Vitamin D3 Attenuates Inflamation And Oxidative Stress Induced By LPS In Asthmatic Mice

objective:To investigate the exacerbating of oxidative damage and inflammation induced by Lipopolysaccharide(LPS)and explore the protection of vitamin D3 on lung tissues through anti-oxidative stress and anti-Inflammatory Responses in asthmatic mice airways.Methods:Thirty-five female specific pathogen-free mice,6 to 8 weeks,were randomly divided into 5 groups:PBS control group(A group,PBS sensitization,PBS challenge and PBS treat);OVA asthma group(B group,OVA sensitization,OVA challenge and PBS treat);OVA/LPS asthma group(C group,OVA sensitization,OVA and LPS challenge,PBS treat);OVA/VD3 group(D group,OVA sensitization,OVA challenge and "Vitamin D3 treat);OVA/LPS/VD3 group(E group,OVA sensitization,OVA and LPS challenge,Vitamin D3 treat).(1)Determination of inflammatory changes in the lung tissue of asthmatic mice,cells in bronchoalveolar lavage fluid(BALF)were counted and lung histopathology was evaluated by HE staining;Detection the inflammation factor expression of TNF alpha,NF-?B,PNF-?B in lung tissue homogenate by Western Blot and IL-17 in homogenate of lungs by Elisa.(2)Detect the oxidative stress levels of which including oxidative damage situation:Comet assay to detect cell DNA damage of mice peripheral blood lymphocytes by ordinary fluorescent microscopy;Analysis the Reactive Oxygen Species(ROS)of lung tissue by Reactive Oxygen Species Assay Kit,Confocal microscope analysis ROS fluorescence expression in lung tissue.(3)Antioxidant levels test includes:measured SOD to detect the degree of anti-oxidant in homogenate of lung tissue by full wave-length by Total SOD activity detection kit(WST);Detection the total glutathione level in lung tissue homogenate by Total glutathione peroxidase detection kit.Results:(1)Compared with the control group,There is a degree of inflammation and oxidative stress change within the lung tissue in asthma groups;leukocytes,neutrophils,eosinophils and lymphocytes in BALF in the asthma group are:14±0.49?6.51±0.28?1.91±0.09?3.17±0.18,Compared with the control group Respectively;Pathological changes in lung tissue revealed worse inflammation and tracheal mucosa thickening,the score of lung tissue pathology:(16±1)(P<0.05);the TNF-??NF-?B?PNF-?B more expression in lung tissue homogenate by Western Blot,(P<0.001):0.26±0.04?0.24±0.03?0.21±0.02;the level of IL-17 in lung tissue was 0.09±0.02.Single cell gel electrophoresis(comet experiments)analysis and DNA damage is significantly higher than the control group:7.69±0.41;detection the ROS fluorescence expression in lung tissue:78.24±7.08,Were higher than the normal control group(P<0.001);the SOD vitality of lung tissue homogenate:1.45±0.07;the level of total glutathione in lung tissue homogenate:35±2.Were lower than the normal control group,(P<0.05).(2)Compared with the PBS group,after the use of LPS can worsen asthma mice lung oxidative damage,inflammation significantly increased,the level of leukocytes,neutrophils,eosinophils and lymphocytes in BALF respectively increased significantly(P<0.05):57.65±2.12?33.45±2.64?2.4±0.1?9.52±0.62;Obviously increase the inflammatory cells infiltration and tracheal mucosa thickening with the lung tissue(p<0.01):22±2;the TNF-??NF-? B?PNF-? B expression significantly increased in lung tissue homogenate by Western Blot(P<0.01):0.4±0.03?0.41±0.02?0.35±0.03;the level of IL-17 in lung tissue was0.16±0.05.Comets experimental analysis,DNA damage increase obviously(P<0.001):12.75±0.79;Significantly enhanced of ROS expression within the lung tissue:144.51±18.64;while the SOD activity decreased obviously within the Lung tissue homogenate:1.01±0.05;the total glutathione of Lung tissue homogenate decreased significantly:29±2.(3)After treated with vitamin D3,compared with the matching of asthma group,the level of inflammatory cells decreased within BALF and lung tissue with statistical significance;the expression of TNF-a?NF-?B?PNF-K B?IL-17 significant decrease:P<0.001 with statistical significance;DNA damage alleviate by the comet experiment:P<0.05 with statistical significance;the ROS expression in lung tissue reduced:P<0.05 with statistical significance;the SOD activity increased obviously within Lung tissue homogenate:P<0.05 with statistical significance;the level of total glutathione increased:P<0.05 with statistical significance.Conclusion:Oxidative stress is involved in the pathogenesis of asthma,LPS can induce oxidative stress and damage and cause symptoms worse while Vitamin D3 reduce damage of lung tissue by inhibiting oxidative stress in asthma mice.