Qingwen Hufei Granules Attenuate Lipopolysaccharide-Induced Acute Lung Injury by Suppressing NLRP3 Inflammasome-Dependent Pyroptosis

Introduction

Acute lung injury (ALI) is a major health burden worldwide and is associated with high morbidity and mortality rates. The presenting features are acute respiratory insufficiency, rapid onset of polypnea, cyanosis resistant to oxygen therapy, and diffuse alveolar infiltrates observed on chest radiography.^1,2 Various etiologies, including acute pneumonia, sepsis, and severe trauma can cause ALI. Lipopolysaccharide (LPS), the main constituent of the cell wall of Gram-negative bacteria, is an infectious agent that causes ALI. As one of the common critical clinical conditions, ALI progresses rapidly and usually degenerates into acute respiratory distress syndrome (ARDS), characterized by acute respiratory distress and refractory hypoxemia, which can cause respiratory failure. The mortality rate of acute lung injury ranges from 33% to 55%, posing a serious threat to human health and putting immense pressure on the global burden of health resources.^3,4 Current clinical treatments for ALI include mechanical ventilation, bronchodilators, corticosteroids, antibiotics, and other supportive therapies. Unfortunately, these methods have several limitations including poor patient tolerance and multiple side effects.⁵

The “holistic concept” and “syndrome differentiation” approaches in TCM provide a particular advantage in the treatment of ALI. TCM can inhibit oxidative stress, reduce inflammation, and improve lung tissue damage, thus playing an important role in treatment of ALI.^6,7 QHG manufactured by Professor Cao Liping, a well-known TCM professional, is derived from the Chinese prescriptions “Yinqiao San”, “Xuanmai Ganjie Decoction”, and “Sijunzi Decoction”. It contains 16 herbs, including Lonicera japonica Thunb. (Jinyinhua), Forsythia suspensa (Thunb.) Vahl (Lianqiao); Isatis indigotica Fort. (Daqingye), Scrophularia ningpoensis Hemsl. (Xuanshen), Prunus armeniaca L.var.ansu Maxim. (Kuxingren), Fritillaria thunbergii Miq. (Zhebeimu), Platycodon grandiflorum (Jacq.)A. DC. (Jiegeng), Perilla frutescens (L.) Britt. (Zisuye), Saposhnikovia divaricata (Turcz.) Schischk. (Fangfeng), Atractylodes macrocephala Koidz. (Chaobaizhu), Poria cocos (Schw.) Wolf (Fuling), Artemisia capillaris Thunb.(Yinchen), Codonopsis pilosula (Franch.)Nannf. (Dangshen) and Lophatherum gracile Brongn. (Danzhuye), Ophiopogon japonicus (L. f) Ker-Gawl. (Maidong) and Glycyrrhiza uralensis Fisch. (Gancao). We analyzed the effective chemicals in QHG collected by UHPLC-Q Exactive FocusMS/MS using our crew: terpenoids, phenylpropanoids, chromogen flavonoids, and organic acids. These components have been shown to exert anti-ALI effects.^8–10

The primary pathological features of ALI include diffuse alveolar injury and breakdown of the endothelial barrier of microcirculation vessels. ALI is a complex inflammatory cascade that involves cell death.¹¹ The NLRP3 inflammasome plays a crucial role in innate immunity and maintaining microenvironmental homeostasis. However, excessive activation can lead to excessive inflammatory responses and worsening of disease progression in various inflammatory diseases.¹² During ALI, abnormally activated NLRP3 inflammasome activates Caspase-1 and Cleaves gasdermin D to form a peptide in the nitrogen-terminal active domain, which causes cell membrane rupture, releases mature IL-1β, and induces pyroptosis in macrophages.^13,14

In this study, we established a fingerprint and content determination method for QHG by HPLC. We then analyzed the effect of QHG on inflammatory changes in RAW264.7 macrophages under LPS stimulation. Additionally, we conducted an experimental study on the pharmacodynamic and anti-inflammatory effects of QHG in an LPS-induced mouse model. Finally, we performed a transcriptomic analysis of mouse lung tissue and verified the mechanism by which QHG alleviates ALI using qRT-PCR, Western blotting, immunohistochemistry, and immunofluorescence.

In conclusion, QHG have shown remarkable efficacy in the clinical treatment of respiratory diseases. They contain multiple active ingredients that are effective against acute lung injury. However, the specific mechanism of action remains unknown, and it is worth exploring whether they exert their effects through NLRP3 inflammasome.

Our study is of great significance for the research on the treatment of acute lung injury with traditional Chinese medicine.

Materials and Methods

Reagents

LPS (Escherichia coli 0111:B4) was procured from Sigma-Aldrich, and Dexamethasone (LB22207, XianJu) was used in this study. The concentrations of IL-6, IL-1β, and TNF-α were quantified using enzyme-linked immunosorbent assay (ELISA) kits (Bioswamp). ROS and Myeloperoxidase(MPO) assay kits were obtained from Nanjing Jiancheng Bioengineering Institute. Antibodies NLRP3 (ab270449), GSDMD (ab219800) were purchased from Abcam. ASC (#67824) was obtained from Cell Signaling Technology, and Caspase-1 (22915-1-AP), IL-1β (26048-1-AP) were obtained from Proteintech. All antibodies were diluted at a ratio of 1:1000. β-Actin was purchased from zenbio (Chengdu, China), dilution ratio is 1:10,000.

Preparation of QHG

Qingwen Hufei granules were self-produced preparations by the Shaanxi Provincial Hospital of Chinese Medicine (Xi’an, China),10g x 6 bags/box, and identification of product 20220506.

Chemical Analysis of QHG

The QHG was finely ground, and 2.0 g was accurately weighed and placed in a 100 mL conical flask with a plug. It was dissolved in 30 mL of methanol, weighed, and treated with ultrasound (50 kHz frequency, 300 W power) for 30 min. After cooling to room temperature, the solution was shaken, filtered, and dried. The total volume was adjusted to 10 mL in a volumetric flask. Subsequently, the solution was filtered through a 0.45 μm microporous filter membrane, and the resulting filtrate was used for subsequent analyses.

Control standards were accurately weighed and placed in 10 mL volumetric flask. The volume was fixed after diluting with 50% methanol. The following compounds were used as standards: neochlorogenic acid (CHB220328), chlorogenic acid (CHB231010), cryptochlorogenic acid (CHB230830), forsythia glycoside A (CHB231017), 3,5-O-dicaffeoylquinic acid (CBH231227), 4,5-caffeoyl quinic acid (CHB240104), and angloside C (CHB240217), with a purity of ≥98%. All control products were purchased from Chengdu Cloma Biotechnology Co. Ltd.

To establish an HPLC chromatographic analysis method were conducted on an Agilent ZORBAX Eclipse Plus-C18 column (250 mm × 4.6 mm, 5 μm). The mobile phase consisted of methanol (A) and 0.1% phosphoric acid (B), with a flow rate of 0.8 mL/min, and an injection volume of 10 μL. The gradient elution sequences are listed in Table 1.

Table 1 Gradient Elution Program

Animals and Treatments

Male C57 mice were acquired from Jiangsu Huachuang Cigna Pharmaceutical Technology Co. Ltd. (certificate no. SCXK 2020-0009). The experimental protocols for animal studies were approved by the Ethics Committee of the Shaanxi Academy of TCM (Animal Approval Number: (2022) Animal Ethical Review Number (15)). All procedures were performed in strict accordance with the Guidelines for Animal Experimentation (ARRIVE).¹⁵ The animal experiment protocol of this study strictly adheres to the 《Guidelines for the Ethical Review of Animal Welfare》 (GB/T 35892-2018). Prior to experimentation, the mice were acclimatized for three days under controlled conditions with 12-hour light and dark cycles. SPF-grade male C57 mice, with an average weight of 22 ± 2 g, were randomly assigned to six groups (n=60): control, LPS (5 mg/kg), LPS + dexamethasone (DXM, 2 mg/kg), LPS + low-QHG (QL, 3.075 g/kg), LPS + medium-QHG (QM, 6.15 g/kg), and LPS + high-QHG (QH, 12.3 g/kg). QHG and DXM were administered via gavage for 7 days, whereas the control and LPS groups received saline. On the final day, 1.5 hours post-gavage, all groups, barring the control, were administered an intratracheal instillation of LPS.^16,17 Subsequently, the mice were euthanized using an overdose of pentobarbital sodium, and BALF, serum, and lung tissues were preserved at −80°C for analysis.

Wet/Dry Weight Ratio

The chest of each mouse was opened and the lung was removed. After separating the trachea and esophagus, the lung lobe was weighed immediately to obtain a wet mass. After euthanasia, the lungs were desiccated at 60°C for 72 h until consistent weight was achieved, and the wet-to-dry (W/D) weight ratio was calculated.¹⁸

Histopathologic Evaluation of Lung Tissue

The lung tissues were preserved in 4% paraformaldehyde, subjected to gradient ethanol dehydration, and embedded in paraffin. After cooling, the tissues were sectioned and stained with hematoxylin and eosin (H&E) for microscopic evaluation of pulmonary changes.¹⁹

Myeloperoxide (MPO) Activity and Reactive Oxygen Species (ROS) Assay

MPO activity in the lung tissues was quantified using spectrophotometric colorimetry. Tissue homogenates were prepared, cooled, and centrifuged at 4°C and 6000 rpm for 15 minutes. This was followed by additional centrifugation at 4°C and 12,000 rpm for 15 minutes. The MPO activity (U/g) was determined using the following formula:

MPO activity (U/g) = (measured OD value − control OD value) / 11.3 × sample weight.

ROS levels in the lung tissue were determined using the double-antibody sandwich method. The lung tissues were homogenized using a chilled tissue homogenizer at 3000 rpm for 20 min, after which the supernatants were separated. ROS levels were quantitatively assessed by analyzing the color intensity, which was directly correlated with the ROS concentration in the samples. OD was measured at 450 nm using a spectrophotometer.²⁰

Bronchoalveolar Lavage Fluid (BALF) Analysis

After blood extraction, the mice were placed on an operating table, their chest was opened, and their lungs were ligated. Pre-cooled PBS buffer (1 mL) was injected into the trachea, and the lungs were lavaged back and forth twice to collect alveolar lavage fluid. The samples were then centrifuged at 3000 rpm for 15 min at 4°C. The resultant supernatant was examined using ELISA kits, following the manufacturer’s protocols, to assess the levels of IL-6, IL-1β, and TNF-α.²¹

Levels of TNF-α, IL-6, and IL-1β in Serum

After a 72-hour period of modeling, mice were euthanized, and their eyeballs were excised. The collected supernatants were centrifuged under the same conditions, and the cytokines IL-6, IL-1β, and TNF-α were measured using ELISA kits, according to the manufacturer’s instructions.²²

Cytokine Assay

RAW264.7 cells, sourced from the Cell Resource Center at the Institute of Basic Medicine of the Chinese Academy of Medical Sciences (fifth passage), were cultured at a density of 2×10⁵ cells/mL. The cell viability assay was conducted using the Cell Counting Kit-8 (CCK-8) method. The experiment included three groups: blank control (without LPS or drug), model (LPS at a final concentration of 100 ng/mL), and LPS + drug treatment (LPS at 100 ng/mL + QHG freeze-dried powder at concentrations of 5, 10, 20, 40, 80, and 160 µg/mL) groups. Each group had three to four replicates and the experiment was repeated three times. For each experimental group, the corresponding drug solution was added for 6 h, followed by addition of LPS to a final concentration of 100 ng/mL. The control group was administered an equal volume of DMEM. After 24 h of incubation, NO release in the supernatant was measured using a NO kit, and ROS production was assessed using a ROS kit. Intracellular ROS levels were quantified using flow cytometry (BECKMAN COULTER, Cytoflex). ELISA kits were used to measure the secretion of TNF-α, IL-6, and IL-1β from the supernatant.²³

Transcriptome Analysis of Lung Tissue

Total RNA was extracted from lung tissue using the TRIzol method (Life Technologies), and RNA quality was assessed by agarose gel electrophoresis. Transcriptome libraries were constructed using mRNA purification and fragmentation, followed by cDNA first-strand synthesis, second-strand synthesis, adapter ligation, and purification of ligation products. The DNA 1000 Assay Kit (Agilent Technologies) was used for quality assessment. Differential gene expression analysis was performed using the DESeq2 package in R software.²⁴

qRT-PCR Analysis

The lung tissue was lysed using Trizol, total RNA from the lung was extracted for concentration assessment, the sample was diluted, and subsequently reverse-transcribed into cDNA (condition: 20μL reaction system). qRT-PCR conditions: pre-denaturation at 95°C for 30 seconds, denaturation at 95°C for 10 seconds, annealing and elongation at 60°C for 30 seconds, with a total of 40 cycles conducted. The mRNA expression levels of IL-18 were quantified using the 2^−ΔΔCt method. The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. The sequences are shown in Table 2.

Table 2 Primers Sequencing Used for mRNA Quantitation

Western Blotting

Lung tissues were lysed in RIPA buffer and protein concentrations were quantified using the BCA assay. Proteins were separated by SDS-PAGE and transferred onto (lyvinylidene difluoride a PVDF) membranes. The membrane was blocked for two hours at ambient temperature and subsequently incubated with primary antibody at 4°C overnight. All the antibodies were diluted using the antibody diluent, with a dilution ratio of 1:1000. After four TBST washes, the membrane was incubated with secondary antibody for two additional hours. The protein bands were visualized using an ECL luminescent reagent, and band intensities were analyzed using the Image J software.²⁵

Immunohistochemistry

Paraffin-embedded sections were sequentially deparaffinized in three different solutions for 15 min each followed by 5 min of anhydrous ethanol. Antigen retrieval was performed using a microwave in the retrieval solution for 20 minutes. After cooling, the sections were washed three times with PBS for 5 min each and treated to block endogenous peroxidase activity. Blocking was performed using bovine serum albumin (BSA) for 20 min at room temperature. The sections were incubated with primary antibodies overnight at 4°C in a humidified chamber. For DAB staining, fresh DAB solution was prepared and applied to the tissue for color development at room temperature. Color development was monitored under a microscope. The sections were counterstained with hematoxylin for 3 min, washed with tap water, and stained with blue water.²⁶

Immunofluorescence

Sections were prepared as described previously for immunohistochemistry. For antigen retrieval, the sections were dipped in the retrieval solution and microwaved for 20 min, followed by endogenous peroxidase blocking. After the primary antibody application, the sections were sealed with BSA for 30 min at room temperature before the application of the secondary antibody. DAPI was used for nuclear staining and the sections were incubated for 10 min. After three PBS washes, the slides were examined and imaged using fluorescence microscopy.²⁷

Statistical Analysis

Data are presented as the mean ± standard deviation (SD). Statistical analysis was conducted using one-way analysis of variance (ANOVA), with statistical significance set at P-value of < 0.05. Principal component analysis (PCA) was performed using an Illumina NovaSeq X Plus system (Gene Denovo Biotechnology Co., Ltd., Guangzhou, China). Graphical representations were produced using GraphPad Prism version 9.0.

Results

Determination of Active Components in QHG

Chromatographic analysis revealed peaks 1–7, which were identified as Neochlorogenic acid, Chlorogenic acid, Cryptochlorogenic acid, Forsythoside A, Isochlorogenic acid A, Isochlorogenic acid C, and Angoroside C, respectively, and aligned with the reference substances (Figure 1A and B).

Figure 1 Chromatogram of QHG and compound structure. (A) QHG sample; (B) Mixed control sample.

Effects of QHG on RAW264.7 Cells

LPS successfully stimulated RAW264.7 macrophages to generate an inflammatory cell model. The effect of QHG on the secretion of ROS, NO, IL-1β, TNF-α, and IL-6 in cellular supernatants was evaluated. As illustrated in Figure 2, the LPS-treated group exhibited a significant increase in ROS, NO, TNF-α, IL-6, and IL-1β levels in the supernatant. Conversely, the QHG-treated groups (20, 40, 80, and 160 µg) showed a significant reduction in ROS, IL-6, and IL-1β levels in a concentration-dependent manner.

Figure 2 Effects of QHG on the secretion of NO, ROS, TNF-α, IL-6 and IL-10 in RAW264.7 cells treated with LPS. (A) Cell viability was determined by LPS; (B) Cell viability was measured by QHG; (C and D) Comparison of ROS activity in six groups; (E) Comparison of NO content in nine groups; (F) Comparison of IL-6 content in six groups; (G) Comparison of IL-1β content in six groups; (H) Comparison of TNF-α content in six groups. These data are represents as mean ± SD (n=3-4). ns P > 0.05. Compared with the control group *p < 0.05, **p < 0.01; compared with the LPS group ##p < 0.01.

Preventive Effects of QHG on ALI in Mice

The control group showed no abnormalities in lung tissue structure, with clear alveoli and chambers, and no hemorrhagic edema or inflammatory cell infiltration. In contrast, the model group exhibited disorganized lung tissue, an altered alveolar structure, pulmonary capillary congestion, cellular bleeding, inflammatory cell infiltration, and significant tissue damage. Compared with the model group, the lung tissue of QM-treated and QH-treated mice showed significant improvements, with near-normal alveolar structure and effective alleviation of inflammatory cell infiltration in the lung interstitium (Figure 3A). On the first day after the model was established, the body weight of the LPS group mice decreased significantly compared to the blank group. The body weight of the treatment group was significantly higher than that of the control group. On the third day, the body weight of the LPS group failed to recover and was significantly lower than that of the control group (Figure 3B). The LPS group showed a significantly increased W/D ratio (Figure 3C), ROS activity (Figure 3D), and MPO activity (Figure 3E), which was reduced in the QM, QH, and DXM treatment groups. These results suggest that LPS induces edema and cellular damage, whereas QM and QH exert significant protective effects.

Figure 3 Protective effects of QHG on LPS-induced ALI in mice. (A) H&E result of lung tissue for histopathologic analysis (scale bar = 200 μm); (B) Body weight; (C) W/D ratio; (D) ROS viability in lung; (E) MPO viability in lung. These data are represents as mean ± SD (n=6). ns P > 0.05. Compared with the control group *p < 0.05, ***p < 0.001, ****p < 0.0001; compared with the LPS group #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001.

QHG Attenuated Inflammation in ALI

LPS-induced ALI is characterized by elevated levels of TNF-α, IL-1β, and IL-6. These cytokines were quantified in the bronchoalveolar lavage fluid (BALF), serum, and lung tissue. ALI can cause an inflammatory response in the lungs, and this local inflammatory response can trigger the migration of inflammatory cells, which in turn leads to a systemic inflammatory response. This experiment measured the levels of inflammatory factors in the serum, lung tissue and BALF of mice. As shown in Figure 4A–C, there was a significant increase in these cytokines in the LPS group, whereas their levels were substantially decreased in the QM, QH, and DXM groups. These results indicate that QHG significantly alleviates ALI; therefore, we measured the levels of inflammatory cytokines in the BALF, serum, and lung tissue of mice. The results revealed that the expression of TNF-α, IL-1β, and IL-6 in the serum, BALF, and lung tissues of mice in the LPS group was significantly increased. The levels of TNF-α, IL-1β, and IL-6 in BALF, serum, and lung tissues were significantly decreased in QM, QH, and DXM (Figure 4A–C), suggesting that QHG can significantly inhibit ALI through anti-inflammatory effects.

Figure 4 QHG suppressed inflammatory cytokine. (A–C) Inflammatory cytokines content in BALF, serum and lung tissues. These data are represents as mean ± SD (n=6). ns P > 0.05. Compared with the control group **p < 0.01, ***p < 0.001, ****p < 0.0001; compared with the LPS group #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001.

Transcriptomics Analysis of QHG

PCA was conducted using the R software. PCA results indicated distinct separation among the control, LPS, and QH groups, revealing notable differences in gene expression in the lung tissues of ALI mice compared with controls (Figure 5A). A marked reversal in gene expression was observed after QH treatment. Differentially expressed genes (DEGs) were identified using DESeq2 software between two different groups with the parameter set to false discovery rate (FDR) < 0.05 and absolute fold change (FC) ≥ 2.²⁸ Compared to the control group, 5433 DEGs were identified in the LPS group, with 1976 upregulated and 3457 downregulated genes. QH and LPS treatment resulted in the identification of 127 DEGs, including 60 upregulated and 67 downregulated genes (Figure 5B). Further analysis included differential volcano plots based on DEG data for each control group (Figure 5C and D). To identify affected pathways, KEGG enrichment circle maps (Figure 5E and F) and bubble maps (Figure 5G and H) were generated. The purple plates, representing metabolism, showed significant increases for both LPS and QH treatments (Figure 5E and F), indicating that QH-induced changes in biological function may concentrate on metabolites in the ALI mouse model. A total of 120 KEGG pathways were enriched by QH and LPS treatments, with the top 13 most significant pathways presented as scatter plots (Figure 5H). Notably enriched pathways included the “NLR signaling pathway”, “hepatitis C”, “measles”, “influenza A”, and “Epstein-Barr virus infection”, all with statistical significance at p < 0.01. These findings suggested that QHG may affect these specific pathways, leading to reduced ALI symptoms in mice.

Figure 5 Continued.

Figure 5 Transcriptomics analysis treated with QH in mice. (A) Show the PCA scores plots of control, LPS and QH groups; (B) Show the differential gene data statistical map; (C and D) Show the volcano map DEGs of control, LPS and QH groups; (E and F) Show the KEGG enrichment diagram of control, LPS and QH groups; (G and H) GO and KEGG enrichment analysis for DGEs of LPS vs QH; (I and J) GSEA enrichment analysis about the NOD-like signaling pathway, in GESA for Con vs LPS, LPS vs QH.

Gene Set Enrichment Analysis (GSEA) of the NLR signaling pathway revealed an overall upregulation trend in the LPS vs control group (Figure 5I and J). After QH treatment, a significant downregulation was observed in most of the upregulated genes. By analyzing these genes Collectively within the NLR pathway, Nampt, Casp4, Nfkbia, IL-6, Cxcl1, Cxcl2, Ikbke, Nod2, Tnfaip3, Nlrp3, Mefv, Birc3, and Gsdmd might play crucial regulatory roles in modulating this pathway. Therefore, it is reasonable to speculate that QH potentially exerts a protective effect against LPS-induced ALI in mice by stimulating the interplay between signaling pathways through its influence on relevant genes within the NLR signaling pathway. However, the precise mechanism by which QH alleviates LPS-induced ALI via the NLR signaling pathway warrants further investigation.

Effects of QHG on NLR Signaling Pathway Activation in ALI Mouse Lung Tissues

GSEA highlighted the involvement of crucial proteins in the NLR signaling pathway, such as Caspase-1, ASC, IL-1β, GSDMD, and NLRP3. Western blot analysis and qRT-PCR analysis, depicted in Figure 6, confirmed that QH significantly suppressed LPS-induced increase in Caspase-1, NLRP3, and IL-1β levels, QH can reduce the expression of IL-18 in mRNA. Protein expression was assessed in mouse lung tissue, and immunofluorescence results (Figure 7) showed a significant reduction in the expression of ASC, GSDMD, IL-1β, NLRP3, and Caspase-1 in the QH group compared to that in the LPS group. Immunohistochemical findings (Figure 8) further supported that the protein levels of ASC, GSDMD, IL-1β, NLRP3, and Caspase-1 were markedly lower in the QH group than in the LPS group, indicating the substantial inhibitory effect of QH on key proteins in the NLR signaling pathway.

Figure 6 Western blots and quantification. (A) Western blots of ASC, GSDMD, IL-1β, NLRP3, Caspase-1 in each group of mice lungs. (B–F) The expression levels of ASC, GSDMD, IL-1β, NLRP3, Caspase-1 proteins in mice lungs. (G) The mRNA expression levels of IL-18 in mice lungs. These data are represents as mean ± SD (n=3). ns P > 0.05. Compared with the control group *p < 0.05, **p < 0.01; compared with the LPS group ##p < 0.01, ###p < 0.001.

Figure 7 Immunofluorescence and quantification. (A and B) Representative immunofluorescence for ASC, GSDMD, IL-1β, NLRP3, Caspase-1 after DAPI staining in mice lungs. Scale bar: 50μm. (C–G) The quantification of positive area in mice lung. These data are represents as mean ± SD (n=3). ns P > 0.05. Compared with the control group **p < 0.01, ***p < 0.001, ****p < 0.0001; compared with the LPS group #p < 0.05, ##p < 0.01.

Figure 8 Immunohistochemical and quantification. (A) Representative immunostaining for ASC, GSDMD, IL-1β, NLRP3, Caspase-1 in mice lungs. Scale bar: 40 μm. (B–F) Quantification of ASC, GSDMD, IL-1β, NLRP3, Caspase-1 fluorescence intensity in six groups. These data are represents as mean ± SD (n=3). ns P > 0.05. Compared with the control group **p < 0.01, ****p < 0.0001; compared with the LPS group #p < 0.05, ##p < 0.01, ####p < 0.0001.

Discussion

Following the onset of ALI, a rapid inflammatory response results in endothelial cell damage and epithelial barrier dysfunction, and further promotes immune cell infiltration into the lung interstitium and alveoli. Macrophages and neutrophils involved in this process produce excessive pro-inflammatory cytokines, leading to a cytokine storm. This reaction increases the permeability of pulmonary microvessels and causes pulmonary edema, resulting in severe lung injury and multiple organ dysfunction.^29–31 Recent research has demonstrated that TCM offers distinct therapeutic advantages in the treatment of ALI.^32–34 Clinically, QHG effectively treats numerous patients with upper respiratory tract infections, particularly influenza. Therefore, it is hypothesized that QHG has potential therapeutic effects on ALI in mice, and its pharmacodynamics and mechanisms of action are currently being investigated through relevant experiments.

HPLC analysis identified seven primary chemical components of QHG. The anti-inflammatory activity of cryptochlorogenic acid may inhibit LPS-induced inflammatory responses via the Nrf2/HO-1 signaling pathway in RAW 264.7 macrophages.³⁵ Similarly, Forsythiin A has been reported to alleviate ALI by reducing inflammation and restoring the integrity of the lung and colon epithelial barriers via the PPAR-γ/RXR-α pathway.³⁶ Isochlorogenic acid A was identified, which has the ability to inhibit the Nf-κB/NLRP3 signaling pathway, thereby reducing ALI.³⁷ In a mouse model of LPS-induced ALI, QHG significantly reduced IL-6, IL-1β, and TNF-α levels in the serum, alveolar lavage fluid, and lung tissues, potentially mitigating of ALI-related injury.^38,39 In this context, pathological findings also demonstrated that QHG remarkably ameliorated ALI-induced pathological changes by mitigating edema and the infiltration of inflammatory cells within the lung tissue, thereby reducing hemorrhage. The rupture of inflammatory factors leads to an imbalance in the oxidative and antioxidant mechanisms of the lungs, which increases oxidative stress and induces apoptosis.^40,41 MPO and SOD levels are indicators of lung tissue inflammation.⁴² In the present study, QHG significantly reduced the ROS and MPO levels in the lung tissues of mice. Thus, the improvement of QHG in LPS-induced ALI may be due to the anti-inflammatory, antioxidant, and anti-apoptotic mechanisms.

Transcriptome sequencing is a powerful tool for exploring RNA expression changes in lung tissue, uncovering disease mechanisms, identifying drug targets, comprehensively understanding abnormal gene expression during ALI, and elucidating potential molecular mechanisms of QHG in ALI treatment.⁴³ Transcriptome analysis in this study confirmed that the NLR signaling pathway was differentially enriched, and that the anti-ALI process of QHG involved factors such as Nampt, Casp4, IL-6, Cxcl2, Nod2, Tnfaip3, Nlrp3, and gsdmd. This may indicate that these factors are crucial for improving ALI. To confirm the results of transcriptome analysis, qRT-PCR, Western blot analysis, immunohistochemistry, and immunofluorescence assays were performed. The experiments further confirmed that the NLR signaling pathway is the key pathway for the QHG treatment of ALI, and its key targets, such as Caspase-1, ASC, IL-1β, GSDMD, and NLRP3, were significantly improved in LPS-induced lung injury caused by LPS. The inflammasome is a protein complex that includes the NLR family and is very important in innate immunity. The NLRP3 inflammasome, a complex containing an N-terminal PYD, is highly representative of this pathway because of its ability to bind ASC and pro-caspase-1, thus forming the NLRP3 inflammasome. To date, activation of the NLRP3 inflammasome has been documented to result in autocleavage of caspase-1, which further cleaves pro-IL-1β and pro-IL-18 into their mature forms, IL-1β and IL-18. Caspase-1 can also promote the cleavage of GSDMD and release the inflammatory subunit GSDMD-N, thereby inducing an inflammatory response.⁴⁴ Furthermore, activation of the NLRP3 inflammasome has been associated with the acceleration of ALI.⁴⁵ This study also showed that QHG exerts its protective role in LPS-induced ALI in mice, mainly by modulating key proteins in the NLR signaling pathway, including the inhibition of inflammasome cell pyroptosis.These results suggest that QHG could be developed into a therapeutic agent for treating ALI (Figure 9). At present, our research team has established a cell pyroptosis model suitable for studying inflammasomes. In the future, we plan to investigate the effects of silencing or overexpressing the key gene NLRP3 to observe the influence of Qingwen Hufei Granules on NLRP3 inflammatory pyroptosis. The results will be verified in NLRP3 knockout model mice based on the experimental findings.

Figure 9 Mechanism of QHG in the alleviation of ALI. “⊣” means QHG can inhibit NLRP3 inflammasome-dependent pyroptosis. “→” means PAMPs/DAMPs can activate key genes on the NOD-like signaling pathway.

QHG was shown to suppress LPS-induced inflammatory responses in RAW264.7, inhibit peroxide reactions, and further reduce inflammatory factor production. Macrophages serve as natural immune cells for the regulation of inflammation and tissue repair, and have drawn significant research interest. Additionally, mesenchymal stem cells (MSCs) from various tissues have been shown to aid in tissue regeneration and inflammation reduction. The primary mode of MSC involvement in tissue repair is via a paracrine mechanism, particularly via exosomes. Studies have shown that alveolar macrophages are the main cellular targets of MSC-derived exosomes in mitigating lung inflammation and injury,^3,46,47 providing new directions for future QHG research. QHG can relieve wind and heat, clear the lungs, alleviate cough, and strengthen the spleen and stomach. Herbs such as DangShen (Codonopsis pilosula (Franch.)Nannf.), ChaoBaizhu (Atractylodes macrocephala Koidz.), and FuLing (Poria cocos (Schw.) Wolf) help strengthen and protect the acquired roots. In the realm of TCM, recent years have seen a growing acknowledgment among experts of the theory concerning the interconnectedness of viscera, especially the concept that “the lung and large intestine are interior-exteriorly related”, in managing respiratory ailments. Stemming from this concept, the “lung-gut axis” theory has been proposed to further understand and treat respiratory diseases. The mechanism of action is primarily reflected in the regulation of bacterial metabolism and immune mechanisms of intestinal microorganisms.^48–50 Therefore, future research on the QHG can be based on the “lung-gut axis” theory.

Conclusion

In conclusion, our study indicated that QHG attenuates lipopolysaccharide-induced acute lung injury by suppressing NLRP3 inflammasome-dependent pyroptosis. Inhibition of the NLR signaling pathway is one of the mechanisms by which QHG affects ALI treatment.