| Objective1. Using the animal experiments of stable bronchial asthma SD rat model for medicine, to confirm the AP-1is a key link for the pathogenesis of bronchial asthma,and to verify the efficacy of Qiweilifei Decoction and get its evidence.2. Through using the methods of molecular biology to test the upstream and downstream factors of AP-1, to improve the mechanism of bronchial asthma treatment by Qiweilifei Decoction,and to explore the details in regulating AP-1.MethodsAfter7days to feed adaptively,70SPF SD rats were randomly divided into7groups: the blank control group, the model control group,the blocker control group, the Chinese medicine low-dose group,the Chinese medicine middle-dose group, the Chinese medicine high-dose group and the western medicine group. Before the model-making of the blocker control group rats, used curcumin to precondition them for3days (100mg. kg-1. d-1,50mg/ml×0.2mlBid, intraperitoneal injection). Then, ea-ch rat except the blank control group sensitized by injecting the mixture with10%OVA/Al (OH)3+0.0023%o Pertussis toxin from Bordetella pertussis lml into five point(intraperitoneal, subcutaneous of the left/right rear toe and the left/right groin,0.2ml each point)in day1and day8to make model. After sensitization,used1%OVA+normal saline to stimulate immunoreaction by inhaled(30min Qd,7days). The blank control group used normal saline to do all above treatment.Intragastric administration began in day9(finished the last injection), lasted14days,Qd. The Chinese medicine low-dose, middle-dose and high-dose groups were given by Chinese medical extractum normal saline solution0.182g/ml,0.363g/ml and0.727g/ml (dosage:low-dose group=1/2middle-dose group, middle-dose group=1/2high-dose group)3ml. The western medicine group was given by dexamethasone normal saline solution50μg/ml3ml. And the blank control group, the model control group and the blocker control group were given by normal saline3ml.After administration,all rats were evaluated pulmonary function under anesthesia. Then taken the samples of bronchoalveolar lavage fluid,arterial blood and the lung tissue to do eosinophil cell count, serum IgE/IL-4/IL-5/IL-13content test,lung tissue sections stained by HE (to observe the morphological alternation) and immumohistochemical (to test the AP-1nucleoprotein),and lung tissue homogenate AP-1mRNA/nucleoprotein content test (by RT-PCR/Western blot).All statistical analysis was performed using SPSS17.0software package. Calculated data:mean±standard deviation(χ±s).Enumeration data:mean±standard deviation(χ±s)(normal distribution), or median±interquartile range (Md±QR)(abnormal distribution).First, used1-Sample K-S Test of non-parametric test to do normality test.Then, if the data fit normal distribution, used One-Way ANOVA to do the homogeneity test of variances, and used LSD(equal variance) or Tamhane’sT2(unequal variance) to do the multiple comparisons; if the data doesn’t fit normal distribution, transformed the data (such as logarithmic transformation) and repeat the above steps,or still doesn’t fit normal distribution,used Kruskal-Wallis to do the multiple comparisons. Regarded P<0.05as statistically difference, and P<0.01as statistically significant difference.Results1.Observation and test results of the model assessment index and the therapeutic effect index.General conditions:After the injection for model-making, rats of the model control group, the Chinese medicine low-dose, middle-dose, high-dose groups and the western medicine group developed asthma symptoms, when rats of the blank control group and the blocker control group did not.Pulmonary function results:Compared with the blank control group, the results of the model control group’s airway hyperresponsiveness (I), tissue sluggish (H), tissue damping (G) and dynamic lung compliance (Crs) were higher (P>0.05, no statistically significant). When the results of the other groups’ hyperresponsiveness (I) and tissue sluggish (H) were both higher.Among all the other groups, the airway hyperresponsiveness (I) of the blocker control group was the lowest (P>0.05, no statistically significant). The rest of results such as inspiratory capacity (IC).Central airway resistance (Rn), tissue damping (G), dynamic lung compliance (Crs) and static lung compliance (Cst) had different trends between every group (P>0.05, no statistically significant).The bronchoalveolar lavage fluid (BALF) smear can recognize some Bosinophil cells (EOS) under the microscope. Preliminary observation the number of them from high to low was the model control group,the Chinese medicine low-dose group, the Chinese medicine middle-dose group, the Chinese medicine high-dose group,the western medicine group,the blocker control group,the blank control group. Count results showed that:Compared with the blank control group,the result of the model control group’s BALF smear EOS count was significant elevated(P<0.01); the blocker control group’s was a little higher(P>0.05,no statistically significant);the Chinese medicine low-dose, middle-dose and high-dose groups’were higher (P<0.01) and showed the concentration-response relationship; and the western medicine group’s was higher too(P<0.01). Compared with the model control group, the blocker control group’s was lower (P<0.01); the Chinese medicine low-dose, middle-dose and high-dose groups’were lower too, but the low-dose group’s P>0.05(no statistically significant), when the middle-dose and high-dose groups’P<0.01; and the western medicine group’s was lower (P<0.01). Compared with the blocker control group,the Chinese medicine low-dose, middle-dose and high-dose groups’were higher (P<0.01); and the western medicine group’s was higher too, but P>0.05(no statistically significant). Compared with the western medicine group, the Chinese medicine low-dose, middle-dose and high-dose groups’were higher, but the high-dose group’s P>0.05(no statistically significant), and the low-dose, middle-dose groups’ P<0.01.Under the microscope view to observe the morphological alternation from lung tissue sections stained by HE:The blank control group had not showed any change and inflammatory cellular infiltration, when he blocker control group had a little, and the other groups all had inflammatory cellular infiltration in the lung tissue, bronchial epithelial cells irregular arrangement and desquamation, thic-kened alveoli septum and bronchiolar spasm, but the pathological damage severity were different. Results of the score by using tissue injury and inflammation pathological scoring criteria showed that:On the item of the inflammatory cellular infiltration, the score from high to low was the model control group=the Chinese medicine low-dose group> the Chinese medicine middle-dose group=the Chinese medicine high-dose group> the western medicine group> the blocker control group=the blank control group. On the item of the bronchial epithelial cells desquamation, the score from high to low was the model control group> the Chinese medicine low-dose group=the Chinese medicine middle-dose group>the Chinese medicine high-dose group=the western medicine group> the blocker control group=the blank control group. On the item of the thickened alveoli septum, the score from high to low was the model control group=the Chinese medicine low-dose group> the Chinese medicine middle-dose group> the Chinese medicine high-dose group=the western medicine group> the blocker control group=the blank control group. On the item of the bronchiolar spasm, the score from high to low was the model control group=the Chinese medicine low-dose group> the Chinese medicine middle-dose group=the Chinese medicine high-dose group> the western medicine group> the blocker control group=the blank control group. And the total points from high to low was the model control group=the Chinese medicine low-dose group> the Chinese medicine middle-dose group>the Chinese medicine high-dose group> the western medicine group> the blocker control group> the blank control group, all above P<0.01.Results of the serum IgE content test showed:Compared with the blank control group, every group’s serum IgE content increased (P<0.01), and the Chinese medicine low-dose, middle-dose and high-dose groups showed the concentration-response relationship. Compared with the model control group, the blocker control group’s was lower (P<0.01); the Chinese medicine low-dose, middle-dose and high-dose groups’were lower too,but the low-dose group’s P>0.05(no statistically significant), when the middle-dose and high-dose groups’P<0.05;and the western medicine group’s was lower (P<0.01). Compared with the blocker control group, the Chinese medicine low-dose,middle-dose and high-dose groups’ were higher(the low-dose group’s P<0.05, while the middle-dose and high-dose groups’ P<0.01); and the western medicine group’s was higher too, but P>0.05(no statistically significant). Compared with the western medicine group,the Chinese medicine low-dose, middle-dose and high-dose groups’ were higher, but the high-dose group’s P>0.05(no statistically significant), and the low-dose group’s P<0.01, the middle-dose group’s P<0.05.2. Results of AP-1mRNA expression quantities by Real-time RT-PCR.c-jun mRNA expression quantities:Compared with the blank control group,every group’s c-jun mRNA expression quantities increased, among them, the model control group and the Chinese medicine low-dose, middle-dose groups’ P<0.05, the others P>0.05(no statistically significant). Compared with the model control group, the Chinese medicine low-dose group’s was a little higher, and the other groups’ were lower,but only the blocker control group’s P<0.05, all of the rest groups’ P>0.05(no statistically significant). Compared with the blocker control group, the Chinese medicine low-dose, middle-dose, high-dose groups and the western medicine group’s was higher, when the the western medicine group and the middle-dose group’s P<0.05, the others P>0.05(no statistically significant). Compared with the western medicine group, the Chinese medicine low-dose, middle-dose and high-dose groups’were higher,but P>0.05(no statistically significant).c-fos mRNA expression quantities:Compared with the blank control group, every group’s c-fos mRNA expression quantities increase.Compared with the model control group, every group’s was lower. Compared with the blocker control group, the Chinese medicine low-dose, middle-dose, high-dose groups and the western medicine group’s was lower.Compared with the western medicine group, the Chinese medicine low-dose, middle-dose and high-dose groups’ were higher,but all above P>0.05(no statistically significant).3. Results of AP-1component content and activity test.①Result of observing the positive expression of AP-1. Under the microscope view, the intensity of positive expression from high to low was the model control group, the Chinese medicine low-dose group, the Chinese medicine middle-dose group, the Chinese medicine high-dose group,the western medicine group, the blocker control group, the blank control group. The positive expression mainly located in bronchial epithelial cells, sometimes in alveolar interval.Compared with the blank control group, every group’s c-Jun nucleoprotein integral optical density(IOD) increased and P<0.01,and the Chinese medicine low-dose, middle-dose and high-dose groups showed the concentration-response relationship. Compared with the model control group, the blocker control group’s was lower (P<0.01);the Chinese medicine low-dose, middle-dose and high-dose groups’were lower too, and the low-dose groups’ P<0.05, the middle-dose and high-dose groups’P<0.01; the western medicine group’s was lower (P<0.01).Compared with the blocker control group, the Chinese medicine low-dose, middle-dose, high-dose groups and the western medicine group’s was higher(P<0.01).Compared with the western medicine group,the Chinese medicine low-dose, middle-dose and high-dose groups’were higher,but P>0.05(no statistically significant).Compared with the blank control group, every group’s c-Fos nucleoprotein integral optical density (IOD) increased and P<0.01,and the Chinese medicine low-dose, middle-dose and high-dose groups showed the concentration-response relationship. Compared with the model control group, every group’s was lower; among them, the blocker control group’s P>0.05(no statistically significant), the Chinese medicine low-dose groups’P<0.05, all of the rest P<0.01. Compared with the blocker control group,the Chinese medicine low-dose,middle-dose, high-dose groups and the western medicine group’s was lower, the western medicine group’s P<0.05, the others P>0.05(no statistically significant). Compared with the western medicine group, the Chinese medicine low-dose, middle-dose and high-dose groups’were higher, but P>0.05(no statistically significant).②Result of AP-1nucleoprotein (c-Jun and c-Fos) expression quantities by Western blot:Preliminary observation:All of the β-actin expressed almost the same, while c-Jun nucleoprotein expression quantities from high to low was the model control group,the Chinese medicine low-dose group, middle-dose, high-dose group, the western medicine group, the blocker control group, the blank control group, and c-Fos nucleoprotein expression quantities from high to low was the model control group, the blank control group, the Chinese medicine low-dose group, middle-dose, high-dose group, the western medicine group, the blocker control group.Compared with the blank control group, every group’s c-Jun nucleoprotein mean gray value increased and P<0.01,and the Chinese medicine low-dose, middle-dose and high-dose groups showed the concentration-response relationship. Compared with the model control group, the blocker control group’s was lower(P<0.01); the Chinese medicine low-dose, middle-dose and high-dose groups’were lower too, and the low-dose and middle-dose groups’ P>0.05(no statistically significant), the high-dose group’s P<0.01; the western medicine group’s was lower (P<0.01). Compar-ed with the blocker control group, the Chinese medicine low-dose, middle-dose, high-dose groups and the western medicine group’s was higher (P<0.01). Compared with the western medicine group,the Chinese medicine low-dose, middle-dose and high-dose groups’were higher,but the high-dose group’s P>0.05(no statistically significant), whlie the low-dose and middle-dose groups’P<0.01.Compared with the blank control group, every group’s c-Fos nucleoprotein mean gray value increased and P<0.01,and the Chinese medicine low-dose, middle-dose and high-dose groups showed the concentration-response relationship. Compared with the model control group,the blocker control group,the Chinese medicine low-dose, middle-dose, high-dose groups and the western medicine group’s was lower(P<0.01).Compared with the blocker control group, the Chinese medicine low-dose, middle-dose, high-dose groups and the western medicine group’s was higher(P<0.01).Compared with the western medicine group, the Chinese medicine low-dose, middle-dose and high-dose groups’were higher, but the high-dose group’s P>0.05(no statistically significant), whlie the low-dose and middle-dose groups’ P<0.01.4.Results of serum IL-4, IL-5, IL-13content test.Compared with the blank control group,every group’s serum IL-4content increased,and the Chinese medicine low-dose, middle-dose and high-dose groups showed the concentration-response relationship, among them, the blocker control group’s P>0.05(no statistically significant) and all of the rest P<0.01. Compared with the model control group, every group’s was lower (P<0.01). Compared with the blocker control group, the Chinese medicine low-dose, middle-dose, high-dose groups and the western medicine group’s was higher (P<0.01).Compared with the western medicine group, the Chinese medicine low-dose, middle-dose and high-dose groups’ were higher (P<0.01).Compared with the blank control group, every group’s serum IL-5content increased,and the Chinese medicine low-dose, middle-dose and high-dose groups showed the concentration-response relationship,but only the Chinese medicine low-dose and middle-dose groups’P<0.01, the others P>0.05(no statistically significant). Compared with the model control group, every group’s was lower (P <0.01).Compared with the blocker control group,the Chinese medicine low-dose, middle-dose, high-dose groups and the western medicine group’s was higher, among them, the high-dose group and the western medicine group’s P>0.05(no statistically significant), while the low-dose and middle-dose groups’P <0.01. Compared with the western medicine group, the Chinese medicine low-dose, middle-dose and high-dose groups’were higher,but the high-dose groups’ P>0.05(no statistically significant), and the low-dose and middle-dose groups’ P<0.01.Compared with the blank control group,every group’s serum IL-13content increased, and the Chinese medicine low-dose, middle-dose and high-dose groups showed the concentration-response relationship, among them, the blocker control group and the Chinese medicine high-dose groups’P<0.05, while the others P<0.01. Compared with the model control group,every group’s was lower,and the low-dose group’s P<0.05, the others P<0.01.Compared with the blocker control group, the Chinese medicine low-dose, middle-dose, high-dose groups and the western medicine group’s was higher,but the high-dose group’s P>0.05(no statistically significant), and the low-dose and middle-dose groups’P<0.01,the western medicine group’s P<0.05. Compared with the western medicine group,the Chinese medicine low-dose and middle-dose groups’were higher but P>0.05(no statistically significant), while the high-dose group’s was a little higher (P<0.01).Conclusion1.The bronchial asthma rat model which was established in this experimient had been successful, and AP-1is a key link for the pathogenesis of bronchial asthma.①The bronchial asthma SD rat model had been successfully duplicated.The method of sensitized by injecting the mixture with10%OVA/Al (OH)3+0.0023%o Pertussis toxin from Bordetella pertussis into five point (intraperitoneal, subcut-aneous of the left/right rear toe and the left/right groin) to make model,and uses1%OVA+normal saline to stimulate immunoreaction by inhaled can successfully duplicat the bronchial asthma rat model.②Curcumin had successful blocked the AP-1expression in the lung tissue of bronchial asthma rat.Through various experimental methods to test the content of AP-1gene expression product (mRNA and nucleoprotein) and downstream factors of Th2cells (IL-4, IL-5and IL-13),we found that the blocker control group’s content of c-jun mRNA and nucleoprotein, IL-4,IL-5and IL-13were close to the blank control group but far below the model control group,while the content of c-fos mRNA and nucleoprotein were close to the model control group but far above the blank control group, thus this blocker group had been blocked successful in general.③AP-1is a key link for the pathogenesis of bronchial asthma.The blocker control group rats did not present with typical symptoms and physical signs of asthma. Results of pulmonary function,BLAF smear EOS count, lung tissue sections stained by HE and serum IgE did not have positive performance of typical asthma. We can consider that this blocker group failed to copy the bronchus asthma model. All above suggest that AP-1is a key link for the pathogenesis of bronchial asthma, and to block it can prevent asthma pathogenesis to some extent.2. By regulating the expression and activity of AP-1, and affecting its expression and activity of downstream factors (IL-4、IL-5and IL-13), Qiweilifei Decoction is effective in the treatment of asthma.①Qiweilifei Decoction is effective in the treatment of asthma.Compare the results of BLAF smear EOS count,lung tissue sections stained by HE and serum IgE had positive performance of typical asthma, the Qiweilifei Decoction low-dose, middle-dose and high-dose groups rats were lower than the model control group but a little higher than the western medicine group. Among them, the high-dose group can be considered equal to the western medicine group,while the low-dose group can be considered close to the model control group. All above indicate that Qiweilifei Decoction is effective in the treatment of asthma, and high-dose gavage can be considered as Dexamethasone, whlie low-dose gavage is ineffective.②The expression and activity of AP-1were regulated by Qiweilifei Decoction.Through various experimental methods to test the content of AP-1gene expression product (mRNA and nucleoprotein), the Qiweilifei Decoction low-dose, middle-dose and high-dose groups showed the concentration-response relationship. The content of AP-1mRNA:Compare with the model control group and the western medicine group, three Chinese medicine groups’differences had no statistical significance. The content of AP-1nucleoprotein:The high-dose group can be considered equal to the western medicine group, while the low-dose group can be considered close to the model control group. The results showed that the effect of the AP-1mRNA regulation by Qiweilifei Decoction is not yet clear from this experiment. However, Qiweilifei Decoction can reduce the content of AP-1nucleoprotein and reduce the synthesis of AP-1.③The expression of downstream factors of AP-1(IL-4, IL-5and IL-13) were affected by Qiweilifei Decoction.The Qiweilifei Decoction low-dose, middle-dose and high-dose groups’content of Th2downstream factors(IL-4, IL-5and IL-13)showed the concentration-response relationship. The low-dose group can be considered equal to the model control group while the high-dose group can be considered close to the western medicine group. We preliminary presumption that Qiweilifei Decoction possibly be block the process of AP-1mRNA served as a template to compound a proteins, then reduce content of AP-1nucleoprotein (c-Jun and c-Fos) in the lung tissue of rats. At last, AP-1itself and the Th2downstream factors (IL-4, IL-5and IL-13) also be reduced. |
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