Stroke is a global health problem. The limitation of the thrombolytic time window makes the development of new neuroprotective agents against the neuronal cell death and neurological deficits caused by ischemic stroke necessary. In the first experiment, we established a IP model to further investigate the regulation mechanism of endogenous TK and found that IP can significantly upregulate the expression of endogenous TK and confer neuroprotection. We then studied the functional mechanism of endogenous TK in the next experiment.
Hypoxia and hypoxia-ischemia have extensive and far-reaching effects on gene expression and protein synthesis. Epigenetic alternation plays an important role in the changes of gene expression induced by ischemia and hypoxia . At present, the research on epigenetic regulation mainly focuses on DNA CG island methylation modification, histone methylation and acetylation modification. Studies have reported that the methylation of DNA CG islands and the histone H3K9 locus can modify the expression of silent genes, while the acetylation of histone and the methylation of histone H3K4 sites activate gene expression [24–26]. Our previous studies have also shown that hypoxia can regulate gene expression by regulating histone methylation and acetylation binding to the neprilysin (NEP) promoter region [27, 28]. In this experiment, we discovered that IP can upregulate the expression of endogenous TK by affecting histone H3 acetylation modification. In addition, we found the first association between histone acetylation modification and the expression of endogenous TK. In recent years, many studies have found that histone deacetylase inhibitors injected at different time points can play a neuroprotective role against cerebral I/R injury in rats [29, 30]. Combined with our research, it is possible that there are similarities between IP and histone deacetylase inhibitors to some extent.
The neuroprotective effect of IP is not a new proposition. Many studies have attempted to achieve a neuroprotective effect by inducing transient ischemia. In addition to a cerebral ischemia tolerance model, remote ischemic preconditioning (RIPC) can also provide neuroprotection. On the one hand, it can reduce brain oedema and the volume of cerebral infarction; on the other hand, it can inhibit neuronal apoptosis in the ischemic penumbra. Recently, our study  demonstrated that remote ischemic post-conditioning (RIPostC) can promote motor function recovery and reduce brain injury in rats with acute cerebral ischemia by upregulating endogenous TK in a cerebral I/R model. Combined with the results of this experiment, maybe it is possible to find that remote ischemic preconditioning can upregulate the expression of TK, thus protecting the brain against injury in rats. In the future, it may also be possible to conduct a series of clinical trials of ischemic preconditioning in a non-invasive way in a high-risk population of stroke patients, so as to explore whether ischemic preconditioning can reduce the incidence or show beneficial effects in patients with stroke. However, research in this area is highly difficult. Because animal models often cannot represent the real clinical situation after stroke due to a lack of comorbidities, the study design is highly important.
Currently, exogenous TK in the acute phase administered by intravenous injection has achieved more positive clinical outcomes. Recent studies have shown that exogenous TK could prevent I/R-induced neuronal injury via the B2R-ERK1/2 pathway [10, 32]. Our latest research results also show that exogenous TK can activate ERK1/2 and its downstream mitochondrial pathway through activation of the β-arrestin-2 assembled B2R-Raf-MEK1/2 signalling module . To explore the signalling pathway of endogenous TK, we investigated the related activation of signal factors after IP. We found that the phosphorylation of Raf, MEK1/2 and ERK1/2 was upregulated after IP and that IP further upregulated the expression of p-Bad, depressed the release of cytochrome c and Bax from mitochondria to the cytosol and inhibited caspase-3 activation. Combined with the increase of endogenous TK after IP, we have reason to believe that endogenous TK can participate in the activation of Raf, MEK1/2, ERK1/2, affect the mitochondrial pathway, inhibit apoptosis and play a neuroprotective role.
It is a pity that our research on the origin of endogenous TK is not sufficiently thorough. As we can see, cresol violet staining cannot stain neurons exclusively, but also stains glial cells. The acetylation of histone H3 is also closely related to oligodendrocytes. Accordingly, in our future work we will further explore the source of endogenous TK from the cellular level.