The Role And Underlying Mechanism Of The Nrf2/Keap1 Signaling Pathway In Ketamine Regulating Airway Hyperresponsiveness

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 concentrationMethods: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+K50 group(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 inj ection 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 hyperresponsivenessMethods: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(ML3 85)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 inflammationMethods: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 antiapoptotic 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.