本文選題：FJ1 + 炎癥�。� 參考：《山東大學(xué)》2014年碩士論文

【摘要】：3β-當(dāng)歸酰氧基-8p,10p-二羥基佛術(shù)烯-7(11)-烯-12,8α-內(nèi)酯(FJ1)是從傳統(tǒng)中藥大吳風(fēng)草中提取的活性化合物。大吳風(fēng)草為多年生草本菊科植物,其藥用全株叫做蓮蓬草,主要分布于中國的東南部和南部、日本、韓國[1],對于例如感冒、咳嗽、炎癥等多種疾病均有良好療效,因此推測其活性提取物具有抗炎癥作用。課題中研究的FJ1是本實(shí)驗(yàn)室由大吳風(fēng)草中提取的多種活性產(chǎn)物經(jīng)篩選后確定的考察對象。 炎癥是機(jī)體對由微生物感染和其他傷害性刺激造成的組織損傷的響應(yīng),是一種十分常見同時(shí)也非常重要的防御機(jī)制,然而,低分辨率和不受控制的炎癥反應(yīng)會導(dǎo)致一種與包括癌癥在內(nèi)的多種人類疾病有關(guān)的慢性炎癥狀態(tài)。本課題旨在驗(yàn)證FJ1的抗驗(yàn)證活性并闡明其作用機(jī)制。實(shí)驗(yàn)采用脂多糖(LPS, Lipopolysaccharide)誘導(dǎo)小鼠單核巨噬細(xì)胞白血病細(xì)胞RAW264.7構(gòu)建炎癥細(xì)胞模型。MTT實(shí)驗(yàn)檢測FJ1的細(xì)胞毒活性以及對LPS損傷細(xì)胞的保護(hù)活性,結(jié)果表明,二者均具有時(shí)間和劑量依賴性,細(xì)胞毒在安全范圍內(nèi),FJ1能夠提高保護(hù)組細(xì)胞活性。利用一氧化氮試劑盒測定NO的產(chǎn)生量,結(jié)果顯示FJ1明顯抑制LPS誘導(dǎo)RAW264.7產(chǎn)生NO量,且呈劑量依賴性。流式細(xì)胞技術(shù)檢測細(xì)胞活性氧水平,結(jié)果與上述結(jié)論相符。蛋白免疫印跡法考察ERK, p38, JNK, p-ERK, p-p38, p-JNK[9], NF-κB, IκB-α, p-1κB-α[10]的蛋白表達(dá)水平變化,結(jié)果表明,ERK, p38, JNK蛋白表達(dá)水平?jīng)]有明顯變化,p-ERK, p-p38, p-JNK, NF-κB, p-1κB-a[12]的蛋白表達(dá)水平隨著FJ1濃度升高而降低,而1κB-α蛋白表達(dá)水平則隨著FJ1濃度升高而增加。RT-PCR檢測炎癥相關(guān)基因iNOS,COX-2, TNF-a的RNA表達(dá)水平,結(jié)果發(fā)現(xiàn),三者均隨著FJ1濃度的升高而顯著降低。綜上所述,FJ1對炎癥細(xì)胞模型具有顯著的保護(hù)作用,提示FJ1的潛在抗炎癥活性可能成為我國傳統(tǒng)中藥對世界醫(yī)藥發(fā)展的又一貢獻(xiàn)。 帕金森病(Parkinson's disease, PD)是一種常見的神經(jīng)系統(tǒng)變性疾病,其臨床表現(xiàn)主要包括靜止性震顫、運(yùn)動遲緩、肌強(qiáng)直和姿勢步態(tài)障礙,同時(shí)患者可伴有抑郁、便秘和睡眠障礙等非運(yùn)動癥狀,給患者及其家屬帶來巨大的痛苦。帕金森癥與氧化應(yīng)激有著密切的關(guān)聯(lián),FJ1在抗炎癥活性探討中表現(xiàn)出來的抗氧化活性提示我們其潛在的抗帕金森癥活性。經(jīng)典的帕金森癥細(xì)胞模型是由1-甲基-4-苯基-1,2,3,6-四氫吡啶(MPTP)誘導(dǎo)褐家鼠腎上腺嗜鉻細(xì)胞瘤細(xì)胞PC12細(xì)胞株構(gòu)建而成,百草枯(PQ)是一種常見的農(nóng)業(yè)毒素,其結(jié)構(gòu)與MPTP的活性部分MPP+相近,因此近年來多被用于構(gòu)建新型帕金森癥模型,本研究采用此種模型,也是本實(shí)驗(yàn)室首次自行構(gòu)建此模型,因此需要對其各項(xiàng)指標(biāo)進(jìn)行檢測。MTT實(shí)驗(yàn)檢測PQ的細(xì)胞毒活性,結(jié)果顯示PQ抑制PC12增殖,且具有明顯的劑量和時(shí)間依賴性。流式細(xì)胞技術(shù)測定細(xì)胞線粒體膜電勢[24]以及細(xì)胞凋亡,結(jié)果表明高濃度PQ使細(xì)胞膜電勢下降,誘導(dǎo)PC12凋亡。上述結(jié)果均與已報(bào)道的MPTP以及其他實(shí)驗(yàn)室構(gòu)建該模型的數(shù)據(jù)相符,可用作后續(xù)研究中的神經(jīng)細(xì)胞保護(hù)實(shí)驗(yàn)。在FJ1對PC12的保護(hù)活性研究中,MTT實(shí)驗(yàn)檢測FJ1對PQ誘導(dǎo)PC12細(xì)胞活性的影響,結(jié)果表明FJ1對受損細(xì)胞具有保護(hù)作用且具有時(shí)間和劑量依賴性。流式細(xì)胞技術(shù)檢測細(xì)胞活性氧水平[26],細(xì)胞線粒體膜電勢以及細(xì)胞凋亡,結(jié)果顯示FJ1能夠抑制由PQ導(dǎo)致的ROS和凋亡的增長,提高M(jìn)MP水平。蛋白免疫印跡法考察ERK, p38, JNK, p-ERK, p-p38, p-JNK, Bcl-2, Bax的蛋白表達(dá)水平變化,結(jié)果表明,ERK, p38, JNK蛋白表達(dá)水平?jīng)]有明顯變化,p-ERK, p-p38, p-JNK, Bc1-2的蛋白表達(dá)水平隨著FJ1濃度升高而降低,而Bax蛋白表達(dá)水平則隨著FJ1濃度升高而增加。 本論文首次研究了本實(shí)驗(yàn)室提取的大吳風(fēng)草活性提取物FJ1的抗炎癥以及抗帕金森癥活性,為其臨床治療炎癥相關(guān)疾病和帕金森癥等神經(jīng)退行性疾病提供實(shí)驗(yàn)依據(jù)。此外,有研究表明,近年來帕金森癥多發(fā)與PQ的廣泛應(yīng)用存在一定關(guān)聯(lián),本實(shí)驗(yàn)采用PQ作為損傷因素,篩選并驗(yàn)證FJ1對神經(jīng)細(xì)胞的保護(hù)作用,使得本研究具有環(huán)境學(xué)與流行病學(xué)的雙重意義。
[Abstract]:3 beta - angelicoyl -8p, 10p- two hydroxybuddle -7 (11) - -12,8 alpha - lactone (FJ1) is an active compound extracted from the traditional Chinese herbal medicine. It is a perennial herb of perennial herb. The whole medicinal plant is called lieneronium. It is mainly distributed in Southeast and south of China, Japan, and [1] in Korea, for example, cold, coughing, and inflammation. So many kinds of diseases have good curative effect, so it is speculated that the active extract has the effect of anti-inflammatory. The study of FJ1 is the subject of screening of various active products extracted from our laboratory.
Inflammation is the body's response to tissue damage caused by microbial infection and other nociceptive stimuli. It is a very common and very important defense mechanism. However, low resolution and uncontrolled inflammatory response can lead to a chronic inflammatory state associated with a variety of human diseases, including cancer. Verify the anti validation activity of FJ1 and clarify its mechanism. The experiment used LPS (Lipopolysaccharide) to induce murine mononuclear macrophage leukemia cell RAW264.7 to construct an inflammatory cell model.MTT test to detect the cytotoxic activity of FJ1 and the protective activity to LPS damaged cells. The results showed that all of the two had time and dose dependence. In a safe range, FJ1 could improve the cell activity of the protective group. The production of NO was measured by the nitric oxide kit. The results showed that FJ1 obviously inhibited the NO amount produced by RAW264.7 induced by LPS, and was dose-dependent. Flow cytometry was used to detect the level of reactive oxygen species in cells. The results were in accordance with the above conclusion. Protein immunoblotting was in accordance with the results of Western blot. ERK, p38, JNK, p-ERK, p-p38, p-JNK[9], NF- kappa B, I kappa B- alpha, and P-1 kappa B- alpha protein expression level. The increase of.RT-PCR detected the RNA expression level of inflammation related genes iNOS, COX-2 and TNF-a. The results showed that the three were significantly decreased with the increase of FJ1 concentration. To sum up, FJ1 has a significant protective effect on the inflammatory cell model, suggesting that the potential anti-inflammatory activity of FJ1 may become a traditional Chinese medicine for the development of the world medicine. A contribution.
Parkinson's disease (Parkinson's disease, PD) is a common degenerative disease of the nervous system. Its clinical manifestations include static tremor, slow motion, myotonic and postural gait disorders, and patients with depression, constipation and sleep disorders, and other non motor symptoms, bring great pain to the patients and their families. Parkinson's disease and oxidation Stress has a close association, and FJ1's anti - inflammatory activity in the anti - inflammatory activity suggests its potential anti Parkinson activity. The classic Parkinson's disease cell model is constructed by 1- methyl -4- phenyl -1,2,3,6- four hydropyridine (MPTP) induced Rattus norvegicus adrenal chromaffin cells PC12 cell lines, and paraquat (P). Q) is a common agricultural toxin, its structure is similar to the active part of the MPTP, so it has been used to construct a new Parkinson's disease model in recent years. This study is the first time that this model is used to construct this model in our laboratory. So it is necessary to detect the cytotoxic activity of PQ by testing its various indexes by.MTT test, and the result shows P. Q inhibited the proliferation of PC12 and had a significant dose and time dependence. Flow cytometry was used to determine the mitochondrial membrane potential [24] and apoptosis. The results showed that the high concentration of PQ reduced the cell membrane potential and induced the apoptosis of PC12. The results were all consistent with the reported data of MPTP and other laboratory construction of the model, which could be used as a follow-up. In the study of neural cell protection experiments. In the study of the protective activity of FJ1 to PC12, MTT test detected the effect of FJ1 on the activity of PC12 cells induced by PQ. The results showed that FJ1 had protective effects on damaged cells and had time and dose dependence. Flow cytometry was used to detect the level of reactive oxygen species ([26]), cell mitochondrial membrane potential and cells of cells. The results showed that FJ1 could inhibit the growth of ROS and apoptosis caused by PQ and increase the level of MMP. The protein expression level of ERK, p38, JNK, p-ERK, p-p38, p-JNK, Bcl-2, Bax protein expression level was examined by Western blot. With the increase of FJ1 concentration, the expression level of Bax increased with the increase of FJ1 concentration.
In this paper, we first studied the anti inflammatory and anti Parkinson activity of FJ1 extracted from our laboratory, and provide experimental basis for its clinical treatment of inflammation related diseases and neurodegenerative diseases such as Parkinson's disease. In addition, some studies have shown that in recent years, many cases of Parkinson's disease are associated with the extensive use of PQ. In this study, PQ was used as a damage factor to screen and verify the protective effect of FJ1 on neurons. This study has the dual meaning of environmental science and epidemiology.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R742.5

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