Abstract

   Introduction: With amazing clinical efficacy, Yangyin Qingfei Decoction
   Plus (YQDP), a well-known and age-old Chinese compound made of ten
   Chinese botanical drugs, is utilized in clinical settings to treat a
   range of respiratory conditions. This study examines the impact of
   Yangyin Qingfei Decoction (YQDP) on lung tissue metabolic products in
   severe Mycoplasma pneumoniae pneumonia (SMPP) model mice and examines
   the mechanism of YQDP in treating MP infection using UPLC-MS/MS
   technology.

   Methods: YQDP’s chemical composition was ascertained by the use of
   Agilent 1260 Ⅱ high-performance liquid chromatography. By using a nasal
   drip of 10^10 CCU/mL MP bacterial solution, an SMPP mouse model was
   created. The lung index, pathology and ultrastructural observation of
   lung tissue were utilized to assess the therapeutic effect of YQDP in
   SMPP mice. Lung tissue metabolites were found in the normal group,
   model group, and YQDP group using UPLC-MS/MS technology. Using an
   enzyme-linked immunosorbent test (ELISA), the amount of serum
   inflammatory factors, such as interleukin-6 (IL-6) and tumor necrosis
   factor α (TNF-α), was found. Additionally, the protein expression of
   PI3K, P-PI3K, AKT, P-AKT, NF-κB, and P-NF-κB was found using Western
   blot.

   Results: The contents of chlorogenic acid, paeoniflorin, forsythrin A,
   forsythrin, and paeonol in YQDP were 3.480 ± 0.051, 3.255 ± 0.040,
   3.612 ± 0.017, 1.757 ± 0.031, and 1.080 ± 0.007 mg/g respectively. YQDP
   can considerably lower the SMPP mice’s lung index (p < 0.05). In the
   lung tissue of YQDP groups, there has been a decrease (p < 0.05) in the
   infiltration of inflammatory cells at varying concentrations in the
   alveoli compared with the model group. A total of 47 distinct
   metabolites, including choline phosphate, glutamyl lysine, L-tyrosine,
   6-thioinosine, Glu Trp, 5-hydroxydecanoate, etc., were linked to the
   regulation of YQDP, according to metabolomics study. By controlling the
   metabolism of porphyrins, pyrimidines, cholines, fatty acids,
   sphingolipids, glycerophospholipids, ferroptosis, steroid hormone
   biosynthesis, and unsaturated fatty acid biosynthesis, enrichment
   analysis suggested that YQDP may be used to treat SMPP. YQDP can lower
   the amount of TNF-α and IL-6 in model group mice as well as
   downregulate P-PI3K, P-AKT, and P-NF-κB expression (p < 0.05).

   Conclusion: A specific intervention effect of YQDP is observed in SMPP
   model mice. Through the PI3K/Akt/NF-κB signaling pathways, YQDP may
   have therapeutic benefits by regulating the body’s metabolism of
   α-Linoleic acid, sphingolipids, glycerophospholipids, arachidonic acid,
   and the production of unsaturated fatty acids.

   Keywords: Yangyin Qingfei decoction plus, Mycoplasma pneumoniae, severe
   illness, metabolomics, PI3K/Akt/NF-κB

1 Introduction

   One of the most frequent infections in children with community-acquired
   pneumonia (CAP) is Mycoplasma pneumoniae (MP), which has a widespread
   pathogenicity and frequently exhibits periodic and regional
   epidemiology. Ten to forty percent of pediatric pneumonia cases are
   caused by Mycoplasma pneumoniae pneumonia (MPP), which is the result of
   MP infection ([40]Kutty et al., 2019). The MP infection is typically
   regarded as a self-limiting illness. It is marked by subtle respiratory
   symptoms, the most notable of which is paroxysmal dry cough, which is
   frequently followed by the expectoration of little amounts of mucus or
   mucopurulent sputum. Although MPP is thought to be self-limiting,
   0.5%–to 2.0% of instances are known to proceed to severe MPP (SMPP).
   This condition is linked to life-threatening consequences such
   respiratory failure and acute respiratory distress syndrome, which can
   be fatal ([41]Izumikawa, 2016; [42]Lee et al., 2021). Although the
   exact pathogenic mechanism of MP infection is unknown, some studies
   think that adhesion and cytotoxic effects cause direct damage to the
   respiratory epithelium during MP pathogenesis ([43]Atkinson et al.,
   2008). When MP infection damages host mucosal epithelial cells, it can
   generate toxic compounds that cause the epithelial cells to die and
   apoptose, which can result in pneumonia and other extrapulmonary
   injuries ([44]Sun et al., 2008; [45]He et al., 2016). Since MP lacks a
   cell wall structure, it is resistant to antibiotics that stop bacteria
   from producing cell walls, but it is susceptible to quinolones,
   tetracyclines, and macrolides. For this reason, macrolides are the
   recommended treatment for MPP in children. Unfortunately, resistance to
   macrolides has grown in many regions since the discovery of
   macrolide-resistant strains of MP in China in 2003. Additionally, the
   side effects of macrolide antibiotics, such as gastrointestinal
   distress and vascular irritation, have made the hunt for more effective
   treatment options necessary ([46]Xin et al., 2005; [47]Zhao et al.,
   2019; [48]Dou et al., 2020). There are benefits to using traditional
   Chinese medicine (TCM) in the treatment of SMPP, and research on the
   targeted mechanism of TCM intervention in SMPP is currently a popular
   topic.

   The “Chonglou Yuyao” book, authored by renowned Qing Dynasty physician
   Zheng Meijian, is where Yangyin Qingfei Decoction Plus (YQDP)
   originated. Ten traditional Chinese medicines are included in it:
   Rehmanniae Radix, Scrophulariae Radix, Ophiopogonis Radix, Paeoniae
   Radix Alba, Moutan Cortex, Fritillariae Thunbergii Bulbus, Menthae
   Haplocalycis Herba, Glycyrrhizae Radix Et Rhizoma, Forsythiae Fructus,
   and Lonicerae Japonicae Flos. In clinical settings, YQDP is frequently
   used to treat bronchopneumonia, post-infection cough, and chronic
   obstructive pulmonary disease, among other conditions ([49]Liu et al.,
   2017; [50]Min et al., 2021). Contemporary pharmacological studies have
   demonstrated that YQDP functions by increasing tissue healing,
   controlling immunological function, and suppressing inflammatory
   reactions ([51]Tang et al., 2020). Forsythoside, a representative of
   Forsythiae Fructus, has shown antiviral, anti-inflammatory, and
   antioxidant properties in contemporary studies ([52]Ma et al., 2020).
   Phenolic acid compounds in Lonicerae Japonicae Flos have
   pharmacological activities such as antiviral, antibacterial,
   anti-inflammatory, and antioxidant effects. Compounds containing
   phenolic acid in Lonicerae Japonicae Flos include antioxidant,
   antiviral, and antibacterial properties ([53]Xiong et al., 2022). As an
   indication component of Lonicerae Japonicae Flos, chlorogenic acid
   possesses a variety of anti-inflammatory properties ([54]Gao et al.,
   2019) and can eliminate reactive oxygen species within cells ([55]Shin
   et al., 2017; [56]Liang and Kitts, 2018). The primary ingredients of
   Forsythiae Fructus, forsythiaside A and forsythin, have potent
   antiviral and anti-inflammatory properties ([57]Qu et al., 2016;
   [58]Runfeng et al., 2020). The functional elements of Moutan Cortex and
   Paeoniae Radix Alba are pheonol and pheoniflorin, respectively. The
   analgesic and anti-inflammatory properties of monoterpenes and their
   glycosides, embodied by Paeoniflorin, are acknowledged as the
   pharmacologically active components of Paeoniae Radix Alba ([59]Wu et
   al., 2018; [60]Zhang et al., 2021). Paeonol has the ability to protect
   against oxidative stress, prevent the production of reactive oxygen
   species, and boost the activity of the enzymes superoxide dismutase and
   hydrogen peroxide ([61]Zhang et al., 2020). In earlier research, we
   established and evaluated a severe pneumonia model in high-load
   MP-infected mice ([62]Wu et al., 2023a) and showed that YQDP regulates
   the TLR2/MyD88/NF-κB signaling pathway, Aquaporin5 (AQP5), and Mucin
   5ac (MUC5ac) to regulate SMPP ([63]Wu et al., 2023b; [64]Qi et al.,
   2023).

   An HPLC method is used in this study to simultaneously determine the
   level of five useful compounds in YQDP for quality control. The
   therapeutic benefits and metabolic pathways of YQDP on SMPP were then
   examined.

2 Materials and methods

2.1 Reagents and chemicals

   Merck Company (USA) provided the acetonitrile (CAS: 75-05-8),
   phosphoric acid (CAS: 7664-38-2), and methanol (CAS: 67-56-1). The
   China Academy of Food and Drug Administration provided the following:
   Chlorogenic Acid (CGA, CAS: 327-97-9), Paeoniflorin (PF, CAS:
   23180-57-–6), Forsythoside A (FTA, CAS: 79916-77-1), Forsythin (FT,
   CAS: 487-41-2), and Paeonol (PAE, CAS: 552-41-0). Proteintech was the
   source of the following antibodies: Anti-GAPDH (60004-1-Ig), Anti-PI3K
   (60225-1-Ig), Anti-AKT (10176-2-AP), Anti-Phospho-AKT (66444-1-Ig), and
   Anti-NFκB p65 (10745-1-AP). Bioss provided the anti-phospho-PI3K
   (bs-3332R) and anti-phospho-NFκB p65 (bs-3543R) antibodies.

   Rehmannia glutinosa (Gaertn.) DC. [Orobanchaceae] (Rehmanniae Radix,
   2202163), Scrophularia ningpoensis Hemsl. [Scrophulariaceae]
   (Scrophulariae Radix, 2203081), Ophiopogon japonicus (Thunb.) Ker Gawl.
   [Asparagaceae] (Ophiopogonis Radix, 2203052), Paeonia lactiflora Pall.
   [Paeoniaceae] (Paeoniae Radix Alba, 2203152), Paeonia × suffruticosa
   Andrews [Paeoniaceae] (Moutan Cortex, 2,203,121), Fritillaria
   thunbergii Miq. [Liliaceae] (Fritillariae Thunbergii Bulbus, 2203151),
   Mentha canadensis L. [Lamiaceae] (Menthae Haplocalycis Herba, 2203241),
   Glycyrrhiza uralensis Fisch. ex DC. [Fabaceae] (Glycyrrhizae Radix Et
   Rhizoma, 2203152), Forsythia suspensa (Thunb.) Vahl [Oleaceae]
   (Forsythiae Fructus, 2204102), and Lonicera japonica Thunb.
   [Caprifoliaceae] (Lonicerae Japonicae Flos, 2203081) were purchased
   from Anhui Puren Traditional Chinese Medicine Drinking Tablets Co Ltd
   (Anhui, China) and authenticated by Professor Leng Yujie from the
   Chinese Herbal Medicine Bureau of the Affiliated Hospital of Liaoning
   University of Traditional Chinese Medicine (Shenyang, China).
   Rehmanniae Radix sample (RGL20220617), Scrophulariae Radix sample
   (SNH20220618), Ophiopogonis Radix sample (OJK20220619), Paeoniae Radix
   Alba sample (PLP20220617), Moutan Cortex sample (PSA20220617),
   Fritillariae Thunbergii Bulbus sample (FTM20220617), Menthae
   Haplocalycis Herba sample (MHB20220617), Glycyrrhizae Radix Et Rhizoma
   sample (GUF20220617), Forsythiae Fructus sample (FSV20220617) and
   Lonicerae Japonicae Flos sample (LJT20220617) were kept in the
   Laboratory of Pharmacognosy, Liaoning Academy of Traditional Chinese
   Medicine.

2.2 Experimental animals and strains

   36 SPF BALB/c mice, 4–6 weeks, weighing 20 ± 2g, were provided by
   Liaoning Changsheng Biotechnology Co., Ltd. [SCXK (Liao) 2020–0001].
   The mice were kept in separate cages at the Experimental Animal Center
   of Liaoning University of Traditional Chinese Medicine [SYXK (Liao)
   2019-004]. The housing environment was kept at (22 ± 2)°C, with a
   12h/12 h light/dark cycle, and a humidity of (50 ± 2)%. The MP standard
   strain FH was stored in the virus room of the Affiliated Hospital of
   Liaoning University of Traditional Chinese Medicine. The Animal Ethics
   Committee of Liaoning University of Traditional Chinese Medicine gave
   its approval to this study (Approval No.: 21000042021131).

2.3 Determination of five active ingredients in YQDP

2.3.1 Preparation of standard solutions

   Precisely weighing CGA, PF, FTA, FT, and PAE, then dissolving them with
   methanol to create a solution with CGA at 0.214 mg/mL, PF at
   0.217 mg/mL, FTA at 0.214 mg/mL, FT at 0.118 mg/mL, and PAE at
   0.068 mg/mL.

2.3.2 Preparation of YQDP sample solutions

   The procedure described in the literature was used to make YQDP ([65]Wu
   et al., 2019). Rehmanniae Radix (15 g), Scrophulariae Radix (11 g),
   Ophiopogonis Radix (9 g), Paeoniae Radix Alba (6 g), Moutan Cortex
   (6 g), Fritillariae Thunbergii Bulbus (6 g), Menthae Haplocalycis Herba
   (4 g), Glycyrrhizae Radix Et Rhizoma (4 g), Forsythiae Fructus (6 g)
   and Lonicerae Japonicae Flos (6 g) were then placed in a 10-fold 70%
   ethanol (v/v). The extract was then extracted using cold reflux for 70
   min, filtered, concentrated to create a paste, and dried at a constant
   temperature to produce YQDP dry powder. Ultimately, 25 mL of methanol
   and 0.5 g of the dry powder were combined, sonicated, and then filtered
   through a 0.45 µm filter membrane for additional usage.

2.3.3 HPLC analysis

   A SHIMADZU-GL C18 (250 mm × 4.6 mm × 5 μm) column was used in an
   Agilent 1260 high-performance liquid chromatograph to determine the
   content. The flow rate was 0.8 mL/min and the column temperature was
   30°C. The following gradient elution conditions applied to the mobile
   phase, which was 0.1% phosphoric acid-water (A) and acetonitrile (B):
   0∼20 min, 10%∼20% (B); 20∼25 min, 12%∼15% (B); 25∼30 min, 15%–18% (B);
   30–50 min, 18%–36% (B); 50∼70 min, 36%∼50% (B); 70∼75 min, 50%∼50% (B);
   75∼80 min, 50%∼60% (B); 80∼85 min, 60%∼70% (B). The following were the
   detecting wavelength and program: 0∼25 min, 330 nm; 25∼37 min, 230 nm;
   37∼45 min, 280 nm. 5 μL was the injection volume of the sample.

2.4 Preparation of YQDP prescription for intervention in SMPP model mice

   Condensation and refluxing with 70% ethanol for 70 min was used to
   extract Rehmanniae Radix (150 g), Scrophulariae Radix (110 g),
   Ophiopogonis Radix (90 g), Paeoniae Radix Alba (60 g), Moutan Cortex
   (60 g), Fritillariae Thunbergii Bulbus (60 g), Menthae Haplocalycis
   Herba (40 g), Glycyrrhizae Radix Et Rhizoma (40 g), Forsythiae Fructus
   (60 g) and Lonicerae Japonicae Flos (60 g). 176.23 g of the extract
   were obtained by drying the extracts at a steady temperature after they
   had been concentrated into a paste. The drug extraction rate was
   24.14%.

2.5 MP cultivation

   After thawing under running water, the MP strain was introduced to PPLO
   liquid medium and cultured at 37°C in an incubator set to a constant
   temperature with 5% CO[2]. The third generation was removed and placed
   aside when the liquid medium turned yellow instead of red. The Color
   Change Unite (CCU) was defined as the highest dilution concentration at
   which the medium turned yellow. The MP concentration was ascertained
   using the CCU/mL method, and the gradient dilution method was used to
   prepare the bacterial solution with a concentration of 1×10^10 CCU/mL.

2.6 Animal grouping and dosing

   Following a week of consistent adaptive feeding, 36 BALB/c mice were
   split into six groups at random (n = 6). Referring to the modeling
   method of previous studies ([66]Wu et al., 2023a), mice were
   anesthetized with isoflurane. Mice in the normal group were given nasal
   drip of 100 μL of physiological saline, while mice in the other groups
   were given nasal drip of 100 μL of MP bacterial solution at a
   concentration of 1×10^10 CCU/mL. All groups received nasal drip for
   three consecutive days.

   According to the “Animal Dose Conversion Table” ([67]Wei, 2010), the
   positive group was administered azithromycin dry suspension at a total
   dose of 0.370 g/kg. The YQDP group received three dosages of formula
   solution (1.552, 3.104, 6.208 g/kg, crude drug), The normal group and
   model group were given an equal amount of physiological saline. Lung
   tissue was utilized for pathological and metabolomic research, and
   serum from each group of mice was obtained for ELISA detection
   following continuous dosing for 7 days.

2.7 Mouse lung index

   To determine the lung index, lung tissues were blotted dry on filter
   paper, washed with 0.9% saline, and then weighed.
   [MATH: <mrow><mtext>Lung index</mtext><mo>=</mo><mrow><mfenced open="("
   close=")"
   separators="|"><mrow><mrow><mtext>lung weight </mtext></mrow><mo>/</mo>
   <mrow><mtext> body weight</mtext></mrow></mrow></mfenced></mrow><mo>×</
   mo><mn>100</mn><mo>%</mo></mrow> :MATH]

2.8 Histopathological observation

   After being fixed with 4% paraformaldehyde, the lung tissue was
   sectioned into thin 5 μm slices using a sectioning machine and embedded
   into wax blocks using a paraffin embedding machine. Under a light
   microscope, the pathological alterations in the lung tissue following
   HE staining were seen.

2.9 Observation of lung tissue ultrastructure

   Two to three pieces of lung tissue (1 mm^3) were removed from each
   group, preserved using 2.5% glutaraldehyde and 1% osmium acid,
   gradually dried with acetone, and then sliced using an epoxy resin
   infiltration and embedding process. The samples were then stained with
   lead citrate and uranyl acetate so that an H-7650 transmission electron
   microscope could be used to observe the ultrastructural alterations in
   lung tissue.

2.10 Metabolomics analysis

   0.1 g of lung tissue samples were weighed and then finely powdered
   using liquid nitrogen. Each group of samples received four times the
   volume of the extraction buffer MeOH/ACN (1:1, v/v), which was added,
   properly vortexed, and then incubated at −20°C for 1 h before being
   centrifuged at 18,000 g for 15 min at 4 °C. After transferring the
   liquid metabolite extraction supernatant to a centrifuge tube, separate
   an aliquot of 10 μL dilution solution from each sample to combine with
   quality control samples in preparation for metabolomics analysis.
   Utilizing the Waters ACQUITY UPLC ultra-performance liquid
   chromatography system in conjunction with the Waters ACQUITY UPLC BEH
   C18 Column (1.7 µm, 2.1 mm × 100 mm) for separation, 10 µL of injection
   volume, 0.40 mL/min of elution, and 40°C column temperature, the
   analysis was carried out.

   The mobile phase A was an aqueous solution containing 0.1% formic acid,
   and the mobile phase B was an acetonitrile-water solution containing
   0.1% formic acid. The liquid phase gradient was set as follows:
   0–11 min, 2%–98% B; 11–12 min, 98% B; 12–12.1 min, 98%–2% B;
   12.1–15 min, 2% B. The metabolites were separated by an
   ultra-high-performance liquid-phase system and injected into an ESI ion
   source for ionization and then analyzed by timsTOF Pro (Bruker) mass
   spectrometry. The ion source voltage was set at 4.5 kV, and both
   precursor ions and its secondary fragments were detected and analyzed
   using high-resolution TOF. The mass spectrometry scan range was set at
   50–1,300 m/z, and the data acquisition mode used Parallel Accumulation
   Serial Fragmentation (PASEF) mode.

2.11 Enzyme-linked immunosorbent assay (ELISA) for the detection of IL-6 and
TNF-α expression in serum

   The serum was divided into sterile EP tubes, centrifuged for 20 min at
   3,000 r/min, and refrigerated at −80°C. Following the kit’s
   instructions, add the sample, enzyme-labeled antibody, chromogenic
   reagent, and stop solution once the reagents have warmed up to room
   temperature. Then, use a 450 nm wavelength to examine the contents.

2.12 Protein expression of PI3K, P-PI3K, AKT, P-AKT, NF-κB, P-NF-κB in lung
tissues by Western blot

   After extracting the total protein from the lung tissue, the
   concentration of the protein was measured quantitatively and adjusted
   to a uniform concentration. 10% SDS-PAGE gel electrophoresis was used
   for protein separation, and the wet transfer technique was used to
   transfer the proteins to a PVDF membrane. The primary antibodies, PI3K
   (1:2000), P-PI3K (1:2000), AKT (1:10,000), P-AKT (1:5000), NF-κB
   (1:5000), P-NF-κB (1:5000), and the internal reference antibody GAPDH
   (1:10,000), were incubated after blocking with 5% non-fat milk powder.
   At 4°C, the incubation was carried out all night. The membrane was
   washed three times with TBST and then incubated with HRP-conjugated
   goat anti-rabbit secondary antibody (1:10,000) at room temperature for
   1 h. Following membrane cleaning, the PVDF membrane was put in a
   chemiluminescence imaging system for detection after the proper volume
   of ECL luminous solution was introduced. The internal control protein’s
   grayscale value served as a mathematical reference while using ImageJ
   software for analysis.

2.13 Statistical analysis

   The statistical software GraphPad Prism six was used for both graph
   processing and statistical analysis. When it came to quantitative data
   that satisfied the requirements for normality and homogeneity of
   variance, the one-way ANOVA test was employed, and the data were
   reported as Mean ± SD. *p < 0.05 was considered statistically
   significant.

3 Results

3.1 HPLC determination of five chemical components in YQDP

   Using acetonitrile (B) and 0.1% phosphoric acid water (A) as mobile
   phases will yield the best peak shape and resolution. [68]Figure 1
   displays the chromatograms of the YQDP sample solution and the mixed
   reference solution under these circumstances.

FIGURE 1.

   [69]FIGURE 1
   [70]Open in a new tab

   HPLC chromatograms of mixed reference solution (A) and modified sample
   solution of YQDP decoction (B) 1. CGA. 2. PF. 3. FTA. 4. FT. 5. PAE.

3.2 Validation

   Tests on linearity, accuracy, stability, reproducibility, and sample
   recovery identified five chemical components in the YQDP recipe.
   [71]Supplementary Table S1 displays all of the component standard
   curves. According to the data, there is a good linear association (r ≥
   0.9996) between the compounds that were tested and their corresponding
   concentration ranges. CGA, PF, FTA, FT, and PAE have respective RSD
   between 0.31% and 0.88%. Within 48 h, all components exhibited good
   stability, with an RSD ranging from 0.56% to 1.25%. consistency,
   ranging from 0.47% to 1.79%. All components had recovery rates between
   98.63% and 100.04%, with an RSD between 0.27% and 1.84%.

3.3 Content of five chemical components in YQDP

   YQDP recipe’s five chemical components’ contents were ascertained by
   the application of the established analytical method. [72]Table 1
   displays the results.

TABLE 1.

   Content determination results of five chemical components in YQDP
   (mg/g, Mean ± SD, n = 6).
   Component CGA PF FTA FT PAE
   Content 3.480 ± 0.051 3.255 ± 0.040 3.612 ± 0.017 1.757 ± 0.031 1.080 ±
   0.007
   [73]Open in a new tab

3.4 Changes in lung index of mice in each group

   The lung index of the model group mice was higher than that of the
   normal group on the seventh day following MP infection (p < 0.01). At
   every dosage, YQDP groups and the positive group shown a decline in
   comparison to the model group. Both the YQDP (3.104 g/kg) and YQDP
   (6.208 g/kg) groups saw a considerable decline. These two YQDP
   groupings are not significantly different from one another. The
   outcomes are displayed in [74]Figure 2.

FIGURE 2.

   FIGURE 2
   [75]Open in a new tab

   Lung index. **p < 0.01.

3.5 Pathological observation and scoring of lung tissue

   The normal group mice had intact lung tissue morphology and structure,
   with no edema or congestion in the tracheal wall, and no infiltration
   of inflammatory cells in the surrounding blood vessels. Most of the
   alveolar structures in model group mice were severely damaged, with
   fewer intact alveoli, significantly widened alveolar septa, and a large
   number of inflammatory cells exuding from the alveolar lumen.
   Inflammatory cell exudation and widened alveolar septa were observed in
   lung tissue of YQDP group mice at various concentrations. Lymphocyte
   infiltration was observed around the bronchioles and blood vessels. The
   positive group mice showed inflammatory cell infiltration around the
   trachea, bronchi, and small blood vessels, with mild pulmonary tissue
   congestion. [76]Figure 3 The results of the pathological score indicate
   that the lung tissue in the model group of mice had higher pathological
   scores than each treatment group (p < 0.01). The positive group
   performed better than the YQDP groups at different concentrations (p <
   0.01). The outcomes are displayed in [77]Figure 4.

FIGURE 3.

   FIGURE 3
   [78]Open in a new tab

   Pathological score. **p < 0.01.

FIGURE 4.

   [79]FIGURE 4
   [80]Open in a new tab

   Pathology of lung tissue (HE, 200×). (A) Normal group. (B) Model group.
   (C) YQDP (1.552 g/kg). (D) YQDP (3.104 g/kg) group. (E) YQDP
   (6.208 g/kg) group. (F) Positive group.

3.6 Ultrastructural observation of lung tissue

   The alveolar wall of the normal group mice is intact, the cytoplasm is
   uniform, the nuclear membrane is intact, and a small amount of
   microvilli can be seen on the free surface. The microvilli on the free
   surface of type II alveolar cells in the model group mice thickened and
   disordered, with mitochondrial swelling and thickening of the thin
   layer of connective tissue and basement membrane between alveoli. YQDP
   groups of mice at different concentrations showed more microvilli on
   the surface of type II alveolar cells and clear nuclear membranes.
   There are organelles in the cytoplasm, such as mitochondria with clear
   cristae and numerous lamellar bodies, and there are cell junctions
   between adjacent cells. The microvilli in the positive group were
   slightly thickened, with a small amount of shedding and irregular
   arrangement. Some mitochondria were swollen, and the connective tissue
   and basement membrane were thickened. Results are shown in [81]Figure
   5.

FIGURE 5.

   [82]FIGURE 5
   [83]Open in a new tab

   Observation of lung tissue ultrastructure (TEM, 120,00×). (A) Normal
   group. (B) Model group. (C) YQDP (1.552 g/kg). (D) YQDP (3.104 g/kg)
   group. (E) YQDP (6.208 g/kg) group. (F) Positive group. Lamina (White
   →), mitochondria (black →).

3.7 Metabolomics analysis

   At 6.208 g/kg, the YQDP group had significant pharmacological effects.
   In conjunction with the normal group, model group, and YQDP group,
   metabolomics analysis was carried out. The three groups’ metabolites
   varied in both positive and negative ion modes, according to PLS-DA
   analysis. As seen in [84]Figure 6, the P-test findings reveal that
   there is no overfitting across the group models. [85]Figure 7 further
   demonstrates a complete separation of the normal, model, and YQDP
   groups based on OPLS-DA data analysis. In [86]Supplementary Figure S1,
   the 200 permutation test chart is displayed. Fold Change >1.5 served as
   the upregulation threshold, Fold Change <1/1.5 served as the
   significant downregulation threshold, and t-test p < 0.05 and VIP >1
   served as the screening criteria for differential metabolites based on
   the VIP values of the developed OPLS-DA model. In the positive ion
   mode, the model group’s lung tissue included 81 metabolites, 64 of
   which were upregulated and 17 of which were downregulated,
   significantly different from the normal group’s lung tissue
   composition. The content of 32 metabolites, including choline
   phosphate, glutamine, lysine, and L-tyrosinamide, changed following
   YQDP intervention. In the negative ion mode, the model group’s lung
   tissue contained significantly less of 49 metabolites (42 upregulated
   and seven downregulated) than the normal group’s. The levels of 15
   metabolites, including Glu Trp, 5-hydroxydecanoate, and 6-thioinosine,
   varied following YQDP medication. The findings are displayed in
   [87]Supplementary Tables S2, S3, [88]Figures 8, [89]9. We displayed the
   differential metabolite data, as seen in [90]Figure 10, to better
   comprehend the metabolic variations in the lungs of several groups of
   mice.

FIGURE 6.

   [91]FIGURE 6
   [92]Open in a new tab

   PLS-DA score and model validation of pulmonary metabolites in SMPP mice
   treated with YQDP. (A) Positive PLS-DA. (B) Positive PLS-DA Validation.
   (C) Negative PLS-DA. (D) Negative PLS-DA Validation.

FIGURE 7.

   [93]FIGURE 7
   [94]Open in a new tab

   OPLS-DA analysis of pulmonary metabolites in SMPP mice treated with
   YQDP intervention. (A) Positive OPLS-DA. (B) Negative OPLS-DA.

FIGURE 8.

   [95]FIGURE 8
   [96]Open in a new tab

   (Continued).

FIGURE 9.

   [97]FIGURE 9
   [98]Open in a new tab

   Multiple comparison analysis of differential metabolites in lung tissue
   under negative ion mode. Compared with normal group, *p < 0.05, **p <
   0.01. Compared with model group, #p < 0.05, ##p < 0.01.

FIGURE 10.

   [99]FIGURE 10
   [100]Open in a new tab

   Heat map analysis of differential metabolites in lung tissue. (A)
   Positive. (B) Negative.

3.8 Enrichment analysis of differential metabolite pathways

   We performed pathway enrichment analysis of metabolites that underwent
   significant changes in mouse lung tissue after YQDP therapy, as shown
   in [101]Figure 11. Each circle in the image represents a different
   metabolic route. As the enrichment factor rises, so does the degree of
   differential metabolite enrichment in this channel. Based on the
   results, YQDP may be used to treat SMPP by controlling the metabolism
   of porphyrins, pyrimidines, cholines, fatty acids, sphingolipids,
   glycerophospholipids, ferroptosis, steroid hormones, and unsaturated
   fatty acids.

FIGURE 11.

   [102]FIGURE 11
   [103]Open in a new tab

   Bubble diagram of KEGG pathway enrichment analysis. (A) Positive. (B)
   Negative.

3.9 ELISA for detecting IL-6 and TNF-α expression in serum

   The mice in the model group had higher serum expression levels of IL-6
   and TNF-α than the mice in the normal group (p < 0.01), and the mice in
   the YQDP administration group had lower expression levels of IL-6 and
   TNF-α at all concentrations than the mice in the model group (p <
   0.01). The results are displayed in [104]Figures 12, [105]13.

FIGURE 12.

   FIGURE 12
   [106]Open in a new tab

   Serum IL-6 expression (pg/mL). **p < 0.01.

FIGURE 13.

   FIGURE 13
   [107]Open in a new tab

   Serum TNF-α expression (pg/mL). **p < 0.01.

3.10 Western blot for detecting PI3K, P-PI3K, AKT, P-AKT, NF-κB, P-NF-κB
protein expression in lung tissue

   As demonstrated in [108]Figure 14, the model group mice’s lung tissue
   showed an increase (p < 0.01) in the P-PI3K, P-AKT, and P-NF-κB protein
   expression ratio when compared to the normal group. In the lung tissue
   of mice in the positive group and YQDP groups for each dose, the ratio
   of P-PI3K, P-AKT, and P-NF-κB protein expression reduced (p < 0.05)
   when compared to the model group.

FIGURE 14.

   [109]FIGURE 14
   [110]Open in a new tab

   Effect of YQDP on the relative content of PI3K/AKT/NF-κB protein in
   SMPP mice. (A) Normal group. (B) Model group. (C) YQDP (1.552 g/kg).
   (D) YQDP (3.104 g/kg) group. (E) YQDP (6.208 g/kg) group. (F) Positive
   group. *p < 0.05, **p < 0.01.

4 Discussion

   MPP is one of the common community-acquired diseases in children caused
   by MP infection ([111]Rogozinski et al., 2017), with unremarkable
   respiratory symptoms and paroxysmal irritating cough as the prominent
   manifestation of MP infection, accompanied by coughing up and vomiting
   small amounts of mucus or mucopurulent sputum. In previous studies, our
   research team applied the theory of “dryness” and the concept of
   “drying toxins” to treat SMPP using the well-known prescription YQDP
   from the book “Chonglou Yuyao.” We conducted experiments from multiple
   angles, including immunoinflammation, epithelial-mesenchymal
   transition, oxidative stress, and water metabolism, and demonstrated
   the advantageous targeting of YQDP in intervening in MP infection.
   First, we conducted a pharmacodynamic component analysis and identified
   five chemical components in YQDP, including chlorogenic acid,
   paeoniflorin, forsythoside A, forsythin, and gallic acid. Through the
   use of experimental techniques such lung tissue pathology,
   ultrastructure observation, and pulmonary index, we assessed the
   effectiveness of the model and YQDP. According to the findings, YQDP
   considerably improved lung tissue pathology and decreased the pulmonary
   index in SMPP mice. In order to investigate the YQDP’s mechanism of
   action on SMPP mice further, we used UPLC-MS/MS technology to do a
   metabolomic study of lung tissue samples from normal, model, and
   YQDP-treated groups. This study screened 47 differential metabolites
   that changed after the addition of YQDP, mostly lipids, nucleosides,
   and fatty acids, which play important roles in inhibiting the immune
   inflammatory process and participating in multiple inflammatory
   processes ([112]Das, 2021; [113]Nieto-Garai et al., 2022). The research
   discovered that while YQDP intervention altered various endogenous
   metabolites in the model group’s lung tissue, it also altered pathways
   including the metabolism of α-linolenic acid, sphingolipids,
   glycerophospholipids, arachidonic acid, and unsaturated fatty acid
   biosynthesis, which had a therapeutic effect on SMPP mice. α-linolenic
   acid belongs to the ω-3 polyunsaturated fatty acid family and can
   generate oxidized lipids under the action of specific enzymes. In terms
   of inflammation, ω-3 polyunsaturated fatty acids can alleviate and
   inhibit inflammation ([114]Simopoulos, 2002; [115]Saini and Keum,
   2018). The synthesis of phospholipase is an important link that affects
   the metabolites of glycerophospholipids, and if the content is reduced,
   it will result in a large number of phospholipid metabolites
   accumulation. Glycerophospholipid metabolism is achieved through the
   hydrolysis of phospholipase liaison with the relevant signaling
   pathways of humoral metabolism, to promote the normal operation of the
   material and energy metabolism, to maintain metabolic balance and
   homeostasis ([116]Kouznetsova et al., 2019). Unsaturated fatty acids
   like arachidonic acid are essential for immunological response,
   inflammation, and antibacterial activities ([117]Yan et al., 2019;
   [118]Das, 2021). The study’s findings demonstrated that giving YQDP to
   SMPP mice decreased the amount of phosphatidylcholine in their lungs,
   which increased the amount of phosphatidylethanolamine—a material
   crucial for the metabolism of arachidonic acid in the
   glycerophospholipid metabolism pathway—and sped up the rate at which
   phospholipase hydrolyzed the lipid ([119]Zengin et al., 2019). YQDP may
   be able to treat MP infection-related lipid metabolic abnormalities by
   controlling glycerophospholipid metabolism. The immune system and
   inflammatory reactions are greatly influenced by the amounts of
   endogenous fatty acids and their metabolites. While saturated fatty
   acids tend to increase inflammation, certain unsaturated fatty acids
   have anti-inflammatory properties ([120]Rodriguez-Perez et al., 2017;
   [121]Kotlyarov and Kotlyarova, 2021). Nucleotide production and
   breakdown are regulated by purine metabolism. The primary cause of
   multiple organ damage and mortality in SARS-CoV-2-infected severe
   COVID-19 patients is cytokine storm. Research has revealed a robust
   association between cytokine storms and dysregulated purine metabolism
   in COVID-19 patients ([122]Sauer et al., 2012; [123]Zardini et al.,
   2020), suggesting a direct connection between hyperinflammation and
   dysregulated purine metabolism in respiratory tract infections.
   Additionally, studies have demonstrated the regulatory effects of the
   chemical components in YQDP, including chlorogenic acid, paeoniflorin,
   gallic acid, glycyrrhizic acid, and forsythoside, on the metabolism of
   glycerophospholipids, arachidonic acid, and sphingolipids ([124]Zhao,
   2018; [125]Cheng et al., 2020; [126]Duan et al., 2020; [127]Feng et
   al., 2020; [128]Li et al., 2022; [129]Wang et al., 2022).

   Phospholipids are essential components of pulmonary surfactants, with
   the ability to modulate local immune and inflammatory responses, lower
   alveolar surface tension, preserve alveolar fluid balance, engage in
   cell membrane protein recognition and conduction, and function as an
   essential secondary messenger in immune metabolic pathways downstream
   ([130]Chao et al., 2020). One important modulator of phospholipid
   conversion is phosphatidylinositide 3-kinases (PI3K), which activates
   protein kinase B (Akt) signaling pathways that control cellular
   activity. In order to induce the expression of inflammatory and
   chemotactic factors, activated Akt can enter the cell nucleus and
   activate downstream factor NF-κB. This can lead to the infiltration and
   aggregation of inflammatory cells at the site of inflammation, speeding
   up the onset and progression of inflammation ([131]Li et al., 2020;
   [132]Saravia et al., 2020). Following modeling, the model group’s lung
   tissues had much higher amounts of PI3K/AKT/NF-κB protein than the
   normal group’s, indicating that the stimulation of inflammatory factors
   had activated the PI3K/AKT/NF-κB signaling pathway. The ratio of
   P-PI3K, P-AKT, and P-NF-κB protein expression in lung tissues decreased
   following YQDP intervention, suggesting that YQDP could prevent the
   PI3K/AKT/NF-κB signaling pathway from being activated, lessen lung
   damage, and restore lung function in SMPP animals. The body releases
   tumor necrosis factor-alpha (TNF-α) as the first cytokine in reaction
   to a potentially harmful stimulus, and it is essential in starting the
   inflammatory cascade of cytokines. Co-stimulation of Interleukin-6
   (IL-6) production can occur through the combined action of TNF-α and
   other inflammatory agents. According to certain research, CGA can have
   anti-inflammatory properties, remove Akt phosphorylation, and decrease
   the expression of TNF-α and IL-6 ([133]Hou et al., 2016). The
   experiment’s findings demonstrated that YQDP intervention led to a
   decrease in the expression of TNF-α and IL-6 in serum, suggesting that
   YQDP can effectively suppress inflammatory factors’ expression and
   reduce the inflammatory response.

5 Conclusion

   In conclusion, YQDP affects SMPP model mice in a certain way. Based on
   the examination of biological markers and their associated metabolic
   pathways, conjecture has it that YQDP primarily functions by modulating
   pathways like the metabolism of α-linolenic acid, sphingolipids,
   glycerophospholipids, arachidonic acid, and unsaturated fatty acid
   biosynthesis. Additionally, it may potentially exert a regulatory
   influence via the PI3K/Akt/NF-κB signaling pathway.

Abbreviation

   Akt, Kinase B; AQP5, Aquaporin5; CAP, Community Acquired Pneumonia;
   CGA, Chlorogenic Acid; ELISA, Enzyme-linked immunosorbent assay; FT,
   Forsythin; FTA, Forsythoside A; IL-6, Interleukin-6; MP, Mycoplasma
   pneumoniae; MPP, Mycoplasma pneumoniae pneumonia; MUC5ac, Mucin 5ac;
   PAE, Paeonol; PASEF, Parallel accumulation serial fragmentation; PF,
   Paeoniflorin; PI3K, Phosphatidylinositide 3-kinases; SMPP, Severe
   mycoplasma pneumoniae pneumonia; TCM, Traditional Chinese medicine;
   TNF-α, Tumor necrosis factor α; YQDP, Yangyin Qingfei Decoction Plus.

Acknowledgments