Anthrahydroquinone-2-6-disulfonate is a novel, powerful antidote for paraquat poisoning

Paraquat (PQ) is a widely used fast-acting pyridine herbicide. Accidental ingestion or self-administration via various routes can cause severe organ damage. Currently, no effective antidote is available commercially, and the mortality rate of poisoned patients is exceptionally high. Here, the efficacy of anthrahydroquinone-2-6-disulfonate (AH2QDS) was observed in treating PQ poisoning by constructing in vivo and ex vivo models. We then explored the detoxification mechanism of AH2QDS. We demonstrated that, in a rat model, the PQ concentration in the PQ + AH2QDS group significantly decreased compared to the PQ only group. Additionally, AH2QDS protected the mitochondria of rats and A549 cells and decreased oxidative stress damage, thus improving animal survival and cell viability. Finally, the differentially expressed genes were analysed in the PQ + AH2QDS group and the PQ group by NextGen sequencing, and we verified that Nrf2’s expression in the PQ + AH2QDS group was significantly higher than that in the PQ group. Our work identified that AH2QDS can detoxify PQ by reducing PQ uptake and protecting mitochondria while enhancing the body's antioxidant activity.


Results
Binding of PQ and AH 2 QDS. Firstly, we used AutoDock Vina 21 to simulate the binding conformation between AH 2 QDS and PQ (Fig. 1A,B). A grid map of dimensions 26 Å × 26 Å × 26 Å with a grid space of 0.375 Å was set. The search space's center was set to − 0.014 Å, − 0.008 Å and − 0.037 Å (x, y, z). One hundred GA (genetic algorithm) runs was placed, and all other parameters were the default option values by AutoDock Vina. Molecular docking results indicate that the crystal structure of the PQ + AH 2 QDS complex contains three intermolecular interactions, with π π stacking between the two benzene rings of AH 2 QDS and the PQ molecule, hydrophobic interactions between the middle of AH 2 QDS and PQ, and the two sides of AH 2 QDS forming a salt bridge with PQ (Fig. 1C,D). The above docking simulation studies demonstrate at a theoretical level that AH 2 QDS is able to bind to PQ to form a complex, thereby eliminating the toxicity of PQ. Next, we constructed in vivo and in vitro models to validate the detoxification of PQ by AH 2 QDS. AH 2 QDS for the treatment of PQ poisoning in vitro. In the in vitro experiment, we first used CCK8 to determine the effects of different concentrations of PQ on the viability of A549 cells 22   www.nature.com/scientificreports/ data, we used this condition in subsequent experiments. At the same time, we measured the effects of different concentrations of AH 2 QDS on the viability of A549 cells ( Figure S1B). It is worth noting that when the concentration of AH 2 QDS is greater than 200 μM, it will also have a toxic effect on cells, so we chose 200 μM AH 2 QDS as the concentration for the follow-up experiment. Given the oxidative damage-related mechanism of PQ, we also used glutathione, which is often used to resist the damage caused by oxidative stress 10 . Here, we chose different concentrations of glutathione to determine its effect on the viability of A549 cells ( Figure S1C). The results showed that glutathione had no toxic effect on cells.
Next, Fig. 2A,B showed that PQ could significantly damage the viability of A549 cells, while glutathione and AH 2 QDS intervention raised the activity of A549 cells. In addition, we pre-intervened AH 2 QDS and glutathione and then administered PQ staining after different treatment times to assay the cell activity for 72 h. The results showed that the Glutathione/AH 2 QDS pretreatment + PQ group still showed a significant rise in cell viability compared to the PQ group ( Figure S2). However, the Glutathione pretreatment group only showed good results at 1 h, while the AH 2 QDS pretreatment group could still exert excellent cytoprotective effects until 12 h. This result reflects the ability of both AH2QDS and Glutathione to induce intrinsic cellular protective effects, however, AH 2 QDS is more protective than Glutathione. AH 2 QDS improve antioxidation in the treatment of PQ poisoning. PQ poisoning often causes oxidative stress damage. Consistent with the literature, the results in Fig. 3A-C showed that the level of GSH-Px in the PQ group decreased, indicating that its antioxidant capacity decreased 23 . In contrast, ROS and MDA levels increased in the PQ group, indicating that oxidative damage was aggravated 24,25 . In this context, the level of GSH-Px in the group treated with AH 2 QDS was significantly higher, and the levels of ROS and MDA were significantly lower than those in the PQ group. The same trend was also confirmed in vivo ( Fig. 7D-F). In summary, AH 2 QDS plays an antioxidant role in the treatment of severe PQ.
Protective effect of AH 2 QDS on cell mitochondria. Many studies have reported that PQ poisoning often causes damage to mitochondria [26][27][28] . To explore whether AH 2 QDS can protect mitochondria, we used a transmission electron microscope to observe the mitochondrial structure under a microscope. From the Fig. 8D-F, we can see that after PQ intervention, the mitochondrial structure of A549 cells was destroyed, vacuoles appeared in the cell body, the cell wall was broken, and a large number of organelles were extruded. However, the morphology of cells in the untreated control group and PQ + AH 2 QDS group was normal, the chromatin was evenly distributed, the morphology of the cells was as expected, and the morphology of the mitochondria was normal. Furthermore, through detection of the mitochondrial membrane potential, we found that the membrane potential of the PQ + AH 2 QDS high-dose group was the highest, followed by the high-dose group and PQ + AH 2 QDS low-dose group, and the mitochondrial membrane potential of the PQ group was the lowest (Fig. 3D-J). The above results indicate the protective effect of AH 2 QDS on cell mitochondria in vitro.
The survival rate in a rat model of PQ poisoning. According to the literature, the single-dose oral LD50 for PQ was 100 mg/kg in rats 29 . Therefore, to determine PQ toxicity, we gavaged PQ at doses of 100, 200, 300, 400, and 500 mg/kg in vivo (Fig. 4A). We found that when the concentration of PQ was more than 300 mg/ kg (3X LD50), the rats showed obvious poisoning symptoms and died within two weeks. When the concentration of PQ was more than 400 mg/kg (4X LD50), the animals died in approximately three days. According to these data, to show the superior detoxification ability of AH 2 QDS, we chose 400 mg/kg (4X LD50) as the dose www.nature.com/scientificreports/ of PQ to evaluate the detoxification effect of AH 2 QDS. Subsequently, we used AH 2 QDS to detoxify the animals at different times after exposure. As shown in Fig. 3A, the 30-day survival rate of rats exposed to 400 mg/kg PQ could reach 100% when they were detoxified with AH 2 QDS within 2 h. However, as the time window for AH 2 QDS treatment was extended, the 30-day survival rate of SD rats gradually decreased (Fig. 4B). According to the above results, we chose 2 h as the detoxification time of AH 2 QDS. Next, we designed different experimental groups to verify the detoxification effect of AH 2 QDS (Fig. 4C). We discovered that the untreated control group's 30-day survival rates, the AH 2 QDS group, and the PQ + AH 2 QDS group were all 100%. The untreated control group's 30-day survival rates and the PQ + "white and black" group were zero, and all of the rats died within one week. The detoxification effect of AH 2 QDS is better than that of the "white and black" scheme. AH 2 QDS mitigates organ damage in a rat model of PQ poisoning. The lung is the main target organ after PQ poisoning 30 . Patients often die of acute lung injury in the early stage, and pulmonary fibrosis often occurs later 31,32 . According to Fig. 5A-P, we also observed alveolar inflammation in the lung tissue of rats in the PQ and PQ + c "white and black" groups at all time points, including destruction of the alveolar structure, oedema in the alveolar cavity, intracapillary hyperaemia, and inflammatory cell infiltration, indicating acute lung injury. On the seventh day, alveolar fusion, alveolar septum thickening, and fibrous tissue hyperplasia were found in the lungs of the two groups, indicating pulmonary fibrosis. However, the lung tissues of rats in the untreated control, AH 2 QDS, and PQ + AH 2 QDS groups were as expected at all periods, with very little infiltration of inflammatory cells, no collapse of the alveolar walls, no thickening of the alveolar septa, no exudation in the alveoli, and no capillary dilation, hyperaemia or other manifestations. The pathological injury score of the lung tissue showed that lung injury in the PQ group and PQ + "white and black" group was significantly worse www.nature.com/scientificreports/ than that in the untreated control, AH 2 QDS, and PQ + AH 2 QDS groups, and the difference was statistically significant (p < 0.001) (Fig. 5Q).
In addition to lung injury, PQ poisoning can also cause severe functional damage to multiple organs, so we measured the liver and kidney function and blood gas of the SD rats in each group [33][34][35] . As shown in Fig. 6A,B, the ALT, AST, CREA, and UREA levels in the PQ and PQ + "white and black" groups increased from the 3rd day and peaked on the 7th day. The ALT, AST, CREA, and UREA levels in the untreated control, AH 2 QDS, and PQ + AH 2 QDS groups were significantly lower than those in the PQ and PQ + "white and black" groups in the first seven days, and the difference was statistically significant (P < 0.001). In addition, we compared the PQ group with the PQ + "white and black" group and found that the ALT, AST, CREA, and UREA levels in the PQ + "white and black" group were significantly lower than those in the PQ group (P < 0.001). However, the hepatic and renal function of the untreated control, AH 2 QDS, and PQ + AH 2 QDS groups was in the normal range during each period, and there was no significant difference between them (P > 0.05). PQ poisoning has been verified to cause functional damage to multiple organs, and AH 2 QDS treatment of PQ poisoning can alleviate liver and kidney function damage. The blood gas analysis results showed ( Fig. 6 C-D) that the pH and PaO2 values in the PQ and PQ + "white and black" groups were significantly lower than those in the untreated control, AH 2 QDS, and PQ + AH 2 QDS groups (P < 0.001). Compared with the PQ group and PQ + "white and black" group, the pH and PaO2 values in the PQ + "white and black" group were significantly higher than those in the PQ group. The PaO2 values in the untreated control, AH 2 QDS, and PQ + AH 2 QDS groups were in the normal range during each period, and there was no significant difference between the three groups (P > 0.05). Contrary to this trend, the PaCO2 values in the PQ and PQ + "white and black" groups showed an increasing trend, indicating that hypoxaemia and carbon dioxide retention occurred in PQ-poisoned rats, which eventually led to type II respiratory failure. In summary, these findings suggest that AH 2 QDS can lower the damage to organ function caused by PQ poisoning.  The survival curve of rats in different treatment groups. The untreated control group did not make any interventions. In the AH 2 QDS group, only 400 mg/kg AH 2 QDS antidote was given by gavage. PQ was given by gavage only at a concentration of 400 mg/kg in the PQ group. In the PQ + "white and black" group, 400 mg/kg of PQ was given by gavage first, and 500 mg/kg was given by gavage 2 h later, with a "white and black" scheme. In the PQ + AH 2 QDS group, 400 mg/kg of PQ was given to the stomach first, and 400 mg/kg of AH 2 QDS antidote was given 2 h later. Kaplan-Meier survival analysis was used to analyze the survival rate of rats in different treatment groups, n = 7, *P < 0.05, **P < 0.001. www.nature.com/scientificreports/ PQ + AH 2 QDS group and PQ + "white and black" group decreased immediately after 2 h and was significantly lower than that in the PQ group (P < 0.001). The difference between 2 and 24 h was significantly smaller in the PQ + AH 2 QDS group than in the PQ + "white and black" group, and the difference was statistically significant. Similarly, the concentration of PQ in the lung tissue and urine decreased significantly in the PQ + AH 2 QDS group. The decrease in PQ drug concentration may have occurred because AH 2 QDS neutralizes PQ in the gastrointestinal tract.

Protection of mitochondria by AH 2 QDS in vivo.
The induction of mitochondrial damage by PQ has been confirmed in in vitro experiments, and we also observed the same phenomenon in vivo experiments. Figure 8B showed that the PQ group's mitochondria were swollen, structurally damaged, vacuolated and empty. Under the electron microscope, the mitochondrial structure was as expected in the rat lung tissues in the untreated control group and PQ + AH 2 QDS group ( Fig. 8A/C). These pictures illustrated that AH 2 QDS protects the structure of mitochondria.
NextGen sequencing. To better understand the detoxification mechanism of AH 2 QDS, we used RNAseq to investigate the differential gene expression patterns of rat lung tissue in the PQ and PQ + AH 2 QDS groups. Firstly, we performed data quality control ( Figure S4A), after which we used principal component analysis (PCA) to identify outlier samples and high similarity samples. As illustrated in the Figure S4B, in this experiment, different samples from the same experimental group are arranged compactly and aggregated into clusters, showing good repeatability. In contrast, different experimental groups are clearly separated from each other, showing reasonable specificity. We can see from Fig. 9A that there were 3325 gene changes in the PQ group compared with the PQ + AH 2 QDS group, including 1455 upregulated genes and 1870 downregulated genes. As shown in Fig. 9B, the most differentially regulated pathways in these two samples are the PI3K-AKT pathway, MAPK pathway, AMPK pathway, etc. Consistent with our previous findings, these pathways are mainly oxidative stressrelated pathways. We investigated the most significant pathway, namely, the PI3K-AKT pathway, to identify the genes with significant changes, and the results showed that Nrf2, Foxo3, Rxra, Itga4, Creb3l2, Angpt1, Egfr, Tnc, Lamc1, and Met were significantly upregulated. Nrf2 is significantly upregulated in tissues, and its function is www.nature.com/scientificreports/ closely related to oxidative stress, so we speculate that Nrf2 may be an essential gene for AH 2 QDS treatment of PQ poisoning. We further verified by western blot and RT-qPCR experiments that in in vitro experiments, as illustrated in the Fig. 10A-C, compared with PQ treatment, glutathione and AH 2 QDS could significantly increase the expression of Nrf2, while the Nrf2 level of PQ + AH 2 QDS group was significantly higher than that of the PQ + glutathione group. The in vivo experiment showed that the levels of Nrf2 in the AH 2 QDS, PQ, and PQ + "white and black" groups were higher than that in the untreated control group, while the Nrf2 level in the PQ + AH 2 QDS group was significantly higher than those in the other groups (Fig. 10D-F). The results indicated that "white and black" scheme did not activate Nrf2. In contrast, glutathione could increase the expression of Nrf2, but its effect was weaker than that of AH 2 QDS, indicating that our antidote, AH 2 QDS, could significantly increase the expression of Nrf2, thus exerting its detoxification effect.

Discussion
In this study, we used AH 2 QDS as an intervention in a rat model of PQ poisoning. Compared with those of the PQ group, the poisoning symptoms of the PQ + AH 2 QDS group were significantly improved, with a lower blood drug concentration, less organ function damage, and a higher survival rate. In the PQ + AH 2 QDS group, mitochondrial damage in lung tissue was alleviated, and a similar phenomenon was found in the cell test. The structure of the mitochondria was intact, the damage was significantly alleviated, and the expression of Nrf2 was significantly increased. These studies have proven for the first time that AH 2 QDS is an effective treatment for PQ poisoning, and Nrf2 plays a crucial role in its detoxification process.
Previous studies have shown that activated carbon or the "white and black" scheme can effectively treat PQ poisoning [6][7][8] . In contrast, our experiments only confirmed that the conventional "white and black" scheme can quickly and effectively degrade the PQ blood concentration but does not affect the survival rate of rats. Specifically, a 4X LD50 dose of PQ was used to construct the poisoning model, and the drug intervention time was as long as 2 h, during which most of the PQ may have been absorbed into the blood, while the "white and black" scheme could only absorb the residual poison in the stomach and accelerate its excretion but had no effect on  www.nature.com/scientificreports/ the PQ already in the blood. Additionally, the results also showed that AH 2 QDS is not only faster than the "white and black" scheme in removing toxins but also plays a specific role in the blood-related effects of PQ. The toxic effect of PQ on mitochondria was proposed as early as 1968 36 . Since then, a large number of studies on the damage of PQ to mitochondria have been published [26][27][28] . Some scholars indicated that PQ could cause accumulation of the hMn-SOD precursor of human manganese-dependent peroxidase and diminish Mn-SOD activity. The conversion of GSH to GSSG leads to a decrease in GSH levels and weakens its antioxidant activity 37 . Other studies have shown that PQ can cause the production of H 2 O 2 and lessen the activity of catalase 38 . H 2 O 2 can induce changes in mitochondrial permeability and affect the mitochondrial membrane potential, resulting in the movement of cytochrome C from the mitochondria into the cytoplasm, and then induce apoptosis by activating caspase9 39 . In our study, the microstructure of the lung tissue and A549 cells in the PQ group was observed under a projection electron microscope. It was found that the structure of the mitochondria was destroyed, vacuoles appeared in the cells, the cell walls were broken, and a large number of organelles were extruded. In contrast, the morphology of the mitochondria in the PQ + AH 2 QDS group was as expected, and the lamellar structure was normal. The cell membrane potential of the PQ + AH 2 QDS high-dose group was the highest, and the membrane potential was positively correlated with the concentration of AH 2 QDS, while the mitochondrial membrane potential of cells treated with only PQ was the lowest. In summary, PQ can destroy the tissue structure of the mitochondria, affect the membrane potential, and eventually lead to cell rupture and death, while AH 2 QDS can prevent this process and protect the function and structure of the mitochondria.
The data show that after PQ is absorbed into the blood, it causes the formation of excess reactive oxygen species (ROS), which leads to imbalance of the redox system, the consumption of NADPH, damage to mitochondria, the destruction of lipids, proteins and DNA, and a decrease in the activity of various antioxidant enzymes 24 . After continuous oxidative stimulation, the body eventually sustains tissue damage. A large amount of ROS produced by PQ may be the leading cause of acute lung injury caused by PQ poisoning. In this study, it was found that after PQ exposure, the levels of ROS and MDA in the PQ group and conventional treatment group increased, while the level of GSH-Px decreased. In the PQ + AH 2 QDS group, the ROS and MDA levels decreased, and the level of GSH-Px increased. The results show that PQ can produce a large amount of ROS to cause lipid peroxidation and oxidative stress injury. AH 2 QDS can inhibit PQ's effect, improve antioxidant ability, and decrease the level of lipid peroxidation. www.nature.com/scientificreports/ After further investigation of the detoxification mechanism of AH 2 QDS, we found that the main differentially regulated pathway was the oxidative stress pathway, in which we found that the nuclear factor Nrf2 was significantly upregulated. Many studies have shown that Nrf2 can be used as a "guard" to protect the body against a variety of toxic effects [40][41][42] . Nrf2 can be activated in a variety of processes involving oxidative stress. Nrf2 was expressed in epithelial cells, macrophages and vascular endothelial cells of normal rat lung tissue [43][44][45] . MDA in the serum of rats poisoned by PQ increased significantly with the prolongation of poisoning time, while the activity of SOD decreased significantly. Nrf2 protein increased significantly in lung tissue injury induced by PQ. It has been found that the Nrf2-ARE pathway protects the lungs against dibutyl hydroxytoluene-induced acute respiratory distress syndrome (ARDS) and hyperoxia-induced lung injury by activating antioxidant enzymes 46,47 . In our experiment, PQ, as a potent stressor, could activate the Nrf2 signalling pathway. Nrf2 was expressed at low levels in normal rat lung tissue and A549 cells, but the expression of Nrf2 was significantly increased after AH 2 QDS treatment. These results show that Nrf2 plays a vital role in the treatment of PQ poisoning by AH 2 QDS.
The direct mechanism of AH 2 QDS in the treatment of PQ poisoning is that AH 2 QDS enters the gastrointestinal tract and comes into contact with the PQ solution. Through a rapid redox reaction, PQ is reduced to a nontoxic green needle-like solid. Thus, detoxification is realized. Energy spectrum analysis showed that the acicular substance was stable and could not be dissolved in strong acids, strong bases, or organic solvents and was extremely stable at room temperature and pressure. At the same time, it was also found in the faeces of SD rats. We were concerned that after administration of AH 2 QDS, a green needle-like solid will be formed in the blood, leading to the formation insoluble thrombi and resulting in thrombotic disease and a series of clinical symptoms. Therefore, we tested the blood and tissues of experimental animal SD rats but did not find this substance. So, after administering AH 2 QDS, green needle-like solids would not be formed in the blood, tissues and organs to cause thrombotic disease.
To prove whether there is an indirect mechanism of AH 2 QDS in PQ poisoning treatment, we constructed animal and cell models. ELISA, WB, and qPCR were performed to detect the levels of GSH-Px, MDA, ROS, and Nrf2, and transmission electron microscopy was performed to observe the microstructure of the mitochondria. The same trend was observed in vivo and in vitro. After the intervention with AH 2 QDS, the expression of nuclear factor Nrf2 was enhanced, mitochondrial damage was relieved, and antioxidant reaction to oxidative stress was improved. Unfortunately, our experiment cannot determine whether the mechanism of AH 2 QDS in the treatment of PQ poisoning is the direct mechanism or the indirect mechanism. Further research is needed.
In this paper, AH 2 QDS was used as an antidote in the treatment of PQ poisoning for the first time and achieved excellent results, but this was verified only in SD rats, and it has not been tested in more advanced mammals; thus, a long and strict clinical study is needed to investigate the use of AH 2 QDS in humans. Additionally, a 4X LD50 dose of PQ was given to SD rats in the poisoning model, and AH 2 QDS was given for detoxification 2 h later. The 30-day survival rate of SD rats in the treatment group reached 100%, but if the time window of www.nature.com/scientificreports/ treatment with AH 2 QDS were prolonged (2.5 h, 3 h, 4 h, or 6 h), the 30-day survival rate of SD rats in the treatment group decreases with the prolongation of intervention time. This may be because 2 h after ingestion of PQ, the rats have rapidly absorbed it into the blood and transported it to various organs through the blood flow. Even if AH 2 QDS can detoxify the absorbed PQ, too high a concentration of PQ causes irreversible toxic damage to the organs in this time. In the follow-up studies, the sequencing results will be further analysed, and mechanistic research will be performed to elucidate the molecular functions of the gene, the cell location, and the biological process involved. At the same time, experiments were carried out on the Nrf2-ARE pathway through gene silencing/overexpression of related proteins to demonstrate the profound relationship between the Nrf2-ARE pathway and AH 2 QDS in the treatment of PQ poisoning.

Conclusion
In summary, paraquat poisoning is still an extremely high clinical mortality disease, and conventional treatments are clinically ineffective. The new antidote we developed, AH 2 QDS, can lower the concentration of PQ by binding it and protect the mitochondria and reduce the oxidative stress damage caused by PQ. The relationship between mitochondrial damage, the expression changes upstream and downstream of the Nrf2-ARE pathway, and AH 2 QDS in PQ poisoning treatment must be further explored.

Methods
Animals and cell lines. All  Cell counting kit-8 (CCK8). A549 cells were incubated with different concentrations of PQ, AH 2 QDS and glutathione for 12 h. After 12, 24, 48 and 72 h, 10 μL of CCK8 solution (Dojindo, Japan) was added, and the cells were incubated in the incubator for 2 h. An enzyme labelling instrument was used to measure the absorbance at 450 nm, and a formula was used to calculate the cell viability.
Mitochondrial membrane potential. The cell culture medium was removed, the cells were washed with PBS, 1 ml of medium was added, and 1 mL of JC-1 staining working solution was added and mixed well. After incubating the cells for 20 min in the incubator at 37 °C, the supernatant was removed, the cells were washed with diluted staining buffer (1x), 2 mL of medium was added, and images were captured under the fluorescence microscope.
Animal experiments. SD rats (~ 300 g) were subjected to gavage 400 mg/kg PQ, and 500 mg/kg "white and black" scheme and 400 mg/kg AH 2 QDS intervention treatment were administered 2 h later. The specific methods used to establish the model is shown in Figure S3. We selected rats without collecting blood after establishing the model and observed and recorded the survival of each group over 30 days. The occurrence of death was recorded as 1, and no death was recorded as 0. Finally, a survival curve was drawn. www.nature.com/scientificreports/ guination, and the lung tissue was collected, washed with PBS and stored at − 80 °C for follow-up analysis. The bodies of the animals were then incinerated.
Histopathology. SD rats were sacrificed at different times, and the lungs of the rats were harvested, fixed in 4% formalin, embedded in paraffin, sectioned, and stained with haematoxylin and eosin (H&E). The lung injury score was determined according to methods that were previously reported in the literature 48 . A score of 0 means there is no alveolitis. 1 point means mild alveolitis, the lesions are limited to local and pleural lesions, accounting for less than 20% of the lung, and the alveolar structure is sound. A score of 2 indicates moderate alveolitis, and the lesion area accounts for 20-50% of the lung. Finally, a score of 3 means severe alveolitis, with diffuse alveolitis involving more than 50% of the lung.
Blood analysis. The collected venous blood samples were placed into a test tube with a coagulant and centrifuged at 3000 r/min for 5 min. Rat serum was obtained and placed into an automatic biochemical function analyser for analysis. After collecting blood from the abdominal aorta with an arterial blood gas sampler and rubbing with both hands for 1 min, 0.1 mL was injected into the blood gas analyser for analysis. Western blotting. Proteins were extracted from tissues and cells with a BCA kit (Biyuntian Biotechnology Co., Ltd.), separated in SDS-PAGE gels, and transferred to cellulose membranes. After sealing, the membranes were incubated with the primary antibody overnight, then incubated with the secondary antibody for 1 h (Table S1), and finally developed by exposure.

Quantitative real-time polymerase chain reaction (RT-qPCR). TRIzol (Biyuntian Biotechnology
Co., Ltd.) was used to extract RNA, and a cDNA reverse transcription kit (Applied Biosystems, cat. no. 4368814) was used to reverse-transcribe the extracted RNA into cDNA. PCR was performed on an ABI Prism 7900HT system (Applied Biosystems, Foster City, CA, USA) using SYBR GREEN PCR Master Mix (Applied Biosystems). Primers were purchased from Sangon Biotech (Shanghai) Co., Ltd. The primer sequences are listed in Table S2.
NextGen sequencing. Total RNA was extracted from rat lung tissue in the PQ and PQ + AH 2 QDS groups and enriched with eukaryotic mRNA using magnetic beads with Oligo(dT). The second cDNA strand was then purified by QiaQuick PCR kit and eluted with EB buffer, followed by end repair, the addition of poly(A) and ligation of the sequencing junction, then agarose gel electrophoresis for fragment size selection, and finally PCR amplification. After that, the library was sequenced on the Illumina NovaSeq6000 platform.
To make sure reads reliable, Illumina paired-ended sequenced Raw reads were filtered using the fastp to remove low quality reads (https:// github. com/ OpenG ene/ fastp). The filtered data is then compared to the reference sequence. Reference genome and gene model annotation files were downloaded from genome website directly. (https:// www. ncbi. nlm. nih. gov/ assem bly/ GCF_ 00000 1895. 5#/ def). The sequenced data were imported into Partek Flow (Partek Inc., St. Louis, MO) and principal component analysis (PCA) images were generated to visualise distribution differences.
Differential expression analysis was performed using the DESeq2 49 . Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) 50 , we used the R package cluster Profiler 51 to perform KEGG functional enrichment analysis of differentially expressed genes.
Statistical analysis. Statistical analyses were performed using GraphPad Prism 8.0 or SPSS 20.0 software. Measurement data are expressed as the mean ± SEM, and significance was tested by single-factor analysis of variance (ANOVA). Kaplan-Meier survival analysis was used to analyse the survival rate of rats in different treatment groups. P < 0.05 indicates that a difference is statistically significant.
Ethical approval. The experiment was carried out according to the guiding principles for animal experiments at Hainan Medical University.