A total of 48 male SD rats (7-8 weeks old) with an average weight of 180-200 g were obtained from Changsha Tianqin Biotechnology Co., Ltd., and the rats were kept at Hainan Medical University's Experimental Animal Center using specific pathogen-free standards, with free access to water and food. The animal room was maintained at 22-24°C with a 12/12-h light-dark cycle. All animal experiments are conducted in the Experimental Animal Center of Hainan Medical University. The rats were randomly divided into the following four groups (n=12) after 7 days of acclimation feeding: Control group, PQ group, PQ + AH₂QDS group and AH₂QDS group (20). To model the poisoning, rats were fasted for 10 h prior to poisoning with reference to previous experiments (19,20). The control group was given 5 ml saline by gavage. The PQ group was administered 200 mg/kg 20% PQ solution per rat by gavage (Henan Lane Pesticide Factory). The PQ + AH₂QDS group was given 5 ml AH₂QDS by gavage at the molar ratio of PQ:AH₂QDS=1:1 (mol:mol) after 2 h of PQ intoxication, which was used to construct the treatment model. The AH₂QDS group was administered 5 ml AH₂QDS by gavage. To adhere to the principle of humane endpoints, the following measures were taken during the experiment to minimize the use of animals while ensuring the scientific rigor and humanity of the experiment. Firstly, sufficient practice was conducted and the modelling techniques were mastered beforehand to significantly reduce the failure rate of animal modelling, thus ensuring the reliability and validity of the experimental results. Secondly, during all experimental operations, it was ensured that animals were anesthetized to minimize potential pain and discomfort during the experiment. In addition, strict adherence to experimental ethical norms was implemented and clear humanitarian endpoints were formulated, tailored to the objectives of the present study. The specific standards were as follows: i) After 72 h of PQ intervention, as the scientific goals of the research were achieved, there was no need to continue the experiment; ii) when animals suffered from pain that was not caused by the experiment itself and was unexpected before the experiment starts, such as animals with inherent defects; iii) if it was anticipated before the experiment that the animals may suffer from pain, but the degree of pain exceeded expectations during the actual process, such as severe nasal and oral bleeding, severely abnormal behaviour, and other situations; and iv) if the pain of the animals was caused by the experiment itself, and this pain had been anticipated before the experiment started. Once any of the aforementioned standards were observed during the experiment, the animals were immediately euthanized to reduce their suffering. All animals in the present study were euthanized before the end of the experiment once these standards were met, ensuring that no animals would succumb due to any unnecessary reasons during the experimental process. The animal welfare was always upheld and the smooth progress of the experiment was ensured while maintaining the accuracy and reliability of the experimental results. The rats were euthanized using an overdose of sodium pentobarbital (>150 mg/kg) injected intraperitoneally following exposure to PQ for 72 h. When the cessation of the heartbeat and breathing of the rats was confirmed, and there were no reflexes, the death of the experimental animals was confirmed painlessly. Whole lung tissue was excised, dried and weighed. The left lung was placed in 10% paraformaldehyde and the right lung was placed in a cryopreservation tube and stored in a -80°C refrigerator.

The experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Ethics Committee of the First Affiliated Hospital of Hainan Medical University [approval no. 2020 (Research) No. (97); Haikou, China].

Rat lung tissue were paraffin-embedded in paraffin after being fixed in 10% formaldehyde at room temperature for 24 h and cut into 4-μm-thick sections. After successful staining with haematoxylin and eosin, pathological changes were detected under a light microscope. Lung tissue damage was quantified using a score of 0 (normal) to 3 (severe) that included alveolar wall oedema, haemorrhage, vascular congestion and polymorphonuclear leukocyte infiltration. As previously reported, lung injuries were categorized according to the sum of the scores (27). Lung injuries were graded on a scale of severity from 0 to 5 (0, normal; 1, mild or very small amount; 2, mild or small amount; 3, moderate or larger amount; 4, severe or large amount; and 5, very severe or extremely large amount).

To determine pulmonary vascular permeability, Evans blue solution (2 mg/kg) was injected into the tail vein of rats, and the rats were euthanized 1 h after the injection. Lung tissue was perfused with PBS until the lungs turned white, resected as a whole, dried and weighed, and the right lung was weighed in half. The lung tissue was homogenized with formamide and incubated at 60°C in a water bath protected from light for 24 h. After incubation, the suspension was centrifuged (4°C, 13,400 × g, 15 min) and 200 μl of the supernatant was added to a 96-well plate, and the absorbance at 620 nm was measured using an enzyme counter. The amount of Evans blue dye was calculated using the standard curve method.

Immunohistochemical staining was used to detect the protein expression of vascular endothelial-cadherin (VE-cadherin), zonula occludens-1 (ZO-1) and CD31 in paraffin sections (4-μm-thick) of lung tissues. Paraffin sections were dewaxed (environmental-friendly dewaxing solution; cat. no. G1128; Wuhan Servicebio Technology Co., Ltd.), rehydrated using descending alcohol series for antigen repair and recovered; and the sections were placed in 3% BSA (cat. no. 36100ES25; Shanghai Yeasen Biotechnology Co., Ltd.) for 30 min at room temperature for blocking, and then incubated overnight at 4°C with VE-cadherin antibody (dilution, 1:50; cat. no. XF354429; Invitrogen; Thermo Fisher Scientific, Inc.), ZO-1 antibody (dilution, 1:1,000; cat. no. 21773-1-AP; Proteintech Group, Inc.) and CD31 antibody (dilution, 1:1,000; cat. no. GB113151; Wuhan Servicebio Technology Co., Ltd.), followed by incubation with HRP-labelled goat anti-rabbit IgG secondary antibody (dilution, 1:200; cat. no. GB23303; Wuhan Servicebio Technology Co., Ltd.) for 1 h at room temperature. Finally, the sections were stained with diaminobenzidine and counterstained with haematoxylin (room temperature, 3 min), and observed under a light microscope. Microscopic images were evaluated using ImageJ software (Fiji distribution of ImageJ; https://imagej.net/software/fiji/), and the area of VE-cadherin, ZO-1 and CD31-positive cells was quantified.

HPMECs were purchased from Otwo Biotechnology Development Co., Ltd. (cat. no. HTX2255) and cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (Procell Life Science & Technology Co., Ltd.) with 5% CO₂ at 37°C for proliferation. Cells were incubated with different concentrations of PQ pure (AccuStandard, Inc.) for 24, 48 and 72 h. The results are shown in Fig. S1A. Cell viability was reduced in a dose-dependent manner, and the reduction in cell viability was more pronounced after 72 h of treatment than at 48 and 24 h. Although the IC₅₀ results ranged between 200 and 300 μM (Fig. 1C), the viability of the cells was <50% after 300 μM treatment. Therefore, a PQ dose of 200 μM was chosen for subsequent experiments to treat the cells for 72 h. HPMECs were then pre-treated with different concentrations of AH₂QDS for 2, 6 and 12 h, and 200 μM AH₂QDS had the least effect on cell viability (Fig. 2A and B). Therefore, 200 μM was selected as the concentration of AH₂QDS for subsequent experiments.

The cells were divided into the following four groups: Control group, HPMECs were treated with medium for 72 h; PQ group, cells were stimulated with PQ (200 μM) alone for 72 h; PQ + AH₂QDS group, HPMECs were treated with AH₂QDS (200 μM) for 2 h followed by stimulation of the cells with PQ (200 μM) for 72 h; and AH₂QDS group, cells were stimulated with AH₂QDS (200 μM) alone for 72 h.

Experiments were performed using Transwell plates with a pore size of 5 μm to assess cell migration. Briefly, 3×10⁴ HPMECs were inoculated into the upper part of the migration chamber containing serum-free medium. The lower chamber was filled with complete medium containing 20% fetal bovine serum as an attractant. After 72 h of incubation, migratory cells were stained with 0.1% crystal violet (Biosharp Life Sciences) at room temperature for 10 min and non-migratory cells in the upper chamber were removed with a cotton swab. Cells were then imaged and counted under a light microscope.

To assess PQ and AH₂QDS cytotoxicity, HPMECs in the logarithmic growth phase were homogeneously inoculated into 96-well plates at a density of 3-5×10³ per cell culture (100 μl/well). After waiting for 12 h of cell apposition, the cells were treated with different concentrations of PQ for 24, 48 and 72 h. The cells were pre-treated with different concentrations of AH₂QDS for 2 h, and then treated with 200 μM PQ for 72 h. Cell Counting Kit-8 (CCK-8) solution (10 μl) was added to each well according to the manufacturer's instructions (cat. no. BS350C; Anhui Biosharp Technology Co., Ltd.). The 96-well plates were further incubated in a 37°C cell incubator for 1-2 h. Absorbance was measured at 450 nm using an enzyme marker.

Intracellular NO levels were measured using the fluorescent indicator diaminofluorescein-FM diacetate (DAF-FM DA; Shanghai Yeasen Biotechnology Co., Ltd.). Cells were inoculated at a density of 1×10⁴ per dish and cultured in confocal dishes, and cells were pre-treated with AH₂QDS for 2 h and stimulated with PQ (200 μM) for 72 h. After treatment, the medium was discarded and the cells were gently washed three times with PBS, and then the cells were treated with 200 μl DAF-FM DA (5 μM) for 30 min in a 37°C cell incubator. Cells were washed three times using PBS and kept in PBS throughout the experiment. Finally, the fluorescence intensity was measured using a laser confocal microscope with an excitation wavelength of 488 nm and an emission wavelength of 515 nm. The measured fluorescence values were analysed using ImageJ software to examine the changes in fluorescence intensity in each group.

The level of intracellular ROS was measured using the Reactive Oxygen Detection kit (cat. no. BL714A; Anhui Biosharp Technology Co., Ltd.). Cells were inoculated at a density of 1×10⁴ per dish and cultured in confocal dishes, and cells were pre-treated with AH₂QDS for 2 h and stimulated with PQ (200 μM) for 72 h. After treatment, the medium was discarded and the cells were gently washed three times with PBS. Subsequently, the cells were treated with 200 μl 2',7'-dichlorodihydrofluorescein diacetate (10 μM) for 30 min at 37°C in a cell culture incubator, and washed three times with PBS, and after that, the whole experiment was kept in serum-free medium. Finally, the fluorescence intensity was measured using a laser confocal microscope with an excitation wavelength of 488 nm and an emission wavelength of 515 nm. The measured fluorescence values were analysed using ImageJ software to examine the changes in fluorescence intensity in each group.

After establishing the cell model, the cells were lysed with RIPA lysis solution and centrifuged at 13,400 g at 4°C for 10 min, and the supernatant was the desired sample. According to the instructions of the MDA (cat. no. BL1481A; Anhui Biosharp Technology Co., Ltd.) and SOD (cat. no. A001-3-2; Nanjing Jiancheng Bioengineering Institute) detection kits, the relevant reagents were added to the reaction, and finally the absorbance values were measured using an enzyme marker at 532 and 450 nm, respectively.

TNF-α, IL-1β and IL-6 levels in the supernatants of HPMECs were detected using a human ELISA kit (Fine Biotech Co., Ltd.) according to the manufacturer's instructions.

The rat lung tissues and HPMECs were lysed with RIPA lysis buffer (cat. no. G2002-100ML; Wuhan Servicebio Technology Co., Ltd.) containing protease inhibitors (cat. no. BL612A; Anhui Biosharp Technology Co., Ltd.) to prepare protein samples. After quantification of the protein concentration using a BCA Protein Assay Kit (cat. no. 20201ES76; Shanghai Yeasen Biotechnology Co., Ltd.), protein samples (40 μg) were separated by 7.5-10% SDS-PAGE, and then transferred to PVDF transfer membranes. The membranes were blocked with 5% skimmed milk powder for 1 h at room temperature, and incubated with primary antibody at 4°C overnight. The following primary antibodies were used: VE-cadherin antibody (dilution, 1:250; cat. no. XF354429; Invitrogen; Thermo Fisher Scientific, Inc.), ZO-1 antibody (dilution, 1:10,000; cat. no. 21773-1-AP; Proteintech Group, Inc.), IL-6 antibody (dilution, 1:1,500; cat. no. bs-0782R; BIOSS), IL-1β antibody (dilution, 1:1,000; cat. no. bs-0812R; BIOSS), TNF-α antibody (dilution, 1:1,000; cat. no. BP4903; Wuhan Boster Biological Technology, Ltd.), PI3K antibody (dilution, 1:1,000; cat. no. 20584-1-AP; Proteintech Group, Inc.), phosphorylated (p-)PI3K antibody (dilution, 1:1,000; cat. no. PA5-104853; Invitrogen; Thermo Fisher Scientific, Inc.), AKT antibody (dilution, 1:1,000; cat. no. 10176-2-AP; Proteintech Group, Inc.), p-AKT antibody (dilution, 1:1,000; cat. no. 28731-1-AP; Proteintech Group, Inc.), eNOS antibody (dilution, 1:1,000; cat. no. 27120-1-AP; Proteintech Group, Inc.), β-actin antibody (dilution, 1:1,000; cat. no. GB11001-100; Wuhan Servicebio Technology Co., Ltd.) and GAPDH antibody (dilution, 1:1,000; cat. no. GB15004-100; Wuhan Servicebio Technology Co., Ltd.). The membranes were washed at least three times with TBS with 1% Tween-20 (TBST) and incubated with secondary antibody conjugated to HRP-labelled goat anti-rabbit IgG (dilution, 1:10,000; cat. no. BL003A; Anhui Biosharp Technology Co., Ltd.) at room temperature for 2 h. After three washes with TBST, images of the bands were captured (Super ECL Detection Reagent-ECL Ultra-Sensitive Chemiluminescent Detection Kit; cat. no. 36208ES60; Shanghai Yeasen Biotechnology Co., Ltd.) and analysed using a chemical gel imager (CHAMP CHEMI TOP 610 PLUS).

All data are presented as the mean ± standard error of the mean (SEM) of at least three independent experiments and were analysed using GraphPad Prism8 software (Dotmatics). One-way ANOVA followed by Tukey's multiple comparison test was used to determine differences between groups. P<0.05 was considered to indicate a statistically significant difference.

To study the cytotoxicity of PQ, cell viability was examined using a CCK-8 assay. Cells were treated with pure PQ (0, 200, 400, 800, 1,200, 1,600 and 2,000 μM) for 24 and 48 h, and cell viability was assessed at the end of culture. As shown in Fig. S1A and B, the inhibition of cell viability by PQ was dose- and time-dependent. After 24 h of PQ treatment, the cell viability was decreased following 1,600 μM PQ treatment (Fig. S1A), and after 48 h of PQ treatment, the cell viability was decreased following 1,200 μM PQ treatment, with a cell viability of >60% compared with the control in all cases (Fig. S1B). However, after 72 h of PQ treatment, the cell viability was significantly decreased following 200 μM PQ treatment, down to 59% compared with the control (Fig. S1C). When calculating the IC₅₀ of PQ, the IC₅₀ of PQ was 200-300 μM; however, at 300 μM, cell viability was <50%. Therefore, 200 μM was selected as the concentration of PQ for subsequent experiments (Fig. 1C). These results indicated that PQ induced cytotoxicity in HPMECs in a dose- and time-dependent manner (Fig. 1A and B).

To investigate the effect of AH₂QDS on the proliferation of cells, cell viability was determined using the CCK-8 assay. Cells were treated with 0, 10, 50, 100, 200, 300, 400, 600 and 800 μM AH₂QDS for 24 h. Cell viability was assessed at the end of the incubation. As demonstrated in Fig. 2A, after 2 h, 0, 10, 50, 100 and 200 μM AH₂QDS treatment had no significant effect on cell viability (Fig. 2A); however, 300 μM AH₂QDS and other concentrations of AH₂QDS had an effect on cell viability. Furthermore, when treating the cells with 200 μM AH₂QDS for 2, 6 and 12 h, cell viability was decreased after 6 h of treatment (Fig. 2B), which demonstrated the time- and dose-dependent effects of AH₂QDS on the viability of HPMEVCs. To assess the protective effect of AH₂QDS against PQ-induced cytotoxicity, cells were pre-treated with 200 μM AH₂QDS for 2 h, and then cell viability was assessed after exposure to 200 μM PQ for 72 h. As revealed in Fig. 2C, pretreatment with AH₂QDS significantly increased the PQ-induced decrease in cell viability (Fig. 2C), but this protective effect was markedly reduced by PQ at concentrations >300 μM.

The intracellular oxidative stress level was assessed by detecting ROS, NO fluorescence intensity, MDA and SOD levels. As shown in Fig. 3A-D, the fluorescence intensity of ROS and NO in PQ-induced HPMECs was significantly higher compared with that in the control group, whereas the fluorescence intensity of ROS and NO in PQ-induced cells was decreased by pretreatment with AH₂QDS, suggesting that AH₂QDS could attenuate the production of ROS and NO in PQ-induced cells, thus reducing endothelial cell damage. As demonstrated in Fig. 3E and F, the levels of SOD and MDA were also examined in HPMECs. MDA levels were significantly higher and SOD levels were lower in the PQ group compared with the control group; while MDA levels were decreased and SOD levels were significantly higher in the PQ-induced cells after pretreatment with AH₂QDS, which indicated that AH₂QDS could attenuate the level of oxidative stress in the PQ-induced cells. As revealed in Fig. 3G-I, ELISA results revealed that AH₂QDS significantly inhibited the expression of TNF-α, IL-6 and IL-1β in PQ-induced HPMECs.

The migration of HPMECs was detected using a Transwell assay. As displayed in Fig. 4A and B, the number of migratory cells in the PQ group was significantly lower compared with that in the control group, while the number was significantly higher after AH₂QDS treatment, which indicated that AH₂QDS could promote the migration of HPMECs. The intracellular protein expression levels of VE-cadherin and ZO-1 were detected by WB, and the results are shown in Fig. 4C-E. The protein expression levels of VE-cadherin and ZO-1 in the PQ group were significantly reduced, and the protein expression levels were significantly increased after AH₂QDS treatment, which demonstrated that AH₂QDS could maintain the normal connectivity of HPMECs. The fluorescence intensity of ZO-1 protein was detected using an immunofluorescence assay. The results in Fig. 4F and G indicated that ZO-1 protein expression was significantly reduced in the PQ group compared with the control group, and some cells were deficient in protein expression. This result was reversed by AH₂QDS treatment.

To assess the effect of AH₂QDS on PQ-induced ALI in rats, a PQ-induced ALI rat model was constructed, and pathological changes in lung tissues were detected by H&E staining. As demonstrated in Fig. 5A, PQ treatment resulted in marked inflammatory infiltrates, destruction of alveolar and alveolar wall capillary structures, and pulmonary oedema in the lung tissues. These symptoms were markedly improved by AH₂QDS treatment. Additionally, as shown in Fig. 5B, the lung injury score was significantly higher in the PQ group compared with the control group, whereas the lung injury score was reduced after AH₂QDS treatment. To verify that AH₂QDS could reduce inflammation in lung tissues, the protein expression levels of IL-6, IL-1β and TNF-α in lung tissues were detected using WB. As indicated in Fig. 5C-F, the protein expression levels of IL-6, IL-1β and TNF-α in the PQ group were significantly increased compared with those in the control group; however, the protein expression levels of IL-6, IL-1β and TNF-α were significantly decreased after AH₂QDS treatment.

To verify that AH₂QDS reduces pulmonary oedema by decreasing pulmonary microvessel permeability and decreasing exudation, pulmonary microvessel permeability was examined using the Evans blue assay (Fig. 6A and B). The protein expression levels of VE-cadherin and ZO-1 in lung tissues were detected by WB, and the results were consistent with the results obtained in HPMECs (Fig. 6C-E). Immunohistochemical staining results showed that the protein expression levels of VE-cadherin, ZO-1 and CD31 were significantly reduced in lung tissues in rats; however, these were significantly increased in the AH₂QDS treatment group (Fig. 6F-I).

To further elucidate the underlying mechanisms of AH₂QDS for the management of PQ-induced endothelial dysfunction, the present study concentrated on the PI3K/AKT/eNOS signalling pathway. As shown in Fig. 7A-C, in rat lung tissues, p-PI3K and p-Akt protein levels were elevated in the PQ group compared with the control group, whereas AH₂QDS treatment reduced the protein levels of p-PI3K and p-Akt. As demonstrated in Fig. 7D and E, PQ increased eNOS expression in rat lung tissues, which was reversed to a certain extent by AH₂QDS treatment. As revealed in Fig. 7F-H, the protein levels of p-PI3K and p-Akt were elevated in HPMECs, whereas AH₂QDS treatment suppressed the protein levels of p-PI3K and p-Akt, which was consistent with the results of the in vitro model. The results demonstrated that the PI3K/AKT/eNOS signalling pathway was involved in the beneficial effect of AH₂QDS on PQ-induced ALI. The mechanism of AH₂QDS reducing PQ-induced pulmonary microvascular permeability is presented in Fig. 8.