A Study Based on Metabolomics, Network Pharmacology, and Experimental Verification to Explore the Mechanism of Qinbaiqingfei Concentrated Pills in the treatment of Mycoplasma Pneumonia

Abstract

Qinbaiqingfei concentrated pills (QB) are a commonly used medicine for the treatment of mycoplasma pneumonia in China, and the mechanism of action of QB needs to be studied further. Therefore, we use a combination of metabolomics and network pharmacology to clarify the mechanism of QB. Nontarget metabolomics studies were performed on rat serum, urine, and lung tissues, and 56 therapeutic biomarkers were found. Subsequently, the components of QB absorbed into the blood and lung tissues were clarified, and based on this finding, the core target of network pharmacology was predicted. The enrichment analysis of biomarkers-genes finally confirmed their close relationship with the NF-κB signaling pathway. By western blotting expression of the proteins in the lung tissue-related signaling pathways, it is finally confirmed that QB inhibits the NF-κB signaling pathway through SIRT1, IL-10 and MMP9, CTNNB1, EGFR, and other targets. It plays a role in regulating immunity, regulating metabolism, and treating diseases.

Keywords: NF-κB signaling pathway; Qinbaiqingfei concentrated pills; metabolomics; mycoplasma pneumonia; network pharmacology.

FIGURE 1

Process design diagram.

FIGURE 2

HE staining of rat lung tissue and the amount of mycoplasma carrier in lung tissue. (A) RtPCR mycoplasma real-time fluorescence detection, using analysis of variance to calculate the significant difference, compared with the MPP group #p < 0.05, ##p < 0.01. (B) The alveolar wall of the control group was composed of a single layer of epithelium with a simple structure; the interstitium, including the connective tissue and blood vessels in the lung, had no obvious abnormalities. In the Mycoplasma pneumoniae group, large areas of alveolar walls are severely thickened, alveolar sizes vary, alveolar walls are infiltrated with a large number of inflammatory cells (black arrows), and few of the foam cells in the alveolar cavity (yellow arrows) and local perivascular inflammatory cells infiltrate into rings (red arrow). In the QB group, the alveolar wall was slightly thickened, with a small amount of inflammatory cell infiltration in the alveolar wall (black arrow). In the AZM group, large areas of alveolar walls were moderately thickened, alveolar sizes varied, and alveolar walls were infiltrated with moderate inflammatory cells (black arrows).

FIGURE 3

(A) T-cell differentiation diagram, the black arrow indicates inhibition and the remaining arrows indicate promotion. (B) Serum TNF-α and IL-10 expression levels, and the Th1/Th2 ratio. The significant difference was calculated using analysis of variance, #p < 0.05, ##p < 0.01 and compared with the MPP group.

FIGURE 4

Urine, serum, and lung tissue OPLS-DA images of rats in CN, MPP, QB, and AZM groups. (A) Four groups of urine positive ion mode OPLS-DA diagram (B) Four groups of urine negative ion mode OPLS-DA diagram (C) Four groups of serum positive ion mode OPLS-DA diagram (D) Four groups of serum negative ion mode OPLS-DA diagram (E) Four groups of lung tissues with positive ion mode OPLS-DA (F) four groups of lung tissues with negative ion mode OPLS-DA. We select 10 samples for each group.

FIGURE 5

Urine, serum, and lung tissue have a tendency to recall the biomarker.

FIGURE 6

(A) Wogonin’s cracking method. (B) The cracking method of Tubeostemoninol A.

FIGURE 7

C-T network diagram, red represents components and yellow represents targets. (A) is the contribution of each component to the C-T network and (B) is the four core components that contribute more than 85%.

FIGURE 8

T-D network diagram. (A) is the graph after the isolated nodes and connected edges are deducted, (B) is the relevant information graph after extracting the mean value greater than a degree (11.463), (C) is the graph of six core targets, (D) is the six core targets The core target network of the remaining connecting nodes.

FIGURE 9

Molecule docking verification diagram. (A) Component-target affinity energy heat map, the greater the absolute value of kJ/mol, the stronger the binding energy. (B) Molecule docking diagram.

FIGURE 10

The core role path diagram. MP is specifically recognized by toll-like receptors and then promotes the activation of the NF-κB pathway by promoting the expression of MyD88 protein. This is also the main path of MPP pathogenesis. Both G-protein–coupled receptors and EGF receptors that can pass the direct or indirect pathway is positively correlated with the NF-κB pathway.

FIGURE 11

(A) Western blot map of each histone. (B) Gray value of each histone.