Ephedrae Herba (No. 20200103), Gypsum Fibrosum (No. 20200327), Rhei Radix Et Rhizoma (No. 20200107), Belamcandae Rhizoma (No. 20200412), Asteris Radix Et Rhizoma (No. 20200310), Farfarae Flos (No. 20200411), Citri Reticulatae Pericarpium (No. 20200511), Pinelliae Rhizoma Praeparatum Cum Zingibere Et Alumin (No. 20200717), Poria (No. 20200904), Armeniacae Semen Amarum (No. 20200414), Cicadae Periostracum (No. 20200813), Fritillariae Thunbergii Bulbus (No. 20200907), Taraxaci Herba (No. 20200528), and Platycodonis Radix (No. 20200816) were provided by Beijing Bencaofangyuan Pharmaceutical Co., Ltd. (Beijing, China). The standards including alanine, caffeic acid, quercetin, β-sitosterol, chrysophanol, amygdalin, and hesperidin were purchased from National Institutes for Food and Drug Control. Lipopolysaccharides (LPS, from Escherichia coli O55:B5, abs47014848, Absin, Shanghai, China) and dexamethasone (D4902, Sigma-Aldrich, St. Louis, MO, United States) were purchased from Absin and Sigma-Aldrich.

QWZK comprises 14 herbs: Ephedrae Herba, Gypsum Fibrosum, Rhei Radix Et Rhizoma, Belamcandae Rhizoma, Asteris Radix Et Rhizoma, Farfarae Flos, Citri Reticulatae Pericarpium, Pinelliae Rhizoma Praeparatum Cum Zingibere Et Alumin, Poria, 6.30 % Armeniacae Semen Amarum, Cicadae Periostracum, Fritillariae Thunbergii Bulbus, Taraxaci Herba, and Platycodonis Radix. The QWZK was acquired as described above. The specimens (No. 20211009) were deposited in the Beijing Research Institute of Chinese Medicine, Beijing University of Chinese Medicine.

The preparation methods of the QWZK powder were as follows: the crude drugs of QWZK accurately weighed 1.43 kg. These drugs were soaked in 14.30 L (10 times, w/v) pure water for 30 min and was then boiled for 2 h. Subsequently, they were boiled in 11.40 L (8 times, w/v) pure water for 1 h twice.

The extracts were filtered through three-layer gauze, then combined and concentrated to 66.50 ml in a rotary evaporator at 75°C. The concentrate was vacuum dried, and 0.446 kg dry powder was obtained. The extract rate (%) = extract dry powder/total quality of crude drugs. Therefore, the extract rate of QWZK powder was 31.20%. The powder was used in the follow-up experiments.

According to the body surface area (BSA) scaling for converting the dose of a test drug from human clinical trials to animal species (Mahmood, 2007; Blanchard & Smoliga, 2015), the test doses of QWZK in animals were 3, 6, and 12 g/kg/day and that of an adult human was 71.5 g/day of crude drugs. The body weight of an adult human is 70 kg, and the convert coefficient is 6. Based on the extract rate, the text dose of QWZK powder were 0.94, 1.87, and 3.74 g/ kg/ day in animals.

Liquid chromatography was performed using a Dionex Utimate 3000 UHPLC Plus Focused Ultra High-Performance Liquid Chromatography System (Thermo Scientific, Santa Clara, CA, United States). Chromatographic separation was achieved through a ACQUITY UPLC C18 column (2.1 mm × 100 mm, 1.7 mm particles) at a flowrate of 0.3 ml/ min, defended by a high-pressure column prefilter (2 mm) (Shimadzu, Kyoto, Japan) at 35°C. Mass spectrometric detection was performed with an LTQ-Oribitrap XL linear ion trap tandem electrostatic field orbital trap mass spectrometer (Thermo Scientific, Santa Clara, CA, United States) in positive and negative ion modes, which was equipped with an electrospray ion source in MRM modes.

QWZK powder was accurately weighed 1.00 g and added into 10.00 ml methanol, ultrasonically treated for 45 min using an ultrasonic cleaning instrument (KQ-500DB CNC, Kunshan Ultrasonic Instrument Co., Ltd., Kunshan, Jiangsu, China), and the solution was filtrated through 0.22 μm microporous membrane. The standards such as alanine, caffeic acid, quercetin, β-sitosterol, chrysophanol, amygdalin, and hesperidin were weighed in precision, dissolved in methanol with a standard solution of 1 mg/ml, and filtered through 0.22 μm microporous membrane. Samples or strands were separated on an ACQUITY UPLC C18 column (2.1 mm × 100 mm, 1.7 mm) at 35°C. The mobile phase consisted of 0.1% formic acid aqueous solution (A) and acetonitrile solution (B). The gradient elution conditions were as follows: 0–6 min (90–60% A), 6–9 min (60–40% A), 9–42 min (40–20% A), and 42–60 min (20–90% A). The flowrate was 0.3 ml/min, and the injection volume was 3.0 μl.

Electrospray ionization mass spectrometry (ESI-MSP) analyses were performed on an LTQ-Oribitrap XL linear ion trap tandem electrostatic field orbital trap mass spectrometer (Thermo Scientific, Santa Clara, CA, United States). Samples of QWZK were detected in positive ion detection mode, and the spray and capillary voltages were set to 4.0 KV and 35.0 V, respectively. The tube lens voltage was 110 V, and the source temperature was set to 350°C. Nitrogen (purity >99.99%) was used as both the sheath gas (40 arb) and auxiliary gas (20 arb). Then, samples were analyzed in negative ion detection mode, with the spray and capillary voltages set to 3.0 kV and 35.0 V, respectively. The tube lens was set to 110 V, and the source temperature was set to 350°C. Nitrogen (purity >99.99%) was used as both the sheath gas (30 arb) and auxiliary gas (10 arb). Data-dependent acquisition (ddms3) of high-resolution Fourier transform (TF, full scan; resolution, 30,000) and CID fragmentation were used for positive and negative ion data acquisition. The compositions of QWZK were authenticated by referring to the retention time of each chemical component, high-resolution precise molecular weight, and MSn multilevel fragment information detected by LC-MS and combined with the extraction of ion flow map and standard product information and related literature.

Adult Wistar rats (4–6 weeks, 180–220 g, male) were purchased from the Vital River Laboratories (SYXK 2016-0006). All the animals were housed in an environment with temperature of 23 ± 1°C, relative humidity of 50 ± 1%, and a light/dark cycle of 12/12 h. All animal experimental procedures were conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of Beijing University of Chinese Medicine. After acclimation for 7 days for 7 days, the rats were randomly assigned into 6 groups (n = 10/group): the control group was treated by saline only, the LPS group was treated by LPS (from Escherichia coli O55:B5, abs47014848, Absin, Shanghai, China) only, the dexamethasone group was treated by dexamethasone and LPS, and the QWZK groups were pretreated with QWZK followed by LPS. QWZK was administrated with 3, 6, and 12 g/kg via intragastric (i.g.) administration once per day for 7 consecutive days. LPS was injected intraperitoneally (i.p.) after final injection of medication for 1 h. At 4-h intervals, the left lung was lavaged with cool phosphate-buffered saline (PBS) to collect the bronchoalveolar lavage fluid (BALF); the middle lobe of the right lung was fixed with 4% paraformaldehyde (PFA), and the upper lobe, lower lobe, and accessory lobes were stored at −80°C for protein expression tests.

The BALF was centrifuged at 3,000 rpm for 5 min, at 4°C. The supernatant was discard, and then, the cell pellet was resuspend in PBS. Whereafter, a Sysmex XS-800iBayer ADVIA120 Hematology System was used for cell counting and classification.

The tissues fixed with 4% PFA were dehydrated, transparent, and immersed in paraffin. Before staining, the slices (3 μm) were dewaxed and soaked. Then, they were stained with hematoxylin aqueous solution and eosin staining solution, respectively. Finally, they were dehydrated and rendered transparent and sealed with neutral gum. Sections were observed under a microscope and photographed. A 20-fold field was selected for the statistics of alveolar wall area (%). The airway wall area was detected and calculated with Image-Pro Plus software according to references (Moon et al., 2021). We used the lung tissue slices of the control group to calibrate the quantitative parameters, randomly select different areas of the lung, and quantify the alveolar wall and blank area. Alveolar wall ratio (%) = alveolar wall area/total area. Lung injury was scored according to the following criteria (Schingnitz et al., 2010; Moon et al., 2021; Zhao et al., 2021): (1) alveolar congestion, (2) hemorrhage, (3) infiltration or aggregation of neutrophils in airspace or vessel wall, and (4) thickness of the alveolar wall. For each subject, a 5-point scale was applied: 0, minimal (little) damage; 1+, mild damage; 2+, moderate damage; 3+, severe damage; and 4+, maximal damage. The total score of each criteria was used for statistics.

To investigate the expression of proteins by Western blot analyses, animal tissue samples were lysed in a protein cell lysis buffer (Applygen, Beijing, China). The protein concentration of the samples was determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, MA, United States). The samples were boiled for 10 min, and proteins were separated by electrophoresis using a 10% or 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel. After the transfer of protein to a polyvinylidene difluoride membrane (PVDF, Millipore, Bedford, MA, United States), the membrane was incubated in blocking buffer [5% non-fat dairy milk in Tween-20 Tris-buffered saline (TBST)] for 2 h at ambient temperature and probed with various antibodies in a blocking buffer overnight at 4°C. The membrane was washed four times with 0.1% TBST, probed with a secondary antibody in the blocking buffer for 2 h at ambient temperature and then washed again with TBST. The membranes were detected with an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Piscataway, NJ, United States). The primary antibody included TLR4 (1:1,000, NB100-56566, Novus, CO, United States), IKKα (1:1,000, #11930, CST, Boston, United States), IKKβ (1:1,000, #8943, CST, Boston, United States), p-IKKα/β (1:1,000, #2697, CST, Boston, United States), IκBα (1:1,000, #4814, CST, Boston, United States), p-IκBα (1:1,000, #2859, CST, Boston, United States), NF-κB p65 (1:1,000, #8242, CST, Boston, United States), p-NF-κB p65 (1:1,000, #3033, CST, Boston, United States), NLRP3 (1:1000, ab263899, Abcam, Cambridge, United States), pro-caspase-1 + p10 + p12 (1:1,000, ab179515, Abcam, Cambridge, United States), ASC/TMS1 (1:1,000, NBP1-78977, Novus, Colorado, United States), and β-actin (1:5,000, #8457, CST, Boston, United States). The secondary antibodies include goat antirabbit IgG H&L (HRP, 1:5,000, ab6721, Abcam, Cambridge, United States), goat antimouse IgG H&L (HRP, 1:5,000, ab6789, Abcam, Cambridge, United States).

The contents of IL-6, TNF-α, IFN-γ, MCP-1, IL-1β, and IL-18 were determined by ELISA method. The lung tissues were crushed with balls at 0–4°C and centrifuged at 12,000 rpm for 20 min. Protein concentration was determined by BCA protein assay kit (Thermo Fisher Scientific, MA, United States). The levels of IL-6, TNF-α, IFN-γ, MCP-1, IL-1β, and IL-18 in lung tissues were measured by using commercially procured ELISA assay kits, including IL-6 ELISA kits (Biolegend, San Diego, United States, Item No. 437107), TNF-α ELISA kits (Biolegend, San Diego, United States, Item No. 438207), IFN-γ ELISA kits (Biolegend, San Diego, United States, Item No. 439007), MCP-1/CCL2 ELISA kits (Genie, London, United Kingdom, Item No. RTFI00038), IL-1β ELISA kits (Genie, London, United Kingdom, Item No. RTDL00552), and IL-18 ELISA kits (Genie, London, United Kingdom, Item No. RTDL00548).

The data and statistical analysis comply with the British Journal of Pharmacology on experimental design and analysis in pharmacology (Curtis et al., 2018). All data were presented as means ± standard error of mean (SEM). All rights reserved based on at least three independent experiments and analyzed on GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, United States). Statistical analysis was undertaken for studies where each group rats were at least n = 8. Statistical data conforming to a Gaussian distribution was performed either with one-way analyses of variance (ANOVAs) followed by Fisher’s least significant difference (homogeneity of variances) and Tamhane T2 (heterogeneity of variance) post-hoc test using SPSS version 25 for windows (IBM^® SPSS^® Statistics, Chicago, IL, United States). Mann–Whitney U test was applied to data analysis of abnormal distribution. p < 0.05 was considered statistically significant.

In this study, the chemical compositions of QWZK were analyzed by UHPLC-LTQ-Orbitrap MS. There were 21 compounds identified in the negative spectrum and 78 compounds were identified in the positive spectrum. A total of 99 compounds were identified, including 33 flavonoids, 23 phenolic acids, 3 alkaloids, 3 coumarins, 20 triterpenoids, 5 anthraquinones, and 12 others (Figure 1). The list of identified compounds was shown in the supplementary material (Supplementary Table S1).

The effects of QWZK on ALI were evaluated by the detection of the number and classification of leukocyte in BALF and the histopathology of lung. As shown in Figure 2A, the number (2.92 ± 0.57 × 10³ cells/μl) of leukocyte in BALF of the rats in LPS group was significantly increased than that in the control group (1.63 ± 0.26 × 10³ cells/μl), and the number of leukocyte in BALF of rats in the dexamethasone group and QWZK 3 g/ kg group, QWZK 6 g/ kg group, and QWZK 12 g/ kg group were 1.24 ± 0.11 × 10³ cells/μl, 1.94 ± 0.35 × 10³ cells/μl, 1.38 ± 0.41 × 10³ cells/μl, and 1.69 ± 0.30 × 10³ cells/μl, respectively, which were declined significantly than that in the LPS group (Figure 2A). The number (2.17 ± 0.32 × 10³ cells/ μl) and proportion (84.17 ± 2.27%) of neutrophils in BALF of LPS group were enhanced significantly compared with those of control group (1.04 ± 0.21 × 10³ cells/μl, 52.77 ± 3.78%). The dexamethasone and QWZK 3 g/ kg, QWZK 6 g/ kg, and QWZK 12 g/kg treatments all decreased the number and proportion of neutrophils in BALF (Figures 2B,C). Moreover, QWZK 6 g/kg decreased the neutrophil number to 0.94 ± 0.16 × 10³ cells/μl, as much as that in control group. Furthermore, the number of lymphocyte and monocyte in BALF of rats in the LPS group were 0.03 ± 0.01 × 10³ cells/ μl and 0.04 ± 0.01 × 10³ cells/ μl, respectively, which declined significantly compared with that in the control group (0.43 ± 0.12 × 10³ cells/μl, 0.19 ± 0.06 × 10³ cells/ μl), and QWZK treatment increased compared with that of the LPS group (Figures 2D,F) and the changes in proportion (Figures 2C,E,G). All these data suggested that QWZK reduced inflammatory response in LPS-induced ALI rats.

Besides that, we observed the changes in pulmonary pathology (Figure 3). The alveolar wall areas were performed to evaluate pathological changes in the lung. LPS treatment showed significant thickening of the alveolar wall, thickening of the septum, infiltration of neutrophils in the septum and alveolar cavity, and obvious bleeding in the lung interstitium (Figures 3A,C). The alveolar wall area of rats in the LPS group were almost twofold more than that in control group. In the dexamethasone group, rats showed mild thickening of the alveolar septum and obvious infiltration of neutrophils. The alveolar wall areas were reduced by 32.1% compared with the LPS group. The rats in QWZK groups showed varying degrees of thickening of the alveolar septum and neutrophil infiltration, and no obvious bleeding lungs were seen. The QWZK of dose 6 g/ kg exhibited the most obvious effect on the anesis of alveolar wall thickness and hemorrhage (Figures 3A,B). These data showed that QWZK could ameliorate LPS-induced ALI by regulating the number and classification of WBC in BALF, and the better effect of QWZK was given at 6 g/ kg.

ELISA was performed to evaluate the inflammation-related cytokines, including IL-6, TNF-α, MCP-1, IL-1β, IL-18, and a lymphokine related to immune regulation, IFN-γ, in the total protein of the lung. As shown in Figure 4, the expression of IL-6, TNF-α, IL-1β, and IL-18 of the rats in the LPS group were 1,036.20 ± 53.57 pg/mg, 34.82 ± 2.51 pg/mg, 1,354.90 ± 155.75 pg/mg, and 123.47 ± 42.21 pg/mg, respectively, which were significantly increased compared with those in the control group (2.41 ± 0.68 pg/mg, 1.40 ± 0.34 pg/mg, 556.53 ± 96.13 pg/mg, 57.17 ± 5.03 pg/mg). The expressions of IL-6, TNF-α, IL-1β, and IL-18 in the other groups were lower than those in the LPS group (Figures 4A,B,D,E).

The level of MCP-1 was significantly decreased in the rats after dexamethasone (39.77 ± 13.21 pg/mg), QWZK 3 g/kg (73.39 ± 3.74 pg/mg), QWZK 6 g/kg (61.22 ± 18.07 pg/mg), and QWZK 12 g/kg (70.25 ± 11.02 pg/ mg) administration, respectively, which was in contrast with that of the LPS group (83.17 ± 4.82 pg/ mg). Similarly, LPS induced IFN-γ production, and dexamethasone (24.28 ± 9.38 pg/mg), QWZK 3 g/kg (46.43 ± 7.64 pg/mg), QWZK 6 g/kg (26.63 ± 10.98 pg/mg), and QWZK 12 g/kg (59.34 ± 6.64 pg/mg) treatments could reverse the IFN-γ level to normal (Figure 4F).

Among them, the QWZK 6 g/kg group showed the optimal effect on the downregulation of proinflammatory cytokines, and the inhibition effect on some cytokines, such as TNF-α, IL-18, and IFN-γ, were better than dexamethasone.

As is well-known, the TLR4/NF-κB signaling pathway is involved in regulating proinflammatory factors (Lawrence and Fong, 2010). Further, we investigated the effect of QWZK on TLR4/NF-κB signaling pathway. As shown in Figure 5, compared with the control group, the expressions of TLR4, p-IKKα/β, p-IκBα, and p-NF-κB were significantly upregulated in the lungs of LPS-induced ALI rats, and IKKα/β, IκBα, and NF-κB expression were significantly downregulated. Compared with the rats in LPS group, TLR4, p-IKKα/β, p-IκBα, and p-NF-κB expression were declined in the dexamethasone group and the QWZK groups. Among them, the expression of TLR4, p-IKKα/β, p-IκBα, and p-NF-κB in QWZK 6 g/kg group was even lower than those in the dexamethasone group and recovered to almost the same level as that in control group. The results indicated that QWZK could inhibit the TLR4/NF-κB signaling pathway.

Studies demonstrated that inflammasome activation could increase the expression of IL-1β and IL-18 (Seoane et al., 2020). Then, we investigated whether QWZK inhibited IL-1β and IL-18 level via activation of NLRP3 inflammasomes. Western blot results showed that the expression of NLRP3, cleaved caspase-1 and ASC increased significantly in the rats of the LPS group compared with those in control group. All of dexamethasone, QWZK 3 g/kg, QWZK 6 g/kg, and QWZK 12 g/kg treatments could downregulate NLRP3, cleaved caspase-1, and ASC expression (Figure 6). Interestingly, the levels of NLRP3 and ASC in the rats treated with QWZK 6 g/ kg were almost the same as those in the rats of the dexamethasone group. QWZK could inhibit the activation of NLRP3 inflammasomes, and QWZK 6 g/kg exhibited better role on inhibition of NLRP3, cleaved caspase-1, and ASC.