| Purposes:To explore the pharmacological mechanism of Tong Bi Zhi Qiu(TBZQ)Formula inhibiting allergic rhinitis, and predict the potential drug targets and new pharmacological effects.Methods:1. Oval albumin-induced allergic rhinitis mice model was established. The mice were randomly divided into six groups: control group, allergic rhinitis group, cetirizine hydrochloride group, low dose of TBZQ formula(L-TBZQ)group, middle dose of TBZQ formula(M-TBZQ) group and high dose of TBZQ formula(H-TBZQ) group. The behavior was recorded and the changes of histopathology in nasal mucosa were detected. The content of immune molecules Ig E, IFN-γ, IL-2, IL-4, IL-10 and ICAM-1 in mice serum and bronchoalveolar lavage fluid was determined by ELISA. The expression of inflammation related genes Ig E, IFN-γ, IL-2, IL-4, IL-10 and ICAM-1 in mice nasal mucosa and lung tissue was determined by RT-PCR. The expression of inflammation related proteins Ig E, IFN-γ, IL-2, IL-4, IL-10 and ICAM-1 in mice nasal mucosa and lung tissue was determined by western blot.2. Using the methods of network pharmacology and bioinformatics, thenetworks of “Ingredients-Targets-Diseases” for single herb and the whole formula in TBZQ formula were built and analyzed.Results:1. Behavior changes Compared with control group, scores of sneeze, nasal itch and nasal secretions were significantly higher in model group(p < 0.05). Compared with model group, the treatment groups significantly improved allergic rhinitis spray behavior, scratching nose behavior and rhinorrhea behavior(p < 0.05).In sneeze change, compared with positive drug group, the improvement was worse in L-TBZQ group(p < 0.05), there was no significant difference in M-TBZQ group(p > 0.05), and the improvement was better in H-TBZQ group(p < 0.05). In nasal itch change, compared with positive drug group, the improvement of L-TBZQ group and M-TBZQ group were worse(p < 0.05),and there was no significant difference in H-TBZQ group(p > 0.05). In nasal secretions change, compared with positive drug group, the improvement of L-TBZQ group and M-TBZQ group were worse(p < 0.05), and the improvement was better in H-TBZQ group(p < 0.05).2. Histopathology In control group mice, pseudostratified ciliated columnar epithelium structure completed in nasal mucosa tissue, neat cilia, smooth and no fault, no inflammatory cell infiltration. In oval albumin-induced allergic rhinitis model mice, severe nasal mucosa thicken, mucosal surface cilia adhesion or falling off, and mucosa lamina propria structure disordered, the gland and vessels expansion and hyperplasia, severe edema, stroma of eosinophilic cells and other inflammatory cells infiltration. In positive drug group mice, a small amount of inflammatory cell infiltration. In L-TBZQ group mice, nasal mucosa moderately thicken, lamina propria structure disordered, cilia fell off,the gland and vessels mild expansion and mild edema, a small amount ofinflammatory cells infiltration, occasional eosinophilic cells. In M-TBZQ group, nasal mucosa mild thicken, cilia fell off, the gland and blood vessels with occasional expansion, mild edema, a small amount of inflammatory cells infiltration, occasional eosinophilic cells. In H-TBZQ group mice, mild nasal mucosa hyperemia, accidentally inflammatory cells infiltration, no obvious hyperplasia, lodging cilia changes.3. ELISA Compared with control group, serum Ig E levels significantly increased in allergic rhinitis model group(p < 0.001). Compared with model group, Ig E levels in the treatment groups decreased(p < 0.001). Compared with positive drug group, the descending degree of Ig E was obviously less in L-TBZQ group(p < 0.001), but there were similar effects in M-TBZQ group and H-TBZQ group(p > 0.05). Compared with control group, serum IFN-γand IL-2 significantly decreased, and IL-4 and IL-10 increased in allergic rhinitis model group(p < 0.001). Compared with model group, serum contents of Th1 and Th2 cytokines all restored in treatment groups. Among them, IFN-γcontent increased significantly(p < 0.001) in all treatment groups except L-TBZQ group. IL-2 levels rise significantly only in positive drug group and H-TBZQ group(p < 0.001 and p < 0.05), and there were no significant differences in the other groups(p > 0.05). IL-4 content changed significantly,decreased obviously in each group(p < 0.001). IL-10 levels also declined, but the effect was weaker in L-TBZQ group(p < 0.05) than other groups(p <0.001). Compared with control group, serum ICAM-1 content significantly increased in allergic rhinitis model group,(p < 0.001). Compared with model group, serum ICAM-1 content decreased in the treatment group(p < 0.05).Compared with positive drug group, the effect was slightly weaker in L-TBZQ group, a little stronger in M-TBZQ group(p > 0.05), and significantly stronger in H-TBZQ group(p < 0.001).The change of cytokines in bronchoalveolar lavage fluid was weaker than in serum. Compared with control group, IFN-γand IL-2 significantly reduced,and IL-4 and IL-10 rise in model group(p < 0.001). Compared with model group, the contents of Th1 / Th2 cytokines in bronchoalveolar lavage fluid restored in the treatment groups, but the callback was significantly less than in serum. Among them, the content of IFN-γ changed significantly, and increased in all treatment groups(p < 0.001). IL-2 levels significantly increased only in positive drug group(p < 0.05). IL-4 content changed significantly only in H-TBZQ group(p < 0.05). IL-10 content decreased in each treatment groups,but there were no statistical difference(p > 0.05).4. RT-PCR Compared with control group, the gene expression level of significantly Ig E increased significantly in model group(p < 0.001). Compared with model group, Ig E gene expression all decreased in treatment groups. Among them, the descending degree was largest in M-TBZQ group and H-TBZQ group(p < 0.001). Compared with control group, IFN-γand IL-2 gene expression significantly reduced, and IL-4 and IL-10 significant rise(p <0.01). Compared with model group, Th1 and Th2 cytokines gene expression restored in treatment groups. IFN-γ gene expression level increased, most obviously in H-TBZQ group(p < 0.001), next in positive drug group(p <0.05), and no statistical difference in L-TBZQ group and M-TBZQ group(p > 0.05). IL-2 gene expression level restored in all treatment groups except L-TBZQ group, but only statistically different in H-TBZQ group(p < 0.01).IL-4 gene expression level showed the different degree of fall back, positive drug group decreased most obviously(p < 0.001). The level of IL-10 also declined in treatment groups, but only statistically different in the positive drug group and H-TBZQ group(p < 0.05). Compared with control group,ICAM-1 gene expression significantly increased in model group(p < 0.05).Compared with model group, the gene expression of ICAM-1 all declined in treatment groups. Among them, the descending degree was largest in M-TBZQ group, similar in other groups and no statistically significant changes(p > 0.05).Compared with control group, Ig E gene expression in lung tissue increased significantly in model group(p < 0.001). Compared with model group, Ig E gene expression had different degrees of decline in treatment groups. Among them, there were the most obvious difference in L-TBZQ group and H-TBZQ group(p < 0.01). Compared with control group, IFN-γand IL-2gene expression significantly reduced, IL-4 and IL-10 significant rise in model group(p < 0.05). Compared with model group, Th1 and Th2 cytokines gene expression have different degrees of callback in treatment groups.IFN-γincreased, but only statistically significant in H-TBZQ group(p < 0.05).IL-2 gene expression restored in all treatment groups, the strongest effect in L_TBZQ group(p < 0.01), next in positive drug group(p < 0.05), and the weakest effect in M-TBZQ group and H-TBZQ group(p > 0.05). IL-4 gene expression also showed the different degree of fall back, but only there was statistically significant in positive drug group(p < 0.05). IL-10 also declined,but only there were statistically difference in the positive drug group and M-TBZQ group(p < 0.05). Compared with control group, ICAM-1 gene expression in lung tissue increased significantly in model group(p < 0.01).Compared with model group, ICAM-1 gene expression have different degrees of decline in the treatment groups. Among them, the descending degree were largest in M-TBZQ group and H-TBZQ group(p < 0.01), next the positive drug group(p < 0.05), smallest in L-TBZQ group(p > 0.05).5. Western blot Compared with control group, the protein expression of Ig E, IL-4 and IL-10 in nasal mucosa significantly increased(p < 0.01), IFN-γ and IL-2significantly reduced(p < 0.01). Compared with model group, the proteins restored in treatment groups except ICAM-1. Among them, the Ig E protein expression decreased, but only statistical different in H-TBZQ group(p <0.05). IFN-γprotein expression increased, statistically significant in M-TBZQ group and H-TBZQ group(p < 0.05). IL-2 protein expression only statistically increased in H-TBZQ group(p < 0.05). IL-4 had different degree of fall back, most obviously in positive drug group and H-TBZQ group(p <0.05). IL-10 protein expression also decreased, most obviously in H-TBZQ group(p < 0.001), next positive drug group and M-TBZQ group(p < 0.01),and most slightly in L-TBZQ group and no statistically difference(p>0.05).Compared with control group, protein expression of Ig E, IL-4, IL-10, and ICAM-1 in the lung tissue significantly increased(p < 0.01), IFN-γ, IL-2significantly reduced(p < 0.01). Compared with model group, the proteins in the lung tissue presented different degrees of callback in treatment groups.Ig E declined, and the effects of M-TBZQ group and H-TBZQ group were strongest and statistically significant(p < 0.05). IFN-γprotein expression only statistically increased in H-TBZQ group(p < 0.05). IL-2 protein expression only statistically increased in M-TBZQ group(p < 0.05). IL-4 showed the different degree of fall back, the largest in positive drug group and H-TBZQ group(p < 0.05). IL-10 protein expression restored weakly, and there were no statistical differences in treatment groups(p > 0.05). ICAM-1 decreased in all treatment groups, most obviously in L-TBZQ group and M-TBZQ group(p <0.05), and most slightly in positive drug group and H-TBZQ group(p >0.05).6. Network pharmacology Through the database mining technology and literature, ingredients in herbs of formula were found, including 94 kinds in magnolia flower, 118 kinds in perilla leaf, 138 kinds in fructus viticis, 97 kinds in bitter apricotkernels, 265 kinds in licorice, 106 kinds in xanthium, 219 kinds in angelica dahurica, 8 kinds in cicada, 103 kinds in rhizoma ligustici wallichii, and 21 kinds in white batryticated silkworm.The network of xanthium contained 48 kinds of compounds,corresponding to 101 targets and 262 kinds of diseases. The network of white batryticated silkworm contained 14 kinds of compounds, corresponding to112 targets and 45 kinds of diseases. The network of angelica dahurica contained 62 kinds of compounds, corresponding to 224 targets and 288 kinds of diseases. The network of cicada contained 4 kinds of compounds,corresponding to 60 targets and 99 kinds of diseases. The network of rhizoma ligustici wallichii contained 149 kinds of compounds, corresponding to 216 targets and 283 kinds of diseases. The network of licorice contained 118 kinds of compounds, corresponding to 301 targets and 335 kinds of diseases.The network of bitter almond contained 47 compounds, corresponding to 272 targets and 332 kinds of diseases. The network of fructus viticis contained 99 kinds of compounds, corresponding to 269 targets and 322 kinds of diseases.The network of lily magnolia contained 67 kinds of compounds,corresponding to 156 targets and 265 kinds of diseases. The network of perilla leaf contained 87 kinds of compounds, corresponding to 288 targets and 310 kinds of diseases. TBZQ formula contained 508 kinds of compounds in network, corresponding to 672 targets and 470 kinds of diseases, including allergic rhinitis and allergic diseases, respiratory allergic inflammation, etc..18 potential protein targets for TBZQ formula inhibiting allergic rhinitis were predicted, including IL-2, IFN-γ, IL-4, IRF-1, IL-6, IL-36 b, ITGA2 B,IL-1a, IL-1b, ICAM-1, ITGB2, IL-13, MMP1, JUN, ALOX5, RELA, IL-10,ITGAM.Conclusions:TBZQ formula could effectively inhibit allergic rhinitis through multipleingredients and multiple targets. The possible mechanisms of TBZQ formula included:(1) mainly by adjusting the expression of Ig E, IFN-γ, IL-2, IL-4,IL-10, ICAM-1 and other factors;(2) through multiple targets synergistically regulating various of disorders. |
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