| ObjectiveYupingfeng powder is a traditional classical prescription of traditional chinese medicine. It has the effect of increasing organism immunity and supporting healthy energy, mainly used to treat vulnerable to wind-cold, debility. Modern medical study found that yupingfeng powder can improve immunity, often used to prevent and treat respiratory tract infection, allergic rhinitis, asthma, and internal and external pediatric diseases in clinical or used as the care medicine for the old. On the basis of previous research, the research project combines the traditional Chinese animal model with modern immunology, further to research the effect of yupingfeng powder on regulating lung-qi deficiency syndrome and its specific mechanism from animal、cell and immune cell factor three aspects, expecting to provide some scientific basis to the treatment of lung-qi deficiency syndrome and the drug research of yupingfeng powder. Methods1. Refering to relevant research literature of LQD animal model, adopting the sulfur dioxide and cold stimulation which simple and short building time to build the LQD rats animal model.2. Experimental animals were randomly divided into four groups: blank group, model group, YPFP group, ren shen bei qi pian+levamisole hydrochloride tablets group, 8 per group. Model group, YPFP group, ren shen bei qi pian+levamisole hydrochloride tablets group were given a gavage of saline, YPFP and ren shen bei qi pian+levamisole hydrochloride tablets respectively with the dose of 1ml / 100 g during the period of modeling. Recording the weight, respiratory rate of each rat before and after modeling. Collecting blood from artery and separate serum, detect the content of serum Ig E by Elisa. Separating the bronchus and lung tissue and make pathological section, HE stain and observe the change of lung and bronchus tissue.3. Using the Sulfur dioxide and cold comprehensive stimulation method to establish the LQD rats model.Separating the spleen lymphocytes, peritoneal macrophages, spleen NK cells respectively, trypan blue staining was used to count the live cell>95%, adding cells to 96-well plates. Cells were divided into three group: the blank group, model group, YPFP group, each group had 5 duplications. YPFP group set seven drug concentration gradient:0.52 mg/ml, 1.04 mg/ml, 2.08 mg/ml, 4.16 mg/ml, 8.32 mg/ml, 16.64 mg/ml, 33.28mg/ml. MTT method was used to calculate the cell vitality, NO2 ˉreduction method was used to determine NO content in cell cultures, LDH method were used to determine NK cell killing activity.Study respectively:(1) The effect of YPFP on spleen lymphocyte activity of LQD rats;(2) The effect of YPFP on spleen lymphocyte proliferation activity of LQD rats induced by Con A;(3) The effect of YPFP on abdominal macrophage activity of LQD rats;⑷ The effect of YPFP on abdominal macrophage phagocytosis function of LQD rats;⑸ The effect of YPFP on abdominal macrophage secrete NO induced by LPS of LQD rats;⑹ The effect of YPFP on spleen NK cell killing activity of LQD rats. Results1.After modeling, the blank group rats were live, active, fur smooth、glossy and in good spirits; model group rats were depression, arch back, hair messy, lacklustre, depilation and appear the symptoms of cough, wheeze, activity amount decreased significantly.The YPFP group and ren shen bei qi pian+levamisole hydrochloride tablets group were active and in good spirit,part of the rats have a cough.2. Compared with blank group, the respiratory rate and serum Ig E content of model group rats increased significantly, the weight sharply decreased, the difference was statistically significant(P<0.05).Compared with model group, the respiratory rate 、 serum Ig E content of YPFP group and ren shen bei qi pian+levamisole hydrochloride tablets group decreased significantly, the weight sharply increased, the difference was statistically significant(P<0.05). Histopathological examination revealed: compared with blank group, model group rats alveolar interval obviously thickening, lung moderate hyperemia, a large number of inflammatory cells infiltration in alveolar walls; Bronchial mucosa columnar epithelium scale, focal loss, a large amount of inflammatory cells in infiltration bronchial wall. The alveolar、bronchus injury and inflammation levels of YPFP group and ren shen bei qi pian+levamisole hydrochloride tablets group decreased significantly, a small amount of inflammatory cells infiltration.3. Compared with the blank group, the spleen lymphocyte activity of model group decreased significantly(P<0.05).Compared with model group, the lymphocyte activity of YPFP group increased at different degrees. When YPFP concentration is 0.52 mg/ml, the lymphocyte activity of YPFP group increased significantly(P<0.05). With the increase of YPFP concentration, the lymphocyte activity gradually strengthened, presenting a dose effect relationship.4. Compared with the blank group, the proliferation index of model group has no significant change, the difference was no statistically significant(P>0.05). When YPFP concentration is 2.08 mg/ml, the proliferation index increased significantly compared with the model group, the difference was statistically significant(P<0.05).With the increase of YPFP concentration, the proliferation index increased gradually, presenting a dose effect relationship.5. Compared with the blank group, the macrophages activity of model group decreased significantly(P<0.05).Compared with model group, when YPFP concentration is 2.08 mg/ml, the macrophage activity of culture 24 h increased significantly(P<0.05); When YPFP concentration is 0.52 mg/ml, the macrophage activity of culture 48 h 、 72 h increased significantly(P<0.05).With the YPFP concentration increases, the macrophage cell activity increased, presenting a dose effect relationship.6. Compared with the blank group, the macrophage phagocytosis of model group rats decreased significantly(P<0.05).Compared with model group, When YPFP concentration is greater than 2.08 mg/ml, the macrophage phagocytosis of culture 24 h enhanced significantly(P<0.05); When YPFP concentration is greater than 4.16 mg/ml, the macrophage phagocytosis of culture 48 h,72h enhanced significantly(P<0.05). With the YPFP concentration increases, macrophage phagocytosis increased, presenting a dose effect relationship.7. Compared with the blank group, the NO content in cell medium of model group has no significant change(P>0.05).Compared with model group, the NO content of LPS group increased significantly(P<0.05); Compared with LPS group, When YPFP concentration greater than 2.08 mg/ml, the NO content decreased significantly(P<0.05). With the increase of YPFP concentration, the inhibition gradually enhance, presenting a dose effect relationship.8. Compared with the blank group, the NK cells killing activity of model group decreased significantly, the difference was statistically significant(P<0.05). The NK cells killing activity of YPFP group increased at different degrees. When YPFP concentration is 2.08 mg/ml, the NK cells killing activity increased significantly(P<0.05).When YPFP concentration is greater than 16.64 mg/ml, the promoting fuction gradually reduced. Conclusions1. Using the sulfur dioxide and cold comprehensive stimulation method can copy LQD rat model successfully.2. YPFP can effectively improve the lungs, bronchus physiological state of LQD rats, reduce the inflammation and damage degree.3. YPFP can improve LQD rat spleen lymphocyte activity,promote lymphocyte proliferate induced by Con A.4. YPFP can improve the activity of macrophages, enhance phagocytosis, inhibit macrophage secrete NO induced by LPS.At the same time, YPFP can improve killing activity of NK cells. |
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