Transcriptomics, metabolomics, and in silico drug predictions to prevent or treat liver damage in young and aged burn victims

doi:10.21203/rs.3.rs-2363178/v1

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Transcriptomics, metabolomics, and in silico drug predictions to prevent or treat liver damage in young and aged burn victims

https://doi.org/10.21203/rs.3.rs-2363178/v1

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published 02 Jun, 2023

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Burns are a leading cause of morbidity and mortality worldwide, affecting individuals of all ages. Burns induce a systemic response affecting multiple organs where the liver is frequently damaged. Since the liver plays a critical role in metabolic, inflammatory, and immune events, a patient with impaired liver often exhibits poor outcomes. The mortality rate after burns in the elderly population is higher than in any other age group, and studies show that the liver of aged animals is more susceptible to injury after burns. Thus, understanding the liver response to burns in young and aged burn victims is fundamental to improving overall health care. Moreover, no liver-specific therapy exists to treat burn-induced liver damage highlighting a critical gap in burn injury therapeutics. In this project, we analyzed transcriptomics and metabolomics data from the liver of young and aged mice to identify mechanistic pathways and in-silico predict therapeutic targets to prevent or reverse burn-induced liver damage. Our study highlights pathway interactions and master regulators that underlie the liver response to burn injury in young and aged animals. The results reveal genes that may represent prospective hallmark signatures for liver damage, especially in the livers of aged burn victims.

Biological sciences/Cell biology/Mechanisms of disease

Biological sciences/Molecular biology/Transcriptomics

Biological sciences/Computational biology and bioinformatics/Gene regulatory networks

Over 450,000 people are affected by burn injuries yearly, resulting in 40,000 hospital admissions in the United States alone¹. Although there have been significant advances in burn injury therapeutics, burns are still a leading cause of morbidity and mortality worldwide². Beyond the skin, burns cause damage to remote organs, and poor outcomes in burn victims increase when multiple organs fail³. Thus, deepening our understanding of how different organs respond to burns can help identify targets to direct organ-specific treatments that may improve the outcomes of burn patients.

The liver is among the critical organs affected after burns, displaying significant morphological alterations, such as hepatomegaly and fatty infiltration⁴. Compared to controls, liver weight increases significantly 2 to 7 days post-burn in rodents⁵. Burn-induced liver damage is associated with edema formation, which, at the same time, may lead to cell damage and the release of hepatic enzymes⁵. Liver enzymes, such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT), are sensitive indicators of hepatocyte injury⁶. Clinical evidence reveals that serum AST and ALT peaked during the first week post-burn ⁷. Because the liver orchestrates metabolic functions, inflammatory processes, and immune events during basal and stress conditions, a burn patient with an impaired liver is more vulnerable to metabolic and infectious complications leading to unfavorable outcomes than one with a healthy liver ⁴. Gong et al. studied the mortality risk of burn patients finding that the survival rates were significantly lower in burn patients with elevated liver transaminases than in those with normal values ⁸. Liver damage after burns has been associated with multifactorial events; hypoperfusion, proinflammatory cytokines, cell death signaling, edema, fatty changes, and mitochondrial dysfunction ⁵. However, burn-induced liver damage pathophysiology is not entirely understood, highlighting a critical gap in burn injury knowledge.

Using a clinically relevant murine burn injury model, our laboratory and others demonstrated that the liver exhibits molecular and histological changes following cutaneous burns^9–11. Also, we showed that these alterations were exacerbated in aged animals, as noted by elevated liver injury markers, increased lipid deposits, heightened oxidative stress, and accentuated histological evidence of hepatic damage¹⁰. These observations highlight changes in the liver of burn-aged animals that indicate increased susceptibility of this organ to acute stressors.

Comprehensive genetic and metabolic studies can help elucidate the nature of the differences in liver response to burns between young and aged individuals. While transcriptomics determines the functional response to burn injury and helps predict its master regulators, metabolomics provides a downstream and closer to phenotype description of the biological processes that occur in the liver ^12,13. In this project, we deepen the study of the hepatic parenchymal cellular interaction, analyzing transcriptomics and metabolomics in the liver of mice after burn injury.

Additionally, we employed an in silico analysis of the databases obtained from the transcriptomics and metabolomics to predict drugs that may prevent or treat the liver pathology observed in mice after burn injury.

Mice

Young 3-4 months old (equivalent to 20-25 human years) BALB/c and C57BL/6 female mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Aged 20-22 months old (equivalent to 65-70 human years) BALB/c and C57BL/6 female mice from the National Institute of Aging (NIA) Colony (Charles River Laboratories, Wilmington, Mass). Before the experiments, all mice were housed at the University of Colorado Anschutz Campus's vivarium for two weeks. The experiments were performed between 9 and 11 AM to decrease variability caused by differences in circadian rhythms. All procedures were approved by The University of Colorado Institutional Animal Care and Use Committee.

Burn Injury model

We distributed mice randomly into four experimental groups (young sham, young burn, aged sham, and aged burn). To create a controlled burn injury, we used the method described by Faunce et al. ¹⁴so. The mice were anesthetized with isoflurane, 55.5 mg/kg of ketamine Intra peritoneal (lP), and 2.6 mg/kg of xylazine IP (Webster Veterinary, Sterling, MA). Each mouse's dorsum was shaved and exposed to 94 degrees Celsius (for the burn groups) or room temperature water bath (for the sham groups) for 8 seconds as described ^14,15. We created a uniform full-thickness 15% total body surface area (TBSA) burn injury as described with minor modifications ^16-18. After this procedure, each mouse received additional pain medication (0.13 mg/kg buprenorphine IP) and 1 ml of normal saline IP for resuscitative purposes. The mice were assessed and monitored through the post-procedure recovery preserving normothermia using warm pads. Lastly, the mice were humanly euthanized 24 hours (for transcriptomics and metabolomics data) and at multiple points 6-, 9-,12-, and 24 hours in a time course experiment (for transcriptomics validation) after the burn injury procedure. Livers were collected, frozen in liquid nitrogen, and preserved at -80 degrees Celsius.

RNA sequencing

The Genomics Shared Resource performed RNA sequencing in the Cancer Center shared resource core at the University of Colorado Anschutz Medical Campus. 100 ng of total RNA was used to construct an Illumina-compatible mRNA library using Tecan Universal Plus mRNA-SEQ kit Cat No. 0520-A01. Sequencing was done on an Illumina NovaSEQ6000 Instrument using an S4 Flow Cell paired-end sequencing 2x150. FASTQ files were delivered after standard demultiplexing procedures were used, converting .bcl files to .seq files using CASAVA software as described ^19,20.

Metabolomic analysis

The Mass Spectrometry Metabolomics Shared Resource Facility at the University of Colorado Anschutz Medical Campus processed and analyzed the metabolomics data. Briefly, mass spectrometry-based metabolomics was performed on frozen liver samples. For polar metabolites, tissues were extracted at 15 mg per mL in ice-cold lysis/extraction buffer (methanol:acetonitrile:water 5:3:2), and 10 µL of supernatants were analyzed using a 5 min C18 gradient in positive and negative ion modes (separate runs) ^21,22. For polar lipids, tissues were extracted at 7.5 mg per mL in cold methanol containing stable isotope-labeled standards (see attached list), and 10 µL of supernatants were analyzed using a 17 min gradient in negative ion mode as previously described ²³. Data was acquired on a Thermo Vanquish UHPLC system coupled online to a Thermo Q Exactive mass spectrometer. Data analysis was performed in Maven against an in-house compound library as described ^22,23.

Statistics and data analysis

The NetworkAnalyst platform was used to perform differential expression analysis with EdgerR test after filtering out genes with variance lower than 15 and abundance lower than 4 and transforming data with upper quantile normalization (Table S1-4). Data visualization for transcriptomic analysis was performed with volcano plots ²⁴. For the transcriptomics analysis, statistical significance was considered with adj. p<0.05 values with n=3 per group. For transcriptomics validation, qPCR data were analyzed in 160 mice (80 Balb/c and 80 C57BL/6) in different time points (6-,9-12-, and 24 hours after burn) n= 5 per study group. We used Graph Pad Prism 9.3.1. The D'Agostino-Pearson normality test was used to check the normal distribution of the variables. For the statistical analysis of variables with a normal distribution, the Student t-test or ANOVA was used, whereas, for variables with a non-normal distribution, the Mann–Whitney U test was used. A p-value <0.05 was considered significant.

Heatmap from metabolomic data was performed with Multiple Experiment Viewer (MeV 4.8) ²⁵; a p-value lower than 0.05 was considered statistically significant with n= 5 per group. Overrepresentation analysis was performed with the g:Profiler ²⁶ using the KEGG database. To perform a functional analysis, construct networks, and predict upstream regulators, we used QIAGEN's Ingenuity® Pathway Analysis (IPA®, QIAGEN Redwood City, www.qiagen.com/ingenuity). In silico drug repurposing was predicted with the Connectivity map platform ²⁷. Graphics were designed with BioRender platform.

The ARRIVE guidelines were used to ensure proper reporting of methods, results, and discussion.

Overview of the unsupervised analysis among the experimental groups

Four groups of liver samples from the young sham, young burn, aged sham, and aged burn groups were collected 24 hours after the burn injury). This time point was selected based on previous animal experiments showing systemic differences, specifically in the liver, between young and aged mice after burn ^10,28. Transcriptional analysis was performed by RNA-seq and targeted metabolomics with mass spectrometry (Fig. 1A). The samples maintained an adequate group separation observed in unsupervised analysis by principal component analysis of transcriptomic data (Fig. 1B). The first component identified and separated the sham and burn treatments (PC1=60.8%). The second component separated young and aged groups (PC2=14.3%).

Hepatic transcriptomics in young and aged sham animals

At first, we focused on characterizing the differences between aged and young sham groups, finding that 75 genes were up-regulated (log2FC>1 and adj. p value<0.05) in aged compared to young mice (Fig. 1C). Among those, 13 genes were related to immunoglobulin production, including Igkv8-19, Ighm, Igkc, Igkj5, and Igkj1 (Supplementary Materials – Tab S4). In overrepresentation analysis, the genes up-regulated in aged mice were associated with two pathways PPAR signaling (Pck1, Angptl4, Plin5, Cyp4a32, Cyp4a14) and "IL-17 signaling" (Cebpb, S100a9, Nfkbia, S100a8, Cxcl1) (Fig. 1D, Tab S7). There were only 20 down-regulated genes (log2FC<-1 and adj. p value<0.05) compared to the aged vs. young mice after sham treatment. No pathway was statistically over-represented among down-regulated genes (Tab S8).

Hepatic transcriptomics, shared response to burn injury by young and aged mice.

Furthermore, we identified a common hepatic transcriptomic profile of burn-injury response in both young and aged animals. We identified genes responding to burn stimulus from the differential expression analysis in young (Fig. 2A) and aged (Fig. 2B) groups and selected the ones shared between the age groups. In the up-regulated genes after burn injury, 157 genes overlapped between young and aged groups (Fig. 2C). Those genes were associated within the 11 KEGG pathways like "protein processing in the endoplasmic reticulum" (30 genes, e.g., Derl3, Hyou1, Pdia3, Pdia4, Pdia6), "protein export' (8 genes identified, e.g., Hspa5, Sec61a1, Sec61b, Sec61g, Spcs2)," fructose and mannose metabolism" (5 genes: Tkfc, Gmppb, Gmppa, Pfkfb1, Akr1b7) and "IL-17 signaling" (6 genes: Lcn2, Hsp90b1, Cxcl1, Hsp90aa1, S100a9, S100a8) (Fig. 2E, Supplementary Materials – Tab S5). In the down-regulated genes, there were 114 genes overlapped in both young and aged groups (Fig. 2D). These down-regulated genes were significantly overrepresented in 18 KEGG pathways, including "chemical carcinogenesis – DNA adducts" (18 genes, e.g., Sult2a3, Gsta4, Cyp3a25, Ugt2b1, Gsta3), "steroid hormone biosynthesis" (13 genes, e.g., Akr1d1, Hsd17b6, Ugt2a3, Cyp2c38, Cyp2c37) and "metabolism of xenobiotics by cytochrome P450" (12 genes, e.g., Gsta2, Cyp1a2, Sult2a8, Cyp2f2, Sult2a1) (Fig. 2F, Supplementary Materials – Tab S6).

Hepatic transcriptomics, differences in response to burn injury by young and aged mice.

After burn, there were 46 up-regulated genes and 89 downregulated genes in the livers of aged mice compared to their younger counterparts (Fig. 2G). No pathway was significantly overrepresented among the up-regulated genes (Tab S9). However, among down-regulated genes, we found eleven overrepresented pathways, including: "chemical carcinogenesis" (11 genes, e.g., Gsta3, Gsta4, Sult1a1, Cyp2c54, Cyp2c38)," steroid hormone biosynthesis" (10 genes, e.g., Akr1d1, Hsd3b3, Cyp2c40, Cyp2b10, Cyp2c29), "bile secretion" (8 genes, e.g., Abcb11, Nr0b2, Hmgcr, Atp1b1, Sult2a5), metabolism of linoleic acid, arachidonic acid and retinol metabolism (5 common genes - Cyp2c54, Cyp2c38, Cyp2c40, Cyp2c29, Cyp2c50), "arginine biosynthesis" (Gpt, Gls2, Ass1), and" metabolism of xenobiotics by cytochrome P450" (Cyp2f2, Gsta3, Gsta4, Sult2a5, Sult2a8) (Fig. 2H, Supplementary Materials – Tab S10). We observed that the abovementioned pathways included many elements of the cytochrome P450 Cyp2 family (Cyp2c54, Cyp2c38, Cyp2c40, Cyp2c29, Cyp2c50, Cyp2f2, Cyp2b10, Cyp2a1) and glutathione metabolism genes (Gsta3, Gsta4).

Transcriptomics validation by qPCR

As Cyp2c genes are associated with multiple essential liver functions, from drug metabolism to inflammatory mediators' production ²⁹, we decided to validate those changes and evaluate their dynamics at 4-time points after burn injury. From the differentially expressed genes in Fig. 2, we selected representatives of the cytochrome P450 Cyp2c family, namely Cyp2c29, Cyp2c38, Cyp2c40, and Cyp2c54. We measured their expression in the 4 study groups at 4 different time points – 6h, 9h, 12h, and 24h after burn injury (supplemental figure). We confirmed the downregulation of their expression at the 24h time point among burn mice in both age groups. The transcriptomics validation of multiple P450 cytochrome genes in a time course study confirms that the downregulation is initially mild and becomes more severe as time progresses postburn (Fig. S1).

Common metabolomic response to burn injury in aged and young mice

Cytochrome P450 genes are closely associated with many metabolic pathways ³⁰. Thus, we performed the metabolite analysis of liver samples in young and aged mice. Metabolomic profiling of liver samples indicated that mice, irrespective of age, responded to burn injury by lowering their levels of many amino acids, including valine, methionine, tyrosine, phenylalanine, and leucine as well as cis-p-coumarate and protein 5-hydroxylysine. We also observed increased levels of glucosamine, UMP (uridine monophosphate), UDP-glucose (uridine diphosphate glucose), thymidine, putrescine, and N-acetylneuraminate (Fig. 3A, Tab S11).

Next, we connected the observed dysregulated metabolites with previously selected commonly differentially expressed genes in both aged and young mice after burn injury (Figure 2). In our network analysis, we looked for a connection between two datasets via the Ingenuity Pathway Analysis tool. The created network showed many associations between differentially expressed metabolites and genes that were commonly dysregulated by burn injury in both aged and young mice (Fig. 3B). Interestingly, Egfr, a membrane receptor, was found to be highly associated with many disturbed metabolites suggesting its crucial role in the development of post-injury metabolomic disturbances ^31,32. Additionally, we looked for upstream regulators of all commonly observed changes in both metabolites and genes (Fig. 3C). We found 9 upstream regulators that were predicted to be activated. Among them, we found two kinases: Eif2ak3 (Eukaryotic Translation Initiation Factor 2 Alpha Kinase 3), Ern1 (Endoplasmic Reticulum To Nucleus Signaling 1); two transcription regulators: Atf6 (Activating Transcription Factor 6), Stat5b (Signal Transducer And Activator Of Transcription 5B); two enzymes associated with glutathione metabolism: Txnrd1 (Thioredoxin Reductase 1), Gsr (Glutathione-Disulfide Reductase); two cytokines: IL1b, IL6, and one hypoxia-inducible gene: S100a4. Inhibition of six upstream regulators was also predicted: two transcription regulators: Zbtb20 (Zinc Finger And BTB Domain Containing 20), Ncoa2 (Nuclear Receptor Coactivator 2); one transporter (Atp7b - ATPase Copper Transporting Beta), one cytokine - visfatin (Nampt), one growth factor - leptin (Lep), and one ligand-dependent nuclear receptor (Ahr - Aryl Hydrocarbon Receptor). Molecular targets of those master regulators found to be dysregulated in our transcriptomic data are shown in Table S12. Interestingly, inhibition of Zbtb20 could be responsible for observed Cyp2 downregulation (by targeting molecules like Cyp1a2, Cyp2c54, Cyp2c8, Cyp2f1) and inhibition of leptin (by targeting: Cyp2c54, Cyp2c8, Cyp2f1, Cyp7a1) ³³.

Metabolomic differences in response to burn injury between aged and young

Having looked at common metabolomic changes after burn injury in both aged and young mice, next, we also looked for the differences.

Firstly, we found that among some of the commonly burn-dysregulated metabolites, three had more pronounced changes in aged mice, including deeper downregulation of phenylalanine and methionine levels and higher up-regulation of UMP levels in aged vs. young mice (Fig. 3D). Additional disturbances, not present in young mice, were identified in aged burn mice and included: downregulation of phosphate, S-adenosyl-L-homocysteine, L-proline, glutathione disulfide and 9(10)-EpOME (epoxyoctadecenoic acid); and up-regulation of guanosine, ADP (adenosine diphosphate), ethanolamine phosphate, acyl-C6-DC (methylglutarylcarnitine) and ectione.

In silico drug predictions

Finally, we searched for drugs able to modify the liver burn injury response. Thus, we perform an in silico analysis of drugs that can cause similar or opposite effects to the transcriptomic differences observed in burn response among both aged and young mice. To do so, we used the Connectivity map tool - Cmap. Providing the list of commonly dysregulated genes from Fig. 2, we predicted which drugs could cause a similar effect to burn injury (Fig. 4A right panel). The predicted drug list included some commonly used drugs like albendazole, dextromethorphan, or valsartan. Assuming that observed expression changes after burn injury are harmful, the abovementioned drugs could exacerbate them. We also looked at drugs that have opposite expression effects. Among them, we found widely used drugs like enalapril, atorvastatin, or cilostazol. Those drugs could cause the opposite transcriptional effect, potentially reversing transcriptomic changes observed in mice liver after burn injury (Fig. 4A left panel).

Additionally, we searched for drugs able to cause similar and opposite expression changes to those observed between aged burn vs. young burn mice (Fig. 4B). Among drugs causing similar transcriptional effects to the one observed in aged vs. young mice after burn injury, we found such commonly used drugs as etomidate, probenecid, flumetazone and nadolol (Fig. 4B – right panel). Among drugs with opposite expressional effect to the one observed in aged burn mice (compared to young burn individuals) were clinically relevant drugs such as melperone, clozapine, dexamethasone, and sertraline (Fig. 4B – left panel).

Burns are a significant cause of morbidity and mortality worldwide, with an estimated 11 million cases yearly and more than 300,000 fatalities ³⁴. Burns affect seemingly every age group ². However, the mortality rate is significantly higher in elderly burn patients ³⁵. Regardless of age, severe burns cause damage to multiple organs distant from the original burn wound, leading to severe clinical problems such as multiple organ dysfunction syndrome (MODS) ³. The liver is particularly affected after burn injury exhibiting hepatomegaly and fatty infiltration ⁵. Burn-induced liver damage is characterized by edema and cellular damage leading to leakage of hepatic enzymes AST and ALT into the circulation ⁴. Clinical studies have demonstrated that the mortality in burn victims is elevated in patients with persistently elevated AST and ALT ⁸. Advanced age is associated with cellular derangements in the liver ³⁶. Several age-related hepatic changes include a decline in liver volume, an increase in the hepatic dense body compartment (lipofuscin), a reduction in drug metabolism, and diminished hepatobiliary functions ^35–37. Based on these correlations, it is not unreasonable to suggest that elderly patients are more susceptible to liver damage after suffering burns. With its metabolic, inflammatory, and immune functions, the liver is fundamental for patient survival and recovery following burn injury ⁴. Therefore, it is logical to study the liver and attempt to elucidate modifiable factors that specific targeted interventions could improve the outcomes of burn victims.

We evaluated the liver omics at a base level comparing young and aged sham animals. In this study, the transcriptomics analysis demonstrated that while the livers from aged sham animals exhibit higher immunoglobulin gene expression and increased activity of IL-17 signaling pathways, they do not present significant downregulation of genes or signaling pathways compared to the livers of younger counterparts. These results match common age-related findings that contribute to the characteristic chronic inflammatory state of aged individuals known as inflammaging ³⁸.

Following a significant trauma such as a burn, the liver physiology drastically redirects its protein synthesis favoring the production of acute phase reactants ⁵. As the liver becomes hyper-functional in producing these essential proteins to survive a burn insult, it reduces the production of constitutional proteins, steroid hormones, bile, and numerous cytochrome p450 metabolic processes ^4,39. As such, in this study, the transcriptomics analysis showed that after burn, the foremost up-regulated pathways in the livers of young and aged mice involved protein processing in the endoplasmic reticulum, protein export, fructose, and mannose metabolism, and IL-17 signaling. The major hepatic pathways downregulated after a burn in young and aged mice were chemical carcinogenesis, steroid hormone biosynthesis, and metabolism of xenobiotics by cytochrome P450.

Additionally, our data demonstrate that after burn, the livers of aged differently from younger mice profoundly downregulate processes such as steroid hormone biosynthesis, bile secretion, linoleic acid, arachidonic acid, and retinol metabolism, arginine biosynthesis, and metabolism of xenobiotics by cytochrome P450. While prioritization of acute phase proteins benefits survival rate at an initial phase post-injury, a severe and prolonged downregulation of other hepatic functions, such as cytochrome metabolism, may be detrimental for burn victims, especially in the elderly ⁴⁰. There is a lack of understanding about the impact of these processes on the outcomes in young and aged burn victims, which can be an important focus of future research.

Particularly distinctive was the downregulation of cytochrome P450 Cyp2 family genes involved in multiple liver functions, including protein and lipid metabolism, oxidative stress buffers, and metabolism of drugs and toxins ⁴¹. These differences can result in a toxin metabolism shift in the liver cells and destabilization of free radical scavenging and DNA replication, recombination, and repair processes in aged animals after burn injury ^42,43. The cytochrome P450 system may also be affected by inflammation and age ⁴⁴. Two cytochrome P450 enzyme families, Cyp2c and Cyp2j, are responsible for metabolizing proinflammatory metabolites (arachidonic acid) to anti-inflammatory compounds (epoxyeicosatrienoic acids) ⁴⁵. In the data presented herein, all 11 members of the Cyp2c family identified in our liver samples (Cyp2c54, Cyp2c38, Cyp2c40, Cyp2c29, Cyp2c50, Cyp2c68, Cyp2c70, Cyp2c39, Cyp2c67, Cyp2c37, Cyp2c69) and one detected CYP2J member - Cyp2j5 - were found to be downregulated among liver of aged burn mice compared to the young burn mice.

Clinical evidence reveals that older patients have an attenuated inflammatory response to burn injury relative to younger injured subjects, followed by a hyper-inflammatory reaction ³⁵. Microsomal oxidation deceleration driven by cytochrome P450 was shown to play a critical role in developing multi-organ dysfunction syndrome (MODS) due partly to the accumulation of endogenous toxic compounds ⁴¹. MODS was found to have increased incidence in the elderly after burn injury, but this was not accompanied by increased infection or sepsis rate ³⁵. Moreover, decreased cytochrome P450 Cyp2c activity could lead to the accumulation of toxic metabolites from burn treatment medications as these cytochrome family members are mainly involved with drug metabolism ⁴⁶. Those observations suggest that inhibiting cytochrome P450 Cyp2 as a component of the hepatic post-burn inflammatory response may have detrimental effects and that increased expression of those enzymes could have a potentially beneficial effect. However, further studies need to establish whether the downregulation of Cyp2c after burn injury is responsible for less favorable clinical outcomes.

Given that the cytochrome p450 system is abundant in the liver playing the role of numerous functions (mentioned in the previous paragraphs) ⁴⁷, we validated our transcriptomics findings by measuring the gene expression of central genes that operate in this system in 2 different mice strains (Balb/c and B57BL) (supplemental figure). The transcriptomics validation of multiple P450 cytochrome genes in a time course study demonstrates that the downregulation becomes more severe as time advances postburn. These findings raise a red flag in treating burn patients, indicating that drug metabolism may be severely affected in the first weeks after burns.

The metabolomic analysis exhibited a common and a different hepatic response pattern to burns between young and aged animals. The common response showed a decrease in amino acid levels, including valine, methionine, tyrosine, phenylalanine, leucine, cis-p-coumarate, and protein 5-hydroxylysine. Most of these metabolites are essential amino acids, and their decrease is commonly associated with a lack of nutritional intake ⁴⁸. Postburn metabolism not only consumes essential amino acids but also decreases appetite, which can result in an essential amino acid deficiency ⁴⁹. Liver injury can cause a decrease in several catabolic pathways resulting in the accumulation of several metabolites ⁵⁰. In the liver of mice postburn, we observed an increased level of glucosamine, UMP (uridine monophosphate), UDP-glucose (uridine diphosphate glucose), thymidine, putrescine, and N-acetylneuraminic, indicating that energy and proliferation catabolic pathways are affected in this pathology (Fig. 4A, Tab S11).

To further evaluate the common (young and aged) hepatic response to burns, we used an Ingenuity Pathway Analysis tool to create a network connecting the metabolomic and transcriptomics changes among the four study groups. The network showed many connections between differentially expressed metabolites and genes that were commonly dysregulated by burn injury in both aged and young mice (Fig. 3B). Epidermal Growth Factor Receptor (EGFR) was found to be highly associated with many disturbed metabolites suggesting its crucial role in the development of post-injury metabolomic disturbances. EGFR is pivotal in hepatocyte proliferation, liver regeneration, liver steatosis, oxidative stress, mitochondria dysfunction, and cell death in different animal models of liver injury ³². EGFR activation is achieved ligand-based or ligand-independent, and the latter is mainly associated with liver injury. Given EGFR's diverse involvement at many levels in hepatic pathophysiological processes, additional research is necessary to determine if EGFR may be used as a therapeutic target to treat liver disorders, including burn induce liver damage ³¹. The IPA analysis of the common hepatic response to burn yield 6, deactivated master regulators: Zbtb20, Ncoa2, Atp7b, vistfatin (Nampt), leptin (Lep), and Aryl Hydrocarbon Receptor (Ahr). Interestingly, inhibition of Zbtb20 could be responsible for observed Cyp2 downregulation (by targeting molecules like Cyp1a2, Cyp2c54, Cyp2c8, Cyp2f1) and inhibition of leptin (by targeting: Cyp2c54, Cyp2c8, Cyp2f1, Cyp7a1 ³³).

The significant differences in metabolic pathways noticed in the livers of young burn and aged burn mice involved a severe downregulation of glutathione metabolism and citrate cycle (TCA cycle) in the aged animal group. These metabolomics results match our previous findings that highlight the presence of a disrupted antioxidant glutathione system in the liver of aged animals, which can lead to increased susceptibility to hepatic damage following burns ¹⁰.

In silico (computer-based) drug prediction and repurposing have become one of the most used methods to make drug discovery more accurate and economical ⁵¹. This method seeks to recognize the relationship network between the target and the drug, which is attained through bioinformatics tools and public databases ⁵². In this study, we used a Connectivity map tool – Cmap, to obtain drugs that can induce opposite and similar transcriptomics and metabolomics changes in hepatocytes as the ones caused by burn injury in our experiments. We hypothesize that harnessing side targets of clinically used drugs might help reprogram liver cells to handle better the stress of responding to a burn injury.

Some predicted drugs, like atorvastatin, enalapril, cetirizine, cilostazol, and venlafaxine, are widely used in clinical practice. Based on their mechanism of action, these drugs should not affect the immune system's response to burn injury, as anti-inflammatory drugs are used to ameliorate organ damage, such as dexamethasone. They might be considered an additional treatment for elderly patients after burn injury. Interestingly, histamine receptor antagonist was found previously to accelerate skin barrier repair. Thus, using cetirizine could have another beneficial effect in treating burns ⁵³.

The list of drugs identified by our in silico search encompasses several FDA-approved compounds used to treat common conditions. Drugs that induce "similar changes" could be harmful to the organ and may be avoided when treating burn injury patients. Drugs that cause "opposite changes" should be further evaluated in animal models of burn-induced liver injury to validate the calculated effect predicted by our analysis.

Our approach via the expression data analysis by Cmap was previously used to predict targeted combination treatment for pancreatic cancer successfully ^54,55. Thus, we expect that some of the predicted drugs will prove to be beneficial for burn victims. However, the recommendation of using any of the predicted medications in a clinical scenario needs to be supported by in-depth in vivo drug studies and clinical trials.

In conclusion, we analyzed the gene expression and metabolic responses to burn injury in the livers of aged and young mice finding multiple similarities and differences. A significant difference in the livers of aged animals after a burn is a profound downregulation of essential hepatic systems such as the cytochrome P450. We predicted commonly used drugs that may rewire the burn-induced liver response to a more favorable one and thus increase the survival of burn victims.

Author contributions:

Beata Malachowska, Israel Muro, Weng-Lang Yang, Elizabeth J. Kovacs, and Juan-Pablo Idrovo conceived and planned the experiments. Devin Boe, Andrea Qualman and Juan-Pablo Idrovo performed the burn injury experiment and collected tissue samples. Beata Malachowska analyzed all transcriptomics and metabolomics data and generated the graphics and figures. Beata Malachowska, Israel Muro, Weng-Lang Yang, Elizabeth J. Kovacs, and Juan-Pablo Idrovo contributed to the analysis and interpretation of the results. All authors provided critical comments and helped in the creation of the manuscript.

Acknowledgments:

The Genomics Shared Resource performed RNA sequencing in the Cancer Center shared resource core at the University of Colorado Anschutz Medical Campus. The Mass Spectrometry Metabolomics Shared Resource Facility at the University of Colorado Anschutz Medical Campus processed the metabolomics data.

Conflict of interest statement:

The authors have no conflicts of interest to declare. All co-authors have seen and agree with the manuscript's contents, and there is no financial interest to report. We certify that the submission is original work and is not under review at any other publication.

Sources of funding:

The research in this publication was supported by K08GM134185 (JPI) and R01AG018859, R01GM115257, R35GM131831, and 1 I01 BX004335 (EJK). The content in this report is the authors' responsibility and does not primarily represent the formal National Institutes of Health opinion.

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Supplementarymaterials20221206.xlsx
Supplementary materials: Figure S1 – qPCR expression measurements in mice liver of 4 cytochrome P450 Cyp2c family members – Cyp2c29, Cyp2c38. Cyp2c40, Cyp2c54 - in four time points (6h, 9h, 12h, 24h) after the burn injury. Table S1 - EdgeR analysis – Differential expression analysis of liver transcriptomics data of aged burn and aged sham groups. Table S2 - EdgeR analysis – Differential expression analysis of liver transcriptomics data of young burn and young sham groups. Table S3 - EdgeR analysis – Differential expression analysis of liver transcriptomics data of aged burn and young burn groups. Table S4 - EdgeR analysis – Differential expression analysis of liver transcriptomics data of aged sham and young sham groups. Table S5 - Over-representation analysis results – KEGG pathway overrepresentation analysis results for genes up-regulated by burn injury in both aged and young mice in comparison to sham conditions. Table S6 - Over-representation analysis results – KEGG pathway overrepresentation analysis results for genes down-regulated by burn injury in both aged and young mice in comparison to sham conditions. Table S7 - Over-representation analysis results – KEGG pathway overrepresentation analysis results for genes up-regulated in aged sham group in comparison to young sham group. Table S8 - Over-representation analysis results – KEGG pathway overrepresentation analysis results for genes down-regulated in aged sham group in comparison to young sham group. Table S9 - Over-representation analysis results – KEGG pathway overrepresentation analysis results for genes up-regulated in aged burn group in comparison to young burn group. Table S10 - Over-representation analysis results – KEGG pathway overrepresentation analysis results for genes down-regulated in aged burn group in comparison to young burn group. Table S11 – Liver metabolomics analysis of aged burn, aged sham, young burn and young sham mice 24 after injury. Table S12 – Ingenuity Pathway Core Analysis results of genes and metabolites commonly dysregulated after the burn injury in both aged and young mice. Upstream regulators with predicted activation or inhibition were shown.

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Transcriptomics, metabolomics, and in silico drug predictions to prevent or treat liver damage in young and aged burn victims

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