EA Alleviates IIRI Symptoms
Compared with sham group, the symptoms including damage to intestinal villi structure (green ring), inflammatory cell infiltration (black arrow), submucosal edema (brown ring) were observed in model group. 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 (Fig. 1B, 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(25,50,100mg/kg) hibit these markers. TNF-α and IL-1β activities were significantly higher in the model group than sham group, which were reversed by EA(25,50,100mg/kg). These results indicated that: EA can significantly alleviated oxidative stress reaction and inflammation reaction (Fig. 2).
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 0.711211 to 1.27859, p = 0.0003), and this level was significantly decreased by EA pretreatment. Expression of apoptosis related protein Bax / Bcl-2 in model group was higher than that in sham group. And decreased expression was reversed by 25, 50 and 100 mg/kg EA (Fig. 3).
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) (Fig. 4A-C). By enrichment, it finally obtained: 20 biological processes (Fig. 4A); 20 Cell composition (Fig. 4B); 20 molecular functions (Fig. 4C). The KEGG enrichment results showed 27 pathways, including PI3K-Akt signalling pathway, AMPK signalling pathway, HIF-1 signalling pathway etc. (Fig. 4D).
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, NFκB1 and SRC (Fig. 5A, 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 increased after EA administration. It indicated that AKT maybe the main target of EA exerted protective effects (Fig. 5C).