新生期地塞米松投藥處理對於海馬迴內BDNF-mTOR訊息傳遞路徑的跨代不良影響

Abstract

重度抑鬱症 (major depressive disorder, MDD) 是全球盛行的精神疾病之一。目前，市面上有許多治療抑鬱症的相關藥物，然而，現行抗憂鬱藥物(antidepressants)的療效有限且有些藥物具有強烈的副作用或成癮性。基於上述情況，憂鬱症的發病機制和相關藥物治療途徑仍然是迫切需要進一步研究的議題。地塞米松(dexamethasone, DEX)是一種人工合成的糖皮質類固醇 (synthetic glucocorticoid)，新生期地塞米松治療(neonatal dexamethasone treatment, NDT)廣泛用於早產兒 (Extremely low body weight infant)，以減輕呼吸窘迫症候群(respiratory distress syndromes) 的嚴重程度。前人研究指出，NDT可能對幼體的發育產生不良影響，並在個體的青少年期時產生類憂鬱症的行為(depression-like behavior)。哺乳動物標靶的雷帕黴素(mammalian target of rapamycin, mTOR)是一種蛋白激酶，研究表明mTOR訊息傳遞鏈與憂鬱症之致病機制有關。本實驗旨在探討BDNF-mTOR訊息傳遞路徑對憂鬱症的影響，並驗證其是否會產生跨代效應 (cross-generational effect)。本實驗使用C57BL/6JNarl品系的小鼠，初代小鼠(F1)於出生的頭三天分別以皮下注射投予遞減劑量(tapering dosage)之DEX並在小鼠週齡為十一週時進行配種。配種所產生的第二代(F2)個體會在週齡為六週時犧牲，取其腦組織進行即時定量聚合酶連鎖反應（qPCR）和西方墨點法（Western blotting）分析，觀察基因和蛋白質表達的變化。懸尾測試（tail suspension test, TST）也在第二代個體成長至六週時進行以測試二代小鼠是否表現出跨代的憂鬱行為。 結果顯示，經過NDT後，初代小鼠（F1）體重顯著下降，而F2個體的體重並無在DxD與SxS組間具有差異，另NDT組的後代 (DXD)在TST測試中表現出類憂鬱症行為。此外，在q-PCR的結果中顯示BDNF-mTOR 訊息傳遞路徑中mTOR與其傳遞路徑下游的Rps6kb1表現量下降，並且在後續的蛋白質表現量測定中，mTOR與下游的p70s6k表現量均顯著降低。最後，再投予具有mTOR專一性的活化劑MHY1485後發現第二代個體之類憂鬱行為會顯著減緩。總結上述的結果，NDT可誘發跨代之長期不良影響，而造成其跨代行為異常的機制可能源於BDNF-mTOR訊息傳遞路徑的失調。綜合上述，本實驗透過行為測試觀察NDT誘發跨代個體之類憂鬱行為，並透過分子生物學技術檢測二代憂鬱個體在BDNF-mTOR訊息傳遞路徑上基因表現之異常，結果顯示BDNF-mTOR訊息傳遞路徑在NDT跨代憂鬱之機轉扮演重要角色。Major depression disorder (MDD) was one of the most prevalent mental health conditions worldwide. However, the commonly used antidepressants had limited efficacy, strong side effects, or even caused addiction. Therefore, based on the above situation, the pathogenesis of depression and related drug treatment paths were still worthy of further exploration. Dexamethasone (DEX) was a synthetic glucocorticoid widely used in treating various diseases for its anti-inflammatory and immunosuppressive properties. Nevertheless, the previous study pointed out that neonatal dexamethasone treatment (NDT) had adverse effects on neonatal development and led to depression-like behavioral responses in mice at a later age. In this study, NDT in mice was applied as the model for studying NDT's possible cross-generational adverse effect. Behavioral tests were used to observe the depressive behavior of the F2 offspring. Biochemical experiments were used to explore whether the cross-generational depression mechanism was associated with the Brain-derived neurotrophic factor (BDNF)-mammalian target of rapamycin (mTOR) signaling pathway. The mammalian target of rapamycin (mTOR) was a protein kinase that plays a crucial role in various cellular processes and was closely connected with thepathogenesis of MDD. The study aimed to reveal the possible involvement of the BDNF-mTOR signaling pathway in the NDT-induced depression-like behavior and observe whether there would be a cross-generational effect. In the pilot experiment, mice of the C57BL/6JNarl strain were administered subcutaneous injections of tapering doses of dexamethasone on postnatal days 1-3, then mated at the eleventh week for breeding second-generation mice (F2). The F2 mice were sacrificed at six weeks old. The dorsal hippocampus was collected for qPCR and Western analysis to observe gene and protein expression changes. The tail suspension test (TST) was subjected to the F2 mice at six weeks old to test whether they exhibited transgenerational melancholic-like behavior.The experiment results showed that the somatic growth of F1 had significantly decreased after NDT; nevertheless, the F2 offspring showed no difference between the DxD and SxS groups somatic growth. The TST test result showed higher immobility duration in the F2 DxD group compared to the SxS group, which represented a higher depressive-like behavior. In the qPCR experiment, the hippocampal expression of Ntrk2, mTOR and Rps6kb1 showed a significant decrease, moreover, according to the western blot result, BDNF-mTOR signaling pathway related protein including mTOR, p-mTOR and its downstream, p70s6k, p-p70sk both show a significance decrease in the F2 DxD group compared to the SxS group. Finally, the cell-permeable mTORC1 and mTORC2 activator, MHY1485, was administrated to the F2 mouse, after administration, the behavioral and protein expression abnormality in F2 mice was rescued.In summary, we evidence that the long-term adverse effect induced by NDT would be passed down to the next generation, and the abnormality might originate from the dysregulation of the BDNF-mTOR pathway. The detailed mechanism was worth further investigation.