Maintaining homeostasis is a key function of the brain, and homeostasis includes blood oxygen levels(van der Velpen et al. 2017). Hypoxia-inducible factor 1α (hif-1α) and vascular endothelial growth factor (vegf) are activated by hypoxia, which then leads to a disruption of the total blood-brain barrier (BBB), resulting in brain edema (Lafuente et al. 2016; Mohaddes et al. 2017). In mammals, hypoxia is a trigger stimulus for vascular remodeling, altered cell permeability, and angiogenesis (Zimna and Kurpisz 2015). Vegf has been proven to be an important determinant of angiogenesis and plays an important role in hypoxic-ischemic brain injury, functioning to promote angiogenesis and neuroprotection (Cao et al. 2018). Vegf could stimulate axonal growth and improve neuronal cell survival. Under pathological conditions, vegf has a protective effect on the central nervous system (Plaschke et al. 2008). Vascular endothelial growth factor-α (vegfa) is a pro-angiogenic member of the vascular endothelial growth factor (vegf) family (Ferrara et al. 2003). Here, we showed that vegfa was up-regulated with decreasing DO concentration. It participates in the AGE-RAGE signaling pathway. In addition, egr1, a gene of the EGR family involved in the AGE-RAGE signaling pathway, showed a decreasing trend. Early growth response 1 (egr1) is considered to be a transcription factor sensitive to ischemia and hypoxia. In the brain tissue after ischemic stroke, the mRNA level of egr1 was significantly increased, while egr1 was overexpressed induced post-stroke inflammatory response and led to secondary brain damage after cerebral infarction (Tureyen et al. 2008). The main mechanism of the AGE-RAGE signaling pathway is that the interaction between AGE and RAGE causes an oxidative stress response, which in turn promotes the increase in diacylglycerol (DAG) synthesis and activates PKC. Activation of PKC increases the expression of vascular endothelial growth factor (vegf), transforming growth factor-β (tgf-β), endothelin-1, and prostaglandin, which proliferates the extracellular matrix, leading to vasomotor dysfunction and capillaries changes in permeability (Ishii et al. 1998; Toth et al. 2008). In our study, under hypoxic stress, vegfa expression was up-regulated and egr1 was down-regulated. It is shown that the brain of T. rubripes might create hypoxia tolerance to resist brain damage caused by hypoxia stress.
HIF-1 signaling pathway is a critical pathway during hypoxia. vegfa not only mediates the AGE-RAGE signaling pathway but also mediates the HIF-1 signaling pathway. Hypoxia-inducible factor 1 (hif1) is a transcription factor expressed in all metazoans and consists of hif-1alpha and hif-1beta subunits. Under hypoxic conditions, hif1 regulates the transcription of hundreds of genes in a cell-type-specific manner and was a major regulator in the HIF-1 signaling pathway (Semenza 2007) . In our study, genes enriched for the HIF-1 signaling pathway: egln2, flt1, sl2a1, vegfa and hk2 were all up-regulated. However, hif1 (and hif1an) did not show a significant change during hypoxia in our results. In Eurasian perch, hif1a mRNA levels were upregulated after acute severe hypoxia exposure in the brain and liver, but not in muscle tissue, whereas significant changes were detected in muscle, but not in the brain and liver after chronic moderate hypoxia exposure (Rimoldi et al. 2012). Ndubuizu et al. demonstrated that despite the lack of hif1 activation, the relative mRNA levels of vegf in the aged cortex significantly increased (Ndubuizu et al. 2010). Fong et al. demonstrated that the knockout of hif1α in colon cancer cells had no effect on angiogenesis (Fong 2008). Liu et al. demonstrated that the large yellow croaker (Larimichthys crocea) has low hypoxia tolerance compared with other fish species, and the mRNA levels of hif1α in its brain did not change markedly under hypoxic conditions (Liu et al. 2018), which was consistent with our results. Hif1α is specific for the hypoxia response, and its degradation mediated by three enzymes egln1, egln2, and egln3 (Zhang et al. 2019). Tcf7l2 positively regulated aerobic glycolysis by suppressing Egl-9 family hypoxia inducible factor 2 (egln2), leading to the upregulation of hif1α (Xiang et al. 2018). Egln2 can hydroxylate foxo3a on two specific prolyl residues in vitro and in vivo. Hydroxylation of these sites prevents the binding of USP9x deubiquitinase, thereby promoting the proteasomal degradation of foxo3a (Zheng et al. 2014). Flt-1 (vegfr-1) and kdr (vegfr-2) were two highly homologous tyrosine kinase receptors of vegf, which can synergize with vegf to promote angiogenesis (Gille et al. 2000). Solute Carrier Family 2 Member 1 (slc2a1) was the most important energy carrier of the brain: present at the blood-brain barrier and assures the energy-independent, facilitative transport of glucose into the brain(Klepper et al. 1999).The hyperpolarization has been proposed to occur via stimulation of Na+/K+ ATPase pumps caused by a glucokinase (gck) induced rise of ATP levels within neurons, leading to inhibition of neuronal activity(De Backer et al. 2016). In summary, hif1-mediated gene expression may be related to hypoxia-induced tolerance (Jones and Bergeron 2001), or it may be related to the different stresses of different tissues on hypoxia stimulation. Hif1 was inhibited in the T. rubripes brain to prevent brain damage and activate related genes of angiogenesis, promotes blood vessel growth, maintains normal blood vessel density, and was protected from damage caused by ischemia and hypoxia. At the same time, hypoxic stress also promoted the brain to reduce energy consumption.
In our research, we observed that when T. rubripes faced with hypoxia, they stopped swimming, laid on the bottom of the tank, and maintained their balance, with only slight swings in the fins. We speculated that the T. rubripes had a certain ability to tolerate hypoxia, and it can be made tolerant to hypoxia by changing its activity mode. The central nervous system of vertebrates controls the body's cognitive functions and autonomous motor activities(Paridaen and Huttner 2014). Axon guidance represents a key stage in the formation of neuronal networks (Negishi et al. 2005). Axons were guided by a variety of guidance factors and these guidance cues are read by growth cone receptors, and signal transduction pathways downstream of these receptors converge onto the Rho GTPases to elicit changes in the cytoskeletal organization that determine which way the growth cone will turn (Govek et al. 2005). Recent work in the D. rerio has shown that developmental hypoxic injury disrupts pathfinding of forebrain neurons in D. rerio, leading to errors in which commissural axons fail to cross the midline. EphrinB2a acts as a ligand for one of the receptor tyrosine kinases (RTK) of the epha3, epha4, or ephb4 families, which in turn sets off an intracellular signaling cascade in the RTK-expressing cell (Stevenson et al. 2012). Hypoxia stimulated the uptake of 5-bromo-2'-deoxyuridine (BrdU) and reduced cell death Coincident with these proliferative changes, both hif1-α and phospho(p)-AKT were increased while ephb3 expression was decreased (Baumann et al. 2013). These reports are consistent with our results. Our results showed that the expression of ephb3, ntng1 and rnd1 was down-regulated with decreasing DO concentration, while epha4, sema5b and nck2 appeared to be up-regulated. Among the down-regulated genes, rnd1 was a small signal transduction G protein and a member of the rnd subgroup of the Rho family of GTPases(Ridley 2006). It contributes to the regulation of the actin cytoskeleton in response to extracellular growth factors(Nobes et al. 1998). Ntng1 serves as an axonal guidance cue during vertebrate nervous system development(Nakashiba et al. 2000). Among the up-regulated genes, sema5b regulates the development and maintenance of synapse size and number in hippocampal neurons (O'Connor et al. 2009). Nck2 adaptor proteins were involved in signaling pathways mediating proliferation, cytoskeleton organization, and integrated stress response (Labelle-Cote et al. 2011). From this, we can speculate that the brain of T. rubripes may be affected by acute hypoxic stress. However, the brain responded promptly by inhibiting the expression of hif1, reducing the damage caused by hypoxic stress and repairing the damaged nerves in time. This may also be the reason why the brain of T. rubripes is able to tolerate hypoxia.
Hypoxia induces bhlhe40 expression independent of hif1α but through a novel p53-dependent signaling pathway, and inhibition of bhlhe40 or p53 may facilitate muscle regeneration after ischemic injuries (Wang et al. 2015). Studies have shown that prenatal hypoxia in mice can cause continuous changes in circadian rhythms in born mice (Joseph et al. 2002). Hypoxia significantly reduced clock, cry2, and per3 in GF and cry1, cry2, and per3 in PDLF (Janjic et al. 2017). Hif-2α increased the expression levels of clock, bmal1, per1, cry1, cry2, and ckiɛ, and decreased the expression levels of per2 and per3 (Yu et al. 2015). The negative circadian regulator cry1 was a negative regulator of hif1a (Dimova et al. 2019). Per3 was one of the primary components of circadian clock system. It was found to play a pivotal role in corticogenesis via regulation of excitatory neuron migration and synaptic network formation (Noda et al. 2019). In our study, the expression levels of both genes cry1 and bhlhe40 showed a decreasing trend due to acute hypoxic stress. Hypoxia has caused brain damage in T. rubripes to some extent, thus causing circadian rhythm disturbance in T. rubripes. In conclusion, the brain of T. rubripes has a certain tolerance to hypoxia, but hypoxia also causes damage to it. Under hypoxic stress, the brain of T. rubripes struggles to maintain its central system from damage. Choose to maintain only basic life activities to resist the effects of hypoxic stress, such as stopping swimming to reduce oxygen consumption.
In addition, through the KEGG network interaction map we found that metabolism-related pathways and genes could be well clustered together, including fatty acid metabolism, phosphate metabolism, nitrogen metabolism, bile secretion, and steroid hormone synthesis. Acl3 encodes a protein that is an isozyme of the long-chain fatty acid coenzyme A ligase family and plays a key role in lipid biosynthesis and fatty acid degradation. And this isozyme is highly expressed in the brain. Furthermore, acadl, the gene encoding LCAD (acyl coenzyme A dehydrogenase), has been shown to consume less energy in LCAD-deficient mice and also suffers from hypothermia, which can be explained by the fact that the reduced rate of fatty acid oxidation correlates with a reduced ability to generate heat(Diekman et al. 2014). From this, we can speculate that regulation of energy metabolism may be another effective way for T. rubripes to cope with hypoxic stress.