 及 75mg/kg 氯胺酮）、OVA+LPS+K100 组（给予小鼠 OVA+LPS 以及 100mg/kg 氯 胺酮）。所有小鼠在最后一次激发后 24 小时进行乙酰甲胆碱激发下气道阻力测定 并接受安乐死，留取肺泡灌洗液以及肺组织进行气道炎症、粘液分泌以及气道重构 评估。通过比较气道阻力、气道炎症、粘液分泌与气道重构情况探究三种浓度氯胺 酮的治疗效果。 结果：在第一部分实验中，K100 组小鼠与 Control 组小鼠表现出相似检测结果， 说明腹腔注射麻醉剂量的氯胺酮不会影响健康小鼠的气道反应性、气道炎症与结构； OVA+LPS 组小鼠对吸入乙酰甲胆碱表现出气道阻力增高的特征，瑞氏-吉姆萨染色 的肺泡灌洗液涂片显示大量中性粒细胞以及嗜酸性粒细胞，苏木素-伊红染色的肺组 织切片显示大量炎症细胞聚集在气道周围，并且肺泡灌洗液中的 Th2 型细胞因子： 白细胞介素 4（Interleukin-4，IL-4）、IL-5 和 IL-13，以及其他炎性细胞因子：IL-6、 IL-1β、肿瘤坏死因子 α（Tumour Necrosis Factor-α，TNF-α）和 IL-8 的表达水平均 显著高于 Control 组小鼠。OVA+LPS 组小鼠的糖原染色肺组织切片亦显示出大量糖 原染色阳性细胞，表明存在气道粘液分泌增多。改良 Masson 染色显示 OVA+LPS 组 小鼠气道周围胶原纤维增多，Western blot 以及免疫荧光结果显示小鼠肺组织中 α平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）的表达增强，提示该模型中存在 气道重构；经过氯胺酮治疗后，结果表明腹腔注射 75mg/kg 氯胺酮最大程度缓解小 鼠的气道高反应及其他症状，说明 75mg/kg 是氯胺酮在该哮喘模型中的最佳治疗浓 度。

第二部分：探究氯胺酮对小鼠肺组织中氧化应激的影响以及氯胺酮缓解气道高反应 的作用机制 方法：课题第二部分主要探索氯胺酮对该模型小鼠肺组织中氧化应激的调节作 用及其作用机制。将 35 只雌性 BALB/c 小鼠随机平分为 7 组（n=5）：Control 组、 K100 组、OVA+LPS 组、OVA+LPS+K50 组、OVA+LPS+K75 组、OVA+LPS+K100 组以及 ML385 组，给予 ML385 组小鼠 OVA+LPS、75mg/kg 氯胺酮以及核因子 E2 相关因子 2（Nuclear factor erythroid2-related factor 2，Nrf2）抑制剂（ML385）。所 有小鼠在最后一次激发后 24 小时进行乙酰甲胆碱激发下气道阻力测定并接受安乐 死，留取肺泡灌洗液以及肺组织进行后续检测：1）通过分析各组肺组织切片二氢乙 锭染色结果以及检测肺组织中丙二醛（Malondialdehyde，MDA）水平、超氧化物歧 化酶（Superoxidase dismutase，SOD）活性以及谷胱甘肽过氧化物酶（Glutathione peroxidase，Gpx）活性，评估模型肺组织中氧化应激以及不同浓度氯胺酮的抗氧化 作用；2）利用 RT-qPCR 和 Western Blot 评估各组小鼠肺组织中 Nrf2/ Kelch 样 ECH 关联蛋白 1（Kelch Like ECH Associated Protein 1，Keap1）信号通路的表达情况（Nrf2、 Keap1、血红素加氧酶 1（Heme Oxygenase-1，HO-1）、Gpx4），探究氯胺酮对 Nrf2/Keap1 信号通路的影响；3）通过给予氯胺酮治疗组小鼠 Nrf2 抑制剂（ML385）， 检测氧化应激、AHR、气道炎症、粘液分泌以及气道重构情况，探究 Nrf2/Keap1 信 号通路是否介导氯胺酮的抗氧化及治疗作用。 结果： 在第二部分实验中，K100 组小鼠与 Control 组小鼠表现出相似检测结 果，说明腹腔注射麻醉剂量（100mg/kg）的氯胺酮不会在健康小鼠肺组织中引起氧 化应激。与 Control 组相比，OVA+LPS 组小鼠表现出显著的氧化应激增强：肺组织 中 ROS 水平和 MDA 含量升高以及 SOD 和 GPx 活性减低。75 mg/kg 氯胺酮治疗显 著降低 ROS 水平和肺组织 MDA 含量，并提高 SOD 和 GPx 酶活性；与 75 mg/kg 氯胺酮相比，50 和 100 mg/kg 氯胺酮抗氧化作用较弱。与 Control 组相比，OVA+LPS 组小鼠肺组织中的 Nrf2 得到激活并导致下游抗氧化酶基因的表达升高，说明 Nrf2/Keap1 信号通路在氧化应激作用下激活，但抗氧化酶表达量不足以中和肺组织 中的氧化物质。与 OVA+LPS 组相比，75mg/kg 氯胺酮治疗显著促进 Nrf2 的活化和 抗氧化基因（GPx4 和 HO-1）的表达，而 50 或 100mg/kg 氯胺酮仅上调 GPx4 的表 达，说明 75mg/kg 是氯胺酮缓解该模型 AHR 和氧化应激的最佳浓度。经过 ML385 干预后，Nrf2 信号通路被抑制，下游抗氧化酶表达降低，氯胺酮对哮喘症状（气道 高反应、粘液高分泌、气道炎症和气道重构）和氧化应激的缓解均受到抑制，表明 氯胺酮在该模型中的抗氧化和治疗作用由 Nrf2/Keap1 信号通路介导。

第三部分：探究氯胺酮对气道炎症中粒细胞凋亡的调控作用及机制 方法：课题第三部分主要探究氯胺酮对气道中粒细胞凋亡的调控作用及机制。 实验动物分组与第二部分相同；本部分主要通过肺组织切片 TUNEL 染色、凋亡与 抗凋亡蛋白免疫组化染色以及肺组织内重要凋亡及抗凋亡蛋白表达水平的检测来 评估各组小鼠肺组织中的细胞凋亡情况以及凋亡细胞位置特征，探究氯胺酮治疗对 哮喘小鼠肺组织中细胞凋亡的影响以及 Nrf2/Keap1 信号通路的作用；本部分利用流 式细胞学检测对肺泡灌洗液中粒细胞进行分类及凋亡检测，进一步探究氯胺酮对嗜 酸性粒细胞和中性粒细胞凋亡的影响。 结果：第三部分结果表明，与 Control 组小鼠相比，TUNEL 染色的肺组织冰冻 切片提示 OVA+LPS 组小鼠肺组织中细胞凋亡增强，Western blot 和 RT-qPCR 结果 提示 OVA+LPS 组小鼠肺组织中半胱氨酸天冬氨酸蛋白酶 3（Caspase-3）分裂体和 髓细胞白血病序列-1（myeloid leukemia cell differentiation protein，Mcl-1）的表达上 调。与 OVA+LPS 组小鼠相比，75mg/kg 氯胺酮显著降低哮喘小鼠肺组织中 Mcl-1 的表达水平，50 或 100mg/kg 氯胺酮则对 Mcl-1 表达无显著影响；三种浓度氯胺酮 治疗均对 Caspase-3、B 细胞淋巴瘤-2（B-cell lymphoma-2，Bcl-2）蛋白和 BCL2 相 关 X 蛋白（BCL2-associated X protein，Bax）的表达无显著影响。由于 Mcl-1 是调 节粒细胞凋亡的关键蛋白，本课题使用连续的三张肺组织切片分别进行 Bax 和 Mcl1 免疫组化染色以及苏木素-伊红染色，初步定为凋亡细胞。结果表明免疫组化阳性 细胞与炎症细胞定位相同，且 OVA+LPS+K75 组的炎症细胞中 Bax 表达增加和 Mcl1 表达减少；流式细胞术检测结果表明 OVA+LPS 组和 Control 组小鼠肺泡灌洗液中 两种粒细胞的凋亡无显著差异；75mg/kg 氯胺酮显著促进两种粒细胞的凋亡。给予 ML385 干预后，氯胺酮对中性粒细胞凋亡的促进作用被部分抑制，但是嗜酸性粒细 胞的凋亡无显著改变。

研究结论： 1. 氯胺酮治疗缓解混合粒细胞型哮喘小鼠的氧化应激、AHR、气道炎症、粘 液高分泌以及气道重构。 2. Nrf2/Keap1 信号通路介导氯胺酮的抗氧化及治疗作用。 3. 氯胺酮具有促粒细胞凋亡作用，并且 Nrf2/Keap1 信号通路介导氯胺酮的促 中性粒细胞凋亡作用。 4. 75mg/kg 是氯胺酮在现有模型中的最佳治疗浓度，麻醉剂量氯胺酮不会引 起健康小鼠肺部细胞凋亡、氧化应激、AHR 及组织学改变。

Background: Airway hyperresponsiveness (AHR) represents a state in which the bronchus responses excessively to inhaled chemical and physical stimuli. AHR is a characteristic symptom of respiratory inflammatory diseases, and can be induced by airway remodeling and inflammation. AHR not only induces the deterioration of lung function, but severe AHR which is not relieved in time will also lead to hypoxemia, acidosis and even cardiac arrest. Oxidative stress is the result of the accumulation of oxidative substances and the weakening or loss of antioxidant capacity. It is not only closely related to the pathogenesis and severity of respiratory inflammatory diseases, but also the reason that some patients with AHR respond poorly to β2-adrenergic receptor agonists and corticosteroid therapy. Ketamine, a traditional anesthetic drug, has received great attention in recent years for its antioxidant and anti-inflammatory effects. Studies have shown that ketamine can relieve AHR that is insensitive to traditional bronchodilators and corticosteroids during severe asthma attacks, but the underlying mechanism remains to be explored. Considering the important role of oxidative stress in the mechanism of AHR, airway inflammation and airway remodeling, the study aims at exploring the key role of anti-oxidative mechanism in ketamine alleviating airway hyperresponsiveness.

Objectives: 1. Establish a murine mixed-granulocytic asthma model and determine the optimal therapeutic concentration of ketamine in the present model. 2. To clarify the regulation and mechanisms of ketamine on oxidative stress and airway hyperresponsiveness. 3. To clarify the regulatory effects and mechanisms of ketamine on granulocyte apoptosis in airway inflammation.

Methods and Results: Part One: The establishment of murine mixed-granulocytic asthma model and exploration of ketamine therapeutic concentration Methods: In the first part of study, ovalbumin (OVA) and lipopolysaccharide (Lipopolysaccharide, LPS) were used to establish a murine model of mixed-granulocytic asthma with mixed- granulocytic airway inflammation, AHR and airway remodeling. The therapeutic effects of three different concentrations of ketamine (50mg/kg, 75mg/kg, 100mg/kg) were detected. A total of 30 female BALB/c mice were randomly divided into 6 groups (n=5): Control group (mice received normal saline), K100 group (mice received 100mg/kg ketamine), OVA+LPS group (mice received OVA+LPS), OVA+LPS+K50group (mice received OVA+LPS and treated with 50mg/kg ketamine), OVA+LPS+K75 group (mice received OVA+LPS and treated with 75mg/kg ketamine), OVA+LPS+K100 group (mice received OVA+LPS and treated with 100mg/kg ketamine). All mice were tested for airway resistance under inhaled methacholine and euthanized 24 hours after the last challenge. Alveolar lavage fluid and lung tissue were collected for airway inflammation, mucus secretion and airway remodeling detection. The optimal concentration of ketamine was determined by comparing the therapeutic effects of three concentrations. Results: In the first part of study, mice in the K100 group showed similar test results with mice in the Control group, indicating that intraperitoneal injection of anesthesia doses of ketamine would not affect the airway responsiveness, inflammation and structure of normal mice. Compared with mice in the Control group, mice in the OVA+LPS group showed increased airway resistance to inhaled methacholine, a larger number of neutrophils and eosinophils in Wright-Giemsa-stained alveolar lavage fluid smears, the infiltration of inflammatory cells around the airways in H&E-stained lung sections, higher levels of Th2-type cytokines (IL-4, IL-5 and IL-13) and other inflammatory cytokines (IL6, IL-1β, TNF-α and IL-8) in lung tissues. A large number of PAS-positive cells in the PAS-stained lung sections can be observed in the OVA+LPS group, indicating increased mucus secretion. Masson-stained sections showed that the airway smooth muscle of the mice in the OVA+LPS group was thickened with increased expression of α-SMA , and the collagen fibers around the airway increased, indicating airway remodeling in the present model; the results of different concentrations of ketamine treatment showed that intraperitoneal injection of 75mg/kg ketamine effectively relieved AHR and other symptoms to the greatest extent, indicating that 75mg/kg was the optimal therapeutic concentration for the present model. Part Two: Exploring the effect of ketamine on oxidative stress and the mechanisms of ketamine alleviating airway hyperresponsiveness Methods: In the second part of study, we mainly explored the regulation and mechanism of ketamine on oxidative stress in the lung tissues of mice. A total of 35 female BALB/c mice were randomly divided into 7 groups (n=5): Control group, K100 group, OVA+LPS group, OVA+LPS+K50 group, OVA+LPS+K75 group, OVA+LPS+K100 group and ML385 group (mice received OVA+LPS, 75mg/kg ketamine and ML385). All the mice were tested for airway resistance under inhaled methacholine and euthanized 24 hours after the last challenge, and alveolar lavage fluid and lung tissue were collected for the following detections: 1) Dihydroethidium-stained frozen lung sections were analyzed for reactive oxygen species (ROS) measurement. The malondialdehyde (MDA) level, the activities of superoxide dismutase (Superoxidase dismutase, SOD) and glutathione peroxidase (Gpx) in lung tissues were detected to evaluate the oxidative substance and antioxidant capacity; 2) RT-qPCR and Western Blot were performed to detect the expression of Nrf2/Keap1 signaling pathway (Nrf2, Keap1, HO-1, Gpx4) in the lung tissues; 3) the Nrf2 inhibitor (ML385) was administered to explore whether Nrf2/Keap1 signaling pathway mediated the antioxidant and therapeutic effects of ketamine in the present model. Results: In the second part of study, mice in the K100 group showed similar test results with the mice in the Control group, indicating that intraperitoneal injection of ketamine at an anesthetic dose did not cause oxidative stress in lung tissues of normal mice. Compared with the Control group, mice in the OVA+LPS group showed a significant increase in the oxidative stress: increased level of MDA, elevated level of ROS and impaired activities of SOD and GPx. Treatment with 75 mg/kg ketamine significantly decreased ROS level and MDA content, and increased the activities of SOD and GPx; compared with 75 mg/kg ketamine, 50 and 100 mg/kg ketamine showed only minor improvement of antioxidant capacity. Compared with the Control group, Nrf2 in the lung tissue of OVA+LPS group was activated and led to an increased expression of downstream antioxidant enzyme genes, indicating that the Nrf2 signaling pathway was activated under oxidative stress, but the downstream antioxidant enzymes were insufficient to neutralize excessive ROS. Compared with the OVA+LPS group, 75 mg/kg ketamine treatment significantly promoted the activation of Nrf2 and the expression of antioxidant genes (GPx4 and HO-1), while 100 mg/kg or 50 mg/kg ketamine only up-regulated the expression of GPx4, indicating that 75 mg/kg ketamine was the optimal concentration for alleviating oxidative stress in the present model. With the administration of ML385, the Nrf2 signaling pathway was inhibited, and the relief of oxidative stress and asthmatic symptoms by ketamine were suppressed to a certain extent, indicating that the antioxidative, anti-inflammatory and AHR-relieving effects of ketamine were mediated by the Nrf2/Keap1 signaling pathway. Part Three: Exploring the regulatory effects and mechanism of ketamine on granulocyte apoptosis in airway inflammation Methods: In the third part of study, we mainly explored the modulatory effects of ketamine on granulocyte apoptosis in the lung tissues. The grouping of mice was the same as in the second part. TUNEL-stained frozen lung sections, immunohistochemical staining of pro-apoptotic and anti-apoptotic proteins, the expression of pro-apoptotic and anti- apoptotic proteins (Caspase-3, Bcl-2, Mcl-1, Bax) in lung tissues were detected to evaluate and locate apoptotic cells in lung tissues, and to explore the effects of ketamine on apoptosis. ML385 was administered to determine the role of Nrf2/Keap1 signaling pathway; Flow cytometry was performed to classify granulocytes and detect cell apoptosis in alveolar lavage fluid, and explore the effects of ketamine on the apoptosis of granulocytes. Results: The results of the third part showed that compared with mice in the Control group, TUNEL-stained frozen sections indicated that the apoptosis in the lung tissues was enhanced, and the results of Western blot and RT-qPCR suggested upregulated expression of Caspase-3 (cleaved) and Mcl-1 in the OVA+LPS group. Compared with mice in the OVA+LPS group, 75mg/kg ketamine significantly reduced the expression level of Mcl-1, while 100mg/kg or 50mg/kg ketamine showed no significant effects on it; there were no significant effects of ketamine on the expression of Caspase-3, Bcl-2 and Bax. Bax- and Mcl-1- immunohistochemical staining and H&E staining were performed on serial lung tissue sections, which was used to initially locate the apoptotic cells: 1) the immunohistochemical-staining-positive cells shared the same location with inflammatory cells; 2)The expression of Bax was increased and the expression of Mcl-1 was decreased in the OVA+LPS+K75 group; The results of flow cytometry showed that there was no significant difference in the apoptosis of granulocytes in the alveolar lavage fluid of mice between the OVA+LPS group and the Control group; 75mg/kg ketamine significantly promoted apoptosis of granulocytes. After intraperitoneal injection of ML385, the proapoptotic effects of ketamine on neutrophils were partially suppressed, but there was no significant change in the apoptosis of eosinophils.

Conclusions: 1. Ketamine treatment relieves oxidative stress, AHR, airway inflammation, mucus hypersecretion, and airway remodeling in mice with mixed granulocytic asthma. 2. The Nrf2/Keap1 signaling pathway mediates the antioxidant and therapeutic effects of ketamine. 3. The Nrf2/Keap1 signaling pathway mediates the pro-apoptotic effects of ketamine on neutrophils. 4. 75mg/kg is the optimal therapeutic concentration of ketamine in the present model. Anesthetic doses of ketamine will not cause apoptosis, oxidative stress, AHR and histological changes in the lung tissues of normal mice.