In this study, we found that the expression of sEH protein and the enzyme activity of sEH were both increased in the hippocampus of LiCl-pilocarpine-induced post-SE rat model compared with Control group. After administering TPPU, the enzyme activity of sEH was decreased and the PUFAs metabolic substrates EpFAs of sEH and ratios of substrates to products were increased in the hippocampus of LiCl-pilocarpine-induced post-SE rat model. This finding indicated that TPPU might take anti-seizure effect through the cellular mechanism of PUFAs metabolic substrates of sEH including EETs. The increased expression of ERK1/2 in the hippocampus indicated it might take part in the cellular mechanism of EETs in the LiCl-pilocarpine-induced post-SE rat model.
In our previous study, we have found that administering TPPU in the LiCl-pilocarpine-induced post-SE rat model reduced the frequency of SRS and alleviated epilepsy-associated depressive behaviors of rats. Another study by Hung et al. also verified that TPPU, the sEH inhibitor, had anti-seizure effect in two mouse models of temporal lobe epilepsy induced by either pilocarpine or electrical amygdala kindling. In addition, Vito et al. demonstrated that the sEH inhibitor had anti-inflammatory effect and prevented tetramethylenedisulfotetramine (a potent convulsant poison) induced mortality in mice by combined treatment with diazepam. The behavioral observation in this study found the frequency of SRS was decreased after administering TPPU, which was consistent with the previous studies.
The sEH is one of the key enzymes for hydrolysing EETs etc. which are metabolisms from PUFAs that are highly stored in membrane phospholipids. The study shows that sEH is extensively expressed in the central nervous system. The expression of sEH protein has been found to be increased in neurological and psychiatric diseases including epilepsy, Parkinson’s disease, Alzheimer’s disease, depression, bipolar disorder, schizophrenia, and autism-like spectrum disease[15, 24–26, 9]. However, the enzyme activity of sEH sometimes showed controversial results. In our previous study, we found the expression of sEH protein was significantly increased in the prefrontal cortex and hippocampus of the LiCl-pilocarpine-induced post-SE rat model. In this study, we demonstrated that the expression of sEH and the enzyme activity of sEH were both significantly increased in the hippocampus of LiCl-pilocarpine-induced post-SE rat model. After administering TPPU, the enzyme activity of sEH significantly decreased while the no significant change was observed for the expression of sEH protein. This result indicated that the action of TPPU mainly contributed to decrease the enzyme activity of sEH but not the expression level of sEH protein. The expression level of sEH protein might be affected by many other pathological factors.
There are several sEH inhibitors developed in recent years, including 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA), 1-adamantan-1-yl-3-(5-(2-(2-ethoxyethoxy) ethoxy) pentyl) urea (AEPU), trans-4-(4-(3-adamantan-1-yl-ureido)-cyclohexyloxy)-benzoic acid (t-AUCB), and TPPU etc.. The ideal sEH inhibitors should have maximal bioavailability and longer half-lives, higher maximum drug concentrations in the blood (Cmax), and larger area under the curve (AUC). Except for that, for drugs targeting the central nervous system, it’s important for them to take effects by successfully passing the blood-brain-barrier (BBB). Studies have demonstrated that TPPU is a potent sEH inhibitor with sufficient solubility in water, vast systemic distribution to tissues, and crosses BBB effectively either after intraperitoneal injection or oral administration[28, 27]. In this study, TPPU was given to the rats via intragastric administration. We measured the concentration of TPPU in the hippocampus by the LC/MS method and showed the concentration was elevated significantly compared with the blank controls, indicating the excellent gastric absorption and BBB permeability of TPPU either.
The anti-seizure effect of TPPU might be contributed by the elevation of EETs and other EpFAs. As demonstrated in this study, we found the sum EETs, the ratio of EETs/DHETs, 14(15) EET, 11(12) EET, and 8(9) EET were all increased significantly in the hippocampus of LiCl-pilocarpine-induced post-SE rat model after TPPU administration. However, there was no significant difference in 5(6) EET between SRS + 0.1TPPU and SRS + PEG400 group. Moreover, other EpFAs such as EpOMEs, 16(17) EpDPA, and 19(20) EpDPA derived from LA and DHA were also elevated significantly in the hippocampus of LiCl-pilocarpine-induced post-SE rat model after TPPU administration. A study showed that combined injection of the sEH inhibitor with EETs but not with epoxy-DHA or epoxy-EPA into the brains of mice delayed the onset of pentylenetetrazol-induced seizures, which might support the major role of EETs in the anti-seizure effect. In the physiological state, the endogenous EETs have important roles in cellular actions, regulation of cerebral blood flow, neurohormone release, and synaptic transmission in the brain. How the level of EETs change in the pathophysiological state of neurological and psychiatric diseases have not been fully elucidated. In this study, we didn’t find any difference of EETs and other EpFAs levels in the hippocampus of LiCl-pilocarpine-induced post-SE rat model compared with Control group. The membrane phospholipids might be damaged under pathological states, PUFAs released from the membrane lipids and were metabolized through COX, LOX, CYP hydroxylases, and CYP epoxygenases pathways, which implied that the levels of EETs and other EpFAs might be influenced by multiple factors but not solely the activity of sEH.
The functions of EETs are complex and have not been very clear yet. Studies indicate that EETs activate K+ channels and have anti-inflammatory effects[31, 32]. In addition, EETs may also have membrane receptor mechanism, which is initiated by EET binding to a plasma membrane EET receptor, and then the signal transduction pathways such as Mitogen-activated protein kinase (MAPK) will be activated[33, 34]. Moreover, EETs block the pathological endoplasmic reticulum (ER) stress response and attenuate oxidative stress. ERK1/2 has been demonstrated to be one of responsive down-stream molecules to ER stress and involved in the defensive effects against ER stress[36, 37]. In this study, the expression level of ERK1/2 was significantly increased after TPPU administration, indicating the cellular mechanism of EETs through ERK1/2 pathway might be responsible for the anti-seizure effect of TPPU.