Role of AhR and Foxo1 in skin inflammation in burn animal model via MAPK signaling pathway

Burn generally refers to thermal damage, including tissue damage caused by hydrothermal (water, soup, oil, etc.), flame, steam, high-temperature gas, hot metal liquid or solid (such as molten steel, ingot). However, little is known about the pathogenesis and inducement of skin inflammation in burned rats. Therefore, this study has carried out an in-depth analysis of the related causes of skin inflammation in burned rats. We analyzed the gene expression and the differentially expressed genes co-expression in burned rats. Subsequently, a set of functional dysfunction modules about inflammation of skin tissue in burned rats were obtained by comprehensive enrichment analysis. In addition, based on related network prediction analysis, we identified a number of regulatory factors, such as endogenous genes, nCRNAs, and transcription factors that have potential monitoring effects on skin inflammation in burned rats. Firstly, we obtained 2679 differentially expressed genes and 7 disease-related dysfunction modules in burned rats. Secondly, we identified a series of regulators related to skin inflammation in burned rats, including 117 ncRNAs (including miR-17-5p, miR-122-5p, and miR-140-5p), 31 transcription factors (including AhR, Foxo1 and Sp1) and 10 endogenous genes (including Il5, Atp5d, and Cox4i1). Core transcription factors AhR and Foxo1 may induce skin inflammation in burned rats through the cascade of MAPK signals. According to the results of this study, we can show a new method for biologists and pharmacists to reveal the inducement of skin inflammation in burned rats and provide a valuable reference for different treatment options.


Introduction
Burns are serious injuries, which may be caused by thermal, radiation, chemical or electrical injuries (1)(2)(3)(4)(5). At the same time, electrical burn is an important cause of trauma in the world. Electrical burn mainly affects adult males who have occupational contact in the world (6). A chemical burn is also the main cause of corneal injury. Oxidative stress, inflammation and angiogenesis after chemical burn may aggravate the corneal injury and lead to a visual loss (7). Heat and chemical burns are the most common types of burns. Severe chemical burns can also cause redness, blistering, skin loss and swelling (8). Burns is not a single pathophysiological event, but a destructive injury that can lead to structural and functional deficiencies in many organ systems (1). Burns may cause physiological changes in numerous organ systems in the body. Burn mortality is usually attributed to pulmonary complications (9). Burns are reported to be one of the major causes of accidental injuries and deaths. In the United States, burns and inhalation injuries cause considerable mortality and morbidity.
Burn size and inhalation injuries are important predictors of post-burn deaths (10)(11)(12). Burns are a common form of injury in childhood, and the greatest risk is in infancy, so burns are also a major form of injury for children all over the world (13,14). Severe physical and psychological complications caused by burns require comprehensive rehabilitation and coordination of acute burn care teams (15). Burns may induce inflammation in blood and wounds. Burn depth and burn size are key determinants in evaluating patients with burns (16,17). Severe burns are more likely to constitute systemic diseases. Severe burns can lead to hypermetabolism, which increases the metabolism of muscles, bones and fat, making patients susceptible to multiple organ dysfunction and sepsis, and even death (18)(19)(20)(21)(22)(23)(24)(25)(26)(27).
A severe burn is a serious injury with a global impact (28). Therefore, many doctors around the world have carried out in-depth research on the nursing and treatment of burns and severe burns. Firstly, scholars believe that it is very important to estimate the severity of burns correctly for patients to get proper treatment and avoid overwork (29). Effective burn care contributes to the burden of surgery, and burn care is also a considerable challenge to the health care system. The treatment of large-scale burn patients is related to social stability, life-saving and disability reduction (30)(31)(32). Healing burn wounds are closely related to wound depth and premature ejaculation, and burns usually require spe-cial treatment at special burn centers (30,33). The study found that burn treatment in a moist environment is more conducive to the recovery of patients (34). The survival of patients after severe burns largely depends on the size of the burn. The modern development of burn care has greatly improved survival and outcomes (35). Burnt patients not only suffer from physical, psychological, social, and spiritual effects but also undergo significant changes in their quality of life related to health, so patients must receive comprehensive treatment and care (36).
In this study, we conducted a series of comprehensive analysis based on the data of burned rats to explore the related factors of skin tissue inflammation in burned rats. Overall, our comprehensive strategy not only provides new insights into effective burn care and treatment but also provides abundant resources and guidance for biologists to design more experiments.

Differential expression analysis
The expression microarray data set collected of burnrelated disease samples from NCBI Gene Expression Omnibus (GEO) database (37), numbered GSE802. The collected disease samples were analyzed by four groups of difference analysis (1 h burn control, 4 h burn control, 8 h burn control, 24 h burn control), using R language limma package (38).

Function modules related to co-expression analysis and recognition
Firstly, we use weighted gene co-expression network analysis (WGCNA) (39) to analyze the correlation phenotype of the expression profiles of these differentially expressed genes and find the gene module of co-expression. Second, using the Nth power of the correlation coefficient, the weighted value of the correlation coefficient was used to calculate the correlation coefficient (individual coefficient) between the two genes. The connection between genes in the network was pursued from a scale-free network, which makes this algorithm more biologically meaningful. The correlation coefficient between genes is then used to construct a hierarchical cluster tree. Different branches of the cluster tree show different gene modules and different colors show different modules.

Functional and Pathway Enrichment Analysis and Identification of Dysfunctional Modules
Studying the functions and pathways of gene signals is usually an effective tool for studying molecular mechanisms of diseases, and the functions and pathways involved in modular genes can determine the mechanism of modular dysfunction in disease processes.
Therefore, we applied R Language Cluster profiler package (40) to supplement and analyze the Go function (p-value Cutoff = 0.01, q value Cutoff = 0.01) and KEGG pathway (p-value Cutoff = 0.05, q value Cutoff = 0.2) respectively. Due to the function and pathway of the modular gene, it has been identified as the main functional pathway for inducing inflammation in the skin tissue of rats.

Identification of transcription factors and regulation of ncRNA on modules
We first downloaded the target data from the TR-RUST V2 (41) database. Then load ncRNA protein data (score> 0.5) from the RAID 2.0 (42) database and perform a centralized analysis based on this interaction data to determine the regulatory role of these transcription and ncRNA factors in the module. Pivot analysis refers to searching at least two driver interaction modules in the target pair and calculating the importance of the interaction between the driver and the module based on the hypergeometric test. TF and ncRNA with P< 0.01 are important pivots of the monitoring module. Finally, through statistical analysis, these pivots are called the main pivots. In addition, Cytoscape (43) is used to display and analyze the network (including connection calculation). Genetic screening is the main molecular regulator that regulates the modular process and is called the intrinsic gene. These endogenous genes may be key detox molecules that can cause burns in mice.

Differentially expressed genes related to inflammation in burned rats
Dysfunction of gene expression often plays an important role in the occurrence of diseases. Therefore, we analyzed the differentially expressed genes based on the expression profiles of four groups of burn diseases at different stages in order to further understand the potential pathogenesis of burn. Combining the four groups, Similarly, transcription factors are equally important in the regulation of gene transcription. Many studies have shown that failure to follow transcription rules can lead to a variety of diseases. Impaired burn function in rats is also closely related to transcription factor dysfunction, and the regulation of transcription factor dysfunction also reflects transcription factor dysfunction. Therefore, we use pivot analysis to predict the module according to the regulation of genes by transcription factors. The results showed that 31 transcription factors had significant transcriptional regulation effects on the potential dysregulation mechanism of burns in rats, involving 36 Pivot-Module interaction pairs (Figure 4). Statistical analysis of these transcription factors showed that both AhR and Foxo1 had a significant regulatory re-2679 differentially expressed genes were obtained. These 2679 differentially expressed genes were considered to be the key genes for the imbalance of burn expression in rats.

Co-expression Behavior of Differential Genes in Burn Rats
By combining the differentially expressed genes, we obtained the genes related to the expression disorder of burns in rats, but the regulatory mechanism and the synergistic relationship between them are still unclear. To this end, we continue to study the differential genes of burns in rats, construct the differential gene expression profiles and conduct co-expression analysis. The expression profiles of burn disease samples were analyzed by WGCNA. Seven modules involving 1445 module genes were excavated. These functional modules may be involved in a variety of functions and pathways and may exhibit a variety of monitoring mechanisms that mediate inflammatory-related diseases in burned mice.

Identification of functional dysfunction module in burned rats
The study of the functions and pathways involved in genes is an important tool for identifying and mediating pathogenesis. In order to evaluate the possible manifestations of the genetic diseases of the modules, we analyzed the high yield and path of each module. The results showed that most modules were enriched with rat-related burning functions and paths. We analyzed GO function and KEGG pathway enrichment in 7 functional modules and obtained 9140 functions and 252 KEGG pathway enrichment results. These include 1522 molecular functions (MF), 624 cellular components (CC) and 6994 biological processes (BP) involving genes (Figure 2A, 2B). It is noteworthy that they are significantly involved in the cascade of MAPK signaling pathways, including stress-activated MAPK cascade, negative regulation of stress-activated MAPK cascade and MAPK signaling pathway, which may be the core signaling pathway to induce burns in rats. From the above data, we can find that the MAPK signaling pathway may be closely related to the inflammation of burn skin tissue in rats.

Key ncRNAs and TFs mediating burn dysfunction module in rats
The scientific prediction of ncRNA, which regulates the genes of the dysfunction module, helps us to further discover the regulatory mechanism of burn transcription. For this purpose, a pivotal analysis was performed based on the targeting relationship between ncRNA and genes to predict the ncRNA regulator that disrupted the modules. It was found that 117 ncRNAs had a significant monitoring effect on the modules, including 131 pairs of ncRNA-module interactions (Figure 3).
Statistical analysis of the results showed that miR-17-5p had a strong regulatory effect on the three dysfunction modules. Therefore, ncRNA, identified as the core, was a key disorder molecule in inducing inflammation of burn skin tissue in rats. MiR-122-5p also plays an important role in the dysfunction of a dysfunctional module. In addition, other ncRNAs also have a certain driving effect on the potential dysfunction mechanism  lationship with one dysfunction module, and they might be involved in the mechanism of burns in rats. These data suggest that transcription factors may play an important role in the mechanism of burns disorder in rats. These transcription factors, which have significant regulatory effects on multiple dysfunction modules, have been identified as the core transcription factors of burns in rats. In addition, we analyzed the network connectivity based on the dysfunction module, and selected the genes with the highest connectivity in the module, and identified them as introductory genes. There were 10 genes, including Il5, Atp5d and Cox4i1. These intrinsic genes have high connectivity in the network module, which may play a key role in inducing burn-related diseases in rats.

Discussion
Burns are a common type of skin injury. Severe burns are usually accompanied by hypermetabolism, characterized by significant muscle atrophy, which leads to considerable morbidity and mortality (44,45). Although in recent years, the research on inflammation of burned skin tissue mainly focused on some genes or proteins and related signaling pathways and achieved some results. However, the global regulation of these genes, proteins and signaling pathways in inducing inflammation in burned skin tissue of rats remains unclear. To further explore the core pathways and regulators that induce inflammation in rat burn skin tissue, we established a multi-dimensional integration method based on gene expression data, co-expression analysis, enrichment analysis, transcriptional and post-transcriptional regulatory data and module interaction.
In order to explore the core pathways and regulators of skin inflammation induced by burns in rats, we obtained 1589 differentially expressed genes based on disease-related gene expression data. Among them, Tat plays an important role in inducing inflammation of burn skin tissue in rats. Tat-Rac1 can increase the proliferation and migration of keratinocytes and dermal fibroblasts in vitro, thus playing a key role in wound healing of burned skin (46). Secondly, we integrate the differentially expressed genes and observe their co-expression behavior in the disease samples. From this, we get seven co-expression modules. The genes contained in the modules are considered to have a co-expression phenomenon. Subsequently, enrichment analysis found that genes in seven functional modules mainly participate in the cascade pathway of MAPK signal, including stress-activated MAPK cascade, negative regulation of stress-activated MAPK cascade and MAPK signaling pathway. Therefore, the cascade of the MAPK signaling pathway is considered to be the core signaling pathway to induce inflammation of burn skin tissue in rats. The inflammation signal transduction pathway of MAPK has been found to play an important role in the host response to injury. Local application of p38 MAPK inhibitors is a clinically feasible treatment after burn (47). Subsequently, we identified transcription factors that significantly regulate these seven dysfunction modules, obtained 31 transcription factors and 36 Pivot-Module interaction pairs. Among them, both AhR and Foxo1 have significant regulatory effects on a dysfunction module, which indicates that they play an important role in the process of inducing burns in rats. This is confirmed by Quintana FJ et al (48,49) that AhR signal transduction is beneficial to solve the inflammation caused by excessive burns, and the overexpression of AhR may also lead to tissue inflammation and autoimmunity. Besides, the Foxo1-mediated gene transcription pathway is a key trigger for protein catabolism in muscle induced by inflammatory reaction (50). Therefore, it can be concluded that AhR and Foxo1 are core transcription factors inducing skin inflammation in burned rats through the cascade of MAPK signals.
In addition, ncRNA has been considered to be an important regulator of disease occurrence and development. We conduct pivot analysis based on the targeting relationship between ncRNA and genes. The predicted results show that 117 ncRNAs have significant regulatory effects on the module. It was found that microRNA-17-5p had significant regulatory effects on three dysfunction modules. It is reported that microRNA-17-5p may inhibit skin inflammation induced by burn injury (51). Mir-122-5p plays a regulatory role in one module, which may promote keratinocyte proliferation and play an important role in the persistent inflammatory response induced by severe burns by down-regulating the expression of SPRY-2 (52). At the same time, we also screened a series of genes with the greatest connectivity as the core molecule of the progress of the regulatory module, that is, the intrinsic gene. In total, there are 10 endogenous genes, which may represent potential disorders that induce inflammation in the burned skin tissue of rats. Among them, Il5 was found to play a major role in local inflammation of burns (53).
A series of regulatory factors predicted in this study have certain regulatory effects on the response process of skin inflammation induced by burns in rats to varying degrees. However, in addition to the above key factors, other unmentioned ncRNA and transcription factors may play a role in the mechanism of inducing imbalance in burned rats, which needs further exploration.
Overall, our study confirms that core transcription factors AhR and Foxo1 are key regulators that can induce inflammation of burn skin tissue in rats through the cascade of MAPK signals. It can not only provide a new way for biologists and pharmacists to study the effect of MAPK signal on burns, but also provide a valuable reference for their follow-up treatment.