IIRI was a well-known surgical complication. IRI has the potential to develop quickly and go undiagnosed in its early stages, making it a surgical emergency. IRI has the potential to develop quickly and go undiagnosed in its early stages, making it a surgical emergency [19]. However, the pathogenesis of IRI involved multiple factors and complex cellular and molecular mechanisms increase the challenge of medical research. Therefore, finding biomarkers that could predict the risk of IIRI in for early prevention was of great value to reduce the high prevalence and mortality of IIRI. In this study, we obtained transcriptional sequence data from 40 small intestinal tissues of C57 mice and screened important biomarkers (Ccl7, Cd14, Cxcl1, Hmox1 and Nfkbia) for the diagnosis and treatment of IIRI by bioinformatics analysis techniques and explored their mechanisms of action.
Chemokine (CC motif) ligand 7 (Ccl7) adsorbed different leukocytes and regulated important immune cells which played an key role in the immune process [20]. Furthermore, Ccl7 was over-expressed in the intestinal lamina propria [21]. Previous researchers found that Ccl7 from intestinal epithelial cells regulated the development of acute liver injury [22]. In a study, researcher found that the over-expression of Ccl7 in intestinal epithelial cells lead to changes in the gut microbial landscape in response to HFD (high fat diet) challenge[23]. In our study, we found an up-regulation of Ccl7 expression in the IRI group. Therefore, we hypothesized that the mechanism by which the regulation of IIRI by Ccl7 might be the result of an altered immune microenvironment in the presence of Ccl7 overproduction. However, the specific mechanism leading to the over-expression of Ccl7 in intestinal epithelial cells affecting the composition and function of the intestinal flora was not known.
Cluster of differentiation 14(Cd14), a 55 kDa cell surface glycoprotein that together with Toll-like receptor 4 and MD-2, acted as a receptor for endotoxin. The mCd14 was mainly expressed on neutrophils, macrophages and dendritic cells. Cd14 on the surface of mononuclear macrophages bind to the LPS-LBP complex and activates immune cells, producing inflammatory mediators and cytokines and promoting tissue damage[24]. The sCd14 might confer LPS responsiveness to cells that do not express CD14[25]. Blood and other body fluids contained sCd14, and the levels were elevated during inflammation and infection. Thus, sCd14 could be considered an acute phase protein [26]. One investigator found that increased Cd14 mRNA levels in the liver after liver I/R corresponded to increased circulating sCd14 levels and that blocking Cd14 by neutralizing antibodies attenuated liver injury and inflammatory responses in C57BL/6 mice after I/R[27]. Analysis of microarray data after cerebral ischemia-reperfusion showed that 15 genes, including Cd14 and Hmox1, showed significant increases after cerebral ischemia-reperfusion injury [28]. This was consistent with the changes seen in our study, where higher expression of Cd14 was also observed in the IRI group in our sequencing data. These results indeed suggest that I/R was a potent stimulus for the up-regulation and release of Cd14 .
Cxcl1 was an important neutrophil chemokine and Cxcr2 was its receptor. Chemokines bind to their receptors played key roles in pathological changes such as cell growth, proliferation, differentiation and apoptosis. They were involved in inflammation, neuronal growth and development, oligodendrocyte proliferation and migration, and have important roles in intestinal and brain development and injury[29]. The level of Cxcl1 in the cerebrospinal fluid of stroke patients were higher than those of healthy patients. The miR-429 protected against OGD/R-induced injury in cerebral microvascular endothelial cells by regulating Cxcl1 [30]. Studies have authenticated that Cxcl1/Cxcr2 was involved in the regulation of intestinal inflammation[31]. Results from some investigators have shown that Cxcr2 and Cxcl1 proteins were up-regulated after IIRI and that Cxcl1 / Cxcr2 signaling is associated with IIRI[32]. Our study also consistently suggested that the expression of Cxcl1 was over-expressed in IRI group samples. In conclusion, Cxcl1/Cxcr2 played a role in IIRI, but its specific mechanism of action was unclear and needs to be further investigated. The signaling pathway of Cxcl1/Cxcr2 might become a new target for clinical treatment of IIRI, reducing the inflammatory response and its secondary damage after IRI and improving clinical outcomes.
Human Heme Oxygenase 1(Hmox1), an isoform of heme oxygenase, is an initiator and rate-limiting enzyme of heme degradation and played a protective role in myocardial IRI injury[33]. Hmox1 was induced by various stimuli that increase oxidative stress and was a homeostatic enzyme that is up-regulated in response to stress[34]. It has been shown that increased expression of Hmox1 was one of the main protective mechanisms after post-ischemic acute kidney injury (AKI) in Wistar rats [35]. The antioxidant and protective role of Hmox1 in myocardial IRI has been confirmed[36]. Our study found that Hmox1 was up-regulated in the IIRI group and that the pathophysiology of IRI includes oxidative stress, leading to varying degrees of overall organ damage. The formation of ROS has been shown to be the most frequently cited disease mechanism in IRI [37]. Therefore, it might play an important role in IRI.
Nfkbia, also known as IKB-, belonged to the family of NF-kappa-B inhibitors and prevented NF-B from becoming activated, hence preventing NF-B from performing its biological role. NF-κB was a transcription factor that played an key role in the pathophysiology of IIRI[38]. Nfkbia was important in myocardial and brain IRI [39].The over-expression of Nfkbia ameliorated H/R-induced myocardial cell injury[40]. NF-kB was highly expressed and IKB-α was lowly expressed in the retina of diabetic (DM) rats [41]. Our study indicated higher expression of Nfkbia in the IRI group, suggesting that Nfkbia plays an important role in the pathophysiology of IIRI. The NF-κB signaling pathway regulated the transcription of a large number of genes associated with cellular emergency states, particularly those involved in immune and inflammatory responses. In turn, IKK phosphorylates IkB at its amino terminus, leading to its dissociation from NFκB and subsequent proteasomal degradation via ubiquitination. NFκB was transferred to the nucleus where it binds to specific recognition sequences and regulates transcription [41]. In a study, liraglutide was found to inhibit nuclear translocation activated by NF-κB pathway, and liraglutide attenuates inflammatory response and apoptosis in intestinal tissues through NF-κB pathway [19], thus preventing IIRI. Our study also found that the screened biomarkers were mainly enriched in NF-κB-related pathway that Inhibition of NF-κB-related pathway might be a protective mechanism for IIRI. We speculated that Nfkbia may regulate the developmental process of IIRI through the NF-κB pathway.
Macrophages were considered as potential targets in inflammatory diseases, and most of the current literature classifies macrophages into two subtypes: classically activated macrophages (M1) and alternative activated macrophages (M2). Macrophages were phenotypically converted to M1 when stimulated by signals such as interferon (IFN) and Toll-like receptors, and polarized to M2 when stimulated by signals such as IL-4 and IL-13, the equilibrium of which is related to the microenvironment[42, 43]. Earlier studies reported that promoting the conversion of M1 macrophages to M2 might reduce IIRI [44]. Lactobacillus plantarum played an key role in intestinal immune function by up-regulating M1/M2 macrophage ratio [45]. In addition, Lactobacillus plantarum CLP-0611 ameliorated colitis in mice by polarizing M1-like macrophages to M2-like macrophages [46]. Th1 and Th2 were the two major sub-types of helper T cells and there was a significant antagonistic effect between them [47]. Under normal conditions, a dynamic equilibrium between Th1 /Th2 was maintained, thus maintaining the normal physiological functions of the body. Once the Th1 /Th2 cell subpopulation is imbalanced, the activation of cytokines such as IL-1β, TNF-α and IFN-γ by the Th1 cell subpopulation overexpresses and induces leukocyte infiltration and thus inflammatory responses, while the immune activation of Th2 cells leads to the secretion of IL-4, IL-10 and other anti-inflammatory cytokines of thereby inhibiting the over-activation of the inflammatory response[48]. Five biomarkers in our results were significantly and positively correlated with M2 macrophages and Th2 cells, and we suggest that M1/M2 and Th1/Th2 cell imbalance is associated with ischemia/reperfusion injury. Theoretically, if this immune imbalance can be regulated, reperfusion injury could be reduced, thus improving patient prognosis.
Dendritic cells (DCs) were efficient antigen (Ag) acquisition, processing cells that were essential for the congenital and adaptive immunity. Damage-associated molecular patterns (DAMPS) stimulated them by activating pattern recognition receptors, which in turn cause inflammatory reactions and elicit an immunological response[49]. After stimulation (inflammation), DCs transform into mature cell types characterized by high levels of MHCII and low phagocytic capacityas well as co-stimulatory molecules. Mature DCs specialize in Ag-specific T-cell activation. Some investigators had found that deletion of DC S1P3 prevents DC maturation and promotes the Th2/IL-4 response, thus acting as a preventive and therapeutic agent in renal IRI [50]. The ablation of DCs reduced inflammation associated with colitis[51]. Short-chain fatty acids (SCFA) modulate the inflammatory process, reduce the maturation of dendritic cells and inhibit the ability of these cells to induce the proliferation of CD4 [+] and CD8 [+] T cells, improving organ function and survival after IIRI[52]. Our results were similar to previously published studies in which five biomarkers were positively correlated with activated DCs and negatively correlated with immature DCs, and determining its exact mechanism requires further study.
We obtained five key biomarkers from transcriptome sequencing data, in which higher expression of Ccl7, Cd14, Cxcl1, Hmox1 and Nfkbia was found in the IRI group.We also analyzed the immune microenvironment and found that these biomarkers were mainly enriched in the ‘reactome activation of NF -kappa- B in B cells’. This laid a certain foundation for further research on the disease mechanism of IRI afterwards. However, we should acknowledged the limitations of this study. The specific regulatory mechanisms of these biomarkers and the NF-κB pathway in IIRI were not further analyzed, whereas previous studies have shown that the inflammatory response can be suppressed and apoptosis reduced through the NF-κB pathway, thereby preventing IIRI [19]. The specific regulatory mechanisms of these biomarkers and the NF-κB pathway in IIRI will be examined in future studies.