發炎環境對粒線體功能及神經細胞活動影響之研究

The study of the effects of inflammatory milieu on mitochondrial function and neural activities

林有上(Lim Yeou-San)

指導教授 ： 陳紹寬

國立政治大學/理學院/神經科學研究所/碩士(2022年)

https://doi.org/10.6814/NCCU202201421

摘要

發炎是當病原體入侵和組織損傷時所反應出的急性保護性反應以幫助生理系統恢復正常狀態。發炎可分為急性和慢性發炎，兩種發炎模式都有各自應對發炎的免疫功能和機制。急性發炎可保護個個體引發對抗外來病原體所必需的炎症反應，但長期慢性發炎所產生的炎症反應則會損害個體產生各種疾病問題。因此發炎是目前在已知的研究中被認為與許多神經系統疾病有間接或直接相關的成因之一。許多研究發現周邊系統的發炎會影響大腦中樞系統進而改變生理功能或其行為模式。目前研究多數在於研究周邊系統的發炎，但大腦中樞系統和大腦神經細胞處在發炎環境下所產生的改變尚未釐清。大腦下視丘弓狀核 (ARC)神經元群中的POMC/CART神經元調節著食慾，睡眠，代謝達到能量平衡等關鍵生理功能。下視丘發炎也是周邊發炎影響到大腦中樞神經系統的首要影響核區。促發炎細胞因子能夠通過血腦屏障進入下視丘中並活化小膠質細胞引發下視丘炎症。本研究旨在探討兩種不同因子造成的的不同發炎環境對POMC神經元中的粒線體的變化及POMC神經元活動的影響。本研究分為兩種不同的促發炎因子兩大部分。第一大部分探討第一種促發炎因子-脂多醣 (lipopolysaccharides) 刺激單核細胞系(monocytic lineage cells)對mHypo-POMC/GPF1神經元處在短期急性或長期慢性發炎環境下的粒線體功能及神經細胞活動之變化。 實驗結果發現POMC神經元在脂多醣的長期慢性發炎環境下導致粒線體出現型態上的變化，包括呈長條狀及出現空洞破壞等型態異變， 以及細胞內的ROS/RNS水平有上升趨勢，SOD1抗氧化酶基因和 GPX4 穀胱甘肽過氧化物酶基因的抗氧化酶的表達下降與 ROS/RNS 上升的結果是同步的。此外，也證實了在長期慢性發炎環境下會誘發黑皮質素（α-MSH）神經免疫調節肽的表現量增加。而在短期急性發炎環境下會導致MFN2 線粒體融合基因顯著下降。但脂多醣並未顯著影響POMC神經元細胞活動。另外我們透過腹腔注射脂多醣來誘導小鼠出現類疾病行為 (sickness behavior)，包括顯著增加小鼠體內的細胞因子和其食慾進食量減少，同時刺激下視丘的c-fos表現。第二大部分探討第二種促發炎因子-干擾素-α (IFN-α)。IFN-α在臨床上常被用於治療自體免疫疾病，病毒性疾病和癌症。然而長期使用IFN-α的治療患者會出現憂鬱症症狀。由於IFN-α也是另一類的促發炎因子和免疫調節因子，並且憂鬱症的症狀與大腦下視丘功能相呼應，如睡眠障礙，食慾降低或增加，疲憊等。因此IFN-α可用於探討在短期急性或長期慢性發炎環境下對下視丘POMC神經元之粒線體功能及神經細胞活動的影響。實驗結果發現IFN-α慢性長期發炎在POMC神經元上會活化STAT1傳遞訊號並促進SOCS1的表現。胰島素是POMC神經元調節代謝功能時的重要調節訊號。IFN-α逆轉了胰島素抑制細胞因子信號抑制因子3 (SOCS3)的表現。SOCS3 可以與胰島素受體結合調節胰島素敏感性，說明在IFN-α的作用下會增加胰島素敏感性。另外，過去研究發現POMC神經元在瘦素和胰島素的作用下會誘發神經活動，在本實驗中發現胰島素POMC神經元激活了神經活動(c-fos)之表現，但在IFN-α的刺激下胰島素卻顯著降低了神經活動。此外在IFN-α長期刺激下POMC神經元粒線體也產生型態上的變化，而且也觀察到POMC細胞內自噬過程，但尚未了解IFN-α對於神經細胞啟動自噬過程的相關機制，後續仍有待進一步研究。IFN-α的腹腔注射也證實誘導出小鼠出現類憂鬱行為，包括強迫游泳及懸尾僵直行為顯著增加。總而言之，本實驗結果顯示在兩種不同的發炎環境下，對下視丘POMC神經元中的粒線體和神經活動都有各別影響，而其中有幾個重要發現問題仍有待進一步研究探討及解決。

關鍵字

下視丘 ； 慢性發炎 ； POMC神經元 ； 粒線體 ； 脂多醣 ； 干擾素-α

並列摘要

Inflammation is the biological response of the immune system to protect the body from the harms mediated by foreign pathogen infection or damaged tissues. Inflammation can be either acute or chronic, both have their own immune responses and underlying mechanisms. Acute inflammation protects our body by eliciting the inflammatory responses necessary to fight foreign pathogens. In contrast, chronic inflammation generates long-lasting inflammation responses and caused various chronic diseases, including cardiovascular and bowel diseases, diabetes, and cancer. Mounting studies have demonstrated that peripheral inflammation affects central nervous system and alters physiological functions or behavioral phenotypes. Peripheral inflammatory cytokines directly or indirectly affect the brain and activate microglia to trigger hypothalamic inflammation. POMC neurons is the neuronal population located in hypothalamic arcuate nucleus (ARC) with regulating several essential physiological functions such as appetite, sleep, glucose metabolism and energy homeostasis. In this study, we aim to investigate how pro-inflammatory milieu or cytokines influence mitochondrial function and neuronal activities in hypothalamic POMC neurons. We compared neuronal alterations induced by two different types of pro-inflammatory stimuli. In the first part, mHypoA-POMC/GFP1 cells were treated with the conditioned medium collected from LPS-activated macrophage to mimic the inflammatory milieu during hypothalamic inflammation. Our in vivo data showed that POMC neurons exposed in CMLPS-chronic inflammation milieu led to morphological changes in mitochondrial elongation. Further, the intracellular ROS/RNS levels were elevated. The reduced expression of antioxidant enzymes was synchronized with ROS/RNS levels upregulation, such as Sod1 and Gpx4 genes during chronic inflammation. In vivo studies found that after intraperitoneal injection of lipopolysaccharide significantly increased cytokines and decreased appetite in mice. The expression of c-fos was also elevated in the hypothalamus, suggesting that neuronal activities were increased. The second type of inflammatory condition is chronic administration of interferon-alpha (IFN-α). IFN-α is often used clinically to treat autoimmune diseases, viral diseases, and cancer. However, chronic administration of IFN-α also cause neurological side effects such as depression. Therefore, chronic administration of IFN-α was employed as an alternative model to investigate the
effects of chronic inflammation on mitochondrial functions and neuronal activities of hypothalamic POMC neurons. In vitro experimental results found that IFN-α activates STAT1 expression in POMC neurons. IFN-α also disrupt insulin signaling, which is a crucial regulatory signal for the metabolism-regulatory functions in POMC neurons. IFN-α treatment reversed the expression of suppressed cytokine signaling inhibitor 3 (SOCS3). Also, IFN-α significantly reduced the neural activity activated by insulin. Additionally, IFN-α-induced chronic inflammation initiated mitochondrial changes and autophagy-like alterations in POMC neurons, and need more observatory time points are necessary to further understand the autophagic status. Intraperitoneal injection of IFN-α also confirmed the induction of depression-like behaviors in mice, including significant increase in forced swimming test and tail suspension test. In summary, these two different pro-inflammatory environments get different alterations on mitochondrial and neural activity in hypothalamic POMC neurons, and several questions remain to be further exploring by more experiments.

並列關鍵字

Inflammation ； Hypothalamic POMC neuron ； Mitochondrial morphology ； Lipopolysaccharide ； IFN-α

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