Dexamethasone (Dex, LB2269) was obtained from Zhejiang Xianju Pharmaceutical Co., Ltd. MLKL Mouse McAb (66675-1-Ig), DFNA5/GSDME Rabbit PolyAb (13075-1-AP), HRP-conjugated Beta Actin Monoclonal antibody (HRP-66009), HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (SA00001-2), CoraLite 488-conjugated Goat Anti-Rabbit IgG (H + L), HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L) (SA00001-1), BCA Protein Assay Kit (PK10026), Hesperidin (CM00691) and Necrostatin-1 (CM00423-) were purchased from Proteintech Group, Inc. Anti-MLKL (phospho S345) antibody (ab196436) was obtained from Abcam Co. Polyinosinic-polycytidylic acid salt (poly (I∶C), PIC, P1530) was purchased from Sigma-Aldrich Trading Co.,Ltd. RIPA lysis buffer (R0010), Protein Phosphatase inhibitor (All-in one, 100×) (P1260), Mounting Medium, antifading (with DAPl) (S2110) and electron microscope fixative (G1102) were purchased from Beijing Solarbio Science & Technology Co., Ltd. Triton X-100 Solution (ST797) and Necroptosis Inducer Kit with TSZ (C1058S) were obtained from Beyotime Biotech Inc commit. CellTiter96® AQueous One Solution Cell Proliferation Assay was obtained from Promega Biotech Co., Ltd. Fetal Bovine Serum (10099141C) and DMEM/F-12 (1:1) (C11330500BT) basic medium were purchased from Grand Island Biological Company.

The medicinal materials of JF were obtained from Yunnan Ci Hui Pharmaceutical Co. Ltd. Except for 12 g of gancao, 36 g of each of the other ten medicinal herbs was measured, all materials were soaked in 4500 mL of distilled water for 30 min, the herb mixture was boiled for 20 min to collect the decoction and filtered. The JF water decoction was evaporated by a vacuum concentrator at 55 °C and concentrated to 15.5 mL. The concentrated liquid is freeze-dried, and the powder weight after drying is 6.98 g. After sealing, it is stored in a refrigerator at 4 °C for use.

C57BL/6J male mice (20–22 g, SPF grade, 8 weeks old) and SD rats (200–220 g, SPF grade, 8 weeks old) were housed in a specific pathogen-free-grade animal facility. The animal studies were conducted with the Guidelines for Animal Experiments of Yunnan University of Chinese Medicine. The study protocol received approval from the Institutional Experimental Animal Ethics Committee of Yunnan University of Traditional Chinese Medicine (No. R-062023125; YNUTCM-XMSS-G-20250016). The mice were housed under a diurnal lighting condition (12/12-h light/dark cycle) for 1 week.

16 mice were randomly divided into two groups (n = 8 each): JF and PIC + JF-treated groups, both groups were treated with 0.45 g JF extract/kg (equivalent to four times of the clinical dosage in human, which equivalent dose calculation is based on body surface area). Mice in the PIC + JF-treated group were administered 400 μg of PIC through the right nostril via slow dripping over 3 days to establish the ALI model (Choi et al., 2010). 6 h post-exposure to PIC, the JF and PIC + JF-treated groups received subsequent administrations via gavage for a duration of 4 days 2 h following the last administration, the serum samples were collected and allowed to stand at room temperature for 30 min. Then the samples were centrifuged at 3,500 rpm for 10 min at 4 °C. Subsequently, 1 mL of serum was mixed with 4 mL of pre-cooled methanol and centrifuged at 4 °C at 12,000 rpm for 10 min. The supernatant was then dried using a nitrogen blowing instrument. Following this, 200 μL of pre-cooled methanol was added for re-dissolution, and the mixture was centrifuged at 4 °C at 12,000 rpm for 10 min. The resulting supernatant was used for the identification of small molecular substances.

A total of 48 mice were divided into five groups: Control (Con, treated with saline by gavage), PIC (treated with saline by gavage), PIC + dexamethasone (PIC + Dex) (treated with Dex at a dosage of 5 mg/kg by gavage), PIC + JF-H (treated with 0.9 g JF extract/kg by gavage) and PIC + JF-L (treated with 0.45 g JF extract/kg by gavage). Mice in the PIC, PIC + Dex, PIC + JF-H, and PIC + JF-L groups were utilized to establish the ALI model, following the methodology described in Section 2.4. 6 h post-exposure to PIC, JF-treated groups received subsequent administrations at the specified doses for 4 days. The Dex-treated group received administrations prior to the initial modeling during sampling. Meanwhile, the Con and PIC groups were treated with saline. After 2 h after the last administration, the mice were euthanized after anesthesia via intraperitoneal injection of zoletil to harvest bio-samples.

Mice were anesthetized, and their tracheas were dissected for intubation. Started animal lung function detection system to detect lung function and set parameter: 0.2 mL max stroke volume; 30 cm H₂O max mouth pressure; 0.75 mL deep inflation max volume; 40 cm H₂O deep inflation max pressure; 140 breaths/min rate; 0 cm H₂O peep.

Serum, nasal lavage fluid (NLF), and bronchoalveolar lavage fluid (BALF) were centrifuged at 3,000 rpm for 10 min. The resulting supernatant was then analyzed for the concentrations of TNF-α, IL-6, LDH and IL-18 using a test kit (Nanjing Jiancheng Bioengineering Institute (Nanjing, China)).

The upper lobes of the left lung of mice were promptly fixed in 4% paraformaldehyde, dehydrated using a gradient of ethanol, and cleared with xylene. The tissue was then embedded in paraffin and sliced them into 4.0 μm thick slices. The slices containing the tissue sections were placed in an oven at 60 °C to dry. Subsequently, the sections were immersed in xylene for dewaxing and rehydrated using a gradient of ethanol. Hematoxylin and eosin (H&E) staining was conducted following the manufacturer’s specifications, and scanned with a scanning microscope (Kunming Naray Technology Co., Ltd., SQS-1000SQS-1000, China).

The upper lobes of the left lung of mice were quickly fixed using an electron microscope fixative composed of 2.5% glutaraldehyde. The tissues were trimmed to 1 mm³ and fixed at 4 °C, then fixed in 1% osmic acid for 1.5 h. Subsequently, the samples were dehydrated using graded concentrations of ethanol and acetone, then, soaked, embedded, trimmed, sliced and stained, and observed under transmission electron microscope.

Total RNA was extracted from the three individual lung tissues of Con, PIC, PIC + JF-L (PIC + JF) groups, using TRIzol®Reagent according the manufacturer’s instructions. RNA purification, reverse transcription, library construction and sequencing were performed at Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China). The RNA-seq transcriptome librariy was prepared following Illumina®Stranded mRNA Prep, Ligation (SanDiego, CA) using 1 μg of total RNA. The sequencing library was performed on DNBSEQ-T7platform (PE150) using DNBSEQ-T7RS Reagent Kit (FCLPE150) version3.0. To identify DEGs (differential expression genes) between three groups, the expression level of each transcript was calculated according to the transcripts per million reads (TPM) method. DEGs with |log2FC|≥1 and p-value < 0.05, were enriched in GO and KEGG at Bonferroni-corrected p-value <0.05 compared with the whole-transcriptome background.

Lung tissues (the source of bio-samples is the same as 2.10) were extracted by a mixture of methanol, acetonitrile and water (v/v/v, 2:2:1) (1 mL), and then placed for 1 h ultrasonic shaking in ice baths. The mixture was placed at −20 °C for 1 h and centrifuged at 14,000 g for 20 min at 4 °C. The supernatant was collected and dehydrated under vacuum conditions. Metabolomics profiling was analyzed using a UPLC-ESI-Q-Orbitrap-MS system (UHPLC, Shimadzu Nexera X2 LC-30AD, Shimadzu, Japan) coupled with Q-Exactive Plus (Thermo Scientific, San Jose, United States). For liquid chromatography (LC) separation, samples were analyzed using a ACQUITY UPLC® HSS T3 column (2.1 × 100 mm, 1.8 μm) (Waters, Milford, MA, United States). The flow rate was 0.3 mL/min and the mobile phase contained: A: 0.1% FA in water and B: 100% acetonitrile (ACN). The gradient was 0% buffer B for 2 min and was linearly increase to 48% in 4 min, and then up to 100% in 4 min and maintained for 2 min, and then decreased to 0% buffer B in 0.1 min, with 3 min re-equilibration period employed. The electrospray ionization (ESI) with positive-mode and negative mode were applied for MS data acquisition separately. The HESI source conditions were set as follows: Spray Voltage: 3.8 kv (positive) and 3.2 kv (negative); Capillary Temperature: 320 °C; Sheath Gas (nitrogen) flow: 30 arb (arbitrary units); Aux Gas flow: 5 arb; Probe Heater Temp: 350 °C; S-Lens RF Level: 50. The instrument was set to acquire over the m/z range 70–1050 Da for full MS. The full MS scans were acquired at a resolution of 70,000 at m/z 200, and 17,500 at m/z 200 for MS/MS scan. The maximum injection time was set to for 100 m for MS and 50 ms for MS/MS. The isolation window for MS2 was set to 2 m/z and the normalized collision energy (stepped) was set as 20, 30 and 40 for fragmentation. The raw MS data were processed using MS-DIAL for peak alignment, retention time correction and peak area extraction. And then, the PCA and PLS-DA were visualized the global metabolic changes, and utilized to select the differential metabolites based on VIP >1.0 (or |log2FC|≥0.58) combined with one way ANOVA (p < 0.05). The KEGG database were used to analyze the related metabolic pathways of differential metabolites.

Quickly fixed the upper lobes of the left lung of mice in 4% paraformaldehyde, then embed them in paraffin and slice them into 3.0 μm thick sections. After dewaxing the addition of a pepsin repair solution, which was incubated at 37 °C for 30 min for antigen retrieval. A 2% Triton X-100 permeabilization solution was then added and incubated at room temperature for 20 min. Tissues were incubated with 5% bovine serum albumin at room temperature for 1 h, followed by overnight incubation at 4 °C with p-MLKL (1:200). Afterward, CoraLite 488-conjugated Goat Anti-Rabbit IgG (H + L) (1:500) was incubated at room temperature in dark for 1 h, and DAPI mounting solution was added for sealing. The tissues were subsequently observed under a fluorescence microscope.

The lung tissues of mice were lysed in RIPA lysis buffer with Protein Phosphatase inhibitor. Protein concentration was measured by BCA Protein Assay Kit. Equal amounts of denatured protein were separated by SDS-PAGE and transferred to PVDF membranes (IPVH00010, Millipore, Billerica, United States). Blocked the membranes with 5% bovine serum albumin or 5% no-fat milk for 2 h, then incubate overnight with the following primary antibody at 4 °C: anti-MLKL (1:2000 dilution), anti-p-MLKL (1:2000 dilution), anti-GSDME (1:2000 dilution), and anti-β-actin (1:2000 dilution). The membranes were then incubated with corresponding secondary antibody for 1 h at room temperature. The bands were visualized using UVP gel imaging analysis system (BOX-F3, Beijing Staples Technology Co., Ltd., China) and quantified and analyzed using ImageJ software.

To further elucidate the role of JF in mitigating ALI, the components in the medicated serum were molecularly docked with TNFR1 and TRPV1. The 3D structures of the primary ingredients of JF were downloaded from the PubChem platform, while TNFR1 and TRPV1 structures were obtained from the Worldwide Protein Data Bank (https://www.rcsb.org/) in PDB format. The large proteins and components were imported to AutoDockTools for hydrogenation, dehydration, and other pretreatments, followed by docking using AutoDock4. Subsequently, the results were visualized using Discovery Studio 2019.

Human lung adenocarcinoma cells A549 were obtained from the Beina Chuanglian Biotechnology Co., Ltd. (Beijing, China). A549 cells were cultured in DMEM medium containing 10% (v/v) fetal bovine serum and 1% (v/v) penicillin-streptomycin solution at 37 °C in a 5% CO₂ atmosphere.

Male SD rats were randomly divided into a control group and a JF treatment group. The control group received physiological saline via gavage, while the JF group was administered 0.68 g of JF extract (equivalent to four times of the clinical dosage in human)via gavage once daily for 7 consecutive days. 2 h after the final administration, the rats were anesthetized with isoflurane, and blood was collected from the abdominal aorta. The blood samples were placed in EP tubes and allowed to stand at room temperature for 30 min before being centrifuged at 3000 rpm for 10 min at 4 °C. The serum was inactivated in a water bath at 56 °C, aliquoted, and stored at −80 °C.

A549 cells in the logarithmic growth phase were seeded at a density of 4 × 10⁴ cells/well in a 96-well culture plate and incubated in a carbon dioxide cell culture incubator. After cell adhesion, the cells were grouped and treated. The experiment was divided into four groups: normal group, model group (NEC), JF-containing serum group (with concentrations of 40%, 20%, 10%, 5%, 2.5%, 1.25%, and 0.625%), and Hes group (with concentrations of 328 μM, 164 μM, 82 μM, 41 μM, 20.5 μM, 10.25 μM, and 5.125 μM). The normal group was maintained under standard culture conditions, while the other groups were treated with a cell necroptosis inducer to establish a cell injury model. Following this, sample working solutions were added for a duration of 24 h. Subsequently, 20 μL of a 5 mg/mL MTS solution was added to each well and incubated at 37 °C for 2 h to ensure complete dissolution. The OD value was then measured at a wavelength of 490 nm using a multifunctional enzyme labeler.

A549 cells were seeded at a density of 1 × 10⁵ cells per well in a 12-well cell culture plate containing glass slides to ensure uniform distribution, and subsequently transferred to a CO₂ incubator for cultivation. The experiment was divided into five groups: the normal group, the NEC group, the inhibitor group (Necrostatin-1 (NEC-1), 20 µM), the JF-containing serum group (2.5%, 5%), and the Hes group (41 μM, 20.5 µM). Except for the normal group, all other groups were treated with a cell necroptosis inducer to establish a cell injury model, alongside the addition of sample working solutions for 24 h. After 24 h of treatment, the medicated medium was removed from the wells, and the wells were washed with PBS. Subsequently, a cell fixation solution was added for 10 min at room temperature, followed by the addition of a pepsin repair solution, which was incubated at 37 °C for 30 min for antigen retrieval. A 2% Triton X-100 permeabilization solution was then added and incubated at room temperature for 20 min. Cells were incubated with 5% bovine serum albumin at room temperature for 1 h, followed by overnight incubation at 4 °C with p-MLKL (1:200). Afterward, CoraLite 488-conjugated Goat Anti-Rabbit IgG (H + L) (1:500) was incubated at room temperature in dark for 1 h, and DAPI mounting solution was added for sealing. The cells were subsequently observed under a fluorescence microscope, and fluorescent images were captured randomly.

All parameters were expressed as means ± SEM. Kruskal–Wallis test and analysis of variance (ANOVA) were performed to compare the means with the control group. Data were analyzed using GraphPad Prism9 software. Statistically significant differences were accepted at p < 0.05.

The results of identification of the transitional components in medicated serum of ALI model intervened by JF showed that a total of 305 components were detected in the negative ion mode, while 535 components were identified in the positive ion mode (Figure 2). By comparing these findings with existing literature and the TCMSP database, the sources of 34 components were analyzed, including 14 flavonoids, 7 coumarins, 8 terpenoids, 3 phenolic acids, 1 phenylpropanoid component and 1 other class components (Table 1). Among these, 10 components are derived from zhiqiao; 9 from fangfeng; 9 from qianhu, 9 from gancao, 8 from duhuo, 8 from qianghuo, 7 from chuanxiong, 4 from chaihu, 3 from jinjie and 1 from jiegeng. The medicinal materials with the most flavonoids was zhiqiao (6), while fangfeng and qianghuo both contained the most coumarins (6 each). In the medicated serum of normal mice after JF intervention, a total of 327 components were detected in the negative ion mode and 522 components were detected in the positive ion mode. Compared with the medicated serum of ALI mice with that of normal mice, there were 358 identical components in positive ion modes and 202 in negative ion modes. The same components were derived from 28 sources, with the content of 14 components increasing in the serum of ALI mice, 13 components decreasing, and the content of 1 component showing minimal difference (Figure 3).

The results of lung function test in mice showed that JF could alleviate airway resistance and respiratory rate (Figure 4A). PIC increased the content of IL-6 and TNF-α in NLF, as well as the TNF-α content in BALF. Notably, JF was shown to reduce the content of IL-6 in NLF, the content of TNF-α in NLF, BALF and serum (Figures 4B–D).

H&E staining and transmission electron microscopy revealed that the lung tissue of Con group mice exhibited normal morphology, while the lung tissue of the PIC group mice displayed the thickened alveolar walls, infiltration inflammatory cells, necrotic exfoliated alveolar epithelial cells of type I, and type II alveolar epithelial cells osmiophilic multilamellar body vacuoles, mitochondrial membrane rupture. JF and Dex intervention significantly ameliorated the pathological changes in the lung tissue in ALI mice (Figures 4E–H).

To explore the molecular basis and mechanism of JF in the treatment of ALI at the genetic level, this study performed transcriptomic sequencing analysis of lung tissues from the Con, PIC group and PIC + JF group in the animal model. According to the results, there were 631 DEGs identified between the PIC and Con groups (605 upregulated and 26 downregulated), and 1041 DEGs between the PIC + JF and PIC groups (280 upregulated and 761 downregulated) (Figures 5A,B). PPI network showed that TNF, IFN-γ, CXCL10 and STAT1 were the core DEGs between the PIC and Con groups, as well as the PIC + JF and PIC groups. Compared to Con group, the mRNA expression of these core DEGs was upregulated in the PIC group, while the PIC + JF intervention significantly downregulated the mRNA expression of these core DEGs compared to the PIC group (Figures 5C–E). All DEGs were functionally enriched, and 10 GO keywords were represented in the bubble plot. The findings revealed that the GO enrichment was primarily associated with immune response, defense response, and response to external biotic stimulus (Figures 5F,G). KEGG enrichment results showed that PIC treatment may affect cell processes such as phagosome and cellular senescence, JF intervention could influence cell processes such as phagosome, apoptosis, and necroptosis (Figures 5H,I).

PCA analysis was performed on all samples, in the PCA model, the distribution of quality control samples was concentrated, indicating that the sample analysis method had good reproducibility and high stability (Figure 6A). There was a significant difference between the PIC group and the Con group in the PC1 direction, indicating that there was a difference in the metabolic profile of the group. After JF intervention, the distribution of metabolites in mice lung tissue was significantly different from that in the PIC group in the PC1 direction (Figures 6B,C). A total of 421 DEMs were identified between the PIC and Con groups comprising 177 upregulated and 244 downregulated metabolites. Additionally, 170 DEMs (79 upregulated and 91 downregulated) were observed between the PIC + JF and PIC groups (Figures 6D,E). Classification of DEMs revealed that the primary regulated lipids and lipid-like molecules differed between the PIC vs. Con group and PIC + JF vs. PIC groups. The expression abundance of certain lipids and lipid-like molecules increased under PIC intervention, but decreased with JF intervention (Figures 6F–H). KEGG enrichment analysis indicated that PIC mainly affected the signaling pathways such as Neuroactive ligand-receptor interaction, Sphingolipid signaling pathway and Oxidative phosphorylation. In contrast, after JF intervention, the primary pathways affected included Neuroactive ligand-receptor interaction, Arginine biosynthesis and Oxidative phosphorylation (Figures 6I,J). Further analysis of the neuroactive ligand-receptor interaction signaling pathway revealed that JF reduced the expression levels of NADA, Citric acid and Psychosine, while increasing the expression levels of ATP, L-Glutamate, L-Aspartate, β-Alanine and Taurine (Figure 6K).

The DEGs and DEMs were utilized to construct a correlation expression heat map, which was subsequently integrated into the “Formula-DEGs-DEMs-pathway” diagram to validate the mechanistic relationship of JF. The role of JF in alleviating ALI was closely related to the regulation of necroptosis, neuroactive ligand-receptor interaction and TCA cycle (Figure 7A). Necroptosis could lead to cell membrane perforation, with p-MLKL and GSDME serving as typical perforins of the cell membrane. Animal immunofluorescence results showed that JF decreased the fluorescence intensity of p-MLKL in lung tissue (Figure 7B), as well as reduced the content of LDH and IL-18 in BALF (Figure 7C), and diminish the protein levels of p-MLKL, GSDME and the conversion rate of p-MLKL/MLKL (Figure 7D). Based on the findings from the omics analysis, it was speculated that TNFR1 and TRPV1 may also be the targets of JF to alleviate ALI. Therefore, the components in the medicated serum were docked with these targets. The results demonstrated that the 7 components represented by baicalin and hesperidin had higher binding affinity with TNFR1 and TRPV1 (Table 2; Figures 7E,F). The animal experiment results showed that necroptosis could occur in lung epithelial cells, so the study was further verified at the cellular level. The experimental results revealed that the IC50 values of JF medicated serum for normal cultured A549 cells and NEC-induced A549 cells were 20.48% and 49.59%, respectively. In contrast, the IC₅₀ for Hes in both normal and NEC-induced A549 cells >328 μM (Figure 7G). Treatment with NEC significantly elevated the fluorescence level of p-MLKL in A549 cells. Following treatment with NEC-1, 5% JF medicated serum, as well as 20.5 μM and 42 μM of Hes, there was a significant decrease in the fluorescence level of p-MLKL in A549 cells (Figure 7H).