In this study, we conducted multi-target screening for co-infection of MG and E.coli based on omics research and investigated the polypharmacology mechanism of NCMC with multiple components. The whole research were based on the establishment of a co-infection model and the subsequent target screening. According to the previous transcriptome studies on co-infection , we obtained a total of 3,115 DEGs and the corresponding KEGG classification. As the previous study showed that oxidative stress transcriptomics and drug discovery approaches could identify and target neurotoxic innate immune populations and lead to the development of selective neuroprotective strategies . However, we hope to screen the representative DEGs from the overall level of in vivo studies. Hence, the DEGs from 33 sub-categories of KEGG have carried out PPI analysis respectively as explained in previous studies [43–45]. The screened target genes cannot be applied directly but are matched to the corresponding target according to the Information in the DRUG BANK database.
Hopkins proposed the concept of network pharmacology in 2007, which aims to design a new generation of drugs by incorporating biological networks rather than single target . Network pharmacology-based strategy to predict therapeutic targets of tanshinone I and cryptotanshinone against inflammation, and further to investigate the pharmacological molecular mechanism in vitro . For complex diseases, the method of applying network pharmacology was also used to reveal the mechanism of the action of TCM [48, 49]. We collected the whole data of TCMSP for the construction of the total network “TCM-component-target”, and then the targets were substituted into the total network for the reverse screening of TCMs. This method of reverse operation is similar to existing relevant studies [50, 51]. However, this is the first attempt to apply the reverse screening method directly to the TCM network.
After obtaining the order of Chinese herbs, we then selected different categories based on the compatibility theory of Chinese medicines. According to the degree of node correlation, the order was as follows: Isatdis Radix (4th, yang tonic) , Forsythia Fructus (11th, Antipyretic and antidote) , Ginkgo Folium (5th, Antitussive and antiasthmatic drugs) , Mori Cortex (3rd, Antitussive and antiasthmatic drugs) , Licorice (1st, reinforcing drugs)  and Radix Salviae (2nd, Drugs with the efficacy of modifying rheological properties of blood) . Aqueous extract of Chinese medicinal compound is the most common method to study compound components [58, 59]. Hence, we sought points that were uniformly scattered on the domain [60, 61], furthermore, we did a multi-nonlinear regression analysis to search for the optimal combination in preliminary experiments. Finally, we obtained the optimal combination of NCMC aqueous extract for subsequent targeted therapy experiments. In the treatment experiment of NCMC aqueous extract for co-infection, we used multiple pathological methods to observe whether a certain curative effect was achieved. Pathological and ultrastructural results showed that the symptoms of co-infection were significantly relieved after treatment, which indicated that NCMC played a significant therapeutic role.
The multi-dimensional validations of targeted therapy were performed at gene, protein, and metabolite levels, as well as computer simulations in vitro, respectively. Afterward the joint analysis of transcriptomics and metabolomics, we obtained the network map of key genes and metabolites. Recent studies show that combined metabolomics and transcriptomics analyses provide sensitive approaches to link infection to biological responses [62, 63]. In our study, the first 12 target genes with the highest correlation degree were selected for RT-PCR quantitative detection. The results showed that the expressions of these related genes were reduced after treatment. In particular, various studies have shown that these genes controlled a broad and flexible network in the transcriptome [64, 65]. However, the expression of CHRM4, MMP2, and VEGFA was shown opposite results. We performed western blot to verify protein level expression, which showed similar trends of the six proteins (MMP2, TLR4, c-FOS, BDKRB1, VEGFA, and EDN-1). The quantitative results of targets indicated that the NCMC aqueous extraction can play a multi-target therapeutic role after the reverse screening by network pharmacology.
In the circulation, as the basis of organism phenotypes, metabolites can help us to understand life phenomena more intuitively and effectively and reveal the essence of life [66, 67]. Our previous metabolic results demonstrated that Arachidonic Acid metabolism is activated in co-infection, and it is recommended that LTC4 in serum acts as a biomarker for detecting poultry respiratory diseases . Based on the results of a joint analysis of transcriptomics and metabolomics, dopamine, γ-Aminobutyric acid, and leukotriene C4 were selected for further study. It had been shown that bacterial infection can cause a dopamine burst in the brain and Dopamine activates NF-κB and primes the NLRP3 inflammasome in primary human macrophages [68, 69]. The study also unveiled that γ-Aminobutyric acid, the principal inhibitory neurotransmitter in the brain, has activation functions in the immune system . These findings are similar to the results of co-infection. Furthermore, the expression of these three metabolites decreased significantly after NCMC treatment.
Finally, to further verify which active components of NCMC aqueous extraction have a multi-target effect, molecular docking was used for in vitro analysis. Affinity was the score for the molecular docking, and when the score was lower, the binding affinity was stronger. An affinity < − 7 indicated a stronger binding activity . Our docking results demonstrated the multi-target action of the main active components of the NCMC. In particular, the targeted therapy of Salvianolic acid A, Liquiritigenin, and Isoliquiritigenin could be further investigated. There are even more naturally derived compounds that are either in clinical trials or have shown potential in pre-clinical studies which merit further investigation . In the present study, we used four methods to verify the multi-target therapeutic effect of NCMC extraction. It can be found that NCMC plays a certain role in the expression of key target proteins, and the corresponding key metabolites have also been confirmed.
However, several potential limitations in our experimental and polypharmacology-based approaches should be acknowledged. First, the incomplete known components of natural products curated from publicly available databases limit our approach to predict NCMC extraction for those without known information. Furthermore, the MetaboAnalyst (v 4.0) was used to simultaneously analyze genes and metabolites of interest within the context of metabolic pathways. But only data from humans, mice, and rats are supported currently . Integrating the target network may help to target the growing potential of co-infection genes via indirectly acting on their neighbor proteins in the human interactome. Lastly, the development of TCM should focus on interdisciplinary, combining research from emerging fields after clarifying the material basis. In terms of pharmacodynamics, it is necessary to clarify how NCMC extraction works, such as analyzing the dialectical relationship between "Chinese herbal medicine, probiotics, and intestinal flora".