Exploring the molecular mechanism of Mahuang decoction against influenza: An integrated computational and experimental study

Abstract

Background: Mahuang Decoction (MHD) is primarily used to treat viral colds and alleviate muscle pain, with extensive clinical application. However, its therapeutic effects against influenza and its anti-inflammatory mechanisms remain unclear.

Methods: Active compounds and targets were identified using the Traditional Chinese Medicine Systems Pharmacology Database platform, OMIM database, and GeneCards database. Protein-protein interaction networks and compound-target networks were constructed to screen core targets and components. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed using the DAVID database, and the results were visualized via the Microbioinformatics platform. Molecular docking was employed to evaluate the binding affinity between potential targets and active compounds. Finally, cytopathic effect assays and reverse transcription polymerase chain reaction experiments were conducted for validation.

Results: Network pharmacology studies revealed that AKT1, EGFR, and SRC are core targets of MHD in treating influenza, with kaempferol, quercetin, and luteolin as key compounds. The mechanism of MHD is closely associated with antiviral, anti-inflammatory, and immune-regulatory effects. Cytopathic effect assays and reverse transcription polymerase chain reaction experiments confirmed the therapeutic effects of key compounds against H1N1.

Conclusion: This study demonstrates that MHD exhibits anti-H1N1 viral activity and comprehensively elucidates its active components, potential targets, and molecular mechanisms in influenza treatment. These findings provide a theoretical foundation for further exploration of MHD's protective effects and anti-inflammatory mechanisms against influenza.

Keywords: Mahuang decoction; RT-PCR; cell experiments; influenza; molecular docking; network pharmacology.

Figure 1.

Mahuang decoction (MHD) and influenza crossover gene Venn diagram. Orange represents MHD, and light yellow represents influenza. MHD = Mahuang decoction.

Figure 2.

STRING diagram of common targets. This diagram was generated by importing common targets into the STRING database. In the network, connection lines represent protein-protein interactions, and the thickness of the lines generally indicates the confidence score of the interactions; the thicker the line, the higher the reliability.

Figure 3.

Protein-protein interaction (PPI) network diagram. The PPI network diagram of MHD and influenza was obtained by visualizing the common targets using Cytoscape. Node size and color were adjusted based on the degree value. Adjust the node size and color based on the degree value. The node size is proportional to the number of neighbor genes interacting with these 67 targets in the background network of the human genome. The node color represents the propensity to bind MHD, and the closer to red, the more inclined to bind MHD. MHD = Mahuang decoction.

Figure 4.

The compound-target (C-T) network diagram. The hexagon represents the pathway of action; the circle represents the compound, and the square represents the target of action. The closer to red indicates the more critical role it plays in MHD influenza. MHD = Mahuang decoction.

Figure 5.

GO functional enrichment analysis. Top 20 GO terms selected based on P values are shown. The x-axis represents GO entries, and the y-axis represents the count number. Green indicates biological process (BP); orange indicates cellular component (CC); purple indicates molecular function (MF). GO = Gene Ontology.

Figure 6.

KEGG pathway enrichment analysis. Top 20 KEGG pathways selected based on P values. The x-axis represents the enrichment factor (calculated as the ratio of the number of target genes in the pathway to the number of all genes in the pathway), and the y-axis represents KEGG entries. The color of the bubbles is associated with −log10 (P value); the closer the color is to red, the smaller the P value. The size of the bubbles is related to the count value; the larger the bubble, the higher the count value. KEGG = Kyoto Encyclopedia of Genes and Genomes.

Figure 7.

The molecular docking diagram illustrates the potential binding site of the key compound on influenza, as well as the detailed amino acid residues within this binding region. The left panel shows the overall docking conformation of the key compound with influenza. The right panel provides an enlarged view of the ligand-binding pocket, displaying detailed amino acid residues. Influenza is represented as a cartoon structure, while the key compound is depicted in stick form. The gray portion represents the protein, blue indicates the ligand, and red highlights the amino acid residues that interact with the ligand.

Figure 8.

Molecular docking-based verification of the top 5 components (based on degree) of MHD and top 3 key targets in the PPI (based on degree) network. In the visualization, darker colors indicate lower binding free energy (stronger binding affinity). MHD = Mahuang decoction.

Figure 9.

In vitro anti-influenza A experiment with key compounds. Label number 1 concentration is TC₀, diluted with maintenance fluid. Label numbers 2 to 4 are dilutions with maintenance fluid multiplicity, respectively (10⁻¹–10⁻³).

Figure 10.

Comparison of Ct values for each concentration of MHD acting on influenza A virus with negative controls. Kaempferol 1, kaempferol 2, kaempferol 3, and kaempferol 4 represent compound concentrations of TC₀, 10⁻¹TC₀, 10⁻²TC₀, 10⁻³TC₀, respectively. Quercetin1, quercetin2, quercetin3 and quercetin4 represent compound concentrations of TC₀, 10⁻¹TC₀, 10⁻²TC₀, 10⁻³TC₀, respectively. Luteolin1, luteolin2, luteolin3 and luteolin4 represent compound concentrations of TC₀, 10⁻¹TC₀, 10⁻²TC₀, 10⁻³TC₀, respectively. MHD = Mahuang decoction.