The aerial parts of Tetrastigma hemsleyanum Diels et Gilg were obtained from Hangzhou China Agrotime Agri-Tech Co., Ltd. SYQP was prepared and characterized in our laboratory as previously reported (Bingqi Zhu et al., 2020).

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (#M2128-100 MG), dimethyl sulfoxide (DMSO) (#V900090), LPS (#l2880-100 mg), dexamethasone (DEX) (#D4902-100 MG) and Pam3CSK4 (P3C) (#tlrl-pms) were purchased from Sigma Chemical Co. (MO, United States). TAK-242 (#HY-11109-10 mg) and C29 (C₁₆H₁₅NO₄) (#HY-100461-5 mg) were purchased from MedChemExpress (MCE) (NJ, United States). Peroxidase-conjugated goat anti-mouse IgG (#115–035–003) and goat anti-rabbit IgG (#111–035–003) were purchased from Jackson ImmunoResearch (PA, United States). RNA-Quick Purification Kit (#RN001) was purchased from ESscience Biotech (Shanghai, China). A BeyoRT™ II First Strand cDNA Synthesis Kit (#D7170M) was purchased from Beyotime Biotechnology (Shanghai, China). PowerUp™ SYBR™ Green Master Mix (#A25742) was purchased from Thermo Fisher Scientific (MA, United States), ROS Assay Kit (2′,7′-dichlorofluorescin diacetate (DCFH-DA) (#S0033S) was purchased from Beyotime Biotechnology (Shanghai, China).

RAW264.7 murine macrophages were purchased from Shanghai Chinese Academy of Sciences cell bank (Shanghai, China). Purified MPMs from Balb/c mice were aseptically harvested based on a reference protocol (Liu et al., 2006). The RAW264.7 cells and extracted MPMs were cultured in DMEM with a high sugar content supplemented with 10% FBS and maintained at 37°C in a humidified 5% CO₂ atmosphere.

MTT assay was used to evaluated cell viability. Briefly, RAW264.7 cells were collected and seeded in 96-well plates (1 × 10⁴ cells/well) for 12 h and then treated with SYQP (0, 0.1, 1,10,100,1000 μg/ml) for 24 h. After incubation, 20 μL MTT solution was added to each well to a final concentration of 0.5 mg/ml. After further incubation for 4h, the supernatant was removed and 150 μL DMSO was subsequently added to each well. The absorbance at 570 nm was measured with a microplate reader.

ROS levels in RAW264.7 cells was detected with ROS assay kit. Briefly, RAW264.7 cells were pre-treated with SYQP (25, 50, 100 μg/ml) or DEX (4 μg/ml) for 2 h, and followed by stimulation with or without LPS (1 μg/ml) for 18 h. After treatment, DCFH-DA was added to the cultured cells for 20 min, and the nucleus were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min in the dark. After washing three times with PBS, cells were photographed by Nikon ECLIPSE Ti-DH inverted fluorescence microscope (Tokyo, Japan).

Healthy male Balb/c mice weighing 20–25 g were purchased from the Laboratory Animal Centre of Zhejiang Chinese Medical University (Hangzhou, China). The ethical approval number of the animal model study is IACUC-20210802–13. The mice were raised under standard conditions. All experimental procedures were in accordance with the People’s Republic of China (PRC) guidelines for the Care and Use of Laboratory Animals and were carried out strictly in accordance with the Guidelines of Zhejiang Chinese Medical University for Animal Experiments.

Animals were divided into six groups: the control group, which was intragastrically administered with normal saline; the LPS group, which was injected intraperitoneally with LPS (20 mg/kg); DEX and three SYQP + LPS groups, which were intragastrically administered with DEX (5 mg/kg), 50 mg/kg SYQP (SYQP of low dose, SYQPL), 75 mg/kg SYQP (SYQP of middle dose, SYQPM) and 150 mg/kg SYQP (SYQP of high dose, SYQPH) for 14 days. LPS was injected intraperitoneally 30 min after the last intragastric administration. Mice were sacrificed 12 h after LPS treatment.

Mice were fixed, and the skin and muscles in front of the neck were cut to expose the trachea. After endotracheal intubation, BALF were collected with 1 ml ice-cold PBS three times. Then BALF were combined and centrifuged at 3,500 rpm for 15 min. The cell pellets obtained after centrifugation were resuspended in 200 μL PBS and subjected to differential cell counter ADVIA 2120i Hematology System (Siemens, Germany).

Blood was used to determine different blood parameters. The blood was collected in EDTA-K₂ contained centrifugal tubes. The whole blood was subjected to automatic blood cell analyzer for detailed hematological analysis.

Plasma was collected by centrifugation (12,000 rpm, 10 min, 4°C) of the whole blood and then stored at −80°C until GSH, SOD and MDA measurement.

The cytokine contents and NO production in the culture supernatants of RAW264.7 cells and plasma of ARDS mice were assayed using a Cytometric Bead Array Mouse Th1/Th2/Th17 Cytokine Kit (BD Biosciences, San Diego, United States) and ELISA kit according to the manufacturer’s instruction, respectively. RAW264.7 cells were processed as follows: cells (2 × 10⁵ cells/well) were seeded in 24-well plates, pre-treated with a series of concentrations of SYQP or DEX (4 μg/ml) for 2 h, and followed by stimulation with or without LPS (1 μg/ml) for 18 h. Alternatively, RAW264.7 cells or MPMs were pre-treated with C29 (25 μM) or TAK242 (10 μM) for 1 h and then cultured with SYQP (100 μg/ml) at 37°C for 18 h. Subsequently, the supernatant was collected by centrifugation (3,000×g, 10 min, 4°C) to determine cytokine and NO production.

Samples of the middle lobe of right lung were fixed in 4% paraformaldehyde for more than 48 h, dehydrated with graded alcohol, and then embedded in paraffin. Paraffin sections stained with hematoxylin and eosin were used for gross morphology and lung damage and inflammation. Images were captured under a microscope (Eclipse TS100; Nikon Corporation, Tokyo, Japan). A quantitative scoring system, which included alveolar congestion, alveolar hemorrhage, neutrophil infiltration, aggregation in the airspace or vessel wall, alveolar wall/hyaline membrane thickness, and inflammatory cell infiltration, was introduced to evaluate lung injury. The grading scale for the lung tissue pathologic findings was as follows: 0 (normal = no inflammation, no airway thickening, no edema), 1 (minimal cellular infiltration, and minimal edema), 2 (mild-moderate cellular infiltration, plus mild airway thickening and mild edema), 3 (severe cellular infiltration, plus diffuse airway thickening and severe edema) in 10–12 fields per mouse (Xian et al., 2021).

Immunohistochemistry was performed as per standard protocol reported earlier (Xian et al., 2021). Lung sections were subjected to incubation with antibodies specific for myeloperoxidase (MPO). Images were captured under a microscope (Eclipse TS100; Nikon Corporation, Tokyo, Japan). Stained areas were quantified using ImageJ software.

The immunofluorescence assay of RAW264.7 cells and lung sections were performed according to previous study (Tian et al., 2021). Briefly, RAW264.7 cells (5 × 10⁵ cells/well) were seeded in Laser confocal Petri dishes, pre-treated with SYQP (25, 50, 100 μg/ml) or DEX (4 μg/ml) for 2 h, and followed by stimulation with or without LPS (1 μg/ml) for 18 h. After treatment, the nuclear translocation of p65 of RAW264.7 cells was subjected to incubation with antibodies specific for NFκB-p65. Lung sections were subjected to incubation with antibodies specific for F4/80. Nuclei were co-stained for 10 min with 0.1 g/ml DAPI. Images were captured under laser scanning confocal microscope (Zeiss, Oberkochen, Germany) and Nikon ECLIPSE Ti-DH inverted fuorescence microscope (Nikon, Tokyo, Japan), respectively. Stained areas were quantified using ImageJ software.

Total RNA was isolated from RAW264.7 cells and lung tissues by RNA-Quick Purification Kit. After determining the concentration of the isolated RNA and reverse transcription, PCR was performed in an ABI-7500 Real-Time PCR System. The mRNA expression of each target gene was normalized to GAPDH. The relative fold change was calculated using the 2^-∆∆Ct method. All primer sequences (Sangon Biotech, Shanghai, China) are listed in Table 1.

Western blot assays were used to investigate the immunomodulatory molecular mechanism of SYQP in LPS-induced RAW264.7 cells and lung tissues of ARDS mice. After incubation, the total proteins were extracted according to the method of literature (Ren et al., 2019). Protein of cell and tissue lysates were quantified by the BCA reagent. Harvested proteins were denatured at 95°C for 10 min, and 50 μg of protein from each sample was electrophoresed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) before transferring onto polyvinylidene difluoride membranes. The membranes were blocked for 1 h at room temperature in 5% non-fat dry milk, and followed by incubation with primary antibodies of iNOS (Cell Signaling Technology [CST], #13120, 1:1,000), COX-2 (Affinity Biosciences, #AF7003, 1:1,000), TLR4 (Sigma-Aldrich, #SAB1300056-100UG, 1:1,000), TLR2 (Sigma-Aldrich, #SAB1300199-100UG, 1:1,000), phospho-NF-κB p65 (CST, #3031L, 1:1,000), NF-κB p65 (Sigma-Aldrich, #ABE347, 1:1,000), p-JAK2 (CST, #3771S, 1:1,000), JAK-2 (Bioss, bs-23003R, 1:1,000), p-STAT1 (Immunoway, #YP0249, 1:1,000), STAT1 (Immunoway, #YT4439, 1:1,000), NLRP3 (Affinity Biosciences, #DF7438, 1:1,000), caspase1 (AdipoGen, AG-20B-0042, 1:1,000), caspase3 (CST, #9662, 1:1,000), caspase8 (AdipoGen, AG-20T-0138-C100, 1:1,000), and β-actin (Proteintech, 66009-1-IG, 1:1,000) at 4°C overnight. Horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000) was incubated for 1 h at room temperature. Finally, the signal was visualized with the ECL Detection Kit, and ImageJ software was used to analysis proteins.

Data are expressed as the means ± SDs, and differences between groups were examined using ANOVA and Tukey post hoc tests. p values less than 0.05 were considered statistically significant.

RAW264.7 cell line was derived from Abelson murine leukemia virus-induced tumors in Balb/c mice. MTT assay was used to evaluate the viability of RAW264.7 cells exposed to different concentrations of SYQP. The result showed that SYQP had no significant effect on the viability of RAW264.7 cells within the concentration range of 0–1,000 μg/ml (Figure 1A).

Different TLRs are the commonly studied innate immune system receptors (Root-Bernstein, 2021). To investigate the effect of SYQP on different TLRs, we measured the mRNA expression of TLR1-TLR9 in RAW264.7 cells by qRT-PCR in this study. As shown in Figure 1B, compared with control group, the mRNA expression levels of the TLR1, TLR2 and TLR6 genes were upregulated in SYQP-treated RAW264.7 cells. But there was no significant difference in TLR4 gene expression level compared with the control group, which was consistent with our previous experiment results (Zhou et al., 2021). In addition, the mRNA expression levels of the TLR3, TLR5, TLR7 and TLR9 genes remained unchanged. Similar results have been found in other studies of polysaccharides, for example, cordyceps sinensis polysaccharides were shown to regulate immune activity by upregulating the expression levels of TLR2, TLR4 and TLR6 in mice (Ying et al., 2020), and Salvia miltiorrhiza polysaccharides activated the mRNA expression of the TLR1, TLR2 and TLR4 genes in T lymphocytes (Chen et al., 2017). These data preliminarily indicated that SYQP could not only stimulate TLR4, but also activate immunity by increasing the gene expression of TLR1, TLR2 and TLR6 on the surface of macrophages.

TLR2 coactivates TLR1 or TLR6, forming heterodimers to evoke effects (Takada and Uehara, 2006). Thus, in this part, we mainly focused on correlation between TLR2/TLR4 and macrophage activation induced by SYQP on TLR2/TLR4 antagonist-treated cells (RAW264.7 and MPMs). TAK-242 and C29, antagonists of TLR4 and TLR2 respectively, have been proven to exhibit effective specific blocking properties (Chen et al., 2018). Firstly, we measured pro-inflammatory cytokines in TLR2/TLR4 antagonist-treated RAW264.7 cells (Figures 2A,B) and MPMs after SYQP stimulation (Figures 2C,D). The known TLR2 and TLR4 ligands Pam3Cys and LPS, respectively, were used as positive controls. In RAW264.7 cells and MPMs, SYQP stimulated the production of IL-6 and TNF-α. Individual application of C29 or TAK-242 decreased the stimulation of IL-6 and TNF-α production caused by SYQP, but cytokine secretion remained at a higher level compared with the control group. Combined application of C29 and TAK-242 almost completely abolished the stimulation of IL-6 and TNF-α production caused by SYQP. In addition, similar to SYQP, LPS induced overexpression of IL-6 and TNF-α in untreated and TLR2 antagonist-treated cells but not TLR4 antagonist-treated cells. In contrast, as expected, P3C was able to induce cytokine overexpression in untreated and TLR4 antagonist-treated cells but not TLR2 antagonist-treated cells.

We further investigated whether C29 or TAK-242 inhibited the transcription of inflammatory cytokines in SYQP-stimulated RAW264.7 cells. As shown in Figures 2E,F, 100 μg/ml SYQP increased the mRNA expression levels of IL-6 and TNF-α in RAW264.7 cells. Compared with SYQP treatment alone, individual treatment and combined treatment of C29 and TAK-242 inhibited the SYQP-induced increase in inflammatory cytokine transcription in RAW264.7 cells. The above experimental results showed that the immunity of SYQP can activate the TLR2 and TLR4 at the receptor level and reveals TLR2/TLR4 modulatory characteristics.

In this part, we further performed multiplex analysis in vitro and in vivo to determine the anti-inflammatory effects and the underlying mechanism. Initially, we examined the effect of SYQP on LPS-induced oxidative stress and inflammatory damage in vitro. RAW264.7 cells were pre-treated with SYQP or DEX for 2 h, and followed by stimulation with LPS for 18 h. The results demonstrated that SYQP played anti-inflammatory roles by suppressing the transcriptions and releases of IL-6 and TNF-α (Figures 3A–D). As shown in Figure 3E, SYQP effectively reduced NO secretion in LPS-induced RAW264.7 cells, and the mRNA and protein results showed that pre-treatment with SYQP (25, 50, and 100 μg/ml) induced significant downregulation of iNOS and COX-2 mRNA and protein levels (Figures 3F–J). The DCFH-DA assay was used to quantify ROS levels. As depicted in Figure 5K, pre-treatment with DEX or SYQP led to marked reduction of reactive oxidative stress in cells exposed to LPS.

To verify the anti-inflammatory effects of SYQP in ARDS mice, we investigated the effect of SYQP on pulmonary morphological damage using HE staining. The results showed that normal pulmonary histology was seen in the control group. In contrast, lung tissues in LPS administration model group were significantly damaged, manifesting as thickening of the alveolar wall, interstitial edema, hemorrhage and infiltration of inflammatory cells, and the destruction was improved by DEX and SYQP treatment (Figures 4A,B). Next, we examined the BALF, and found that the total cells and white blood cells (WBC) in LPS-treatment group were elevated significantly. Whereas, SYQP and DEX treatment significantly inhibited the elevation of these cell counts in BALF (Figures 4C,D). As shown in Figures 5A,B, compared with model group, DEX and SYQP significantly reduced the expression level of myeloperoxidase (MPO), which is a key inflammatory enzyme (Shuang Chen et al., 2020) in the lung tissues of ARDS mice. In addition, the F4/80 immunofluorescence results indicated that pre-administration with DEX or SYQP could inhibited the macrophages in lung tissues of LPS-indued mice (Figures 5C,D). Moreover, whole blood analysis results showed that SYQP caused the reduction of the WBC, neutrophils cells, lymphocytes cells, the neutrophil-to-lymphocyte ratio (NLR) and platelets in LPS-induced ARDS mice compared with model group (Figures 5E–I). According to the BALF and whole blood quantitative analysis data, the anti-inflammatory function of SYQP was equivalent to or even better than DEX.

To investigate the effect of SYQP in LPS-induced ARDS mice, plasma and lung tissues samples were collected and GSH, SOD, MDA and cytokines were determined. As shown in Figures 6A–C, IL-6, TNF-α and IFN-γ levels were significantly increased in the plasma of the LPS group. Treatment of SYQP (50, 100, 150 mg/kg) to LPS-induced mice effectively reduced the cytokine secretion levels. In addition, SYQP significantly improved the levels of MDA, SOD and GSH in plasma of LPS-induced ARDS mice (Figures 6D–F). Additionally, we examined the mRNA levels of cytokines in lung tissues. The mRNA results showed that SYQP or DEX induced significant downregulation of IL-6, TNF-α, IL-1β, COX-2 and iNOS mRNA levels (Figures 6G–K).

To determine whether the anti-inflammatory effect of SYQP is related to the expression of TLR2/TLR4, we investigated the effects of SYQP on TLR2 and TLR4 in LPS-activated RAW264.7 cells and lung tissues of ARDS mice using Q-PCR and western blotting analysis. As illustrated in Figure 7, LPS strongly induced the expression levels of TLR4 and TLR2 in vitro and in vivo. and SYQP could significantly decreases TLR4 and TLR2 expression levels.

TLR2 and TLR4 signalling pathways culminate in activation of NF-κB and result in nuclear translocation of NF-κB p65 (Kawai and Akira, 2007). Hence, immunofluorescence analysis was performed to investigate whether SYQP could inhibit LPS-induced NF-κB p65 nuclear translocation in RAW264.7 cells. As shown in Figure 8A, SYQP prevented the NF-κB p65 transcription factor moving into the nucleus. We further observed the effects of SYQP on the phosphorylation of p65 NF-κB expression in LPS-induced RAW264.7 cells and lung tissues of ARDS mice using western blotting analysis. We found that SYQP inhibited the phosphorylation of p65 NF-κB expression levels in vitro and in vivo (Figures 8B,C). In addition, SYQP markedly inhibited the LPS-induced MyD88, phosphorylation of IKK-β and IκB-α expression in RAW264.7 cells (Supplementary Figure S2).

According to the results of in vitro and in vivo experiments, SYQP could effectively inhibit the expression levels of NO and iNOS in LPS-induced RAW264.7 cells, moreover, the secretion levels of TNF-α and IFN-γ in plasma of ARDS mice were significantly reduced by SYQP. Study results have shown that synergistic increase of TNF-α and IFN-γ could induce cell death through activating JAK/STAT1 axis, inducing nitric oxide production and driving caspase-8 -mediated PANoptosis (Karki et al., 2021). Therefore, we speculated that SYQP was effective in the protection of tissue damage in ARDS mice, and western blotting assay was used to confirm this viewpoint. As shown in Figures 9 A,B, the phosphorylation of JAK2 and STAT1 were significantly inhibited by SYQP, which were related to IFN-γ signaling pathway. What’s more, SYQP reduced the overexpression of NLRP3, caspase-1, caspase-3 and caspase-8 in lung tissues of ARDS mice (Figures 9C–F). These results revealed that SYQP could protect ARDS mice by reducing cell pyroptosis and even apoptosis.