北蟲草與蜜環菌複方液態膳食補充品於急性與慢性壓力動物模式之抗憂鬱功效評估

Abstract

根據世界衛生組織統計資料指出，全球有超過三億人口罹患憂鬱症 (depression)，並預測2030年憂鬱症將成為全球疾病負擔之首。此外，台灣地區常見精神疾病盛行率自1990年11.5%上升至2010年23.8%，且衛生福利部統計2017年服用抗憂鬱藥物已高達127萬人，憂鬱症對患者生活品質及社會成本負擔已不容忽視。然而，抗憂鬱藥物之副作用、症狀緩解率低等因素，導致服藥依順性低下。由此可知，治療憂鬱症之藥物無法滿足目前醫藥需求，積極建立具有實證之輔助與替代療法確有其必要性，例如：以食療為基礎所進行的抗憂鬱保健食品研發，即為本研究之主軸。北蟲草 (Cordyceps militaris) 為冬蟲夏草之近緣種，其活性成分與冬蟲夏草相似，傳統用於治療疲倦、乏力、呼吸系統、腎臟與心臟疾病等，市場上逐漸取代與應用；蜜環菌 (Armillaria mellea) 在傳統漢方中有治療失眠、鎮靜、抗驚厥等效用。文獻指出兩者皆能抑制發炎反應，且於動物實驗中呈現抗憂鬱功效。腦部因壓力造成發炎被認為是憂鬱症的成因之一，雖然過去多數神經與精神疾病研究著重在神經細胞本身的功能障礙或缺損，近年研究指出，於神經元周圍負責支持作用的膠質細胞 (glia cell) 被認為與神經精神疾病的病理致病原因息息相關。其中，微膠細胞 (microglia) 為大腦中監督協調與執行者，負責神經元與其他腦細胞環境中的發炎反應、保護作用及修復功能等，研究指出腦部微膠細胞受不同環境刺激後，可活化成促發炎因子分泌之M1型及抑制發炎與促神經保護之M2型。當M1/M2不平衡即可能造成腦部血清素與多巴胺路徑相關之神經網絡連結與功能失調，引起情緒沮喪、喪失樂趣等憂鬱症狀。故本研究採用急性壓力之強迫游泳試驗 (forced swimming test, FST) 與慢性壓力之慢性不可預期溫和壓力 (unpredictable chronic mild stress, UCMS) 動物模式探討北蟲草與蜜環菌複方液態膳食補充品之抗憂鬱功效。結果顯示，給予大鼠複方補充品於FST試驗期間，不會影響體重、攝食、飲水量及開放空間試驗之總移動距離、穿越次數與待在中央區域時間，且可顯著降低FST不泳動時間；此外，介入複方補充品可改善UCMS造成的體重、攝食與飲水量下降，並顯著提升大鼠糖水偏好程度。分析大鼠腦部神經傳導物質，顯示介入複方補充品可降低FST後大鼠海馬迴中血清素代謝率與前額葉皮質多巴胺代謝率及UCMS後大鼠前額葉皮質、海馬迴血清素與多巴胺代謝率。酵素連結免疫吸附法分析結果顯示其具有降低UCMS後血清皮質固酮濃度，並於免疫組織化學染色海馬迴中離子鈣結合銜接分子1 (ionized calcium binding adapter molecule 1, Iba-1) 分析其形態學變化，具有降低微膠細胞活化態的效果，於西方墨點法中結果亦顯示複方補充品可降低M1型相關促發炎細胞激素表現量，且增加抗發炎之介白素10 (interlekin, IL-10)、乙型轉化生長因子 (transforming growth factor beta,TGB-β) 以及M2型分化簇CD206 (mannose receptor) 表現量。綜合上述結果，北蟲草與蜜環菌複方液態膳食補充品可藉由平衡M1/M2 微膠細胞平衡狀態、降低發炎反應、調節神經傳導物質及改善大鼠類憂鬱行為，達抗憂鬱之功效。

Major depressive disorder (MDD) is a heterogeneous mental disease characterized by low mood, loss of interest and pleasure in normally enjoyable activities. The World Health Organization (WHO) ranks MDD as the third leading cause of disability worldwide and projects that by 2030, it will jump to the first place. However, due to the unpleasant side effects, MDD patients tend to have low compliance of taking antidepressants. Thus, the treatment of depression deserves greater attention by public health officials in order to avoid the already significant burden of this disease on both patients and society. Seeking for alternative approach such as dietary therapy to prevent the incidence of depression or ameliorate the disorder which concept is similar to preventive medicine will be an urgent issue. Cordyceps militaris which contains similar constituents and functions of Cordyceps sinensis has been used as a folk medicine for the treatment of fatigue, asthenia after severe illness, renal dysfunction, respiratory and heart disease for a long time. Armillaria mellea is also widely used in traditional Chinese medicine for the treatment of dizziness, headache, neurasthenia, insomnia, and convulsant. Many studies showed that both of these Chinese herbal medicines could exert anti-inflammatory and anti-depressive effects. Notably, chronic inflammation is now considered to be central to the pathogenesis of major depression. Recent studies also indicate that impairment of the normal structure and function of microglia caused by inflammatory activation can lead to depression and associated impairments in neuroplasticity and neurogenesis. Microglia cells are the resident mononuclear phagocytes in the central nervous system (CNS), also the major orchestrator of the brain inflammatory response, displaying functional polarization states similar to macrophages. M1 polarized microglia can produce pro-inflammatory cytokines and mediators. In contrast, M2 polarized microglia tends to produce anti-inflammatory cytokine and express several receptors that are implicated in inhibiting inflammation and restoring homeostasis. The imbalance between M1 and M2 polarization states was suggested to contribute to dysfunction of neural network, activities of monoamine neurons and symptoms in major depressive disorder. Therefore, the aim of this study is to investigate the antidepressive effects of the liquid dietary supplement of Cordyceps militaris and Armillaria mellea formula in acute and chronic stress animal models. The results of forced swimming test (FST) showed that four weeks of treatment could significantly decrease the immobility time of rats in FST (p<0.05) and ameliorate the turnover rate of serotonin in hippocampus and dopamine turnover rate in prefrontal cortex (p<0.05). On the other hand, unpredictable chronic mild stress (UCMS) showed that after four weeks of treatment the sucrose preference of rats in dose group was significantly higher than negative control group (p<0.05). Further analysis of open field test (OFT) showed that rats treated with liquid dietary supplement of Cordyceps militaris and Armillaria mellea formula have higher total travel distance, crossing number and time in center area when compared to the negative control (p<0.05). In addition, liquid dietary supplement of Cordyceps militaris and Armillaria mellea formula also ameliorate the turnover rate of serotonin and dopamine in hippocampus and in prefrontal cortex and corticosterone level in serum (p<0.05). Moreover, I examined the microglial activation and morphology by Iba-1 IHC staining in the hippocampus. The microglia in the stressed rats markedly exhibited the activated M1 phenotype characterized by enlarged cell bodies, hyporamified and fewer processes. Comparatively, medium and high dose group could improve this status. Results of Western blot analysis indicated that treatment of liquid dietary supplement of Cordyceps militaris and Armillaria mellea formula had significant lower M1 type related protein expression of proinflammatory cytokines like IL-1β and TNF-α in hippocampus and higher M2 type antiinflammatory cytokines IL-10, TGF-β and CD206 expression. In summary, my research demonstrated the liquid dietary supplement of Cordyceps militaris and Armillaria mellea formula reversed depression-like behavior in acute stress and chronic stress animal models, which may be mediated by the regulation of monoamine neurotransmitters turnover rate in prefrontal cortex and hippocampus, cytokine production and the phenotypic switch from M1 to M2 microglia in hippocampus. These results revealed the liquid dietary supplement of Cordyceps militaris and Armillaria mellea formula could be a potential complementary and alternative medicine for prevention and treatment of depression.