The Effect And Mechanism Of Andrographolide On Allergen-Induced Airway Inflammation In Mice

Abstract: Objective:Traditional Chinese herbal medicine Andrographis (Andrographolide) in clinical research has been proven to have strong anti-inflammatory activity and treatment of allergic pulmonary inflammation potential. In the present study, were we measured in mice after treatment of andrographolide in airway responsiveness, airway inflammatory cell infiltration, immunofluorescence and Western blot tests of nuclear factor NF-kB in lung tissue expression, and further study will investigate whether Andrographolide can inhibit allergen-induced airway inflammation and airways hyperresponsiveness. To explored the mechanism of Andrographolide on allergen-induced airway inflammation and airways hyper-responsiveness. Methods:24 BALB/c mice were randomly divided into three group, control group, asthma group and treatment group, each group include 8 mice. Mice were anesthetized by ether. For asthma group and Andrographolide treatment group, the mice were sensitized with intraperitoneal injection of ovalbumin (OVA) hydroxide solution on day 1 and day 8. Normal control group mice were only injected with PBS at the same time. From 15 days to 22 days, asthma group and Andrographolide treatment group mice were boosted again with intranasal instillation of 0.2mg/ml of ovalbumin solution, but this time Androqrapholide treatment group mice were administered intraperitoneally at a dose of 5ug/g body weight for 7 days at the same time. Twenty four hours after the last challenge by Methacholine (Mch), all the mice airway hyperresponsiveness is recorded by whole-body plethysmograph (Buxco,USA). Mice were killed after the separation and removal of the the lung tissue, part of lung tissue is taken and put with 10% formaldehyde, embedded in paraffin and cut into 6-um slices stained with hematoxylin-eosin (HE) staining. The tissues can be assessed for general morphology and cellular infiltration was performed as described previously. The standard of this method discriminates between the presence of mononuclear cells around brochioles,blood vessels and the number of patchy cellular infiltration (ScoreO-3).The remaining frozen lung tissue sections is taken by adding NF-kB antibody and incubated with biotinylated secondary antibodies. Later slides were incubated with SABC-Cy3 labeled Goat Anti-Rabbit IgG and DAPI(a nuclear counter stain can be used to evaluate nuclear localization of NF-kB.).AT last slides were scanned by laser scanning confocal fluorescence microscope. Lung tissue weighing 200mg from each group of mice was homogenated with RIPA lysis buffer. Sample is centrifuge for 30 min on high speed (10,000 rpm) at 4℃and the supernatant was collected. BCA Concentration measurement kit was used for measuring the level of the three group sample protein and loading of 20ug protein is done from each group sample.20ug protein was separated by 10% SDS-PAGE (100v,1.5h) and electrotransferred into a nitrocellulose membrance with Amersham Ecl Semi-Dry Transfer Unit (15 v 30 min). The membrance was blocked with 3% BSA (3 g BSA was dissolve in 100ml PBS) for 1 h at 37℃and incubated with polyclone rabbit anti-NF-KB (1:300) overnight at 4℃. The membrance was washed with TBST for 10min×2 times. Incubated with HRP- labeled secondary monoclone antibody (1:1200) for 1.5 h at 37℃, later it is washed with TBST 2×10min. Chemilumiriescent substrate (supersignal, Pierce) was added to the membrance and exposed strip from Kodak Image Station 4000MM (USA), the strip is saved after exposure. Results:1. The general behavior observed during the model experiment: Control mice coat color gloss, with quick actions, Methacholine induced asthma is not sensitive, no apparent breathing performance. Asthma with inhaled methacholine dose appears to increase flexible grasping limbs, irritability, accompanied by tachypnea, abdominal retraction fluctuations and other symptoms of breathing difficulties. Andrographolide treatment group also appears the above, symptoms, but less frequently than asthma, or mild.2. Andrographolide airway hyperresponsiveness in mice shows its impact in the analysis according to Fine Point software that is charted in the results, we can draw the following conclusions.①In the PBS control group of mice the baseline Penh value in the low concentration of methacholine showed little change in airway responsiveness with methacholine concentration increased slightly.②Asthma mouse model has a clear airway hyperresponsiveness, the Penh curve compared with the control group P<0.05, statistically significant.③Treatment group penh curve value is lower than the asthma group, but was higher as compared with the asthma model group, P<0.05, statistically significant.3. Andrographolide on allergen induced airway inflammation:Lung tissue HE staining was observed in control mice①Lung tissue around the bronchioles, blood vessels, and alveoli shows very small amount of inflammatory cells.②Contrast to control group,the asthma group histological results showed extensive infiltration of inflammatory cells around bronchioles, blood vessels and alveoli, such as eosinophils.③Andrographolide treatment group:the scope of airway inflammation and cell infiltration is expect to be reduced as compared with asthma group, P<0.05 statistically significant. But the treatment group and airway inflammation and cell infiltration area as compared with control group were still evident in P<0.01.4. Andrographolide expession in airway epithelial cells NF-κB:mice were stimulated with allergen, NF-κB factor rapidly translocate from the cytoplasm of airway epithelium to the nucleus as observed in the confocal microscope of the control group mice①Immunofluorescence experiments showed that in cells around the bronchioles there is only a very few cells stained red fluorescent of nuclear factor NF-κB②Asthma model group showed airway epithelial cells of obvious purple fluorescence (blue color mix with red color).③Andrographolide treatment group by airway epithelial cells show only a small amount of overlap purple fluorescence. These data indicate that andrographolide can interfere with NF-κB expression in the nucleus.5. Detection of NF-κB in the lung tissue by Western blot:Western blot experiments is used in the method, with semi-quantitative detection of mouse lung tissue NF-kB protein. Control group, NF-κB protein band shows most light, asthma group NF-κB protein bands were significantly enlarged, the treatment group of protein bands lies in between. Therefore, we conclude that the andrographolide treated mice show reduce in the epithelial cells activity of NF-κB. Conclusion:1.By OVA intraperitoneal injection and continuous nasal excitation, experiment succeeded in producing of asthma group model and Andrographolide treatment group model.2. Andrographolide can inhibit airway hyperresponsiveness. Asthma group are by far higher than the control mice penh curve and also in comparison to the treatment group. After treatment, the mice penh curve value was lower than the asthma group, P<0.05. Therefore, we infer that andrographolide reduces allergen-induced airway hyperresponsiveness.3. Andrographolide could suppress the allergen-induced airway inflammation in asthmatic lung tissue. HE staining showed bronchioles, blood vessels, and alveoli inflammatory infiltration with most extensive (EG, granulocyte macrophage acidic) infiltration. By Andrographolide treatment in mice, the scope of airway inflammation and cell infiltration was inhibited, according to the standard score statistics P<0.05, with the treatment still evident in the control group. The experimental data show that andrographolide can only partially inhibit the allergen-induced airway inflammation.4. Andrographolide can inhibit airway epithelial cell NF-κB activity:The cells around bronchiole were scanned by confocal microscopy, the airway epithelial cells of asthma group has obvious purple color in which case red and blue merge to create. But in Andrographolide group picture, it only showed less NF-κB activated existing in the nuclear, compared to asthma group. The data suggested that Andrographolide interrupted NF-κB to express in cell nucleus. Meantime, NF-κB expression level was analyzed by Western blot. The NF-κB in the asthma group was increased as compared with that in control group and treatment group. The level of NF-κB expression of treatment group was inhibited by Andrographolide, so we conclude that Andrographolide treatment of mice exhibits reduced airway epithelial NF-κB activation.