探討第一介白素訊號與NMDA受體於PTZ誘發癲癇與睡眠障礙中所扮演的角色

Abstract

癲癇為一常見的神經疾病之一，此疾病常伴隨著睡眠障礙並影響所有年齡層，然而，睡眠及癲癇之交互作用，如今仍然尚未釐清。第一介白素 (interleukin-1 beta; IL-1β) 為一睡眠調節物質，並參與許多病理機制，包括癲癇及帕金森氏症。在先前的研究，指出於不同之光照時間 (zeitgeber times; ZTs) 下，癲癇發作對於睡眠的改變也有所不同，且發現第一介白素於ZT13癲癇導致的睡眠變化中具有一定重要性。NMDA受體 (N-methyl-D-aspartate receptors; NMDARs) 已被證實在癲癇形成之過程中，扮演著重要的角色。因此，本研究探討第一介白素與癲癇誘發睡眠障礙之相關性。 本研究分別於野生型 (Wildtype) 小鼠組別及第一介白素第一型受體基因剔除 (IL-1 receptor type I knockout; IL-1RI KO) 小鼠組別採腹腔注射環戊四氮(pentylenetetrazol; PTZ) 以誘發自發性全身性癲癇發作，並分析兩組別之睡眠醒覺之變化與其癲癇發作之閾值。在腹腔注射PTZ後，wildtype小鼠組別產生之自發性癲癇高於IL-1RI KO小鼠組別；在睡眠層次上之變化，wildtype小鼠組別減少了非快速動眼期睡眠 (non-rapid eye movement; NREM) 睡眠，IL-1RI KO小鼠組別則沒有顯著改變，此結果顯示第一介白素訊號於癲癇及睡眠失調上扮演著一定的角色。另一方面，於海馬迴及下視丘中，經 PTZ注射後，發現IL-1RI KO小鼠中NR1亞單位之表現和NR2B亞單位Tyr1472位點之磷酸化，皆顯著低於wildtype小鼠。相較之下，於前腦中經PTZ注射之wildtype小鼠中NR1亞單位表現與NR2B亞單位Tyr1472位點之磷酸化皆顯著高於IL-1RI KO小鼠。初步研究結果顯示，癲癇的發生受到NMDA受體之向上調節，且第一介白素之訊號參與其中。NR1蛋白表現與 NR2B 蛋白之磷酸化的增加可能介由 NF-κB 或 Src之路徑傳導。 經由給予Src 抑制劑注射於腦室中，wildtype小鼠組別相比未注射之wildtype小鼠組別，於 ZT1-ZT6 顯著增加了NREM 及REM睡眠，而 NR2B亞單位Tyr1472位點之磷酸化於wildtype小鼠之海馬迴及下視丘中，在 ZT24 顯著減少表現量；而給予 Src 活化劑注射於腦室中之IL-1RI KO小鼠組別相比未注射之IL-1RI KO小鼠組別，於 ZT1-ZT6 顯著減少NREM 睡眠，而 NR2B亞單位Tyr1472位點之磷酸化於IL-1RI KO小鼠之海馬迴及下視丘中，顯著增加了表現量；進一步相比注射Src 抑制劑之 wildtype小鼠組別與注射Src 活化劑之IL-1RI KO小鼠組別 wildtype小鼠組別之癲癇發作比例則顯著降低。 總結以上，此研究結果顯示，於PTZ誘發之癲癇發生及癲癇誘導之睡眠障礙中，第一介白素訊號的增加有助於NMDA受體之活化，並可能經由 Src 酪氨酸激酶之路徑直接磷酸化 NR2B (Tyr1472) 引起癲癇發生而導致睡眠障礙。

Epilepsy is one of the common neurological disorders that affect people of all ages and is often associated with sleep disorders, but the interaction between sleep and epilepsy is still not clear. Interleukin-1 beta (IL-1β) is a sleep regulatory substance (SRS) and participates in many pathological disorders, such as epilepsy and Parkinson’s disease. Previous studies have demonstrated that seizure occurred at different zeitgeber times (ZTs) alter sleep differently and IL-1 mediates the sleep alteration induced by the ZT13 epilepsy. There is evidence that N-methyl-D-aspartate receptors (NMDARs) play a key role in epileptogenesis. Therefore, we study the relationship between IL-1 and NMDA receptors in epileptogenesis and epilepsy-induced sleep disruption. In this study, the spontaneously generalized seizures were induced by intraperitoneal injection of pentylenetetrazol (PTZ), the sleep-wake activity was analyzed, and the seizure threshold was determined in both the wildtype and IL-1R1 knockout (KO) mice. We found that the occurrence of spontaneous seizure was higher in the wildtype treated with PTZ than that in the IL-1R1 KO mice treated with PTZ. Furthermore, the non-rapid eye movement (NREM) sleep decreased in wildtype mice treated with PTZ, but it was not altered in IL-1R1 KO mice. These results indicate the role of IL-1 signal in both epileptogenesis and sleep disturbance. The expression of NR1 subunit protein and the tyrosine phosphorylation of NR2B (at Tyr1472) in the hippocampus and the hypothalamus were significantly lower in the IL-1R1 KO mice when comparing to those in the wildtype mice. In contrast, the expression of NR1 and the phosphorylated-NR2B in the forebrain were significantly higher in the IL-1R1 KO mice treated with PTZ when comparing to those wildtype mice. These findings suggested that the epileptogenesis is attributed to the up-regulation of NMDA receptors, which is mediated by the IL-1 signal. The increase of NR1 expression and phosphorylated-NR2B maybe mediated by the NF-κB or Src signal cascades. We further investigated whether if the Src signal has been involved in IL-1 signal and epilepsy-induced sleep disruption. Our preliminary data showed that the percentage of seizure was significantly lower in the wildtype mice treated with PTZ and src inhibitor than IL-1R1 KO mice treated with PTZ and src activator. In addition, PTZ-induced seizure of IL-1R1 KO mice was treated with src family activator has increased the NREM sleep and the tyrosine phosphorylation of NR2B protein expression in the hippocampus and hypothalamus of the light period at ZT24. On the contrary, PTZ-induced seizure of wildtype mice was treated with src inhibitor has significantly decreased both in NREM and REM sleep; the tyrosine phosphorylation of NR2B protein expression was also significantly decreased at ZT24. In conclusion, our results indicated that the increase of NMDA receptor activity by the IL-1 signal contributes to the PTZ-induced epileptogenesis and the epilepsy-induced sleep disruption.