姜黃素對魚藤酮誘導的帕金森病SH-SY5Y細胞模型的抗氧化應激作用及其機制
發(fā)布時間：2018-08-03 07:46
【摘要】：背景與目的帕金森病(parkinson disease,PD)是一種神經系統(tǒng)變性疾病,其主要病理特征是黑質多巴胺(Dopamine,DA)神經元細胞死亡和黑質紋狀體通路退化。由于黑質-紋狀體通路的耗盡,黑質對丘腦產生明顯的抑制作用使對肌肉運動和肌張力的控制減弱,最終出現靜止性震顫、肌強直和行動遲緩等臨床癥狀。目前研究表明,多種因素如氧化應激、線粒體功能損傷、炎癥反應、蛋白酶體功能障礙等參與PD的發(fā)生,但具體機制尚未完全清楚。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誘導Bcl-2的表達來抑制MPP+誘導的細胞內活性氧累物質的生成,從而維持線粒體膜電位的穩(wěn)定性,減少細胞色素C的釋放,最終起到對抗氧化應激的作用。同時姜黃素能增加谷胱甘肽的水平,抑制脂質過氧化反應等起到保護多巴胺神經元的作用。賴氨酸殘基乙�；瘜Φ鞍追g后修飾和調節(jié)起到重要作用,它在生物體內普遍存在。有文獻報道,線粒體內超過20%的蛋白存在乙�；F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、腎臟及肝臟等細粒體含量豐富的組織器官中,是Sirtuin家族成員之一。近年來,人們發(fā)現Sirt3能夠對那些乙�；木�粒體蛋白進行脫乙�；瘡亩鸬骄S持線粒體正常生理功能的作用,能夠有效降低氧化應激負荷引起的疾病的發(fā)生。而其降低氧化應激損傷的機制可能通過乙�；饔眉せ罴せ畈骖^框轉錄因子O亞族3a(FOXO3a,forkhead box O 3a)從而上調ROS清除系統(tǒng)如MnSOD和過氧化氫酶catalase(CAT)的表達有關。前期實驗已經證實,姜黃素能夠通過調控Sirt3的表達對魚藤酮誘導慢性PD SD大鼠模型起到保護作用。那么姜黃素能否通過激活Sirt3激活FOXO3a進而上調MnSOD和CAT表達來抑制魚藤酮誘導的SH-SY5Y細胞的損傷或者凋亡,是本實驗研究所要探討問題之一。為此,本研究選擇姜黃素處理魚藤酮誘導的SH-SY5Y的細胞模型,檢測細胞活力,ROS含量,SIRT3、FOXO3a、Mn-SOD和CAT蛋白的表達情況,探討姜黃素對PD細胞模型的保護作用及姜黃素、SIRT3、FOXO3a與ROS清除系統(tǒng)的關系及其所介導的相關機制,為臨床治療提供理論依據和新的思路。材料與方法首先建立SH-SY5Y細胞模型,再將SH-SY5Y細胞,按隨機區(qū)組法分為對照組(不加藥物處理)、魚藤酮(終濃度0.1uM)模型組、姜黃素預處理組(終濃度分別為0.5μM、1.0μM、5.0μM、10.0μM)共6組。采用MTT檢測細胞活力,流式細胞儀檢測ROS含量,Western-blot法檢測細胞內SIRT3、FOXO3a和Mn-SOD及CAT蛋白的表達。結果1.MTT檢測細胞活性:姜黃素為0.5umol/L-1.0umol/L時開始出現對魚藤酮致細胞損傷的保護作用,細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時作用最強;5.0umol/L時其保護作用開始下降,單獨作用于SH-SY5Y細胞使其活力降低,但和魚藤酮共同作用于SH-SY5Y細胞則使其活力較魚藤酮損傷組增加,仍然發(fā)揮了保護細胞的作用,而沒顯示出協(xié)同損傷作用,但差異無統(tǒng)計學意義(P0.05)。2.流式細胞儀檢測細胞內ROS含量的影響:0.1μmol/L魚藤酮處理細胞24h后,細胞內ROS水平較對照組升高(P0.01);0.5μmol/L、1.0μmol/L姜黃素處理組較空白對照組和魚藤酮組細胞內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統(tǒng)計學意義(p0.05)3.Western-blot檢測SIRT3、FOXO3a、Mn-SOD及CAT蛋白表達:魚藤酮處理組較對照組表達降低,0.5μmol/L、1.0μmol/L姜黃素處理組較對照組、魚藤酮組表達升高,差異有統(tǒng)計學意義(P0.01);5.0μmol/L、10.0μmol/L組較魚藤酮組差異無統(tǒng)計學意義(P0.05)。結論1.魚藤酮具有明顯的神經毒性,其毒性作用呈濃度依賴性:適宜濃度能用來制備SH-SY5Y細胞的PD模型,較高濃度時會直接造成細胞的死亡。2.魚藤酮誘導的SH-SY5Y細胞的PD模型中細胞的活力降低,細胞內ROS含量增多,Sirt3、FOXO3a、Mn-SOD和CAT的表達降低。3.姜黃素可能通過誘導SIRT3表達,使其去乙�；せ頕OXO3a,繼而上調MN-SOD及CAT的表達使細胞內ROS含量減少,從而參與了PD的神經保護。
[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.
【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R742.5
本文編號：2161081
[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.
【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R742.5
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