Ellagic Acid Alleviates Mice Intestinal Ischemia‐Reperfusion Injury: A Study Based on Transcriptomics Combined with Functional Experiments

Background: It has been reported that intestinal ischemia‐reperfusion injury (IIRI) is closely related to inflammatory response, apoptosis and oxidative stress. Ellagic acid (EA) has been proved to have antioxidant and anti‐inflammatory effects and can inhibit tumor angiogenesis. The purpose of this study was to investigate the protective effects of EA on IIRI in mice. Methods: A mouse model of IIRI was established by clamping the mesenteric artery. Effects and mechanisms of EA on IIRI were investigate by transcriptomics combined with functional experiments. Results: The symptoms of IIRI were reflected in significant increases in inflammatory factors such as TNF‐α and IL‐1β; significant increases in oxidative stress indicators such as MDA and GSH and decreases in SOD and promotion of the apoptotic protein Bax/Bcl‐2. These indicators were significantly alleviated by EA. And after EA treatment, transcriptomics results identified AKT1 differentially expressed mRNAs mainly enriched in PI3K/AKT signalling pathway. Conclusion: This study illustrates the protective effects against IIRI, the possible mechanisms were also studied. This study provides new scientific information for the application of EA in IIRI therapy.


Introduction
Intestinal ischemia-reperfusion injury (IIRI) is common pathological processes associated with various clinical emergencies, such as trauma, mesenteric vascular occlusion, volvulus, intestinal obstruction. It can eventually progress to multiple organ failure and often death due to local and systemic injuries. [1] Although awareness of Acute Mesenteric Ischemia (AMI) has improved and treatment has progressed significantly over the last four decades, the significant morbidity (25-100 %) and mortality (16-80 %) of AMI remain a challenging clinical issue. [2] Therefore, there is a great need for innovative therapeutic strategies to improve IIRI.
A number of clinical agents are available for the prevention and treatment of ischemia-reperfusion injury, such as adenosine, adenosine triphosphate and oxytocin.
Although these drugs are effective, they are prone to adverse effects such as chest pain, arrhythmias, urticarial, joint pain and hypotension. Other drugs such as verapamil and montelukast are also commonly used to treat ischemia-reperfusion injury, but these drugs are prone to serious adverse reactions such as severe lower limb swelling, muscle weakness, osteoporosis and cardiac arrhythmias. [3,4] Herbal medicines have great potential for the prevention and treatment of IIRI due to their multi-targeting, low cost and low adverse effects. Therefore, it is valuable to screen or develop new drugs to expand therapy options.
More and more researchers are focusing on natural plants as the best choice for disease treatment because they are non-toxic, have few side effects and are effective in treatment, making substantial contributions to the promotion of national pharmacology. Pomegranate peel is herbal medicine with anti-inflammatory [5] and antioxidant properties in China. Ellagic acid (2,3,7,8-tetrahydroxychromeno[5,4,3-cde]chromene-5,10-dione) (EA: As shown in Figure 1A) is widely found in berries, grapes, tea, nuts and other foods as a natural polyphenolic compound. [6] EA has been shown to exert a variety of biological activities in vitro and in vivo, including antibacterial, antioxidant, anti-hepatotoxic, anti-atherosclerotic, anti-inflammatory, anti-HIV, anti-insulin and anticancer effects,anti-aging, insulin-promoting and anticancer effects. [7] EA has been found to protect against ischemia-reperfusion induced damage in the ovaries, stomach, lungs and kidneys. [8][9][10] However, the protective effect of EA against IIRI remains unclear. Therefore, the aim of this study was to determine the ameliorative effect of EA on IIRI.
In this study, transcriptomics and functional experiments are used to uncover the detailed mechanisms of EA-induced therapeutic effects on IIRI. RNA sequencing (RNA-seq) is a deep-sequencing approach in transcriptome profiling that provides an impartial and accurate method for measuring the levels of transcripts and their isoforms, [11] the experimental idea is shown in Figure 1.
Animals adapted to laboratory conditions (23°C, 12 h/ 12 h light/dark, 50 % humidity, free access to food and water) for a period of 2 weeks. Animal studies were conducted using the NIH guidelines for the care and use of laboratory animals. Animal studies were conducted using the National Institutes of Health guide for the care and use of laboratory animals. Studies in which animals were conducted following the ARRIVE guidelines. [25] All applicable institutional and/or national guidelines for the care and use of animals were followed.

Experimental Design
For the IIRI model, mice were divided randomly into five groups of eight mice each. The mice were treated as follows: sham group and model group with gavage administration of saline; and other three groups were treated with the following: low-dose EA (25 mg/kg body weight, gavage administration, dissolved in saline), middle-dose EA (50 mg/kg bodyweight, gavage administration, dissolved in saline), and high-dose EA (100 mg/ kg body weight, gavage administration, dissolved in saline), respectively. After an 18 h fast on the evening of the seventh day, models were made and intestinal tissue taken on the eighth day. A mouse IIRI model was established as previously described. [12] To establish the IIRI model, the clamp was removed after 45 min of occlusion and the blood supply was recovered for 2 h of reperfusion.

RNA-Seq and Bioinformatics
There were three groups of C57BL/6 mice subjected to mRNA profiling, three in each group: sham group, I/R group, and EA group (100 mg/kg). Mice were anesthetized with urethane and the ileum was removed from the sham group, I/R group, and EA group, respectively. Total RNA was extracted using Trizol reagent (Invitrogen) and quality was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA).
Sequencing libraries were generated using the NEB Next® Ultra™ RNA Library Prep Kit for Illumina (NEB, United States) following the manufacturer's instructions, and index codes were added to attribute sequences to each sample for identification. Briefly, purified mRNA was synthesized as double stranded cDNA, the ends were repaired and adenylated, adaptors were ligated and enriched fragments were amplified with PCR. Library quality was assessed using the Agilent Bioanalyzer 2100 system. Libraries were sequenced on the Illumina platform.
Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. Quality and reliability of data analysis was ensured by removing reads with adapters, removing reads containing N (where N indicates that base information cannot be determined), and removing low-quality reads (where the number of bases with a Qphred < = 20 represents more than 50 % of the entire read length).
The number of reads mapped to each gene was counted by using Feature Counts. FPKM (expected number of fragments per kilobase of transcript sequence per million base pairs sequenced) was calculated for each gene based on the read counts mapped to that gene and the gene length. Differential expression analysis between the two groups was performed using the DESeq R package (1.16.1). False discovery rate (FDR) was adjusted by the Benjamin and Hochberg method by adjusting the resulting pvalues. Differential expression was expressed as adjusted P < 0.05 and log2 (fold change) > 1. To test statistical enrichment of gene functions and biological pathways, the Cluster Profiler R package (http:// bioconductor.org) were used to analyze differentially expressed genes through the Kyoto Encyclopedia of Genes and Genomes (KEGG) (https://www.kegg.jp) and Gene Ontology (GO) (http://www.geneontology.org). An adjusted P < 0.05 was considered as statistically significant enrichment.

Construction of the EA Target PPI Network
The predicted intersection of EA targets and IIRI RNAseq genes was compared and analyzed in Venny 2.1. (http://bioinfogp.cnb.csic.es/tools/venny/index.html). Common potential targets for EA and I/R were uploaded on STRING (http://String-db.org) online software, and study species were limited to Homosapiens to reflect the role of target proteins in the system. Set the confidence to 0.700 (A higher score gives a higher degree of confidence in the protein interaction), and P value < 1.0 e -16. The obtained protein interaction information was imported into Cytoscape 3.7.0, and the topological network was analyzed by using Network Analyzer. [13,14] Histological Staining The intestinal tissues were fixed in 4 % paraformaldehyde, dehydrated in conventional gradient ethanol, transparent in xylene, embedded in paraffin, and sectioned 5 μm thick. The pathological morphology of the intestine was observed after staining with hematoxylin and eosin.

Statistical Analysis
All statistics were completed in Prism 8.0, and normally distributed measures were expressed as the mean � standard deviation, and one-way ANOVA was used for comparisons between multiple groups. Further evaluations were carried out using the Kruskal-Wallis rank-sum test. All experiments were repeated at least three times, and P < 0.05 was considered significant. P < 0.05 was considered statistically significant.

EA Alleviates IIRI Symptoms
Compared with sham group (No ischemia-reperfusion injury), the symptoms including damage to intestinal villi structure (green ring), inflammatory cell infiltration (black arrow), submucosal edema (brown ring) were observed in model group (IIRI). The Chiu's score of model group was higher than that in sham group. All the pathological changes and increased Chiu's score were reversed by 25, 50 and 100 mg/kg EA ( Figure 2B, C).

EA Suppressed Oxidative Stress and Inflammation
Compared with sham group, model group showed significantly lower levels of SOD and GSH, and significantly higher levels of MDA. Gavage administration of EA (25, 50, 100 mg/kg) these markers. TNF-α and IL-1β activities were significantly higher in the model group Chem. Biodiversity 2022, 19, e202200345 www.cb.wiley.com (4 of 10) e202200345 than sham group, which were reversed by EA (25, 50, 100 mg/kg). These results indicated that: EA can significantly alleviate oxidative stress reaction and inflammation reaction (Figure 3).

Effects of EA on Intestinal Epithelial Apoptosis
Compared with the sham group, the Bax/Bcl-2 level was significantly increased in the model group (from    Figure 4).

Mechanisms of EA Exerted Protective Effects Against IIRI
We used RNA-seq to investigate the possible mechanisms of ellagic acid for the treatment of IIRI. According to the RNA-seq results, the possible mechanism of EA in IIRI therapy was analyzed. GO enrichment results showed the top 20 were analyzed from small to large according to the P value (< 0.05) ( Figure 5A-C). By enrichment, it finally obtained: 20 biological processes ( Figure 5A); 20 cell composition ( Figure 5B); 20 molecular functions Chem. Biodiversity 2022, 19, e202200345 www.cb.wiley.com (6 of 10) e202200345 ( Figure 5C). The KEGG enrichment results showed 27 pathways, including PI3K-Akt signalling pathway, AMPK signalling pathway, HIF-1 signalling pathway etc. (Figure 5D).

Construction of PPI Network and Target Validation
To further explore the mechanism of EA in IIRI, and map the PPI network relationship of the disease targets of EA.
In PPI network diagram, the larger the node display, the more obvious the interaction between the nodes, and the more important the node (gene/protein) plays a regulatory role. Select the larger nodes in PPI network, namely HSP90AA1, AKT1, NFкB1 and SRC ( Figure 6A, B). According to relevant literature, AKT1 is closely related to three other genes and is the core gene of the pathway. Therefore, we selected AKT1 for verification by western blot experiment. Compared with Sham group, p-AKT/ AKT in I/R group was significantly decreased (from 0.852577 to 0.4051, p = 0.0436) and significantly in-creased after EA administration. It indicated that AKT maybe the main target of EA exerted protective effects ( Figure 6C).

Discussion
This study explored the protective effect of EA on IIRI in vivo, which may resist apoptosis by promoting the expression of AKT. The results include: EA significantly improves oxidative stress by regulating SOD, MDA and GSH; Improve the inflammation induced by IIRI by reversing the levels of inflammatory factors (TNF-α, IL-1β); Improve IIRI by inhibiting apoptosis; RNA-seq identified AKT1 as the main differential gene, which regulates IIRI in vivo by activating PI3K/AKT pathway ( Figure 7). Oxidative stress is the main pathological feature of intestinal ischemia in its pathological process. Hypoxia can lead to decreased ATP in mitochondria of epithelial cells of intestinal mucosa, resulting in decreased activity Chem . Biodiversity 2022, 19, e202200345 www.cb.wiley.com (7 of 10) e202200345 of scavengers (such as GSH and SOD), and affected their capacity of scavenging free radicals, resulting in continuous increase of oxygen free radicals. [15] Polyunsaturated fatty acids are very vulnerable to the attack of reactive oxygen species produced by free radicals, which imbalance the content of polyunsaturated fatty acids in the intestinal epithelial cell biofilm, resulting in the increase of intestinal permeability and the production of a variety of lipid peroxides, which will cause more serious damage to the intestinal tissue. Apoptosis is one of the basic characteristics of cell life, and it is an important mechanism for the body to maintain internal balance and development process. It is a process of cell active death regulated and completed by related genes. Apoptosis is regulated by a variety of genes, including Bcl-2, Bcl-xl, and BHRF1 that inhibit apoptosis, Bax, P53, and Bcl-xs that promote apoptosis, and A1 and LMW5-HL of Mcl-1 that participate in the regulation of cell survival, among which Bax/Bcl-2 plays a major role. Based on RNA-seq analysis, we identified 27 significant pathways involved using the KEGG-target network. Some of these pathways have previously been shown to be involved in IIRI. For example, IIRI via activation of the PI3K/Akt signalling pathway. [16] The expression of PI3K and mTOR mRNA was increased by decreasing the level of miR-199a-3p. This suggests that miR-199a-3p may play a key role in the anti-apoptotic mechanism of HRS. [17] HIF-1 mediates the inflammatory response to pathogenic IIRI. [18] In the previous introduction, it was mentioned that ellagic acid has anti-inflammatory and antioxidant effects, and at the same time, ellagic acid is further metabolized by the microbiota into a bioavailable compound called urolithin, which has been shown to be a Powerful oxidative stress regulators and drugs have potential anti-inflammatory, anti-proliferative, and antiaging properties. [27] However, ellagic acid has not been reported in intestinal I/R via these pathways. We predicted a possible mechanism for ellagic acid protection against IIRI and demonstrated validity in preparation for the next step of pathway validation.
Using PPI network diagram among the 27 pathways analyzed by KEGG, we found that HSP90AA1, AKT1, SRC, and NFкB1 were the four most differential targets. HSP90AA1, one of the subtypes of HSP90, is inducible and involved in cell protection and cell cycle regulation under stress. HSP90 is a highly conservative molecular chaperone highly dependent on ATP, which can utilize the energy generated by ATP hydrolysis to participate in the correct folding of the protein, stabilize the protein structure, participate in the responses of cell signal transduction hormone response and transcriptional regulation, and the regulation of tumor apoptosis and proliferation-related pathways. It is also the molecular chaperone of protein kinase B (AKT). SRC is a nonreceptor tyrosine protein kinase that plays an important role in maintaining the normal physiological function of the body. It has been reported in the literature that SRC is an upstream molecule that regulates the AKT/mTOR pathway. [19] A large number of studies have shown that NFкB and AKT pathways have a strong biological link in the regulation of inflammation. [20][21][22] AKT1 is closely related to three other targets and is the core gene of the pathway, suggesting that the protective effect of EA on intestinal I/R injury is likely to be exerted through the AKT1 gene. In previous studies, ginsenoside Rb1 reduced inflammation and oxidative stress caused by intestinal ischemia/reperfusion by activating PI3K/AKT/Nrf2. [23] Intestinal ischemia-reperfusion injury is alleviated in mice through the AKT/GSK-3/ NRF2 pathway. [24,26] After EA administration, we significantly increased the protein content of p-AKT, which preliminarily indicated that EA could alleviate the injury caused by intestinal I/R by regulating AKT1. [28,29] However, this article does not delve into how EA regulates AKT1, and we will continue to explore this later.   Training Program for College Students' Innovation  and  Entrepreneurship  (202110161006X,  202010161004X, 201910161064, S202010161001X, 2017161025).