Research On Mechanisms Of Lifei Tang Treating COPD Rat Model By Regulating NF-KB

Objective:To study the therapy effect of Lifei Tang on the oxidative-stress COPD rat model from two levels, one includes physiological status, lung function, lung tissue inflammatory manifestations, another includes the expression level of NF-KB, IkBa. In order to confirm the therapy effect of Lifei Tang on oxidative-stress COPD rat model. And then elucidate the regulation of NF-KB/p65, IkBa expression may play the key role in the therapy effect of Lifei Tang by controlling airway inflammation.Methods:60 rats were randomly divided into six groups. (normal group, model group, Dex group, Lifei tang high-dose, medium-dose and low-dose group), and we fed them for a week, sensitized them by 200μg lipopolysaccharide for intratracheal instillation (on day 1 and day 15,2 times per day), ozone exposion (once a week), and passive smoke (5 cigarettes each time, twice a day in the am and pm for 0.5 hours,12 weeks), and we got the COPD rat model. Then the rats were given intragastric administration for 28 days.Lifei tang groups were given Lifei tang in concentration of 1.825g/kg,3.65g/kg and 7.3g/kg from low dose to high dose. Dex group were given 0.81mg/kg dexamethasone solution. The other group was given the same amount of 0.9% saline. Then we observed generally condition of the rats, measured their pulmonary function (FVC, FEV1, FEV1/FVC%), and collected the lung tissue for HE staining analysis for assessing lung injury in rats, taken right lung leaf for PCR, Western Blotting detection NF-KB/p65 and IkBa mRNA, protein expression, immunohistochemistry detecting NF-KB/p65 expression in COPD oxidative-stress rat lung tissue.Results:1.Pulmonary function:In terms of FVC, All TCM groups were higher and statistically significant (p<0.05) compared with the model group. Meanwhile, Dex group had the same influence on FVC with TCM low-dose group and high-dose group, reaching agreement(p>0.05); In terms of FEV1, FEV1/FVC%, three TCM groups had lower FEV1, FEV1/FVC% level compared with model group(p<0.01), but the dose-correlation in three groups was not clear.2.HE staining evaluation:normal rats airway epithelium were complete, cilia arranged in neat rows, and partial airway inflammatory cell infiltration was visible, mainly lymphocytes, smooth muscle was intact with no fibrosis, alveolar tissue is uniform with less inflammatory cell infiltration. COPD model group rats had small bronchial stenosis, thickened luminal epithelium, and the lumen was small, in the terminal portion may even atrophy. We also could see that bronchial epithelium was partially detached and thickened, and normal cilia structure was changed that showed adhesions, lodging of cilia; hyperplasia of epithelium cell, goblet cells and glands was significant, a large number of inflammatory cells (lymph, neutrophils, macrophages) infiltrate mucous, all levels of the bronchial wall and interstitial lung. And other manifestation like hyperplasia, fibrosis of pulmonary arterial smooth muscle, bronchial smooth muscle; partially ulcerated, transparent alveolar wall which results in alveoli reduction and alveolar integration. Dex group, three TCM groups alleviate the situation like model group,so that level of bronchial lumen thickening, mucus plug, fibrosis, inflammatory infiltration decreased, and the number of alveolar damage reduced. But these change in low-dose TCM group is not obvious compared with COPD model group.3. The fluorescence quantitative RT-PCR:P65 mRNA in each group showed no significant difference (p>0.05); IKBamRNA each group showed no significant difference (p> 0.05).4. Western blot:Compared with the normal group, the P65 protein average relative parameters generally increased in all groups that the parameters were significantly statistical different from normal group(p<0.01) except the high-dose group. Model group has the highest average relative parameters, and the parameters of normal group, high-dose group and Dex group showed significant difference(p<0.01), compared with the model group. High-dose TCM group did not show a significant difference with the Dex group(p> 0.05). IKBa protein average relative parameter in each group were significantly decreased (p<0.01) compared with normal group, and the model group decreased the most. Compared with the model group, the high-dose TCM group and the Dex group showed significant difference (p <0.05), but the middle-dose and the low-dose group showed no significant difference (p> 0.05); high-dose group and Dex group also showed no statistical difference (p> 0.05).5. Immunohistochemistry staining:NF-KB/p65 protein positive strength showed in the following order:model group, low-dose group, mid-dose group, high-dose group, dex group and normal group. In normal group, the lung tissue of rats were intact, cilia arranged in neat rows, alveolar and bronchial morphological changes were obscure with almost no protein expression. In dex group, the visible signs of p65 expression scattered in the lung tissue, bronchi, alveolar. Positive expression of p65 are more obvious in three TCM group, and dosage of Lifei tang and intensity of expression was negatively correlated, and the P65 expression in high-dose group is the least in three groups and more concentrated in the peripheral part of the alveoli. In mid-dose and low-dose group the expression of p65 were more diffuse and positive, accompanied by cell arrangement disorder, falling into pieces. Low-dose group expression intensity only second to the model group. IKBa expression level in each group showed the opposite trend with P65. IKBa expression is most abundant in three groups:normal group>Dex group>high-dose TCM group, There is slightly difference in the latter two groups. Like inflammatory cell infiltration in lung tissue, centrilobular emphysema pathology, In dex group the inflammatory infiltration mainly gathered near the bronchial smooth muscle. Mid-dose and low-dose group showed similar IKBa protein staining condition with model group. The proportion and the color depth of brown area were poor in full-field vision and not obvious. But inflammatory infiltration, the degree of airway structural damage were higher than the other three groups, airway collapse and fusion were partially visible.The average integral optical density(IOD) of p65 level from low to high were as follows: Normal group<dex group<high-dose group<mid-dose group<low-dose group<model group. This coincided with the results of immunohistochemistry microscopic pictures. Except dex group, each group has statistically significant difference(p<0.05) with the normal group. Compared with model group, each group were significantly different(p<0.01) except low-dose group. Compared with dex group, high-dose group was slightly higher but there was no statistically significant difference (p> 0.05), while mid-dose group, low-dose group and model group had significant differences(p<0.01). IKBa manifestation variance is more obvious among groups, the normal group had the highest level of IKBa expression, followed by dex group, two groups were significantly different with the other groups(p<0.01). Compared with the model group, IKBa expression level in high-dose group was higher and had a significant difference (p<0.01). Although low-dose and mid-dose group differed from model group, but there was no significant difference between the three mutual.Conclusions:1. Lifei tang could have beneficial effects on the symptoms, lung function and HE staining, so it has therapeutic effect on COPD rats model.2. Lifei tang could inhibit NF-KB expression and the IKBa degradation,these offer the possible mechanism of for the anti-inflammatory effect of Lifei tang on COPD rats.