Discussion
To probe the mechanism of ischemia-reperfusion injury, we used the Metacore™ software to analyze the overlapping and non-overlapping pathways as well as cellular and molecular network processes, and reconstructed networks at 3 h, 12 h, and 24 h post injury. The number of activated pathways and cellular and molecular processes at different time points was found to be quite different.
Several common and unique pathways were observed in the 3 h, 12 h, and 24 h groups. A literature review about ischemia-reperfusion injury revealed a close relationship with 21 pathways across time points post injury [28-42], but G-protein signaling_TC21 regulation pathway that was activated at 24 h has not been previously reported to have a direct relationship with ischemia-reperfusion injury. Studies on the TC21 regulation pathway mostly focused on its role in cancer, which mediates its effects via the PI3K-Akt pathway, NF-κB, and cyclin D1 that are all related with cerebral ischemia reperfusion injury [43]. This may be the reason why it has a higher degree in cerebral I-R, and more attention needs to be focused on the role of the TC21 regulation pathway in cerebral I-R.
Changes in six pathways over time were not consistent with those reported in previous studies, including those involved in cytoplasmic/mitochondrial transport of proapoptotic proteins Bid, Bmf, and Bim, FAS signaling cascades, activation of Erk by ACM1, ACM3, and ACM5, VEGF signaling via VEGFR2-generic cascades, MDA-dependent postsynaptic long-term potentiation in CA1 hippocampal neurons, and G-protein mediated regulation of MARK-ERK signaling. In previous studies, these pathways were reported to be expressed at all observed time points [44-48]. The disparity between our findings and previously published data may be resulted from the use of different models, different observed time points, different genes, or different tissues.
According to enrichment analysis of pathways at different time points, signal transduction, immune response, and apoptosis were identified as the main molecular processes in the 3 h, 12 h, and 24 h groups. However, several differences were observed amongst these three time points. At 3 h, extracellular signals binded to cognate receptors, initiating the immune response. At 12 h, apoptosis was triggered by the differential expression of several pathways, as a result of activation by enzymes and signal transduction. At 24 h, more pathways were activated, especially the G-protein coupled receptor protein signaling pathway (Figure 6).
Figure 6  Visualized dynamic change in the course of pathways at 3 h, 12 h, and 24 h. Squares filled with color represent the receptor, while ovals represent the pathway. The content not within squares or ovals represents a pathway that is related to a pathway activated in our study. Squares not filled with any color represent a biological function. The red-, blue-, and green-colored squares or ovals represent 3 h, 12 h, and 24 h after cerebral ischemia-reperfusion injury, respectively.
Process network analysis revealed that apoptotic and anti-apoptotic pathways existed in both 3h and 12h groups, consistent with the findings from a previous study [49]. Our study also found that apoptosis existed in the top 10 processes at 24 h, whereas anti-apoptotic process was absent. Both apoptotic and anti-apoptotic processes might determine the prognosis of injured neurons. Perhaps at 24 h after injury, the anti-apoptotic process was weakened while apoptosis played a leading role, and therefore cell death reached a peak at 24 h post injury. These results demonstrate that early treatment that may activate the process of anti-apoptosis within 12 h has the potential to decrease cell death. This observation is also supported by other study findings. Bcl-xL, belonging to Bcl-2 family, can inhibit cell death [49]. It was differentially expressed at 3 h and 12 h, but not at 24 h, as revealed by analyzing the process of apoptosis stimulation by external signals, which was a pathway common to all three time points. This role of inhibition of anti-apoptotic processes remains to be elucidated.
Based on published literature [50-52], a minority of nodes in a large variety of real world networks is a hub, i.e. a node having a much higher number of neighbors. Hub nodes are important components for shared networks, providing more information than non-hub nodes. The degree is a factor to evaluate the hub node, and a higher degree represents a more important node [53,54]. By analyzing the node degree in the network at different time points, Pyk2 (FAK2) and PKC were identified to be the most important nodes in the 3h, 12h and 24h groups. Pyk2 (FAK2) and PKC, the important proteins in PLC/PKC/Pyk2/Src signaling pathway, can enhance NMDA receptor function in hippocampal neurons [55], and NMDA receptor may lead to Ca2+ internal flow, resulting in cerebral ischemic reperfusion [56]. However, other nodes including E2A/HLF fusion protein, TFIID, and GRB2 have never been implicated in the pathogenesis of I-R previously and merit further investigation to understand their functions. Prior studies on E2A/HLF fusion protein mainly focused on its relationship to leukemia; in the context of leukemia, it may induce T-cell apoptosis, and is considered as an very important protein [57]. Besides, TFIID plays a critical role in RNA polymerase II (Pol II) pre-initiation complex (PIC) formation, and therefore, it may affect the process of transcription during cerebral I-R [58]. The relationship between GRB2 and cerebral I-R may be owed to GRB-2-associated binder 1 (Gab1), which is essential in preventing against I-R oxidative injury via mediating survival signaling [59].
Overall, most of earlier studies limited their analysis to a detailed investigation of just a few pathways; while our study provides a comprehensive report of the time course of a differential gene expression profile at 3 h, 12 h, and 24 h post cerebral ischemia injury. Being a method of systematic analysis, it allows for observing changes across 22 different pathways at each time point. Such a method can aid in identifying new important pathways, genes, proteins, or cellular processes by tracking dynamic changes over the course of pathogenesis. One caveat of this method is its limitation in further in-depth study of a specific pathway. Another limitation is that we only observe the changes in gene expression, which may miss post-translational mechanisms. Based on the findings of this study, we propose an in-depth experimental analysis of a few candidate pathways.