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標題: | 以急性發炎小鼠模式探討山苦瓜及其萃物對於腦部發炎反應的影響 The effects of Momordica charantia L. and Momordica charantia L. extracts on neural inflammation in LPS-induced acute inflammation murine model. |
作者: | Chia-Ying Chung 鍾嘉瑩 |
指導教授: | 林璧鳳 |
關鍵字: | 山苦瓜,山苦瓜乙酸乙酯萃物,LPS致急性發炎反應,腦部發炎反應,Th1免疫傾向, Wild bitter gourd,ethyl acetate extract,LPS-induced acute inflammation,neuroinflammation,Th1 polarization., |
出版年 : | 2012 |
學位: | 碩士 |
摘要: | 發炎反應是免疫系統中為抵抗並消除外來物質的一種反應,但細菌感染引起全身性的發炎反應過劇亦可能產生敗血症(sepsis),甚至引起死亡;阿茲海默症(Alzheimer's disease)和多發性硬化症(multiple sclerosis)等腦部疾病亦與腦部的發炎反應有關。已知山苦瓜具有調控血糖、改善代謝症候群及減緩呼吸道發炎反應等功效。因此,本研究探討花蓮三號(H3)及四號(H4)山苦瓜對全身性及腦部發炎指標之影響。
首先,以H3或H4的正己烷(HEX)、乙酸乙酯(EA)及水萃物(W)處理已轉染3倍NF-B結合位質體的RAW264.7巨噬細胞株,結果顯示HEX與EA萃物皆能顯著降低NF-B轉錄活性; H3及H4的EA萃物皆能夠降低ConA刺激下初代脾臟細胞IL-2及IL-4的分泌,其中H4-EA更可顯著降低IFN-及IL-10的分泌量。 接著以LPS致急性發炎小鼠模式,探討山苦瓜及其萃物對小鼠存活率與發炎介質生成之影響。將9週齡BALB/c雌鼠分成六組,分別為控制組、藥物組(PDTC)、5%之H3或H4山苦瓜凍乾粉組(H3-BGP、H4-BGP)及H3或H4乙酸乙酯萃物組(H3-EA、H4-EA),分別餵食小鼠七天後,腹腔注射LPS (15mg/kg BW)後,即進行存活率觀察;藥物組則在LPS腹腔注射前一小時,腹腔注射PDTC (50 mg/kg BW)。結果顯示,兩品種山苦瓜凍乾粉組顯著降低急性發炎小鼠的存活率,乙酸乙酯萃物皆無顯著影響。 因此接續僅探討H4凍乾粉與H4-EA對於急性發炎和正常小鼠全身性與腦部發炎指標的影響,以相同模式在腹腔注射LPS後九小時犧牲小鼠,分析脾臟與腦部組織相關mRNA表現量。結果顯示,H4-BGP組小鼠的脾臟細胞IL-1、IL-6及IFN-的分泌量顯著上升,IL-2分泌量則顯著下降;H4-EA組的脾臟細胞IL-2分泌量顯著較低,IFN-分泌量有上升的趨勢。H4-BGP組的腦部組織MCP-1及MIP-2表現量顯著高於控制組,TNF-及IL-6的表現量也有較高的趨勢;乙酸乙酯萃物部分,則皆與控制組無顯著差異。餵食H4-BGP或H4-EA一週後,山苦瓜對正常小鼠發炎指標的影響結果顯示,H4-BGP組脾臟細胞在未加刺激劑下, TNF-及IL-6的分泌量顯著上升;以ConA刺激下,H4-BGP及H4-EA組的IFN-分泌皆有上升的趨勢,H4-BGP組的TNF-及IL-2分泌量則是顯著降低。H4-BGP組的腦部組織IL-6表現量顯著較高,TNF-表現量有增加的趨勢,H4-EA組只有IL-6表現量有升高的趨勢。 綜合以上結果,H4-EA對正常小鼠的免疫傾向較無顯著的影響,因此對LPS致急性發炎小鼠的存活率與腦部發炎反應無顯著影響。而有別於H4-EA,H4-BGP對於急性發炎與正常小鼠皆促進脾臟細胞IFN-的分泌,顯示H4-BGP促進Th1免疫反應,因此在LPS致急性發炎小鼠模式中,促進小鼠全身及腦部發炎反應,而降低小鼠存活率。 Inflammation is a mechanism to resist invader and protect self from harm in the immune system. But when the reaction was too severe and secrete too much pro-inflammatory cytokine, it might be harmful to the host.Bacterial infection, for example, can cause severe inflammation even inducing sepsis and death. Brain diseases such as Alzheimer’s disease (AD) and multiple sclerosis (MS) arise in association with neural inflammation now. Therefore, how to regulate systemic inflammatory response and neuroinflammation has been an important issue now. In preveous study, Momordica charantia L.(wild bitter gourd) possess anti-inflammatory ability in OVA-sensitized mice. In this study, we want to investigate the effects of wild bitter gourd or its extract in flammation by using in vitro experiments and in murine model of LPS-induced acute inflammation. First, Raw264.7 macrophage cell line was transfected with a plasmid containing reporter gene luciferase with NF-κB binding sites promoter. Transfected Raw264.7 were cultured with two stains of wild bitter gourd(H3 & H4) ethyl acetate extract (EA) , hexane extract (HEX) or aqueous extract (W).The results showed that the transcriptional activities were significantly decreased by EA and HEX in both two strains. Second, primary splenocyte from BALB/c were treated with EA、HEX、W of two strains separately for 48 hours. The results showed that H3-EA and H4-EA both could reduce IL-2 and IL-4 secretion and H4-EA could even significantly decrease IFN-and IL-10 secretion by ConA-stimulated splenocytes. In LPS-induced acute inflammation mice model,9 wk-old female BALB/c mice were grouped to six groups (Control、PDTC、H3-BGP、H4-BGP、H3-EA and H4-EA), supplemented with 5% wild bitter gourd powder (BGP) or EA extract (tube-fed, effective dose derived from in vitro exp.) for seven days before injected intraperitoneally (i.p.) with LPS (15 mg/kg BW). Mice in PDTC group were injected intraperitoneally with 50 mg/kg BW PDTC an hour before LPS injection. The result showed that H3 and H4-BGP could significantly reduce survival rate of mice. Next, we continued to investigate the effects of H4-BGP and HA-EA in inflammation by conducting the same model, but mice were sacrificed 9 hours after LPS injection and their spleens and brains were collected for analysis. The results showed that H4-BGP could significantly increase pro-inflammatory cytokine IL-1、IL-6 and Th1 cytokine IFN-secretion but decrease IL-2 secretion by splenocytes. H4-EA increased IFN-secretion in trend and significantly decreased IL-2 secretion by splenocytes. In addition, H4-BGP significantly increased MIP-2 and MCP-1 mRNA expression in brain. Another part of in vivo experiments, mice were sacrificed after seven days supplement with H4-BGP or H4-EA extract. The spleens and brains were collected for analysis as previous. The results showed that H4-BGP could significantly increase TNF-and IL-6 secretion by splenocyte which also secreted small amounts of IFN-. Under mitogen stimulation, IFN-secretion splenocytes is increased in trend both in H4-BGP and H4-EA group while IL-2 and TNF-secretion is decreased significantly. In addition, H4-BGP could significantly increase IL-6 mRNA expression and increase TNF-mRNA expression in trend in brain. H4-EA extract only increased IL-6 mRNA expression in trend in brain. In conclusion, H4-EA extract might have no significant influence on systemic inflammatory response and neuroinflammation in mice in the LPS-induced acute inflammation model. H4-BGP might aggravate systemic inflammatory response and neuroinflammation by promoting Th1 polarization in mice then cause reduction of mice’s survival rate in this model. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/16524 |
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