| Background:Zukamu granule is a key variety of Uygur drugs included in the National Catalogue of Essential Drugs(2012 edition)and the National Catalogue of Model Drugs for Direct Procurement of Urgent Rescue Drugs.It is one of the representative uygur patent drugs.As the first choice medicine for the treatment of common cold in the theory of Uygur medicine theory,it has unique anti-inflammatory and cough efficacy,and clinical studies have shown that its efficacy is accurate,safe and reliable.However,the experimental design of applying scientific methods to explain the anti-inflammatory mechanism of its prescription is relatively insufficient.In this study,supported by the National Natural Science Foundation of China(81860803),the combination of Chinese medical language for functional expression and prescription drug classification,and the reference of modern medical biological indicators for characterization of action targets,will be conducive to improving the theoretical value and clinical value of Zukamu granule.Objective:Based on the predicted potential mechanism of the anti-inflammatory activity,Zukamu granule and its different combinations of traditional efficacy drugs in the granule formulation were observed for their different effects on AM cell phagocytosis,AM cell number,TLR4/MyD88/NF-κB signaling pathway and apoptosis,then analyzed and verified the differences in anti-inflammatory mechanism of different pharmaceutical compositions with different efficacy in Zukamu granule formulation.Methods:(1)The forecast of Zukamu granule anti-inflammatory potential targets:① Retrieval and screening of the potential effective chemical constituents by using TCMSP analysis platform and the journal websites open source database.Based on the systematic pharmacology platform of TCMSP and Stitch,etc.,the potential targets of the above effective chemical components were selected and predicted,and the data set of "active ingredient-potential target of action" of Zukamu granule was constructed.②The potential targets related to inflammation were retrieved by using Genecards database,then the the common targets of Zukamu granule were summarized and introduced into the String database to construct the protein interaction network diagram of "potential target-inflammation",and the pathway analysis and functional enrichment analysis were further carried out.③Based on the above experimental results,the differences in anti-inflammatory mechanism of different pharmaceutical compositions with different efficacy in Zukamu granule formulation were further compared,which includes scattering and dissipating external wind and heat,relieving cough and dissolving phlegm,supplementing the stomach and lungs(2)Verification of the anti-inflammatory activity and target of Zukamu granule:Taking AM phagocytosis activity as the entry point,the inflammatory intervention effect of Zukamu granule on bleomycin-induced pulmonary fibrosis in rats at different time points was studied.①In terms of the pathological state of the body,the effects of Zukamu granule on the body weight,body temperature,respiratory rate,lung tissue pathology and hydroxyproline content in rats were investigated to verify its anti-inflammatory activity.② In terms of efficacy targets,Based on the effect of Zukamu granule on the phagocytic intensity of AM,the intervention of AM cell number,TLR4/MyD88/NF-κB signaling pathway,apoptosis and autophagy process were investigated.To some extent,the prediction of anti-inflammatory targets and related pathways was verified.③ Analyzed and verified the differences in anti-inflammatory mechanism of different pharmaceutical compositions with different efficacy in Zukamu granule formulation,which includes scattering and dissipating external wind and heat,relieving cough and dissolving phlegm,supplementing the stomach and lungs.Results:(1)The forecast of Zukamu granule anti-inflammatory potential targets:① Combined with database screening and literature resource supplement,165 effective active components of Zukamu granule were obtained after eliminating common components of drugs.The TCMSP,Stitch and other platforms were comprehensively used to eliminate the potential targets of multiple active components,and 235 potential targets were obtained.②A total of 695 potential targets related to inflammation and Zukamu granule were retrieved using Genecards database.Using the String database for a total of targets "potential targets-inflammation" protein interaction analysis,enrichment of path analysis and function analysis,it has been preliminarily confirmed that the mechanism of the anti-inflammatory activity of Zukaru granule may be related to Toll-like signaling pathway and cell proliferation,and involves cell phagocytosis,apoptosis,autophagy and other cellular procedures,laying a foundation for the selection of indicators in subsequent biological effect verification tests.③The results of comparative analysis of anti-inflammatory mechanism of the 3 groups of different efficacy drug combinations showed that,the anti-inflammatory effects of the scattering and dissipating external wind and heat group may be related to the HIF-1 signaling pathway,and that of the supplementing the stomach and lungs group may be related to Toll-like receptor signaling pathways.In addition,the downstream of most anti-inflammatory signaling pathways of these 3 groups may be associated with apoptosis.(2)Verification of the anti-inflammatory activity and target of Zukamu granule:①In terms of the pathological state of the body,on the 3rd day,the body temperature of the rats in the group,which treats the root and branch simultaneously,was significantly decreased(P<0.01),and the respiratory rate of the rats in the group,which scatters and dissipates external wind and heat,was significantly decreased(P<0.01),the hydroxyproline content in the lung tissues of the rats in the two groups was statistically different from that in the model group(P<0.05);on the 14th day,the hydroxyproline content in Zukamu granule was significantly decreased(P<0.01);on the 28th day,the weight in the group,which treats the root and branch simultaneously,was significantly different from the model group(P<0.01),the content of hydroxyproline in the group which supplements the stomach and lungs decreased and the difference was statistically significant(P<0.05).The results of HE staining in lung histopathological sections also showed that Zukamu granule and its different drug combinations could control the inflammatory infiltration and fibrosis progression to a certain extent.② In terms of efficacy targets,for the phagocytic intensity of AM cells,compared with the model group,Zukamu granule and its different drug combinations group were significantly improved on the 3rd day(P<0.01);on the 14th days,the 5 groups were improved,and the difference was statistically significant(P<0.05).For AM cell count,compared with the model group,on the 3rd day,the number of AM cells in the group,which treats the root and branch simultaneously,was lower than that in the model group(P<0.05);and in 3 treatment groups which includes dissolving phlegm,supplementing the stomach and lungs,and dissipating external wind and heat,the number of AM cells was significantly low(P<0.05);on the 14th and 28th days,the relative number of AM cells in the 5 groups was lower than that in the model group(P<0.05).For TLR4/MyD88/NF-κB signaling pathway,the mRNA expression levels of TLR4 and MyD88 in the 5 groups were significantly lower than those in the model group on the 3rd and 7th days(P<0.01).The mRNA expression levels of NF-κB in Zukamu granule group and 2 treatment groups,which includes dissolving phlegm,relieving cough and supplementing the stomach and lungs,were lower than those in the model group on the 7th days(P<0.05).On the 7th days,The expression levels of NF-κB mRNA in Zukamu granule group and the treatment groups except for dissolving phlegm and relieving cough were lower than those in the model group(P<0.05).For the balance between apoptosis and autophagy,the results showed that Zukamu granule and its different drug combinations could significantly reduce the apoptosis rate of alveolar macrophages(P<0.01),and the mechanism might be related to the intervention of the binding of the Bcl-2 to Beclinl and the inhibition of the activation of Caspase 3.③ For the different combinations of traditional efficacy drugs in Zukamu granule formulation,further comparative analysis found that the regulation of apoptosis and autophagy in the group,which dissipates external wind and heat,is significant(P<0.01).Meanwhile,the Toll-like receptors signaling pathways regulating effect in the group,which supplements the stomach and lungs,is obvious(P<0.05).In addition,3 different pharmaceutical compositions can play good regulatory roles in phagocytosis and proliferation of AM cells,transcription and secretion of pro-inflammatory factors,which is consistent with network pharmacology prediction results.Conclusion:With the combination of traditional and modern multidisciplinary research methods,the anti-inflammatory potential mechanism of Zukamu granule and its different combinations of traditional efficacy drugs in the granule formulation were successfully predicted and validated.In addition,this paper took the intensity of AM phagocytosis as a clue,the anti-inflammatory mechanism of Zukamu granule were investigated,which includes the number of AM cells,the TLR4/MyD88/NF-κB signaling pathway and the balance of apoptosis and autophagy.The regulation of apoptosis and autophagy in the group,which dissipates external wind and heat,is significant.Meanwhile,the Toll-like receptors signaling pathways regulating effect in the group,which supplements the stomach and lungs,is obvious.This suggests that different pharmaceutical compositions with different efficacy may have different anti-inflammatory mechanisms and targets,which need to be further explored. |
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