Analysis of abnormal expression of signaling pathways in PQ-induced acute lung injury in SD rats based on RNA-seq technology

Abstract Background: Paraquat (PQ) plays an important role in agricultural production due to its highly effective herbicidal effect. However, it has led to multiple organ failure in those who have been poisoned, with damage most notable in the lungs and ultimately leading to death. Because of little research has been performed at the genetic level, and therefore, the specific genetic changes caused by PQ exposure are unclear. Methods: Paraquat poisoning model was constructed in Sprague Dawley (SD) rats, and SD rats were randomly divided into Control group, paraquat (PQ) poisoning group and Anthrahydroquinone-2,6-disulfonate (AH2QDS) treatment group. Then, the data was screened and quality controlled, compared with reference genes, optimized gene structure, enriched at the gene expression level, and finally, signal pathways with significantly different gene enrichment were screened. Results: This review reports on lung tissues from paraquat-intoxicated Sprague Dawley (SD) rats that were subjected to RNA-seq, the differentially expressed genes were mainly enriched in PI3K-AKT, cGMP-PKG, MAPK, Focal adhesion and other signaling pathways. Conclusion: The signaling pathways enriched with these differentially expressed genes are summarized, and the important mechanisms mediated through these pathways in acute lung injury during paraquat poisoning are outlined to identify important targets for AH2QDS treatment of acute lung injury due to paraquat exposure, information that will be used to support a subsequent in-depth study on the mechanism of PQ action.

Paraquat (PQ) (1,1 0 -dimethyl-4,4 0 -bipyridine dichloride) is a water-soluble herbicide that is widely used in industrial applications due to its high safety, few negative effects on the environment, and effective weed control (Subbiah and Tiwari 2021).Aqueous paraquat manufacturing and sales have been outlawed in China because of the prevalence of death after commercialization due to ingestion, which led to fatality rates higher then 50% (Wang et al. 2020).When absorbed into the body, paraquat can harm the heart, lungs, liver, kidneys, and other organs.Due to a strong dopamine uptake system in the lungs, PQ accumulates mainly in the lungs because it has a chemical structure similar to that of polyamines (Li et al. 2021).Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) are the most common clinical signs of respiratory paraquat poisoning, with pulmonary fibrosis occurring in later stages (Yuan et al. 2022).Although the exact mechanism underlying PQ-induced ALI is still unknown, PQ has been acknowledged to play a role in the control of oxidative stress, inflammation, epigenetic modulation, apoptosis, autophagy, and the development of pulmonary fibrosis in the lung (Zhang et al. 2021(Zhang et al. , 2022)).As a result, understanding the precise mechanism of paraquatinduced ALI will aid in the development of powerful medications to treat paraquat toxicity (Zhang et al. 2021).This line of research is crucial for the clinical management of paraquat poisoning-induced lung damage.
Anthrahydroquinone-2,6-disulfonate (AH 2 QDS) is a strong reducing agent derived from the reduction of quinone groups in an anaerobic environment.Dibasic pyridinium ions in paraquat receive electrons because of the dibasic acid group structure, which functions as an electron donor.In an in vitro saline bottle test, hydroquinone and paraquat combined to form a green, nontoxic compound that resembles a needle and is insoluble in water, waterbased solvents, strong acids, and bases.It was also insoluble at room temperature and pressure (Wu et al. 2019).Performing cellular tests, our group discovered that AH 2 QDS intervention may increase PQ-induced A549 cell viability and antioxidant capacity and safeguard the functional integrity mitochondrial structures to reduce mitochondrial disruption.These findings were based on reaction product features.In vivo tests revealed that PQintoxicated Sprague Dawley(SD) rats treated with AH 2 QDS showed a considerably higher the 30-day survival rate of rats (the 30-day survival rate) (Qian et al. 2021;Li et al. 2022).According to follow-up studies, AH 2 QDS bound to PQ to reduce its absorption into the gastrointestinal tract.It also activated pathways in tissues that protect the structural and functional integrity of mitochondria, improve energy metabolism, and inhibit the aggregation of inflammatory cells, which attenuated acute lung inflammation and pulmonary fibrosis.
The changes in signaling pathways at the genetic level in the lung tissue of the SD rats in the PQ poisoning group and AH 2 QDS treatment group were not clear.Therefore, for this study, we established an animal experimental model and sequenced lung tissues of the rats in a poisoned and poisoned with treatment groups(group)using promoter next-generation DNA sequencing technology.Thus, the signaling pathways were identified at the genetic level, and the description of the mechanism of AH 2 QDS action was more accurate.We will try to identify the key targets of action in subsequent experiments and then provide new ideas for clinical treatment.

Model building
To investigate the main signaling pathways of AH 2 QDS that ameliorate the acute lung injury due to paraquat poisoning, PQ poisoning group and AH 2 QDS treatment group models were constructed with SD rats.Total RNA was extracted, and all mRNA was reverse transcribed into a cDNA library, which was then sequenced using the Illumina HiSeq TM platform.The raw data were collected, screened, and optimized, and KEGG analysis of enriched pathways was performed on the basis of the differentially expressed genes identified.In the PQ group, 1854 genes showed differential expression, including 843 genes that were upregulated and 1011 genes that were downregulated (Figure 1a).In the AH 2 QDS group, 1643 genes were differentially expressed, including 961 genes with upregulated expression and 682 genes with downregulated expression (Figure 1b).The differentially expressed genes that were upregulated in the PQ group were primarily enriched in PI3K-AKT signaling pathway, cGMP-PKG signaling pathway, Rap1 signaling pathway, and MAPK signaling pathway, according to the results of the KEGG enrichment analysis (Figure 2a and 2b).PI3K-AKT signaling pathway, cGMP-PKG signaling pathway, and focal adhesion signaling pathway were primarily enriched with the differentially expressed genes in the AH 2 QDS group (Figure 2c and 2d).
The data shoed that the PI3K-AKT signaling pathway, cGMP-PKG signaling pathway, Rap1 signaling pathway, MAPK signaling pathway and focal adhesion signaling pathways played important roles in acute lung injury from paraquat poisoning.They also indicated important therapeutic targets for paraquat detoxification.An overview of various important pathways is described in the following section.

The PI3K-AKT signaling pathway
The phosphatidylinositol-3 kinase (PI3K)/serine/threonine protein kinase (AKT) signaling pathway is an important signaling pathway in the body (Hou et al. 2022).Through the control of downstream effector molecules and other signaling pathways, it plays a role in a variety of physiological processes, including cell proliferation, metabolism, and survival (Wang et al. 2020;2021;Wu et al. 2022).The PI3K-AKT signaling pathway was connected to the pathogenesis of ALI in a number of studies.According to Luo et al. rosmarinic acid (RA) inhibited the formation of ROS and lowered the rate of alveolar epithelial cell death by triggering the PI3K-AKT signaling pathway (Luo et al. 2022).The anti-inflammatory effects of Fengqing Oral Liquid (FOL) was leveraged by Rao et al. to reduce IL-1, IL-6, and TNF-a levels in bronchoalveolar lavage fluid (BALF) and suppress the activation of the PI3K-AKT and NF-jB signaling pathways (Rao et al. 2022).According to Zheng et al. (2018), Ghrelin treatment of endothelial cells prevented lipopolysaccharide-induced ARDS by triggering the PI3K-AKT signaling pathway, upregulating the antiapoptotic protein Bcl-2 and downregulating the proapoptotic protein Bax in LPSinduced lung damage.Notably, the PI3K-AKT signaling pathway plays an important role in paraquat poisoning and has also been shown to play a role in the treatment of paraquat poisoning.PQ poisoning frequently causes significant lung damage, with mitochondrial damage being a major factor.The main potential mechanisms suggest that PQ exposure leads to mitochondrial dysfunction and reactive oxygen species(ROS) production, acting through the redox cycle, PQ affects electron transport chain function and increases superoxide production in mitochondria, disrupting lung surfactant synthesis and damaging important cellular components (Ji et al. 2022).Liu et al. (2022) discovered that mitoquinone (MitoQ) promoted Mitofusin 1 (MFN1)-/ Mitofusin 2 (MFN2)-mediated mitochondrial fusion to reduce ROS generation and mitigate PQ-induced lung epithelial cell damage.It has been demonstrated that preventing the generation of ROS and maintaining the integrity of mitochondria attenuates the lung damage caused by PQ (Pang et al. 2019;Huang et al. 2021;Zheng et al. 2021).According to Ju et al.PQ treatment of relevant cells greatly lowered the phosphorylation of PI3K and AKT, which resulted in cell death.The drug also inhibited the effect of PQ on cells by increasing AKT-PI3K signaling (Ju et al. 2019).According to Liu et al.Kawarazin reduced the level of oxidative stress by activating the PI3K-AKT-mTOR signaling pathway, ultimately causing a delay in the extent of paraquat-induced lung fibrosis (Liu et al. 2019).Ahmed et al. (2019) found that paraquat-induced lung injury was characterized by enhanced oxidative stress and inflammatory responses with upregulation of PI3K-AKT and b-catenin protein expression.Febuxostat inhibited the harmful effects of paraquat on the lung by inhibiting xanthine oxidase activity and the associated oxidative stress, downregulating the activation of the PI3K-AKT pathway, and inhibiting the expression of the b-catenin protein and its downstream inflammatory mediators.In a study on the detoxification of paraquat with 4-phenylbutyric acid (4-PBA), Hoshina et al. (2018) discovered that 4-PBA reduced the cytotoxicity of PQ through the activation of ERK2 mediated by PI3K.After ERK2 phosphorylation was inhibited, AKT phosphorylation increased noticeably.When ERK activation was inhibited, phosphorylated AKT compensated through its interaction within the MEK/ERK and PI3K-AKT signaling pathways, partially protecting cells.In conclusion, the PI3K-AKT signaling pathway plays an important role in the development of paraquat poisoning.It is involved in oxidative stress, cell proliferation, apoptosis and other pathways affecting the course of ALI/ARDS.The involvement of the PI3K-AKT signaling pathway in the mechanism inducing ALI is shown in Figure 3.

The cGMP-PKG signaling pathway
Cyclic guanosine monophosphate (cGMP) is a prevalent second messenger in the body and brain.It affects numerous downstream pathways, leading to a variety of cellular and tissue outcomes (Hofmann 2020).The role of cGMP has been extensively studied in outside the brain, where it has been found to play important physiological roles in the vascular system, heart, pulmonary arteries and gastrointestinal tract.The cGMP-PKG signaling pathway is another important pathway for various physiological and pathophysiological processes in the human body (Jehle et al. 2022).cGMP is produced by the heterodimeric alpha/beta hemoglobin-soluble guanylate cyclase (sGC) upon activation via its endogenous ligand nitric oxide  reduced fibroblast proliferation (Beyer et al. 2015;Sandner et al. 2017).Thus, the cGMP-PKG signaling pathway acts on inflammatory factors to achieve anti-inflammatory effects in the presence of the toxin PQ.The mechanisms through which the cGMP/PKG signaling pathway is involved in lung disease are described in Figure 4.

The Rap1 signaling pathway
Rap1 is a small GTPase in the Ras superfamily and it regulates a number of cellular processes, including extracellular matrix (ECM)-cell adhesion, cell-cell junction formation, cell migration, and cell polarity (Yamamoto et al. 2021).Its receptors and downstream signaling pathways share structural domains with Ras proteins.The Rap1 signaling pathway is primarily regulated by the precise regulation of guanine nucleotide exchange factors (GEFs) and GTPaseactivating proteins (GAPs) to complete the GTPase transition of GDP/GTP.Functionally, Rap1 can either interfere with Ras protein-mediated ERK activation or activate ERK in a manner independent of the Ras protein or cell context.Controlling cell adhesion and intercellular junctions is among classical roles played by Epac/Rap1 (Aslam et al. 2014).Rap1 has been shown to play multiple roles in the development and function of the vascular system, and in  endothelial cells, Rap1 is required to maintain vascular tone.One of the primary pathological characteristics of ALI is damage to the alveolar capillary endothelium.Drugs that enhance vascular barrier function thus can attenuate the pathology of ARDS.Therefore, drugs that activate Rap1 signaling in endothelial cells may be used to treat ARDS.In ALI, the Rap1 signaling pathway is involved in intercellular adhesion and attachment, apoptosis, proliferation and differentiation (Yamamoto et al. 2021).Rap1 is also a downstream target of Epac-1, which is critical for inhibiting proliferation (Hartopo et al. 2013).According to research by Steven K. Huang et al. lung fibrosis can advance more slowly when lung fibroblast proliferation is suppressed via activation of the Rap1 signaling pathway (Luo et al. 2020).Thus, in ALI induced by PQ toxicity, vascular endothelial cell adhesion and death may be mediated through the Rap1 signaling pathway.Alveolar capillary exudation has been found to be decreased via the Rap1 pathway, which in turn ameliorated pulmonary edema and ALI.The mechanism through which lung injury is caused by the Rap1 signaling pathway is illustrated in Figure 5.

The MAPK signaling pathway
Mitogen-activated protein kinase (MAPK), an important transmitter of signals from the cell surface through the  cytoplasm and to the nucleus, is a serine/threonine protein kinase that is widely found in eukaryotic cells (Li et al. 2022).A three-tier kinase cascade comprises MAPK kinase kinase (MKKK), MAPK kinase (MKK) and MAPK, which are activated sequentially and together regulate a variety of important cellular physiological/pathological processes, such as cell growth, differentiation, and proliferation and inflammatory responses (Cornell et al. 2012;Kong et al. 2016;Li et al. 2021).According to previous research, LPS-induced ALI was accompanied by increased inflammation and programmed cell death when the MAPK and NF-jB pathways were activated (He et al. 2019;Pooladanda et al. 2019).Blocking the MAPK pathway or inhibiting MAPK phosphorylation markedly reduced the transcription and release of proinflammatory mediators6 (Barboza et al. 2018;Zhang et al. 2018;Cong et al. 2020).ALI/ARDS have been linked to the MAPK1/2, Jun amino-terminal kinase (JNK1/2/3), and p38 protein (p38a/b/c/d) enzymes in the MAPK family (Ying et al. 2015;Yao et al. 2017;Zhang et al. 2018).Proinflammatory cytokines induced VEGF expression directly through MAPK1/2-and p38-dependent pathways (Lin et al. 2019).Moreover, PI3K-AKT and MAPK pathways are thought to be crucial for ginseng effects in the treatment of ALI/ARDS, as supported by a pathway enrichment analysis based on the GO and KEGG databases (Ding et al. 2020).Inhibition of LPS-induced inflammatory responses in ALI downregulates Toll-like receptor 4 (TLR4) expression and inhibited extracellular signal-regulated kinase (ERK)1/2 and p38MAPK activation (Sivanantham et al. 2019) In addition, p38 MAPK was activated in lung tissue after PQ exposure, participating in inflammatory responses, cellular stress induction and apoptosis, and the transcription of various cytokines has been shown to be regulated by p38 MAPK (Liu et al. 2015).In recent years, increasing evidence has shown that that PQ-induced apoptosis involves the MAPK signaling pathway and that PQ exposure leads to cell damage in lung tissue, activating p38MAPK and JNK and inducing apoptosis (Jin et al. 2021).Paraquat enhanced the phosphorylation of JNK and p38MAPK, according to Zhang et al.The antiaging protein Klotho suppressed p38MAPK activation, decreased IL-1 and IL-6 expression, and attenuated the inflammatory damage caused by paraquat (Zhang et al. 2020).According to Huang et al. (Huang et al. 2016), paraquat increased the migration and epithelial-mesenchymal transition (EMT) of mouse alveolar epithelial cells by activating the MAPK signaling pathway.It is rational (rationale) to hypothesize that activation of the MAPK signaling pathway may be involved in the initiation and progression of paraquat-induced pulmonary fibrosis because the EMT is a crucial phase in the development of pulmonary fibrosis.The diagram below shows the involvement of the MAPK signaling pathway in the pathogenesis of lung injury (Figure 6).

Focal adhesion signaling pathway
Focal adhesion refers to the association between the extracellular matrix and the actin cytoskeleton of a cell consisting.They work through integrins.It plays an important role in human physiology, regulating cell adhesion, mechanosensing and the signals that control cell growth and differentiation.Focal adhesion kinase (FAK) is a cytoplasmic nonreceptor protein tyrosine kinase that integrates many extracellular signals, such as integrins and mechanical traction.FAK is involved in the regulation of several cellular signaling pathways and is a central molecule in extracellular and intracellular signaling (Alanko and Ivaska 2016).It can receive signals from extracellular stimuli to mediate cell adhesion and other processes to mediate cell growth and metabolism by regulating MAPK, PI3K, p53 and other signaling pathways.FAK is closely associated with cell cycle proliferation, apoptosis, migration, invasion, metastasis and regulation (Chen et al. 2022) and mediates cell-to-cell matrix and cell-to-cell signaling.It also plays a role in regulating adhesion to the ECM.By regulating the proliferation and differentiation of fibroblasts, FAK triggers the EMT and inflammatory cell release of inflammatory factors to promote lung fibrosis.ALI is characterized by disruption of the endothelial barrier that leads to increased vascular permeability (Lederer et al. 2018).As FAK is a nonreceptor protein tyrosine kinase involved in endothelial cell (EC) barrier regulation, it has been clearly to be an important mediator of EC responses in ALI.In addition, increased ROS levels induced by reduced nicotinamide adenine dinucleotide phosphate (NADPH) activation has been identified as a pathogenic mechanism in FAK-mediated ALI.The FAK pathway is involved in the TGF-b-induced conversion of lung fibroblasts to myofibroblasts.A FAK-related nonkinase (FRNK) is an inhibitor of FAK.FRNK expression was reduced in idiopathic pulmonary fibrosis (IPF) in mice.Deficiency in FRNK led to amplification of TGF-b signaling, and FRNK inhibited TGF-b-mediated responses (Klossner et al. 2013).The FAK inhibitor PF-573228 exerted a significant therapeutic effect on pulmonary fibrosis (Zhao et al. 2016).Cell assays suggested that FAK inhibitors inhibited platelet-derived growth factor BB (PDGF-BB)-induced FAK activation and fibroblast migration in a dose-and timedependent manner.Animal studies suggested that FAK inhibitors attenuated pulmonary fibrosis by inhibiting the activation and expression of proteins that differentiate myofibroblasts.The mechanism of action of the focal adhesion signaling pathway in lung disease is shown in Figure 7.

Other signaling pathways
Apelin/APJ signaling pathway In addition to the abovementioned pathways, other signaling pathways identified by RNA-seq analysis play important roles in PQ effects, such as the apelin signaling pathway.Apelin is a ligand of the G protein-coupled receptor APJ, which is expressed in the heart, liver, lung and brain (Wang et al. 2022;Yuan et al. 2022).Apelin plays a regulatory role in lung tissue, participating in processes such as inflammatory responses, oxidative stress and apoptosis (Zhou et al. 2016).Apelin signaling pathway activation has been shown to reduce intracellular ROS levels to maintain mitochondrial integrity, reduce oxidative stress and inhibit apoptosis (Fan et al. 2015;He et al. 2021;Kong et al. 2021;Yuan et al. 2022).Apelin can reduce PQ-induced lung injury, protecting lung function.Apelin plays a beneficial role in a variety of respiratory diseases, and studies have demonstrated that the apelin-APJ pathway can inhibit the fibrotic process in various organs.Wang et al. (2022) found that Apelin mRNA and protein expression levels were significantly reduced in the lung tissue of LPS-induced pulmonary fibrosis mice.Treatment with apelin-13 significantly inhibited LPS-induced collagen deposition in lung tissue and induced the epithelial-mesenchymal transition in the lung microvascular endothelium, thereby alleviating LPS-induced lung fibrosis.
Hedgehog signaling pathway The Hedgehog pathway is essential for lung development, and when activated, the Hh pathway has been shown to be associated with fibrosis in lung, liver and kidney tissue (Wang et al. 2018;Zeng et al. 2022).Increasing evidence suggests that Sonic Hedgehog (Shh) adult lung tissue is involved in tissue migration and the regulation of EMT-related genes.During pulmonary fibrosis and injury (Yang et al. 2022), Shh is reactivated.Overexpression of Shh in mice caused fibrosis, increased the amount of deposited collagen and induced airway injury (Yin et al. 2022).Increased Hh signaling was detected in a mouse model of ALI during when the expression of the proinflammatory cytokine TNF-a was reduced and the lung injury scores were low.Inhibition of Hh activation by cyclopamine treatment after LPS treated increased TNF-a mRNA expression and damage scores in lung tissue.These findings suggested that Hh signaling may be involved in a protective mechanism to mitigate lung injury through its anti-inflammatory effects (Chen et al. 2015;Lau et al. 2021).

Conclusion
In summary, the pathogenesis of ALI/ARDS due to paraquat poisoning is complex and involves multiple cytokines and signaling pathways.It is not a result of the action of one or a few signaling pathways but the result of multiple signaling pathways that interact with each other.Activated signaling pathways affect organisms mainly through oxidative stress, cell proliferation, apoptosis, mitochondrial activity and reduced ROS production.Inhibition of the activation of inflammatory pathways such as the NF-jB signaling pathway reduces the secretion of inflammatory factors.The Rap1 signaling pathway acts on alveolar epithelial sodium channels (ENaC), thereby reducing vascular exudation and pulmonary edema.Activation of the PI3K-AKT or cGMP-PKG signaling pathway can influence downstream pathways to reduce lung injury and delay disease progression.Recently, our team discovered that, in SD rats, AH 2 QDS bind PQ.On the one hand, AH 2 QDS reduces PQ absorption into the body, and on the other hand, it protects lung tissue.For instance, AH 2 QDS reduces oxidative stress and intracellular ROS levels in cells.Additionally, it prevents apoptosis, reduces exudation and pulmonary edema, and prevents endothelial cells from transitioning in to the mesenchymal state.It also protect lung tissues in a variety of ways to attenuate acute lung injury due to paraquat intoxication.These protections include maintaining the structural integrity of alveolar epithelial cells, vascular endothelial cells, mitochondria, and other cellular components.In individuals with paraquat poisoning, AH 2 QDS can increase the survival rate and enhance organ function.However, more research into the precise mechanism of action is still needed, and subsequent studies steadily improve the understanding of theAH2QDS mode of action; this information will give the clinical development of a paraquat antidote a theoretical foundation.

Figure 1
Figure 1.a: Heatmap showing differentially expressed genes in the control vs. PQ group.b: Heatmap showing AH 2 QDS differentially expressed genes in the PQ vs. AH 2 QDS group.Each row in the heatmap represents a gene, and each column represents a sample with red indicating gene upregulation and blue indicating gene downregulation.

Figure 2
Figure 2. a: KEGG pathway classification bar graph of the control group vs. the PQ group.b: KEGG pathway classification bubble diagram showing the control group vs. the PQ group.c: KEGG pathway classification bar graph of the PQ group vs. the AH 2 QDS group.d: KEGG pathway classification bubble diagram of PQ group vs. the AH 2 QDS group.The horizontal coordinate is the Rich factor, indicating the percentage of enriched genes among those annotated.The vertical coordinate indicates the entries in an enriched pathway.The size of a dot indicates the number of enriched genes, and the color indicates the Q value; the larger the Rich factor is, the greater the enrichment.The smaller the Q-value is, the more significant the differential gene enrichment.

Figure 3 .
Figure 3.The involvement of the PI3K-AKT signaling pathway in ALI mechanism.

Figure 4 .
Figure 4.The mechanisms by which the cGMP/PKG signaling pathway is involved in lung disease.

Figure 5 .
Figure 5.The mechanistic rationale for the involvement of the Rap1 signaling pathway in lung injury.

Figure 6 .
Figure 6.The involvement of the MAPK signaling pathway in the pathogenesis of lung injury.

Figure 7 .
Figure 7.The mechanism of action of the focal adhesion signaling pathway involved in lung disease.