| Objective: Based on a network pharmacology approach to investigate the mechanism of olfactory disturbance after severe acute respiratory syndrome coronavirus 2(SARS-Co V-2) pseudovirus infection by applying Manna Sterilizing Tang to intervene in the olfactory disturbance after upper respiratory tract virus infection(POVD)caused by SARS-Co V-2 pseudovirus drops in K18-h ACE2 transgenic mice,we observed the olfactory We observed the histomorphological changes of olfactory mucosa and olfactory bulb,the expression of olfactory marker protein(OMP)and the differences of related tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)inflammatory factors in the mouse model of POVD,and elucidated the role of post-injury repair mechanism of Manna Antiseptic Soup intervention in POVD.MATERIAL AND METHODS: The active compounds and drug targets of the seven core drugs in Ganlu Disinfectant Soup,including Yin Chen,Shi Calamus,Patchouli,Peppermint,Radix Scutellariae,and Scutellariae,were collected through the TCMSP database and input into the Uni Prot database,and the target protein was corrected to the standard name.The keyword "Anosmia" was entered into the Geencards database to screen the predicted targets of olfactory disorders,and the active compound targets of the core drugs and POVD disease targets were entered into the Venny2.1 platform to obtain the intersection of drug and disease targets.Cytoscape 3.7.2 software was used to construct the drug-compound component-disease target network map,and the protein type "Homo sapiens" was entered into the String database to import the protein interaction information.Cytoscape 3.7.2 software was used to construct the key target protein interaction network(PPI).GO analysis of key target gene functions and KEGG pathway enrichment analysis were performed using Metascape database and DAVID database.Next,72 K18-h ACE2 transgenic mice were randomly divided into normal,model,and manna sterilization soup groups,with a total of 24 mice in each group.Six mice were randomly executed on days 3,5,7 and 10 after modeling,and tissue specimens were collected from the nasal cavity,olfactory bulb and peripheral blood.The expression of OMP in the olfactory epithelium(OE)and OB was compared between different groups by immunohistochemistry.The expression of inflammatory factors TNF-α and IL-6 in mice serum was detected by ELISA.protein levels in OB.Results:1.280 active compounds were retrieved from the TCMSP database,with 191 target proteins for Inula,82 for Acorus calamus,177 for Patchouli,125 for Scutellaria,57 for Mucuna pruriens,105 for Peppermint,and 68 for Scutellaria.126 gene targets were intersected by Venn analysis.125 protein nodes were involved in the PPI network,with key targets including JUN,AKT1,RELA TP53,MAPK1,ESR1,FOS,IL6,CAV1,TNF;GO enriched functional analysis was mainly related to regulation of promoting gene expression,estradiol regulation,MAPK cascade activation,cell proliferation migration,lipopolysaccharide regulation,vascular contraction and regeneration;125 KEGG pathways,and pathways closely related to the treatment of POVD by mann sterilization soup were 20 intracellular calcium signaling pathway,HIF-1 signaling pathway,P53 signaling pathway,c AMP signaling pathway,NF-κB signaling pathway,etc.2.Under light microscopy,compared with the normal group,the number of inflammatory cells infiltrating under the OE increased significantly in the model group,the mucosal thickness thickened,the cell arrangement was disorganized and irregular,and their nerve cilia appeared to be significantly absent and detached and fragmented.In the Chinese medicine group,the number of mucosal inflammatory cells was significantly reduced compared with the model group,and the swelling of the olfactory mucosa decreased with the treatment time,the mucosal thickness gradually returned to normal,and the histomorphology of olfactory sensory neuron(ORN)cells tended to be regular and complete.3.Compared with the normal group,the serum level of IL-6 in mice increased significantly(P< 0.01),and the highest level of IL-6 was measured on day 5(P < 0.01);compared with the model group,the serum level of IL-6 in each administration group decreased,and the serum level of IL-6 in the Chinese medicine group peaked on day 5,and the inflammation in the Chinese medicine group decreased significantly from day 7;on day 10 The level of IL-6 in the model group decreased significantly,but the level of IL-6 in the model group and the Chinese medicine group was still higher than that in the normal group.4.Compared with the normal group,the TNF-α level in the serum of infected mice generally increased and reached the highest value on day 5;compared with the model group,there was a significant difference(P < 0.05)in the changes of TNF-α level between the Chinese medicine group and the model group in each administration group except for day 5,indicating that the treatment with Ganlu Disinfection Tang had the effect of reducing the TNF-α level in the serum of mice;on day 5,the treatment group reduced the TNF-α level significantly(P <0.05)compared with the model group.The level of TNF-α in the treated group was significantly lower than that in the model group on the 5th day of administration(P<0.05).5.In the normal group,a large amount of brownish-yellow OMP was positively expressed in the OE and lamina propria of the olfactory mucosa,while in the model group,the intensity of expression of brownish-yellow positive sites was significantly weakened or even disappeared,and the level of OMP expression was down-regulated in the discontinuous distribution of olfactory mucosa,which was statistically significant compared with the normal group(P <0.01).After treatment,the OMP brown-yellow positive expression was enhanced in the Chinese medicine group compared with the model group,and the mean value of IOD increased,but there was no significant difference between the groups(P>0.05).6.The brownish-yellow positive expression of OMP was mainly distributed in the outermost olfactory nerve layer(ONL)and synaptic glomerular layer(GL)of the olfactory bulb under light microscopy,and brownish-yellow globular structures were seen in the GL part.After modeling,the level of OMP brown expression was down-regulated in the model group,which was statistically significant compared with the normal group(P < 0.01).The mean value of IOD was increased in the Chinese medicine group compared with the model group,but there was no significant difference(P>0.05).7.Western blot showed that the OMP level in OB of mice after modeling decreased compared with the normal group(P>0.05),and the OMP level in OB of mice in the Chinese medicine group slightly increased at all time points(P>0.05),but the difference between all groups was not statistically significant(P>0.05).Conclusion:1.Mannu Disinfectant Soup can regulate intracellular calcium signaling pathway through JUN,AKT1,RELA,TP53,MAPK1,ESR1,FOS,IL6,CAV1,TNF and other key targets and inhibit related inflammatory signaling and other pathways to achieve the pharmacological mechanism of action for the treatment of POVD.2.Mannu sterilization soup can down-regulate IL-6 and TNF-α inflammatory factor expression,reduce inflammatory damage of supporting cells,alleviate the degree of swelling of olfactory mucosa,increase OMP expression in olfactory mucosa and OB,promote the maturation of ORNs and accelerate the repair effect of olfactory mucosa,which may be a target for the treatment of POVD. |
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