Collectively, lipidomics and gene expression analysis indicated that myriocin treatment induced complex responses from multiple interconnected metabolic pathways (Fig. 8). We can better understand the potential causal mechanisms of cerebral I/R injury in rats by analyzing the metabolic network.
Initially, in this study we verified that myriocin could significantly decrease cerebral infarction, neurological deficits and pathological changes in cerebral I/R rats. It is interesting to note that myriocin was found to significantly reduce SPTLC2 but not SPTLC1 after myriocin administration, indicating that SPTLC2 is more critical under the cerebral ischemia conditions.The above studies showed that myriocin could effectively ameliorate SPTLC2 activity in the cerebral ischemic cortex, providing stable samples for consequent lipidomics analysis.
Sphingolipids play a critical physiological role in maintaining the structure of cell membranes and are extensively involved in regulating various biological signal transduction processes, such as cell growth, apoptosis, and signal transduction(Grösch et al., 2018; Sun et al., 2016). Meanwhile, sphingolipids are mainly composed of ceramide (Cer), sphingomyelin (SM) and sphingosine(Hannun et al., 2001). Among these lipids, ceramide is the central component of sphingolipid metabolism, which is emerged as a second messenger involved in regulating the physiological activities and metabolism of cells (Hannun and Obeid, 2008).The accumulation of ceramide in the pathogenetic mechanism of cerebral I/R injury has been observed in previous associated studies, which is consistent with our observation(Liu et al., 2000; Takahashi et al., 2004; Yu et al., 2007). In our study, we found that several long-chain Ceramide, Cer (d18:1/16:0) and Cer (d18:1/18:0), were significantly increased in the I/R group when compared with the sham group. However, notably, we also found the very-long-chain ceramide, Cer (d18:1/24:1), was markedly decreased in the I/R group. Our data showed that myriocin could improve the disturbances of these three ceramides in I/R group. It has been reported in the literature that distinct chain lengths of ceramide that synthesized by different ceramide synthases regulated different physiological processes(Cha et al., 2016; Chan and Goldkorn, 2000).Thus, we believe that different ceramide species showed different responses to ischemic injury, but further researches are required.
Ceramide levels are regulated by complex metabolic pathways, in the research, we tried to clarify the effects of inhibition the de novo synthesis of Cer by myriocin on other metabolic mechanisms of Cer in rats with cerebral I/R injury. Ceramide is produced from sphingomyelin (SM) hydrolysis by sphingomyelinases (SMases), along with this, ceramide is converted to sphingomyelin by sphingomyelin
synthase(SGMS)(Tian et al., 2009). Moreover, in brain tissue, ceramides are hydrolyzed into sphingosine under the action of ceramidase encoded by four different genes, which are acid ceramidase(ASAH1)、neutral ceramide (ASAH2), alkaline ceramidase2 (ACER2), and alkaline ceramidase3 (ACER3). In our results, a decreased trend of SM(d18:0/18:0) and SM(d18:0/20:0) was observed after cerebral I/R injury in rats. Additionally, there was a concomitant rise in C18 Sphinganine levels in the I/R group, which is consistent with previous literature reports (Sun et al., 2010). In the present study, we detected that key sphingomyelin cycle-related enzyme (ASMase, NSMase) mRNA expression levels tended to increase and SGMS1 mRNA expression levels tended to decrease after cerebral I/R injury in rats, which explains the reduction in ceramide/sphingomyelin ratio. Similarly, in view of the increased ceramide conversion-related enzyme (ASAH1, ACER2, ACER3) mRNA expressions after cerebral I/R injury in rats, it may account for the accumulation of C18 sphingosine.Remarkably, myriocin could ameliorate the dysregulation of sphingolipid metabolism at the level of the above metabolic enzyme genes, as compared to untreated animals.
Meanwhile, another finding in this study is that an increasing level of DAG (16:0/18:1) was observed in I/R group. It has been shown that SMS enzymes convert Cer and phosphatidylcholine (PC) into SM and diacylglycerol (DAG), respectively. According to our result, a higher level of DAG (16:0/18:1) might be associated with increased mRNA expression of SMS2, which needs to be further investigated.
Glycerophospholipids are the principal lipid components of biological membranes, which are involved in diverse signal transduction processes such as apoptosis and membrane fusion(Farooqui et al., 2000). In animal tissues, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are the most abundant and important intermediates in glycerolphospholipid metabolism(Vance, 2015). In our study, PC (16:0/18:1), PC (18:1/18:1), PC (18:0/20:4), PE(P-18:0/20:1), and PE (16:0/22:6) was significantly decreased, whereas LPC (20:4) and LPE (18:0) was significantly increased in the I/R group. After myriocin treatment, the levels of these metabolites showed a trend toward the normal status. Liu et al(Liu et al., 2017)identified PC(5:0/5:0) and LysoPE(18:2) showed great potential to serve as biomarkers of distinguishing acute ischemic stroke patients from healthy individuals. Rabiei and colleagues (Rabiei et al., 2013)found that the PC content was inversely associated with infarct volumes in a rat model of focal cerebral ischemia injury, which strongly supported the role of PC in ischemic injury. Phospholipids and lysophospholipids can convert to each other through the “Lands cycle” to maintain lipid homeostasis(Wu et al., 2016). When activation of phospholipase A2 (PLA2), PC and PE in organisms can be catalyzed hydrolysis to produce the corresponding single-stranded lysophospholipids LPC and LPE, respectively. Previous reports has been demonstrated that activation of PLA2 plays a pivotal role in acute cerebral ischemia linked to neuronal cell death, which is in line with the results of our study (Sun et al., 2009). Notably, we showed that myriocin could ameliorate the dysregulation of glycerophospholipid metabolism as a result of PLA2, which are related with the balance of land cycle in the cerebral I/R model rats .
Importantly, apoptosis is the key factors of neuronal cell death in cerebral I/R injury (Radak et al., 2017). It is recognized that sphingolipids like ceramides are known to be involved in apoptosis (Hannun, 1996). Based on our present results, we speculated that ceramide causes apoptosis-induced cell death in cerebral I/R injury. We observed the morphological changes of neuronal cells in the cortical brain regions through TUNEL staining, validating our hypothesis. Then, we further explored the underlying mechanisms of myriocin on inhibiting cortical neuronal apoptosis. Our results clearly confirmed that myriocin downregulated the protein levels of Bax, caspase-3 and cleaved caspase-3 while upregulating Bcl-2, thus promoting the survival of neural cells after cerebral ischemia.
However, it is a limitation that this study determined only the mRNA expressions of enzymes. Thus, the enzyme protein levels will be further measured.