Research Of Kechuanning On The Immunoregulatory Mechanisms Of The Rats With Bronchial Asthma

Objective: On the basis of establishing the rat model with bronchial asthma, through the animal experiments to study the role and mechanism of Kechuanning to immuno-regulation of asthmatic rats. Which were to provide experimental evidence for the clinical medication on treating the bronchial asthma.MethodsPartⅠ: Effects of Kechuanning on regulatory role of Th1/Th2 cytokines and patho-morphological of the asthmatic rats.40 clean grade SD rats, half and half for either sex and were 180-200g on weights. These animals were randomly divided into five groups: namely, the normal group, the model group, high dose Kechuanning group (high dose group for short) low dose Kechuanning group (low-dose group for short), Guilong Kechuanning control group (Guilong Kechuanning group for short) with 8 rats for each group. The drugs were given through stomach-perfusion to each treatment group from the beginning of the 1st asthma provocation (third weeks after making model) to the day before killing and continued for four weeks. The dose was: 27g/Kg for the high-dose group and 13.5g/Kg for the low dose group, 0.41 g/Kg for Guilong Kechuanning group and 0.5% sodium carboxymethyl cellulose were given to the normal and model group through stomach-perfusion with one time per day.According to the report, in addition to the normal group, in the first and eighth days after experiment, 1 ml mixture of 10% of egg protein and the aluminum hydroxide were injected intra-peritoneally to the rats from each group, one point was prepared from both sides of waist with subcutaneous injection was 0.5 ml for per point and it was 2 ml in total. All these were to sensitize rats. From 15th days, 50 ml of 3% egg protein was used for spray to provoke the rat asthma; the aerosol flow was 5ml/minutes with 20 minutes for each time, once for every other day and continued for four weeks in total. The manifestations of the provocative success were that there were fast breathing, cyanotic lips, abdominal muscle cramps, nodding breathing and unstable standing etc. In the normal group, the normal saline was used to replace the egg protein for intraperitoneal injection and spray inhalation.Histopathological observation: after complete anesthesia, the thoracotomies were given to the animals and the right main bronchi were ligated rapidly. The middle lobe of the right lung was taken and fixed with 4% paraformaldehyde and gave the dehydration, embedding and sections, then, the hematoxylin - eosin HE staining. The pathological changes were observed microscopically and NYD1000 computer image processing software was used.IL-4 and IFN-γlevel test: after complete anesthesia, the thoracotomies were given to the animals and the right main bronchi were ligated rapidly. The upper lobe of right lung was taken and put into the glass homogenate tube after weighing, adding the normal saline which were 2 times of the weight of lung tissue and gave the full levigation and make the tissue to be homogenized. Then, the prepared homogenate was given the centrifugation with 3500r/min for 10 minutes, after that, the supernatants was extracted and gave subpackage. IL-4 and IFN-γlevel were measured with the double-antibody sandwich ABC-ELISA method. The enzyme label plates were coated with IL-4 (IFN-γ) monoclonal antibody and the plates were washed with PBS for 4 times. Then, 1%BSA (bovine serum albumin) was added to enzyme label plate (200μl/ hole) and kept 4°C overnight. Later, adding the samples and the standard with different concentrations (100μl/ hole) and gave 120 minutes incubation at room temperature. Thus, it formed the immune compounds and connected with the plate, then, the plates were washed with PBS for 4 times and added horseradish peroxidase labeled anti-IL-4 monoclonal antibody (100μl/hole) and gave 60 minute's incubation at room temperature, the plates were washed again with PBS for 4 times and added OPD substrate solution and kept in room temperature for 10-30 minutes with being away from light, there was yellow. At this point, added the sulfuric acid of stopping solutions and mixed evenly, the solution became darker in color in five minutes. Absorbance OD value was measured at 490 nm with enzyme sign analyzer and drew the specimen's curves and searched out the specimen's concentration.The data was expressed with mean±standard deviation (±s) and was processed with the statistical software- SPSS for Windows 11.5. Single factor variance analysis was used to compare the each group. Then, Student-Newman-Keuls Test was used for the comparison between each two groups and the significant difference level took 0.05 and 0.01 as the standard.Part II: the influence of Kechuanning on the inflammatory mediators NO and ET-1 of rats with bronchial asthma.Histopathological observation: after complete anesthesia, the thoracotomies were given to the animals and the right main bronchi were ligated rapidly. The middle lobe of the right lung was taken and fixed with 4% paraformaldehyde and gave the dehydration, embedding and sections, then, the hematoxylin - eosin HE staining. The pathological changes were observed microscopically and the area of the rats airway wall were measured in the light of the literature and the intratracheal perimeter were standardized. NYD1000 image analysis software was used to measure the internal circumference (Pi)of intact bronchial lumen, bronchial wall area (WA), bronchial smooth muscle area. Pi was used for standardization. WA/Pi and the bronchial smooth muscle area/ Pi expressed the bronchial wall thickness and bronchial smooth muscle thickness respectively. At the same time, the number of EOS and the lymphocytes from each high-power field (400 times) were measured.NO and ET-1 content test: after complete anesthesia, the thoracotomies were given to the animals and the right main bronchi were ligated rapidly. The upper lobe of right lung was taken and put into the glass homogenate tube after weighing, adding the normal saline which were 2 times of the weight of lung tissue and levigating it fully and making it homogenizate. Then, the prepared homogenate was centrifugated with 3500r/min for 10 minutes, after that, the supernatants was extracted and the NO content of lung tissue were measured with nitric acid reductase method. The ET-1 content of lung tissue was measured with radio immunity and was calibrated with the protein content. Animal grouping, making model, drug administration and the statistical methods of data are same as the former.Part III: The influence of Kechuanning on the nuclear factor-kB(NF-kB) of rats with bronchial asthma.Animal grouping, making model, drug administration and the statistical methods of data are same as the former.EOS counts of airway wall: the method of making pathological tissue section was the same with before and NYD1000 computer image-processing software was used. A rather complete structure of the bronchial wall was selected and five high-power fields (×400) were chosen randomly, and the number of infiltrating EOS from the bronchial wall was calculated by the same observer,and the averaged value represent as the value of the section.The measurement of NF-kB expression: after complete anesthesia, the thoracotomies were given to the animals and the right main bronchi were ligated rapidly. The middle lobe of right lung was taken and fixed 24 hours with 4% paraformaldehyde and gave the dehydration, paraffin embedding and serial sections with 4μm in thickness. ABC staining of immunohistochemistry was made .The primary antibody was mouse anti-rat P65 monoclonal antibody (F-6), the secondary antibody was the biotinylated horse anti-mouse IgG. Main operation steps are as follows: (1) through xylene dewaxing and gradient alcohol dehydration, the paraffin sections were rinsed with PBS for 5 minutes×2 times; (2) incubating it in 3% hydrogen peroxide and methanol solution for 5-10 minutes at room temperature in order to remove the activity of endogenous peroxidase, and rinsing it with PBS for 3 times; (3) heat retrieval antigen: the slice were immersed in 0.01M citrate buffer solution (pH 6.0) and after heating it to boil with the microwave oven, turning power off with 5-10 minute's interval, repeating 1-2 times. The material was washed with PBS for 2 times after cooling; (4) dropping 5% normal goat serum for blocking and being incubated for 20 minutes at room temperature. Then, pouring the redundant liquid; (5) dropping NF-kBⅠantibody (1:100 dilution)for wet case incubation. Keeping it in 4℃refrigerator overnight and washing in PBS for 5 minutes×2 times;(6) dropping the secondary antibody working fluid with biotinated label and incubating it in 37℃for 45 minutes, then washing it in PBS for 5 minutes×2 times;(7) Dropping SABC reagents (1:100 dilution), then incubating it for 20 minutes and washing it in PBS for 5 minutes×2 times; (8) giving DAB color development for 5-15 minutes, then, stopping the color development; (9) Hematoxaylin staining, the conventional mounting slices, dryness, dehydration, vitrification and mounting. The slice was observed with the optical microscope. A rather complete structure of the bronchial wall was selected and five high-power fields (×400) were chosen by the same observer randomly, which was to count the positive cell percentage of the karyon NF-kB from the bronchial wall and took its average value as the representative value of the section.Part IV: The influence of Kechuanning on the eosinophilic granulocyte apoptosis and bcl-2 and bax expression of the relative gene from the rats with asthmaAnimal grouping, making model, drug administration and the statistical methods of data are same as the former.Tissue sample making:After complete anesthesia, the thoracotomies were given to the animals and the right main bronchi were ligated rapidly. The middle lobe of the right lung was taken and fixed for 24 hours with 4% paraformaldehyde and gave the dehydration, paraffin embedding and serial sections with 4μm in thickness, then, the hematoxylin - eosin HE staining and TUNEL marking and immunohistochemical ABC staining.Eosinophilic granulocyte apoptosis detection: this experiment is carried out in the light of TUNEL reagent kit and the major steps as follows: (1) through xylene dewaxing and gradient alcohol dehydration, the paraffin sections were rinsed with PBS(0.01M, pH7.4) for 5 minutes×2 times (2) proteinase K (2mg/L)digested it in 37℃for 15 minutes, then rinsed in PBS for 5 minutes×3 times (3) after balancing the positive contrast with DNA equilibrium liquid for 2 minutes, added DNase I for reaction in 37℃for 10 minutes (4) TUNEL working fluid (1 : 19 dilution) 50μl were added respectively to the experimental group and positive contrast and was incubated in 37℃for 45 minutes, then, rinsing them with PBS for 5 min×3 times. (5)thus, adding 50μl anti-fluorescein antibody with alkaline phosphatase (AP) label and being incubated in 37℃for 30 minutes, then , rinsing them with PBS for 5 min×3 times. (6) Through Buffer III balance and NBT/BCIP color development, the color development in dark at room temperature got to positive and good control color development, the color reaction can be stopped (7) mounting is taken with glycerol buffer (PBS: glycerol 1:3) and observating.The detection of bcl-2 and bax protein expression: immunohistochemical ABC staining (same as the former method) was used.Results verdict and image processing: it was observed with the optical microscope, the apoptostic eosinophilic granulocyte was the blue-purple binuclear or mononuclear, which was located in the nuclei with no staining for the cytoplasm. Bcl-2 and Bax protein staining showed brown yellow granules and were in the cytoplasm. Five fields were chosen by the same observer randomly from each section under high-power microscope (×400), then, the number (HE) of eosinophilic granulocyte from the bronchial wall and lung tissue, the apoptostic index of staining and eosinophilic granulocyte (TUNE L method, the percentage of the apoptostic eosinophilic granulocyte that occupied in the corresponding eosinophilic granulocyte AI) and the percentage of strong positive cells of Bcl-2 and Bax nuclei were calculated respectively, the averaged value was taken as the representative value of the section.Part V: The influence of Kechuanning on the IL-5 mRNA expression of lung tissue from the rats with bronchial asthmaAnimal grouping, making model, drug administration and the statistical methods of data are same as the former.After complete anesthesia, the thoracotomies were given to the animals and the right main bronchi were ligated rapidly. The right lung that was adjacent to the porta was taken for about 50-100mg, then puting it into the homogenizer, adding 1 ml Trizol and homogenatizating by homogenizer. The IL-5 mRNA expression changes of the lung tissue in rats from each group were detected by RT-PCR method.ResultsPartⅠ: The regulatory role and the patho-morphological effects of Kechuanning to Th1/Th2 cytokines from the asthmatic rats.1. The pathological effects of Kechuanning to the bronchial-lung tissue of rats with bronchial asthma.Normal groups: there was no obvious inflammatory cell infiltration from the bronchus of lung tissues and surroundings in rats with intact wall, the thickness of wall and smooth muscle were normal with no congestion and edema in the tissue mucosa, the alveolar septum was narrow and the alveolar cavity was clear and spacious.Model group: there were many inflammatory cells infiltration in bronchial wall and lumen as well as around blood vessel and bronchus, which mainly were the eosinophilic granulocyte and lymphocyte, accompanied with bronchial mucosal edema, thickening, epithelial exfoliation, microvascular leakage, the secretion increased within lumen stromal cell hyperplasia, thickened smooth muscle, the congestive edema of tissue mucosa, widened alveolar septum and narrowed alveolar lumen.Guilong Kechuanning groups: there were slight decrease for the eosinophilic granulocyte and other inflammatory cell infiltration from lung tissue, also, there were slight decrease for the smooth muscle wall thickness with the congestion and edema from tissue got slight reduction.Low-dose Kechuanning group: the eosinophilic granulocyte and other inflammatory cell infiltration from lung tissue decreased, also, there were decrease for the smooth muscle wall thickness with the congestion and edema from tissue got slight reduction, the alveolar lumen widened with the narrowing of alveolar septum.High-dose Kechuanning group: the eosinophilic granulocyte and other inflammatory cell infiltration from lung tissue decreased obviously and almost disappeared, also, there were marked decrease for the wall and smooth muscle thickness with the congestion and edema from tissue got obvious reduction, the alveolar lumen widened with the narrowing of alveolar septum.2. The influence of Kechuanning on the homogenate IL-4 content of lung tissues from the rats with bronchial asthma.Compared with the normal group, IL-4 content of rat lung tissues from the model group increased significantly with very significant difference (P<0.01). Compared with the model group, IL-4 content can be lowered from each treatment group and it was even more obvious for the high-dose Kechuanning group with very significant difference (P<0.01). The low-dose Kechuanning group and Guilong Kechuanning group had significant difference (P<0.05). Compared with the control group, IL-4 content from the high-dose Kechuanning group was significantly lower than the control group with significant differences (P<0.05).3. The influence of Kechuanning on the homogenate IFN-γcontent of lung tissues from the rats with bronchial asthma.Compared with the normal group, IFN-γcontent of rat lung tissues from model group had slight increase(P>0.05). Compared with the model group, IFN-γcontent can be increased from each treatment group and it was even more obvious for the high-dose Kechuanning group with very significant difference (P<0.01). IFN-γcontent can be elevated for the low-dose Kechuanning group and Guilong Kechuanning group with the significant difference (P < 0.05). Compared with the control group of Guilong Kechuanning, IFN-γcontent from the high-dose Kechuanning group was significantly higher than control group with significant differences (P<0.05).4. The influence of Kechuanning on IFN-γ∕IL-4 ratio of lung tissues from the rats with bronchial asthma.Compared with the normal group, IFN-γ∕IL-4 ratio of rat lung tissues from the model group decreased obviously with very significant difference (P<0.01). Compared with the model group, IFN-γ∕IL-4 ratio can be elevated from each treatment group with very significant difference (P<0.05). It was even more obvious for the high-dose Kechuanning group and it was with very significant difference (P<0.01). Compared with the control group, IFN-γ∕IL-4 ratio from the high-dose Kechuanning group was significantly higher than that of control group with significant differences (P<0.05).Part II: The influence of Kechuanning on the inflammatory mediators NO and ET-1 of rats with bronchial asthma.1. The histopathological observation and the quantitative analysis for the rats with bronchial asthma and treating with Kechuanning There were less inflammatory cell infiltration in the lung tissue of rats from normal group with intact vessel wall, there was no congestion and edema in the mucosa, the thickness of vessel wall and the smooth muscle were normal. In the rats from model group, there were plenty of eosinophilic granulocyte and lymphocyte and other inflammatory cell infiltration(P<0.01) from the bronchial wall and around blood vessel and bronchus, also, there were bronchial mucosal edema, thickening, epithelial exfoliation, microvascular leakage, the secretion increased within lumen,stromal cell hyperplasia ,smooth muscle thickening(P<0.01), Compared with the model group, there were obvious decrease for the eosinophilic granulocyte and lymphocyte and other inflammatory cell infiltration from each treatment group, the congestion and edema of the lung tissue were also mitigated and the thickness of vessel wall and the smooth muscle were reduced obviously(P<0.01).The improvement on histopathological indices from high-dose group were better than control group(P<0.05 or P<0.01).2. The influence of Kechuanning on NO content of lung tissues from the rats with bronchial asthma.Compared with the normal group, NO content of rat lung tissues from the model group increased obviously with very significant difference (P<0.01). Compared with the model group, NO content can be decreased from each treatment group with very significant difference (P<0.05). The result was even more obvious for the high-dose Kechuanning group and it was also with very significant difference (P<0.01). Compared with the control group of Guilong Kechuanning group, NO content from the high-dose Kechuanning group was significantly lower than that of control group with significant differences (P<0.05).3. The influence of Kechuanning on ET-1 content of lung tissues from the rats with bronchial asthmaCompared with the normal group, ET-1 content of rat lung tissues from the model group increased obviously with very significant difference (P <0.01). Compared with the model group, ET-1 content can be decreased from each treatment group with very significant difference (P<0.05). The results were even more obvious for the high-dose Kechuanning group and it was also with very significant difference (P<0.01). Compared with the control group of Guilong Kechuanning group,ET-1 content from the high-dose Kechuanning group was significantly lower than that of control group with significant differences (P<0.05).Part III: The influence of Kechuanning on the nuclear factor-kB (NF-kB) of rats with bronchial asthma1. The comparison of EOS infiltration of airway wall of rats with bronchial asthma with Kechuanning.Compared with the normal group, the EOS count in airway wall of rats from the model group increased significantly(P<0.01),compared with model group,the EOS count of airway wall in the rats from the treatment group decreased significantly(P<0.05),the improvements from the high and low dose Kechuanning groups were more significant(P<0.01) ,better than control group(P<0.01).2. The influence of Kechuanning on the NF-kB expression of lung tissue of rats with bronchial asthmaLess positive expression of NF-kB cell can be seen in the lung tissue and airway wall of rats from normal group ,the cell proportion of karyon NF-kB expression in the lung tissue and airway wall of rats from model group were significantly higher than that of the normal group, which were manifested by the unsymmetric brown yellow particulates(P<0.01), however, in the rats from the treatment group, the proportion of positive cell of karyon in the lung tissue and airway wall were significantly lower than that of the model group(P<0.05), the improvements from the high and low dose Kechuanning groups were more significant(.P<0.01), which were better than that of Guilong Kechuanning group(P<0.01).Part IV: The influence of Kechuanning on the eosinophilic granulocyte apoptosis and Bcl-2 and Bax expression of the relative gene from the rats with asthma1. The influence of Kechuanning on EOS apoptosis index of lung tissue from the rats with bronchial asthma. There was little EOS infiltration in lung tissue and airway wall of the rats from normal group. Compared with the normal group, the EOS count in airway wall of rats from the model group increased significantly(P<0.01);compared with the model group , the EOS count in airway wall of rats from the treatment group decreased significantly(P<0.05), the improvements from the high and low dose Kechuanning groups were more significant(P<0.01),which were better than control group (P<0.01). There was little EOS apoptosis in lung tissue and airway wall of rats from the normal group. Compared with the model group, the EOS apoptosis index in airway wall of rats from the treatment group increased significantly (P<0.05), the improvements from the high dose Kechuanning group were more significant (P<0.01),which were better than control group (P<0.01).2. The influence of Kechuanning on Bcl-2 proteins expression of the lung tissue of rats with the bronchial asthmaLess Bcl-2 positive expression cells can be seen in the lung tissue and airway wall of rats from normal group, the proportion of cell that expressing Bcl-2 in kytoplasm in the lung tissue and airway wall of rats from the model group were significantly higher than that of the normal group ,which were manifested by the unsymmetric brown yellow particulate(sP<0.01), however, in the rats from the treatment group, the proportion of positive kytoplasm cell in the lung tissue and airway wall were significantly lower than that of the model group(P<0.05), the improvements from the high dose Kechuanning groups were more significant(P<0.01),which were better than that of Guilong Kechuanning group(P<0.01).3. The influence of Kechuanning on Bax proteins expression of the lung tissue of rats with the bronchial asthma.More Bcl-2 positive expression cells can be seen in the lung tissue and airway wall of rats from normal group, which were manifested by the unsymmetric brown yellow particulates .The proportion of cell that expressing Bcl-2 in kytoplasm.In the lung tissue and airway wall of rats from model group were significantly lower than that of the normal group, which were manifested by the shallow brown yellow particulate(sP<0.01), however, in the rats from the treatment group, the proportion of positive kytoplasm cell in the lung tissue and airway wall were significantly lower than that of the model group(P<0.05), the improvements from the high dose Kechuanning groups were more significant(P<0.01),which were better than control group (P<0.01).Part V: The influence of Kechuanning on IL-5 mRNA expression of rats with the bronchial asthmaCompared with the normal group, IL-5 mRNA expression of lung tissue in asthmatic model group increased significantly; compared with the model group, IL-5 mRNA expression of lung tissue decreased significantly from each medication group(P<0.05),which was more significant for the high dose Kechuanning group(P<0.01), the level of IL-5 mRNA expression from the high dose Kechuanning group were much lower than control group (P<0.01).Conclusion1. Kechuanning could effectively improve the pathological changes of lung tissue in bronchial asthma,reduce EOS infiltration in lung tissue, decrease the tissue congestion and edema, reduce the thickness of the bronchial wall and smooth muscle, effectively reduce asthmatic airway inflammation and airway remodeling.2. Kechuanning could significantly reduce the IL-4 content in the lung tissue of rats with bronchial asthma, increase IFN-γcontent and the ratio of IFN-γ? I L-4, adjust effectively the asthmatic cytokines network system, improve asthmatic airway inflammation.3. Kechuanning could significantly reduce NO and ET-1 content in lung tissue of rats with bronchial asthma, reduces the secretion of inflammatory mediators in asthma, improve airway inflammation in asthma and airway remodeling.4. Kechuanning could significantly reduce NF-kB expression of lung tissue of rats with bronchial asthma and inhibit EOS apoptosis and improve airway inflammation in asthma through adjusting the signal transmission route.5. Kechuanning could significantly reduce bcl-2 protein expression and increase bax protein expression of lung tissue of rats with bronchial asthma and inhibit EOS apoptosis and effectively improve airway inflammation in asthma through adjusting the expression of apoptosis-related genes.6. Kechuanning could significantly reduce the IL-5 mRNA expression of lung tissue of rats with bronchial asthma, reduce the content of related cytokines by transcription level, reduce EOS infiltration in the lungs, inhibit EOS apoptosis and effectively improve airway inflammation in asthma.