The Effect And Mechanism Of N-acetylcysteine On Airway Inflammation And Oxidative Stress In Asthmatic Mice

Background and objective:Asthma affects about 300 million people of all ages around the world.It is considered as a very serious public health problem that growing at a rate of 50%every decade.Airway inflammation is also often associated with increased production of reactive oxygen species,and the biochemical environment in the airways of asthma is conducive to free-radical-mediated responses.A growing number of clinical and experimental evidences show there are defects in the production of reactive oxygen species and endogenous antioxidant defense mechanism in asthma.Therefore,antioxidant has better clinical value in the treatment of asthma.N-acetylcysteine(NAC)is an antioxidant,acts in both direct and indirect ways,that has anti-inflammatory properties.At present,there are few studies on the specific role of N-acetylcysteine in asthma.The main purpose of this study is to explore the possible mechanism about effects of N-acetylcysteine on airway inflammation and oxidative stress in asthmatic mice.Methods:BALB /c female mice with SPF were divided into three groups by random number table method: control group(n = 6),asthma group(n = 6)and NAC intervention group(n = 6).Asthma model mice were established by sensitization and aerosol stimulation with ovalbumin(OVA).Sensitization: on the 1th,8th and 15 th day,the asthmatic mice were intraperitoneally injected with the mixture of 0.2mL/mouse(including 100μgOVA and 2 mg aluminum hydroxide gel + 0.2mL aseptic phosphate buffer);Stimulation: on the 22 nd day,2.5%OVA aerosol inhalation was given for 45 min for a week.All the mice in the control group were replaced by the same volume of PBS,and the mice in the NAC group were given intragastric administration of NAC(300mg/kg)1 hour before each atomization.The general condition of mice was observed during this period.The airway hyperresponsiveness(AHR)was measured by noninvasive pulmonary function meter in mice.The serum,lung tissue and bronchoalveolar lavage fluid(BALF)of mice were collected.The levels of serumtotal IgE and cytokines IL-13,IL-5 and IFN-γ were measured by ELISA.Pathological staining of lung tissue: HE staining,PAS staining,evaluation of airway inflammatory infiltration and goblet cell mucus secretion,colorimetric measurement of lung tissue MDA,GSH and SOD content.The expression of Nrf2 and HO-1mRNA genes in lung tissue was measured by qRT-PCR.BALF used whole blood cell counter and Swiss Giemsa staining smear to count and classify cells under light microscope.Results:1.Model mice behavior: In OVA group,there were itching and scratching of scalp,bulbar conjunctival edema,shortness of breath,abdominal muscle convulsion and lip cyanosis in different degrees during nebulization,and the more times of excitation,the more serious the symptoms.The above symptoms in the NAC group were better than those in the OVA group,but there was no obvious above-mentioned performance in the challenge process of the mice in the control group.All the mice survived in the experiment.2.Airway responsiveness: The effect of NAC on AHR induced by OVA in mice was observed by measuring Penh.When the mass concentration of Mch was continuously increased,the airway responsiveness index(Penh)of mice in each group showed an increasing trend.Compared with the control group,when the concentration of Mch was greater than or equal to 3mg/mL,the Penh of mice in OVA group increased significantly after inhaling Mch(P < 0.05).Compared with OVA group,when the concentration of Mch was greater than or equal to 6 mg/mL,the Penh of mice in NAC group decreased significantly after inhaling Mch(P < 0.05).NAC treatment can reduce airway hyperresponsiveness in asthmatic mice.3.Inflammatory cells and cytokines(I)The number of inflammatory cells in BALF: The number of inflammatory cells in the asthma group was meaningfully higher than that in the control group(all P< 0.05).Compared with the asthma group,NAC could significantly improve the number of inflammatory cells in mice(all P<0.05).(II)The level of cytokines in serum: Compared with the control group,the level of Th2 cytokines in asthmatic mice increased significantly,and the level of Th1decreased(all P<0.05).Compared with the asthma group,the level of Th2 cytokines in NAC group decreased significantly,and the level of Th1 increased significantly(all P<0.05).NAC could significantly change the balance of Th1/Th2.4.Pulmonary histopathology: The asthma group showed that there were a large number of inflammatory cells around the airway,bronchial structure disorder and destruction,mucus exudation and goblet cell hyperplasia obviously.The NAC group alleviated the above airway pathological manifestations,while the control group had intact airway structure,no obvious inflammatory infiltration and less mucus secretion.5.Oxidative stress index: Compared with the control group,the MDA content in the asthma group were significantly increased,and the GSH and SOD content were significantly reduced(all P<0.05).NAC significantly inhibited the oxidative stress induced by OVA,which showed that the MDA content decreased and the GSH and SOD content increased(all P<0.05).6.The change of Signal path:NAC could significantly activate Nrf2/HO-1 gene expression,which was significantly higher than that in control group and asthma group(all P<0.05).Conclusion:1.The mice induced by OVA showed typical acute asthma symptoms and significant pathological inflammatory changes,indicating that the mouse asthma model was successfully established.2.There is an imbalance of oxidative stress in asthmatic mice.3.NAC intervention could improve abnormal biological behavior in asthma model mice,reduce airway inflammation,reduce AHR,change the balance between cytokines Th1/Th2,increase antioxidant substances,and reduce lipid oxidative damage products.4.NAC intervention can stimulate the expression of Nrf2/ HO-1 gene in mice,suggesting that the mechanism of NAC may be regulated by this signaling pathway.