The PLE was from the same preparation (same batch number 20170622) of our previous research (Yang H. et al., 2020); the standardization of the PLE was also the same as before. Briefly, the dried Perilla frutescens (L.) Britt. leaves were ground into 40 meshes, then refluxed, and extracted twice by 5-fold (w/v) 70% alcohol, for 1.5 h per time. The combined extraction liquid was filtered and concentrated in a rotary vacuum evaporator, then ultrasonically mixed with water 3 times (w/v) to make a suspension and extracted twice by ethyl acetate. After the ethyl acetate in the water layer was volatilized, anhydrous ethanol was slowly added to the water layer and the mixture was stirred rapidly until the alcohol content was 80%, and the water layer was precipitated at room temperature for 24 h. Finally, the suspension was filtered to obtain the precipitant and supernatant. The precipitant was washed with 80% ethanol, and the washing solution was combined with the supernatant; then, the mixture was concentrated and dried to produce the PLE.

Wild-type male Balb/c mice (6–8 weeks old, 20–22 g, Experimental Animal Center, Vital River Laboratory Animal Technology Co., Ltd, Beijing, China) were housed in standard environment-controlled conditions with chow diet and water provided ad libitum. The animal experiments were approved by the Animal Care & Welfare Committee of Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College (Beijing, China) and conformed to internationally accepted ethical standards.

LPS/CS-induced COPD airway inflammation of mice was induced, as described previously (Fan et al., 2019). Briefly, mice were divided randomly into seven groups: the control group, model group, PLE (100, 200, and 400 mg/kg) group, dexamethasone (DEX, 0.5 mg/kg) group, and roflumilast (RFST, 20 mg/kg) group. Except for the control group, mice were intratracheally instilled with LPS (40 µg in 50 µl phosphate buffer saline, PBS) on day 1 and day 14 and from day 2–27 (except on day 14), challenged with CS daily for 1 h in a microenvironment system for small animals (MBL-1; Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing, China). Animals were orally administrated daily from day 14–27, 1 h before air or CS exposure. 24 h after the last treatment, the mice were anesthetized and sacrificed. The timeline of the experiment was shown in Figure 1A.

Twenty-four hours after the last administration, mice were anesthetized. Briefly, the BALF was collected by triple intratracheal instillation with 0.7 ml PBS buffer and centrifuged at 1,500 rpm for 10 min at 4°C to separate cells from the supernatant, and the enumeration for total white cells, neutrophils, monocytes, eosinophils, and lymphocytes was performed via a hemocytometer (Mindray BC-5000 vet, Shenzhen, China). The supernatants were removed and stored at −80°C for IL-6, IL-4, TNF-α, IFN-γ, and IL-17A detection, using commercial ELISA kits (R&D Systems) according to the manufacturer’s protocol with optical density (OD) values measured at 450 and 570 nm using a microplate reader (PowerWave XS2, BioTek, United States).

Lung tissue was harvested and fixed in 10% (v/v) formalin solution, then embedded in paraffin, sectioned at 4-μm thickness, applied to glass slides, and stained with hematoxylin and eosin (H&E) solution. Afterward, they were examined and photographed under a light microscope (100×). A four-point scale was used, as described previously (Yang H. et al., 2020). Generally, from each section, five blood vessels with a diameter of 100–200 μm or bronchi with a diameter of 150–300 μm were selected for examination. Histological examination was performed in a single-blind fashion.

RAW264.7 murine macrophage cells were cultured in Dulbecco’s modified Eagle medium supplemented with 10% (v/v) FBS, 100 U/mL penicillin, and 100 μg/ml streptomycin in a 37°C incubator with 5% CO₂. 2 × 10⁵, 8 × 10⁴, or 2 × 10⁴ cells/well were seeded into 48-well plates for nitric oxide (NO), IL-6, or TNF-α determination, respectively. The cells were pretreated with the PLE (50, 100, 200 μg/ml), DEX (1 μM), TLR4 inhibitor (TAK242, 5 nM), Syk inhibitor (BAY61-3,606, 10 μM), PKC inhibitor (Rottlerin, 2.5 μM), and NF-κB inhibitor (BAY11-7,082, 5 μM) for 1 h and afterward, stimulated with LPS at 20 ng/ml for NO production, 5 ng/ml for IL-6 production, and 2.5 ng/ml for TNF-α production for 24 h. The culture supernatants were collected and used for detection.

RAW264.7 cells were seeded at 2 × 10⁵/well in 96-well plates and were allowed to attach for 4 h before treatment with the PLE (50, 100, 200, 400, 800, 1,600, and 3,200 μg/ml) at 37°C for 24 h. Then, MTT (250 μg/ml) was added and incubated for 2 h. The OD values were measured at 570 nm using a microplate reader (Power Wave XS2, BioTek, Virginia, United States).

The RAW264.7 cells, cultured in 24-well plates at 4 × 10⁵ per well, were used for intracellular ROS detection. The cells were pretreated with the PLE (50, 100, and 200 μg/ml) and DEX (1 μM) for 1 h and then stimulated with LPS (1 μg/ml) for 24 h. The cells were then incubated with 10 μM (final concentration) of the oxidant-sensing probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA, Invitrogen, United States) at 37°C for 30 min in the dark. After cold PBS washing, their DCF fluorescence distribution were detected by using a flow cytometer (ACEA NovoCyte 2060R, China) and analyzed by FlowJo software (TreeStar Inc, Ashland, OR, United States). Images of DCF fluorescences in cells were captured by using an inverted research microscope (Nikon Eclipse Ti2, Tokyo, Japan) and analyzed by ImageJ software (National Institutes of Health, Maryland United States).

Mice lung tissues were lysed with RIPA buffer (Applygen, Beijing, China). The supernatants were collected and quantified with a BCA assay kit (Solarbio, Beijing, China) Then, protein extracts from tissues were separated on a 10% sodium dodecyl sulfate–polyacrylamide gel and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, United States). After being blocked with 5% nonfat milk, the membranes were probed with specific primary antibodies against β-actin, TLR4, Syk, phospho-Syk (p-Syk) (Y525/526), PKC, p-PKC, NF-κB p65, and p-NF-κB p65 (Ser536) (all from Cell Signal Technology, United States) separately overnight at 4°C and incubated with horseradish peroxidase–conjugated anti-rabbit secondary antibody (Thermo Fisher, United States) for 2 h. The labeled protein bands were visualized using an enhanced chemiluminescent (ECL) substrate kit (YEASEN Biotech, Shanghai, China), and digital images were captured by a chemical imaging system (Clinx Science Instruments Co., Ltd, China). The density of each band was quantified by ImageJ.

By using Trizol (Transgen, Beijing, China), total RNA was isolated from RAW264.7 cells, which were pretreated with the PLE (50, 100, 200 μg/ml), DEX (1 μM), TLR4 inhibitor (TAK242, 5 nM), Syk inhibitor (BAY61-3,606, 10 μM), PKC inhibitor (Rottlerin, 2.5 μM), and NF-κB inhibitor (BAY11-7,082, 5 μM) for 1 h, respectively, and then stimulated with LPS (1 μg/ml) for 24 h. Random mixture primers and reverse transcriptase were used to synthesize the first-strand cDNA. Commercial primers were synthesized by XBiotech (Actb: DMM404349, Tlr4: DMM286110, Prkcd: DMM575032, Syk: DMM519482, Rela: DMM880760). qRT-PCR was performed in a real-time PCR machine (MYGO PRO, IT-IS, Ireland), following the instructions of the top green quantitative PCR SuperMix (Transgen, Beijing, China). A 2^−ΔΔCt method was employed to analyze the relative gene expressions by comparing with the internal control gene (β-actin).

Values were presented as the mean ± standard error of mean (SEM). Statistical analysis was performed by the Graphpad Prism 7.0 (GraphPad, La Jolla, CA) software and analysis of one-way ANOVA. Differences were considered statistically significant if p < 0.05.

H&E staining and an inflammatory score were used to evaluate the inflammatory changes in the lung tissue of LPS/CS-induced COPD mice (Figure 2B). Extensive infiltration of inflammatory cells into peribronchial and perivascular regions was observed in COPD mice with significantly increased inflammatory scores, while, they were substantially ameliorated with PLE treatment (200 and 400 mg/kg, p < 0.01) and with RFST (20 mg/kg, p < 0.05 or p < 0.01) and DEX (0.5 mg/kg, p < 0.01) treatment.

Furthermore, as shown in Figure 1C, cell enumeration in the BALF also presented that PLE treatment and positive control RFST and DEX treatment markedly decreased the number of total white blood cells, lymphocytes, neutrophils, and macrophages (p < 0.01) dramatically, which were increased in COPD mice.

The levels of inflammatory cytokines in the BALF were measured by an enzyme-linked immunosorbent assay. As shown in Figure 2, in agreement with the histological inflammatory changes, a memorably higher level of IL-6 (Figure 2A), TNF-α (Figure 2B), IL-17A (Figure 2C), IL-4 (Figure 2D), and IFN-γ (Figure 2E) was observed in COPD mice (p < 0.01), while they were significantly decreased with PLE treatment (p < 0.05 or 0.01), showing a dose-dependent manner in the decrease of IL-6, TNF-α, and IL-4. The ratio of IFN-γ/IL-4 was increased slightly but with no significance in COPD mice when compared with the control group. As to positive control, significant inhibitions were shown in TNF-α (p < 0.01), IFN-γ (p < 0.05), and IL-17A (p < 0.01) with RFST treatment, but no significance was observed with DEX treatment, the exact reason of which still needs to be explored. These results suggested that the PLE was imperative for ameliorating inflammation in LPS/CS-induced COPD mice and exhibited a better outcome in decreasing the levels of inflammatory factors with PLE treatment than RFST and DEX treatment.

In vitro LPS-stimulated RAW264.7 cells were used to assess the anti-inflammatory effects of the PLE. As shown in Figure 3A, no obvious cytotoxicity was observed in PLE concentrations less than 1,600 μg/ml in vitro; therefore, 50, 100, and 200 ug/mL concentrations of the PLE were chosen for experiments in vitro. As shown in Figure 3B, PLE treatment significantly inhibited LPS-induced RAW264.7 cell intracellular ROS production (p < 0.01) manifested both in fluorescent cell counts and their representative fluorescent shift by flow cytometry detection, which was further confirmed by using an inverted research microscope observation (Figure 3C). As shown in Figures 3D–F, PLE treatment can significantly attenuate the secretion of NO (p < 0.01), IL-6 (p < 0.01), and TNF-α (p < 0.01) in LPS-stimulated RAW264.7 macrophages in a dose-dependent manner. By these, it is suggested that the PLE possessed an antioxidant and anti-inflammation potential in vitro.

In vivo significant increases of TLR4 (Figure 4A), Syk (Figure 4B), p-Syk (Figure 4C), PKC (Figure 4D), p-PKC (Figure 4E), NF-κB p65 (Figure 4F), and p-NF-κB p65 (Figure 4G) expressions were observed in lung tissues of LPS/CS-induced COPD mice (p < 0.05 or <0.01), while all of these increases were notably suppressed with PLE treatment (p < 0.05 or <0.01), acting dose dependently except that of the NF-κB p65 expression. These results indicated that the anti-inflammatory effects of the PLE on COPD might associate with inhibition of TLR4/Syk-related signaling pathways.

In line with the in vivo expression of TLR4, Syk, PKC, and NF-κB p65, in vitro PLE treatment notably decreased the mRNA expression of TLR4 (Figure 5A), Syk (Figure 5B), PKC (Figure 5C), and NF-κB p65 (Figure 5D) which appeared to be as effective as DEX treatment (p < 0.05 or <0.01), suggesting its in vitro anti-inflammatory effect which is also associated with TLR4/Syk-related signaling.

To further verify whether the PLE attenuated COPD airway inflammation via inhibition of the TLR4/Syk pathway, inhibitors of TLR4 (TAK242, 5 nM), Syk (BAY61-3,606, 10 μM), PKC (Rottlerin, 2.5 μM), and NF-κB p65 (BAY11-7,082, 5 μM) were employed to confirmatory study in vitro. Compared to individual treatment of PLE, TAK242, BAY61-3,606, Rottlerin, and BAY11-7,082 in LPS-stimulated RAW264.7 macrophages, the PLE co-treated with other inhibitors collaboratively suppressed the production of NO (Figure 6A), IL-6 (Figure 6B), and TNF-α (Figure 6C) (p < 0.01). Furthermore, by co-treating the PLE, TLR4 inhibitor (TAK242) obviously synergistically reduced TLR4, Syk, PKC, and NF-κB p65 mRNA expressions (Figure 6D); Syk inhibitor (BAY61-3,606) synergistically lessened the expression of Syk, PKC, and NF-κB p65 mRNA with no effect on TLR4 (Figure 6E); PKC inhibitor (Rottlerin) synergistically decreased PKC and NF-κB p65 mRNA levels with no effect on TLR4 and Syk (Figure 6F); and NF-κB inhibitor (BAY11-7,082) collaboratively inhibited the NF-κB p65 mRNA expression only (Figure 6G). Collectively, these results demonstrated that the PLE might mediate anti-COPD inflammatory response partly through TLR4/Syk/PKC/NF-κB p65 signals.