【摘要】：背景與目的帕金森病(parkinson disease,PD)是一種神經(jīng)系統(tǒng)變性疾病,其主要病理特征是黑質(zhì)多巴胺(Dopamine,DA)神經(jīng)元細(xì)胞死亡和黑質(zhì)紋狀體通路退化。由于黑質(zhì)-紋狀體通路的耗盡,黑質(zhì)對(duì)丘腦產(chǎn)生明顯的抑制作用使對(duì)肌肉運(yùn)動(dòng)和肌張力的控制減弱,最終出現(xiàn)靜止性震顫、肌強(qiáng)直和行動(dòng)遲緩等臨床癥狀。目前研究表明,多種因素如氧化應(yīng)激、線粒體功能損傷、炎癥反應(yīng)、蛋白酶體功能障礙等參與PD的發(fā)生,但具體機(jī)制尚未完全清楚。姜黃素是姜黃的有效成分,后者已被廣泛用作食物調(diào)料和傳統(tǒng)草藥。姜黃素是從姜黃根莖中提取的一種酚性色素,具有多種藥理作用如抗炎、抗氧化、清除自由基等研究。研究表明姜黃素能夠通過(guò)誘導(dǎo)Bcl-2的表達(dá)來(lái)抑制MPP+誘導(dǎo)的細(xì)胞內(nèi)活性氧累物質(zhì)的生成,從而維持線粒體膜電位的穩(wěn)定性,減少細(xì)胞色素C的釋放,最終起到對(duì)抗氧化應(yīng)激的作用。同時(shí)姜黃素能增加谷胱甘肽的水平,抑制脂質(zhì)過(guò)氧化反應(yīng)等起到保護(hù)多巴胺神經(jīng)元的作用。賴氨酸殘基乙酰化對(duì)蛋白翻譯后修飾和調(diào)節(jié)起到重要作用,它在生物體內(nèi)普遍存在。有文獻(xiàn)報(bào)道,線粒體內(nèi)超過(guò)20%的蛋白存在乙�；F(xiàn)象。Sir T3是一種依賴于煙酰胺腺嘌呤二核苷酸(NAD)的Ⅲ類去乙�；�,主要存在于心臟、腦、腎臟及肝臟等細(xì)粒體含量豐富的組織器官中,是Sirtuin家族成員之一。近年來(lái),人們發(fā)現(xiàn)Sirt3能夠?qū)δ切┮阴；木�粒體蛋白進(jìn)行脫乙酰化從而起到維持線粒體正常生理功能的作用,能夠有效降低氧化應(yīng)激負(fù)荷引起的疾病的發(fā)生。而其降低氧化應(yīng)激損傷的機(jī)制可能通過(guò)乙�；饔眉せ罴せ畈骖^框轉(zhuǎn)錄因子O亞族3a(FOXO3a,forkhead box O 3a)從而上調(diào)ROS清除系統(tǒng)如MnSOD和過(guò)氧化氫酶catalase(CAT)的表達(dá)有關(guān)。前期實(shí)驗(yàn)已經(jīng)證實(shí),姜黃素能夠通過(guò)調(diào)控Sirt3的表達(dá)對(duì)魚藤酮誘導(dǎo)慢性PD SD大鼠模型起到保護(hù)作用。那么姜黃素能否通過(guò)激活Sirt3激活FOXO3a進(jìn)而上調(diào)MnSOD和CAT表達(dá)來(lái)抑制魚藤酮誘導(dǎo)的SH-SY5Y細(xì)胞的損傷或者凋亡,是本實(shí)驗(yàn)研究所要探討問(wèn)題之一。為此,本研究選擇姜黃素處理魚藤酮誘導(dǎo)的SH-SY5Y的細(xì)胞模型,檢測(cè)細(xì)胞活力,ROS含量,SIRT3、FOXO3a、Mn-SOD和CAT蛋白的表達(dá)情況,探討姜黃素對(duì)PD細(xì)胞模型的保護(hù)作用及姜黃素、SIRT3、FOXO3a與ROS清除系統(tǒng)的關(guān)系及其所介導(dǎo)的相關(guān)機(jī)制,為臨床治療提供理論依據(jù)和新的思路。材料與方法首先建立SH-SY5Y細(xì)胞模型,再將SH-SY5Y細(xì)胞,按隨機(jī)區(qū)組法分為對(duì)照組(不加藥物處理)、魚藤酮(終濃度0.1uM)模型組、姜黃素預(yù)處理組(終濃度分別為0.5μM、1.0μM、5.0μM、10.0μM)共6組。采用MTT檢測(cè)細(xì)胞活力,流式細(xì)胞儀檢測(cè)ROS含量,Western-blot法檢測(cè)細(xì)胞內(nèi)SIRT3、FOXO3a和Mn-SOD及CAT蛋白的表達(dá)。結(jié)果1.MTT檢測(cè)細(xì)胞活性:姜黃素為0.5umol/L-1.0umol/L時(shí)開始出現(xiàn)對(duì)魚藤酮致細(xì)胞損傷的保護(hù)作用,細(xì)胞活力較魚藤酮損傷組增加差異均具有顯著意義(P0.01),且1.0umol/L時(shí)作用最強(qiáng);5.0umol/L時(shí)其保護(hù)作用開始下降,單獨(dú)作用于SH-SY5Y細(xì)胞使其活力降低,但和魚藤酮共同作用于SH-SY5Y細(xì)胞則使其活力較魚藤酮損傷組增加,仍然發(fā)揮了保護(hù)細(xì)胞的作用,而沒(méi)顯示出協(xié)同損傷作用,但差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。2.流式細(xì)胞儀檢測(cè)細(xì)胞內(nèi)ROS含量的影響:0.1μmol/L魚藤酮處理細(xì)胞24h后,細(xì)胞內(nèi)ROS水平較對(duì)照組升高(P0.01);0.5μmol/L、1.0μmol/L姜黃素處理組較空白對(duì)照組和魚藤酮組細(xì)胞內(nèi)ROS降低(P0.01);5.0μmol/L、10μmol/L姜黃素處理組與魚藤酮組比較差異無(wú)統(tǒng)計(jì)學(xué)意義(p0.05)3.Western-blot檢測(cè)SIRT3、FOXO3a、Mn-SOD及CAT蛋白表達(dá):魚藤酮處理組較對(duì)照組表達(dá)降低,0.5μmol/L、1.0μmol/L姜黃素處理組較對(duì)照組、魚藤酮組表達(dá)升高,差異有統(tǒng)計(jì)學(xué)意義(P0.01);5.0μmol/L、10.0μmol/L組較魚藤酮組差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論1.魚藤酮具有明顯的神經(jīng)毒性,其毒性作用呈濃度依賴性:適宜濃度能用來(lái)制備SH-SY5Y細(xì)胞的PD模型,較高濃度時(shí)會(huì)直接造成細(xì)胞的死亡。2.魚藤酮誘導(dǎo)的SH-SY5Y細(xì)胞的PD模型中細(xì)胞的活力降低,細(xì)胞內(nèi)ROS含量增多,Sirt3、FOXO3a、Mn-SOD和CAT的表達(dá)降低。3.姜黃素可能通過(guò)誘導(dǎo)SIRT3表達(dá),使其去乙酰化激活FOXO3a,繼而上調(diào)MN-SOD及CAT的表達(dá)使細(xì)胞內(nèi)ROS含量減少,從而參與了PD的神經(jīng)保護(hù)。
[Abstract]:Background and objective Parkinson disease (PD) is a neurodegenerative disease whose main pathological feature is the death of the neurons of the substantia nigra (Dopamine, DA) and the degeneration of the nigrostriatal pathway. The depletion of the nigrostriatal pathway and the obvious inhibitory effect of the substantia nigra on the thalamus to the muscle movement and muscle tension The results show that many factors such as oxidative stress, mitochondrial dysfunction, inflammatory response, and proteasome dysfunction are involved in the occurrence of PD, but the specific mechanism is not completely clear. Curcumin is the effective component of curcumin and the latter has been widely used. Used as a food seasoning and traditional herbal medicine. Curcumin is a phenolic pigment extracted from the rhizomes of turmeric. It has a variety of pharmacological effects such as anti-inflammatory, antioxidant, and free radical scavenging. The study shows that curcumin can inhibit the formation of active MPP+ induced intracellular oxygen by inducing the expression of Bcl-2 to maintain the mitochondrial membrane. The stability of the potential, reducing the release of cytochrome C, eventually plays a role in antagonism to oxidative stress. Meanwhile curcumin can increase the level of glutathione, inhibit lipid peroxidation and protect the dopamine neurons. Lysine residue acetylation plays an important role in the modification and regulation of protein translation. It is in vivo It has been reported that more than 20% of the mitochondria in mitochondria have acetylation,.Sir T3 is a kind of class III deacetylase dependent on nicotinamide adenine dinucleotide (NAD), which is one of the members of the Sirtuin family, which is one of the members of the Sirtuin family, which are mainly in the heart, brain, kidney and liver. Sirt3 can deacetylation of those acetylated mitochondrial proteins to maintain the normal physiological function of mitochondria, and can effectively reduce the occurrence of diseases caused by oxidative stress load. The mechanism for reducing oxidative stress damage may activate the O subgroup 3A (FOXO3a) by acetylated activation. Forkhead box O 3a) up regulate the expression of ROS scavenging system such as MnSOD and catalase catalase (CAT). Earlier experiments have proved that curcumin can protect the rat model of rotenone induced chronic PD SD rat by regulating the expression of Sirt3. The expression of T to inhibit the damage or apoptosis of rotenone induced SH-SY5Y cells is one of the problems to be discussed in this study. Therefore, this study selected curcumin to treat the cell model of rotenone induced SH-SY5Y, and detected the expression of cell vitality, ROS content, SIRT3, FOXO3a, Mn-SOD and CAT protein, and explored the model of curcumin to the PD cell model. The protective effect of curcumin, the relationship between curcumin, SIRT3, FOXO3a and ROS scavenging system and its related mechanism, provide theoretical basis and new ideas for clinical treatment. Materials and methods first set up SH-SY5Y cell model, and then divide SH-SY5Y cells into control group (without drug treatment), and rotenone (final concentration 0.1uM) model. Group, curcumin pretreatment group (the final concentration was 0.5 mu M, 1 mu M, 5 mu M, 10 M) in 6 groups. The cell viability was detected by MTT, the content of ROS was detected by flow cytometry, and the expression of SIRT3, FOXO3a and Mn-SOD and CAT protein in cell was detected by Western-blot method. The protective effect of ketone induced cell damage was significantly higher than that of rotenone injury group (P0.01), and the effect of 1.0umol/L was the strongest. When 5.0umol/L, the protective effect began to decrease, and the activity of SH-SY5Y cells was reduced, but with rotenone, the activity of rotenone in SH-SY5Y cells was more than the damage of rotenone. Group increased, still play the role of protective cells, but did not show synergistic damage, but the difference was not statistically significant (P0.05).2. flow cytometry to detect the effect of intracellular ROS content: after 0.1 u mol/L rotenone treated cells 24h, the level of intracellular ROS was higher than the control group (P0.01); 0.5 mu mol/L, 1 mu mol/L curcumin treatment group is more blank. The cell ROS in the control group and rotenone group decreased (P0.01), and the difference was not statistically significant between the 5 mu mol/L, 10 mol/L curcumin treatment group and the rotenone group (P0.05) 3.Western-blot detection SIRT3, FOXO3a, Mn-SOD and CAT protein expression: the rotenone treatment group was lower than the control group, 0.5 Mu mol/L, 1 mu mol/L curcumin treatment group compared with the control group, the fish Teng group. The expression of ketone group was higher, the difference was statistically significant (P0.01). There was no significant difference between 5 and 10 mol/L groups compared with rotenone group (P0.05). Conclusion 1. rotenone has obvious neurotoxicity, its toxic effect is concentration dependent: the appropriate concentration can be used to prepare PD model of SH-SY5Y cells, and the cell death will be directly caused by high concentration. .2. in the PD model of rotenone induced SH-SY5Y cells, the activity of cells decreased, the content of ROS in the cells increased. The expression of Sirt3, FOXO3a, Mn-SOD and CAT reduced.3. curcumin by inducing SIRT3 expression by inducing its deacetylation activation FOXO3a. Protect.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R742.5

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