- 學位論文
松杉靈芝對於PM2.5造成子代大鼠記憶損傷之保護作用
Protective Effect of Ganoderma Tsugae in PM2.5 Caused Memory Impairment of Postnatal Rat
摘要
隨著科技的發展，環境汙染越來越嚴重，舉凡土地、水質、空氣都被人類破壞殆盡，而近年最受重視的問題便是空氣汙染。懸浮微粒 (particulate matter, PM) 即為空氣汙染的主要因子，大多以碳為核心，表面附著重金屬及有機物。其中，粒徑小於2.5 μm的懸浮微粒，被稱為細懸浮微粒 (fine particulate matter, PM2.5)，可經由呼吸進入人體，再經由肺泡微血管進入血液循環，對全身造成危害。PM2.5進入體內後會引發人體的發炎反應、增加活性氧物種 (Reactive oxygen species, ROS) 的產生並提高氧化壓力 (oxidative stress)，造成脂質、蛋白質及DNA的損傷；長期暴露PM2.5更可能導致腦部損傷，罹患阿茲海默症、帕金森氏症等疾病。近年研究也證實這些傷害會透過懷孕母體影響到下一代的健康，本實驗室先前研究顯示，孕婦暴露PM2.5會造成母體發炎反應，且基因微陣列分析顯示PM2.5會提高胎兒出生後罹患神經相關疾病的風險。為解決孕婦暴露PM2.5造成腹中胎兒損傷，本研究選用市面上常見的保健食品—靈芝來進行探討。靈芝作為中醫用藥已有數千年的歷史。靈芝具有極佳的抗氧化及抗發炎效果，而本篇選用抗氧化效果最好的松杉靈芝 (Ganoderma Tsugae) 萃取物 (GTDE) 來進行測試，探討松杉靈芝是否可以減緩母鼠懷孕期間暴露PM2.5對其子代所造成的影響。懷孕母鼠依照灌食GTDE的有無以及是否暴露兩種不同劑量的PM2.5分為6個組別，並分為胎鼠及子代鼠兩部分進行探討：(1)在母鼠懷孕第18天時將其犧牲，量測其各項生理參數及胎鼠體重。取出胎鼠腦部並分析腦中miRNA變化、測量胎腦中抗氧化及免疫相關蛋白表現量、分析懷孕母鼠血漿及羊水中細胞因子濃度變化、觀察胎腦皮質型態、神經細胞遷移分層狀態、新生細胞數量、發炎及星狀膠細胞型態；(2) 子代鼠部分則是於出生40天後進行行為學分析，行為學選用物體辨識、工作記憶測試及水迷宮測試，用以觀察子代鼠之認知能力，於出生後75天犧牲並分析其生理參數及血漿中細胞因子濃度差異。結果顯示，GTDE可以降低PM2.5造成白细胞分化抗原68 (Cluster of Differentiation 68, CD68) 在胎腦中增加的情況並顯著增加血鐵質氧化酶-1 (Heme Oxygenase-1, HO-1) 在胎腦中的表現量。暴露PM2.5後，懷孕母鼠血漿中的發炎因子TGFβ、MCP-1、IL-1β、IL-6及IL-15有上升的情況，給予GTDE的組別則在發炎因子MCP-1、IL-15有顯著下降。在羊水中則發現GTDE有降低因暴露PM2.5所提升的發炎因子IL-1α、IL-6及IL-15濃度的效果。經由蘇木精-伊紅染色發現了給予GTDE的組別在腦室下區/腦室區 (subventricular zone/ ventricular zone, SVZ/VZ) 層的厚度比例有顯著的增加，並隨著暴露PM2.5劑量的增加而降低。免疫螢光染色法結果顯示，給予GTDE的組別其神經細胞遷移分層狀態有所改善，在暴露高劑量PM2.5的組別中特別顯著，且GTDE確實可以改善PM2.5造成新生神經細胞數量下降、大腦皮質發炎、星狀膠細胞減少等問題。在子代鼠的白血球數量及細胞因子檢測中可以發現，給予母體GTDE可以顯著改善PM2.5造成子代鼠體內發炎的情況。行為學測試中則顯示，GTDE可以顯著增加子代短期記憶的能力，並改善PM2.5造成的長期記憶損傷的情況。因此，松杉靈芝確實可以透過降低母體及胎兒的發炎反應改善PM2.5造成子代大鼠記憶損傷的問題。
並列摘要
Environmental pollution becomes more serious, starting from the industrial revolution in the 19th century. Since there are no efficient ways to solve air pollution, it has become the most urgent issue in recent years. Particulate matter (PM) is the primary harmful factor in air pollution. It is carbon-based particles with heavy metals and organic matter on the surface. The particles which are smaller than 2.5μm are called fine particulate matter (PM2.5). PM2.5 travels down the respiratory tract, penetrates the lungs, and enters systemic blood circulation. Once PM2.5 enters the human body, it might cause the inflammatory response, increase the production of reactive oxygen species (ROS) and oxidative stress that damage to lipids, proteins, and DNA. Long¬-term exposure to PM2.5 might induce neurological diseases, including Alzheimer's disease and Parkinson's disease. Recent epidemiological studies, including our previous lab data, indicated that PM2.5 increases the prevalence of dementia not only in adults but also in newborn infants. Ganoderma is a common health supplement available on the market. It has been used as a traditional Chinese medicine for thousands of years, and it is known for its antioxidant and anti-inflammatory effects. In this study, Ganoderma Tsugae was chosen as it has the highest antioxidant effect to determine whether Ganoderma Tsugae decrease the risk of PM2.5 induced damages in the offspring rats. According to the dosage of PM2.5 and the Ganoderma Tsugae DMSO Extracts (GTDE), the animals were divided into six different groups. Pregnant rats were sacrificed on the 18th day of gestation, and their physiological parameters and fetal weight were measured. The extracts of fetal brains were collected to analyze the changes in the expression of miRNA, antioxidant-related and immunity-related proteins, plasmatic and amniotic cytokines. The histochemical and histoimmunol analysis of fetal cortical sections were used to compare the morphological difference in the fetal cerebral cortex, neuronal migration, neurogenesis, neuroinflammation, and astrocyte. Forty days after birth, object recognition, working memory testing, and Morris water maze was performed to observe the cognitive abilities of the offspring. The offspring were then sacrificed on day 75 after birth, and physiological parameters and concentration of cytokines in plasma were measured. Results showed that GTDE could reduce Cluster of Differentiation 68 (CD68) in the fetal brain caused by maternal exposure of PM2.5, and significantly increased the expression of Heme Oxygenase-1 (HO-1). Maternal plasmatic cytokines, including TGFβ, MCP-1, IL-1β, IL-6, and IL-15, were elevated in the group exposed to PM2.5. However, GTDE was able to decrease the concentration of MCP-1 and IL-15. In the amniotic fluid, GTDE caused reduced levels of IL-1α, IL-6, and IL-15 against PM2.5. Hematoxylin and eosin staining show an increased ratio of subventricular zone/ ventricular zone (SVZ/VZ) in the cortical layer of the fetal brain of pregnant rats exposed to GTDE. Based on immunohistochemistry imaging, the neuronal migration was improved in the GTDE group. GTDE was found to ameliorate PM2.5 caused consequences, including the reduction in neurogenesis and inflammation of the cerebral cortex. Furthermore, GTDE resulted in a reduction of white blood cell number and serum cytokine concentration in offspring. Behavioral study of the offspring demonstrated a positive association between GTDE and short-term memory as well as improvement in long term working memory. Hence, this study showed that GTDE can ameliorate neuro-damages caused by PM2.5-induced inflammation in the offspring of pregnant rats.