TW201838635A - Ａβ誘發型傷害的抑制或緩解劑 - Google Patents
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- G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
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Abstract
本發明係有關針對海馬迴中之β類澱粉蛋白(Aβ)誘發型傷害之抑制或緩解劑，其包含來自經牛痘病毒接種之發炎組織的萃取物。本發明亦係有關來自經牛痘病毒接種之發炎組織的萃取物於製備用於抑制或緩解海馬迴中之Aβ誘發型傷害之用途。
Description
本發明係有關針對海馬迴中之β類澱粉蛋白(Aβ)誘發型傷害之抑制或緩解劑，其包含來自經牛痘病毒接種之發炎組織的萃取物(以下可以稱為“萃取物”)。
根據2016年世界阿茲海默症報告，由於預期壽命延長，全球人口正在迅速高齡化。據報導，全世界有4700萬人患有失智症，到2050年這一數字預計將增加到超過1.31億。在中國，這一數字預計將在2030年增加到超過1600萬。顯然，失智症的盛行率對人們生活質量和經濟產生巨大影響。
阿茲海默症(Alzheimer’s disease；AD)是老年人中最常見的漸進性失智症。到目前為止，由於AD複雜的機制，沒有有效的抗失智症藥物可用於AD的管理。更重要的是，已知對於新的抗失智症藥物的發現、發展以及臨床試驗需要巨大的研究成本和努力。例如，最近在花費數百萬美元進行末期試驗之後，尋找阿茲海默症藥物索拉尼珠單株抗體(solanezumab)已終告失敗。因此，有必要 重新評估目前有前景的藥物，以阻止漸進性認知功能障礙，並建立AD的藥物安全性(safety profile)。神經妥樂平(Neurotropin)(商標；Nippon Zoki Pharmaceutical Co.,Ltd.之產品)(後文中稱為“NTP”)係眾所周知用於治療慢性疼痛(如下背痛、帶狀皰疹後神經痛、頸肩臂症候群、亞急性脊髓視神經病變(SMON)之過敏症以及纖維肌痛症)的含有萃取物作為活性成分的鎮痛藥。最近，據報導，NTP(大多數實驗是使用較市售產品“神經妥樂平”含有更高濃度的萃取物的實驗產品進行。然而，在這種情況下，為了方便起見，本發明也使用了“萃取物”一詞)刺激SH-SY5Y細胞中的BDNF表現，並且NTP之重複的口服投藥(200NU/kg/天，持續3個月)抑制海馬迴BDNF表現的下降，同時改善作為唐氏症模式之Ts65Dn小鼠的空間認知(參見非專利文件1)。此外，Nakajo等人證明慢性NTP治療減少了梗塞病灶的體積、腦水腫以及神經缺陷的程度，並且還增強了C57BL/6J小鼠的空間學習(參見非專利文獻2)。NTP臨床應用的初步研究已顯示NTP能夠改善老年失智症患者的臨床症狀(參見非專利文獻3)。此外，Hoshino等人發現NTP經由提高氧化還原調節分子、穀胱甘肽過氧化物酶以及過氧化氫酶的表現，特別是硫氧還蛋白-1表現，而對過氧化氫和香煙煙霧的細胞保護效果。NTP也增加細胞硫氧還蛋白-1含量並調節硫氧還蛋白-1從細胞釋放，減抑細胞內氧化活性(參見非專利文獻4)。然而，NTP對神經元傷傷的潛在抗氧化作用仍有待確定。此外，氧化壓力在AD 的發病機制中有關鍵作用。因此，吾等研究了NTP對HT22細胞和APP/PS1小鼠的抗氧化能力，以獲得投予NTP用以治療AD的證據。
在本申請的發明人進行的本研究中(後文稱為“本研究”)，吾等使用永生化小鼠海馬迴神經元(HT22細胞)作為細胞模型來探討NTP是否具有緩解Aβ25-35-誘發之神經元傷害的潛在能力。據報導，Aβ25-35是最短的活性片段，與全長Aβ具有相似的神經毒性作用。此外，Aβ25-35可以很容易地合成，並且在科學研究中廣泛使用。因此，吾等在吾等研究中選擇此片段。針對在體內，吾等研究在投予NTP後APP/PS1小鼠海馬迴中之抗氧化劑活性和Aβ沉積之變化，並進一步探討其根本的機制。
1. Fukuda Y, Berry TL, Nelson M, et al. Stimulated neuronal expression of brain-derived neurotrophic factor by Neurotropin. Mol Cell Neurosci 2010; 45:226-233.
2. Nakajo Y, Yang D, Takahashi JC, Zhao Q, Kataoka H, Yanamoto H. ERV enhances spatial learning and prevents the development of infarcts, accompanied by upregulated BDNF in the cortex. Brain Res 2015;1610:110-123.
3. Kimura H, Nakamura S, Okamoto K, Toyama I, Watanabe M, Ikeuchi K. A pilot study for clinical applications of Neurotropin to senile patients with dementia. Jpn Pharmacol Ther 1987;15: 407-423.
4. Hoshino Y, Nakamura T, Sato A, Mishima M, Yodoi J, Nakamura H. Neurotropin demonstrates cytoprotective effects inlung cells through the induction of thioredoxin-1. Am J Resp Cell Mol 2007; 37:438-446.
在一個態樣中，本發明係有關一種包括來自經牛痘病毒接種之發炎組織之萃取物作為活性成分之藥劑，其特徵在於該藥劑具有作用抑制或緩解海馬迴中之Aβ誘發型傷害。
在另一態樣中，本發明還有關一種用途，其係將來自經牛痘病毒接種之發炎組織之萃取物用於製備用以抑制或緩解海馬迴中之Aβ誘發型傷害之製劑。
在一較佳具體例中，Aβ誘發型傷害是氧化傷害。或者，Aβ誘發型傷害係由Aβ沉積誘發。
在另一較佳具體例中，該藥劑的作用係藉由HIF-1α及/或Bax的表現的減抑而誘發。
在另一較佳具體例中，該藥劑是用於預防、緩解或治療阿茲海默症之藥劑。
在另一具體例中，該發炎組織係兔子的皮膚組織。
在又另一具體例中，該藥劑係注射劑或口服劑。
第1圖：將HT22細胞以各種濃度的NTP預處理16小時，然後與40μM Aβ25-35共同培養24小時，接著用CCK8測定法評估細胞存活率。數據係以對照組的相對百分比表示，並顯示為平均值±SE(n=6)。相較於對照為##P<0.01，相較於Aβ25-35群組為*P<0.05和**P<0.01。
第2圖：(A)對照組。(B)將HT22細胞暴露於40μM Aβ25-35 24小時。(C-E)將HT22細胞以各種濃度的NTP(0.001、0.01、0.1UN/mL)預處理16小時，然後與40μM Aβ25-35共同培養24小時。細胞凋亡係藉由流式細胞儀評估。(F)細胞凋亡率的統計結果。(G-K)HT22細胞係以Hoechst 33342和碘化丙啶(PI)染色，並在處理後在螢光顯微鏡下觀察。如圖中所證實，Aβ25-35誘發的細胞凋亡的特徵為濃縮、強烈的螢光核。NTP®明顯減少凋亡細胞的數目。數值係以對照組的相對百分比表示，並顯示為平均值±SE(n=6)。相較於對照組為##P<0.01，相較於Aβ25-35群組為*P<0.05和**P<0.01。
第3圖：(A)對照組。(B)將HT22細胞以40μMAβ25-35處理24小時。(C-E)將HT22細胞以各種濃度的NTP(0.001、0.01、0.1UN/mL)預處理16小時，然後與40μM Aβ25-35共同培養24小時。將細胞以H2DCFDA染色20分 鐘，並且使用流式細胞儀評估細胞綠色螢光。(F)DCF螢光強度的統計結果。(G-K)將HT22細胞以H2DCFDA染色20分鐘，並且在處理後於螢光顯微鏡下偵測到細胞綠色螢光。數值表示為平均值±SE(n=6)。相較於對照為##P<0.01，相較於Aβ25-35群組為*P<0.05和**P<0.01。
第4圖：(A)對照組。(B)將HT22細胞以40μMAβ25-35處理24小時。(C)將HT22細胞以0.01UN/mL之NTP預處理16小時，然後與40μM Aβ25-35共同培養24小時。粒腺體膜電位(MMP，△Ψmt)係以JC-1染色測量。(F)JC-1紅/綠螢光比的統計結果。(E)上述處理後之HIF-1α、Bcl-2以及Bax的蛋白質表現。結果顯示為來自至少三次獨立實驗的平均值±SE。相較於對照為##P<0.01，相較於Aβ25-35群組為*P<0.05和**P<0.01。
第5圖：經NTP處理(TG+NTP)之小鼠、對照(TG)APP/PS1轉基因小鼠以及對照野生型(WT)小鼠。經處理之APP/PS1小鼠藉由口服灌胃給藥每天施用NTP(200NU/kg/天)達3個月。將對照(TG)APP/PS1轉基因小鼠和對照野生型(WT)小鼠以鹽水(0.9%NaCl)處理。(A)超氧化物歧化酶(SOD)活性。(B)過氧化氫酶(CAT)活性。(C)穀胱甘肽(GSH)濃度。(D)丙二醛(MDA)濃度。數據以均值±標準差(SE)表示，各組中n=6的海馬迴。相較於WT小鼠為#P<0.05和## P<0.01，相較於TG小鼠為* P<0.05和** P<0.01。
第6圖：(A)Aβ斑塊係以Bielschowsky銀染偵測。(B) 使用免疫組織化學之HIF-1α定量。(C)HIF-1α、p-ERK1/2、p-JNK1/2以及p-P38之西方墨點及定量分析。結果以至少三次獨立實驗的平均值±SE表示。相較於WT小鼠為## P<0.01相較於TG小鼠為* P<0.05以及** P<0.01。
關於萃取物的基本萃取步驟，例如係使用以下步驟。
(A)採集經皮內接種牛痘病毒之兔子、小鼠等的發炎皮膚組織，並將發炎組織弄碎。在弄碎之組織中加入水、苯酚水、生理食鹽水或加入苯酚之甘油水等萃取溶劑，以進行幾天的萃取處理。然後，將混合物過濾或離心，以得到從其中去除組織片段的粗萃取物(濾液或上清液)。
(B)將在(A)中得到的粗萃取物調整至酸性pH，加熱，之後過濾或離心分離以進行去蛋白處理。之後，將去蛋白的溶液調整至鹼性pH，加熱，然後過濾或離心以得到去蛋白的濾液或上清液。
(C)將在(B)中得到的濾液或上清液調整至酸性pH，並用吸附劑如活性碳或高嶺土吸附。
(D)在(C)中得到的吸附劑中加入水等萃取溶劑，將混合物調整至鹼性pH，洗提吸附成分，以得到從經牛痘病毒接種之兔子、小鼠等的發炎皮膚的萃取物(本提取物)。
如兔子、牛、馬、綿羊、山羊、猴、大鼠、小鼠等可經牛痘病毒感染的各種動物可以用作接種牛痘病 毒，並獲得發炎組織的動物。其中，較佳選擇兔子的發炎皮膚組織作為發炎組織。
任何屬於兔形目(Lagomorpha)之兔子皆可使用。其實例包括穴兔(Oryctolagus cuniculus)、家兔(馴化的Oryctolagus cuniculus)、野兔(日本野兔)、鼠兔和白靴兔。其中，使用家兔是合適的。在日本，有一種叫做“加藤”的兔子家族，其自古以來就被飼養，且經常被用作家畜或實驗動物，且為家兔的另一個名稱。家兔有許多品種，且有利地使用被稱為日本白和新西蘭白的品種。
本文中所用的牛痘病毒可為任何株。其例子包括李斯特(Lister)株、大連(Dairen)株、池田(Ikeda)株、EM-63株和紐約市衛生局(New York City Board of Health)株。
例如，在WO2016/194816的第[0024]至[0027]、[0031]段等中描述了關於製造萃取物的方法的更詳細描述。
由上海生工生物工程技術服務有限公司(中國上海)合成Aβ25-35。從Gibco(New York，USA)獲得胎牛血清(FBS)、培養基(DMEM)、神經基礎培養基以及N2補充物。從Dojin Kagaku(Kumamoto，Kyushu，Japan)獲細胞計數套組-8(cell counting kit-8；CCK-8)。從eBioscience(San Diego，CA，USA)購得細胞凋亡偵測套組。從碧云天生物技術(中國上海)購買有JC-1的ROS偵測套組和粒腺體膜電位測定套組。從Invitrogen/Life Technologies (Carlsbad，CA，USA)獲得Hoechst 33342和碘化丙啶(PI)。由健城生物工程研究(中國南京)供應SOD、GSH、MDA以及CAT套組。從Cell Signaling Technology(Danvers，MA，USA)獲得針對p-Erk1/2、p-P38、p-JNK、Erk1/2、P38、JNK、Bcl-2和Bax之一級抗體及二級抗體辣根過氧化物酶-(HRP-)接合的山羊抗兔子IgG。從Abcam(Cambridge，MA，USA)獲得針對HIF-1α的一級抗體，並且從Sigma-Aldrich(St.Louis，MO，USA)購買針對Aβ1-42的一級抗體。從Millipore(Billerica，MA，USA)購買化學發光辣根過氧化物酶受質。從Thermo Fisher、Invitrogen以及MR Biotech獲得所有其他常規實驗供給物和試劑。
(1)細胞培養、分化、Aβ
25-35
製備和處理
用於培養和分化HT22細胞的方法已於先前詳細描述(參見Liu J，Li L and Suo WZ.HT22 hippocampal neuronal cell line possesses functional cholinergic properties.Life Sci 2009；84：267-271.，和Zhao ZY，Luan P，Huang SX et al.,Edaravone protects HT22 neurons from H2O2-induced Apoptosis by Inhibiting the MAPK Signaling Pathway.CNS Neurosci Ther 2013；19：163-169.)。簡言之，將HT22細胞在補充有10%FBS、100U/mL青黴素以及100μg/mL鏈黴素的DMEM培養基中培養，並在處理前用補充N2的神經基質培養基分化24小時。將Aβ25-35於0.5mM之濃度之無菌生理鹽水中稀釋，並在37℃保持7天以使胜肽預熟 化。根據吾等先前的研究(參見Fan S，Zhang B，Luan P，et al.,PI3K/AKT/mTOR/p70S6K Pathway is involved in Aβ25-35-indauced autophagy,Biomed Res Int 2015；2015：1-9)，當細胞曝露於40μM Aβ25-35 24小時，HT22細胞的存活率可顯著降低。因此，吾等選擇40μM作為Aβ25-35的最佳濃度，以進行吾等後續的研究。將HT22細胞用特定劑量的NTP預處理16小時，隨後使用或不使用40μM Aβ25-35處理24小時。
(2)細胞毒性測定法
將CCK-8分析用來評估HT22細胞的生活率。簡而言之，在不同的治療干預後，將10μL/孔的CCK-8試劑加入細胞中，之後將HT22細胞在黑暗條件下在37℃和5%CO2培養1.5小時。用多功能微盤讀取機(SpectraMax M5，USA)在450nm偵測樣品的吸光度。
(3)藉由流式細胞儀(FCM)的細胞凋亡測定法
如先前詳述，使用膜聯蛋白V-FITC和PI細胞凋亡偵測套組，以流式細胞分析來測量HT22細胞的凋亡性細胞死亡(參見Zhao ZY，Luan P，Huang SX et al.,Edaravone protects HT22 neurons from H2O2-Indund Apoptosis by suppression MAPK Signaling Pathway.CNS Neurosci Ther 2013；19：163-169.)。簡言之，HT22細胞係用磷酸鹽緩衝鹽水(PBS)洗滌兩次，並在存在或不存在NTP下，曝露於Aβ25-3524小時後用膜聯蛋白V-FITC和PI在結合緩衝液中 培養。將細胞懸浮液用流式細胞儀(FACSCalibur；BD，Franklin Lakes，NJ)進行流式細胞分析。經測量，每個樣品有10000個細胞。與流式細胞術分析平行，執行Hoechst 33342和PI染色以進行形態學評估。將HT22細胞以4%多聚甲醛固定10分鐘。用PBS沖洗三次後，將細胞以5mg/L Hoechst 33342和1mg/L PI染色。在螢光顯微鏡下觀察細胞(BX51WI，Olympus，USA)。
(4)細胞內ROS產生的測量
如先前報導的，藉由氧化-敏感性螢光探針(DCFH-DA)測量細胞內ROS(參見Bao FX,Shi HY,Qi L,et al.,Mitochondrial Membrane Potential-dependent Endoplasmic Reticulum Fragmentation is an Important Step in Neuritic Degeneration.CNS Neurosci Ther 2016；22：648-660.)簡而言之，將HT22細胞以NTP預處理16小時，然後曝露於40μM Aβ25-3524小時。處理後，藉由移液收集細胞，以PBS洗滌兩次，以及以10μM DCFH-DA在37℃培養20分鐘。使用FACSCalibur流式細胞儀偵測到每個樣品有10,000個細胞。陽性細胞中的幾何螢光強度表示ROS的濃度。此外，亦在螢光顯微鏡下觀察DCF螢光強度。
(5)粒腺體膜電位的估計(MMP，△Ψmt)
根據製造商的說明，使用具有JC-1的粒腺體膜電位測定套組評估MMP。處理後，將細胞以PBS洗滌兩次，然後以5μl/mL JC-1在37℃染色20分鐘。以JC-1染色緩衝液沖洗兩次後，收集細胞懸液並使用流式細胞儀監控 MMP。於流式細胞儀分析中使用來自每個樣品約10,000個事件。
(6)動物與投藥
從傑克森實驗室(ME，USA)獲得六個月大的雄性APP/PS1轉基因小鼠，並維持自由取用食物和水。本研究中使用的所有小鼠均根據孫逸仙大學實驗動物照護及使用委員會批准的實驗方案(protocol)進行處理。本研究的受試者係由六個月大的APP/PS1轉基因小鼠(TG，n=16)和野生型同窩小鼠(WT，n=8)組成。將動物隨機分為三組：經NTP處理(TG+NTP)之小鼠、對照(TG)APP/PS1轉基因小鼠以及對照野生型(WT)小鼠。經處理之小鼠藉由口服灌胃給藥每天接受NTP(200NU/kg/天)3個月。經鹽水(0.9%NaCl)處理之剩餘的小鼠作為安慰劑對照。
(7)SOD、MDA、GSH以及CAT之測量
根據製造商的說明書，使用商業套組測定超氧化物歧化酶(SOD)、穀胱甘肽(GSH)以及過氧化氫酶(CAT)的活性以及丙二醛(MDA)的含量。將海馬迴組織於0.9%冷NaCl中(9mL)超音震盪而均質化，於4℃和4000g離心10分鐘，取上清液且將其於80℃保存備用。上清液中的蛋白質濃度係用微BCA蛋白質測定套組評估。
(8)Bielschowsky銀染色和免疫組織化學
將先前公開的方法用在固定切片上進行Bielschowsky銀染色(參見Schwab C,Steele JC,McGeer PL.Dystrophic neurites is associated with the majority of early stage extracellular neurofibrillary tangles in parkinsonism dementia complex of Guam.Acta Neuropathol 1997；94：486-492.，和Schwab C，Hosokawa M，Mcgeer PL.Transgenic mice overexpressing amyloid beta protein are an incomplete model of Alzheimer disease,Exp Neurol 2004；188：52-64.)。對於免疫組織化學染色，根據製造商的說明書，用DAB套組對小鼠腦切片進行過氧化物酶標記染色。從螢光顯微鏡獲得影像。用於免疫組織化學染色的第一抗體是小鼠抗HIF-1α(1：800；Abcam，MA，USA)。測量老年斑塊和HIF-1α陽性細胞的表面積，並用Image J軟體比較齒狀回的百分比。
(10)西方墨點分析
處理後，將HT22細胞以補充蛋白酶抑制劑混合物(cocktai)的適量沸騰的變性裂解物緩衝液(1%SDS，1mM原釩酸鈉，10mM Tris-Cl，pH7.4)裂解。在快速免疫沉澱測定緩衝液(50mM Tris[pH 7.4]，150mM NaCl，1%Triton X-100,1%脫氧膽酸鈉，0.1%十二烷基硫酸鈉[SDS])中提取小鼠海馬迴的樣品蛋白。西方墨點法和半定量分析係藉由之前描述的程序進行。所使用之一級抗體和稀釋率係如下所列：HIF-1α，1：2000；Bcl-2，1：1000；Bax，1：1000；Aβ1-42，1：1000；p-JNK，1：500；p-P38，1：500；p-ERK1/2，1：500；JNK，1：1000；P38，1：500；ERK1/2，1：2000以及β-肌動蛋白，1：1000。
(11)統計分析
所有數據均以平均值±SE表示，而且所有統計分析均使用SPSS 16.0軟體(SPSS Inc.,Chicago,IL,USA)進行。有事後比較檢定(post hoc test)之單因子變異數分析(ANOVA)係用於群組間之變異數分析，而且群組間之差異係採用學生t檢驗進行比較。P<0.05時，認為差異有統計學意義。
(12)結果
(i)以NTP進行之體外神經保護
如第1圖所示，以多種濃度(0.001至0.1UN/mL)NTP之預處理減抑由Aβ25-35誘發的細胞毒性(P<0.05)。細胞凋亡的流式細胞儀分析證實，投予0.001至0.1UN/mL的NTP可顯著抑制由Aβ25-35引起的細胞凋亡(P<0.05)(第2A至2F圖)。通過螢光顯微鏡，以Hoechst 33342和PI染色觀察到類似的作用(第2G至2K圖)。吾等接下來探討了NTP是否緩解ROS濃度。NTP處理降低了幾何平均值DCF螢光，而且在0.001和0.01UN/mL的濃度時效果顯著，但在0.1UN/mL的劑量沒有在ROS產生方面觀察到顯著差異，暗示高濃度的NTP可能對減抑細胞內ROS產生沒有影響(第3A至3F圖)。螢光顯微鏡觀察ROS濃度與流式細胞儀結果一致(第3G至K圖)。此外，與Aβ25-35組相比，0.01NU/mL NTP減弱JC-1染色顯示粒腺體膜電位(第4A至4D圖)。
(ii)HIF-1α和細胞凋亡相關的分子之體外NTP調節
為了進一步探討NTP對HT22細胞中之Aβ25-35神經保 護作用的機制，吾等測定了HIF-1α、Bcl-2以及Bax的蛋白表現。如第4E圖所示，西方墨點分析揭示NTP顯著增強Bcl-2的表現。與Bcl-2誘發相反，NTP抑制HIF-1α和Bax的表現(P<0.01)。
(iii)NTP之體外抗氧化作用
在經NTP處理的APP/PS1小鼠的海馬迴中評估氧化壓力標記物。與對照APP/PS1小鼠群組相比，NTP投藥可以增加超氧化物歧化酶(SOD)、過氧化氫酶(CAT)、穀胱甘肽(GSH)的濃度(分別為P<0.01、P<0.05、P<0.05)，並且減少經NTP處理之APP/PS1小鼠的海馬迴中之丙二醛(MDA)含量(P<0.01)(第5圖)，暗示NTP展現調節抗氧化系統平衡的能力。
(iv)於海馬迴中之Aβ沉積之體外NTP抑制
為了探討NTP處理對APP/PS1小鼠海馬迴中之Aβ沉積的潛在作用，使用Bielschowsky銀染測定對照野生型小鼠、對照和經NTP處理之APP/PS1小鼠的海馬迴之冠狀面切片，並且收集海馬迴組織以用於測量Aβ蛋白濃度。定量分析顯示，與APP/PS1小鼠群組相比，經NTP處理之群組中之Aβ斑塊沉積物的表面積顯著降低(第6A圖)(P<0.01)。根據此結果，吾等觀察到經NTP處理之群組之海馬迴中之Aβ1-42蛋白濃度降低(第6C圖)。
(v)由NTP進行之HIF-1α和MAPK家族活化之體外減抑
為了進一步探討NTP神經保護作用的根本機制，吾等首先藉由免疫組織化學和西方墨點法偵測了HIF-1α在海 馬迴中的表現。結果顯示經NTP處理之小鼠中抑制HIF-1α表現(第6B圖)(P<0.05)。如第6C圖所示，吾等還使用西方墨點分析檢驗經有絲分裂原活化之蛋白激酶(MAPK)路徑的活化。與野生型群組相比，吾等發現APP/PS1小鼠中促進ERK1/2、JNK以及P38的磷酸化。然而，NTP處理明顯降低了p-ERK1/2、p-JNK以及p-P38的表現(P<0.01)。
吾等來自體外實驗的結果提供了NTP保護免於發生Aβ25-35誘發之氧化傷害和細胞死亡的直接證據。在HT22細胞中，ROS濃度和HIF-1α顯著降低。同時，粒腺體膜電位增加，事實上，粒腺體作用被認為是AD致病機制中的關鍵作用。粒腺體累積膜損傷可以協助在細胞中和AD小鼠模型中增加ROS產生。經發現，粒腺體功能障礙早在三個月後就開始了。特別地，Chen等人證實氧化壓力和硝化壓力誘發的粒腺體損傷可能經由細胞色素c的釋放引起凋亡蛋白酶之級聯反應，導致海馬迴中之細胞凋亡。
來自體內研究的發現為NTP對提高抗氧化劑之活性和減少Aβ沉積的形成具有保護作用。為了進一步研究NTP的機制，吾等研究了HIF-1α表現和壓力相關的經有絲分裂原活化之蛋白激酶(MAPK)訊號。MAPK是絲胺酸/蘇胺酸蛋白激酶家族，且調節細胞生長、分化和細胞死亡的關鍵訊號傳遞路徑。Aβ誘發之氧化壓力可以改變此等細胞訊號傳遞路徑，並誘發細胞中之磷酸化反應。JNK 和P38的活化的向上調節與AD大腦有關，並且MAPK訊號傳遞路徑可響應於Aβ累積而活化。郭等人觀察到MAPK訊號可由茴香黴素活化，並在神經母細胞瘤細胞中誘發細胞內Aβ產生。因此，MAPK之級聯反應的失調進一步支持Aβ與AD中之氧化壓力之間的病理學關聯。本研究中，吾等發現APP/PS1小鼠中之ERK1/2、JNK以及P38的活化增加。然而，經NTP處理之APP/PS1小鼠中之MAPK的活化減少，表示NTP可以減抑磷酸化反應。
綜上所述，吾等研究結果證實Aβ25-35可以誘發海馬迴神經元損傷，包含ROS濃度的向上調節、粒腺體膜電位的向下調節以及細胞凋亡的促進。相反地，NTP治療可逆轉HT22細胞中之Aβ25-35介導的損傷。此外，NTP可改善APP/PS1小鼠之海馬迴中之Aβ斑塊的沉積，並改變抗氧化劑的活性。NTP的神經保護能力可能與HIF-1α和MAPK訊號傳遞路徑的減抑有關，簡言之，NTP可作為欲用於未來AD的預防和治療之自由基清除劑。
Claims (10)
- 一種藥劑，其包括來自經牛痘病毒接種之發炎組織之提取物，該提取物係作為活性成分，該藥劑之特徵在於：該藥劑具有於海馬迴中抑制或緩解Aβ誘發型傷害之作用。
- 如申請專利範圍第1項所述之藥劑，其中，該Aβ誘發型傷害為氧化傷害。
- 如申請專利範圍第1項所述之藥劑，其中，該Aβ誘發型傷害係藉由Aβ沉積誘發。
- 如申請專利範圍第1至3項中任一項所述之藥劑，其中，該作用係藉由HIF-1α表現之減抑而誘發。
- 如申請專利範圍第1至3項中任一項所述之藥劑，其中，該作用係藉由Bax表現之減抑而誘發。
- 如申請專利範圍第1至3項中任一項所述之藥劑，其中，該藥劑係用於預防、緩解或治療阿茲海默症之藥劑。
- 如申請專利範圍第1至6項中任一項所述之藥劑，其中，該發炎組織係兔子之皮膚組織。
- 如申請專利範圍第1至7項中任一項所述之藥劑，其中，該藥劑係注射劑。
- 如申請專利範圍第1至7項中任一項所述之藥劑，其中，該藥劑係口服劑。
- 一種提取物的用途，其係將來自經牛痘病毒接種之發炎組織之提取物用於製備用以抑制或緩解海馬迴中之 Aβ誘發型傷害之藥劑。
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| EP3592369A1 (en) | 2020-01-15 |
| US20200289581A1 (en) | 2020-09-17 |
| CA3051019A1 (en) | 2018-09-13 |
| JP7445236B2 (ja) | 2024-03-11 |
| WO2018161211A1 (en) | 2018-09-13 |
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| IL268090A (en) | 2019-09-26 |
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| TWI780123B (zh) | 2022-10-11 |
| EP4074323A1 (en) | 2022-10-19 |
| AU2017402155B2 (en) | 2022-04-14 |
| JP7417217B2 (ja) | 2024-01-18 |
| CN116626310A (zh) | 2023-08-22 |
| CN110381966A (zh) | 2019-10-25 |
| US11235006B2 (en) | 2022-02-01 |
| EP3592369A4 (en) | 2020-09-09 |
| AU2017402155A1 (en) | 2019-08-01 |
| JP7445236B6 (ja) | 2024-04-09 |
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