本文選題：帕金森病 + 人參皂苷Rg3��； 參考：《揚(yáng)州大學(xué)》2013年碩士論文

【摘要】：帕金森病(Parkinson's disease,PD)是一種常見于中老年人的神經(jīng)系統(tǒng)退行性疾病,其主要病理特征為黑質(zhì)(Substantia Nigra,SN)致密部多巴胺(dopamine,DA)能神經(jīng)元的進(jìn)行性變性死亡和路易氏小體(Lewy body,LB)的形成,引起黑質(zhì)紋狀體通路中DA水平的降低,最終導(dǎo)致整個(gè)基底神經(jīng)節(jié)環(huán)路功能的改變�；颊叽蠖啾憩F(xiàn)為運(yùn)動(dòng)遲緩、肌肉僵直、震顫和步態(tài)不穩(wěn)等。PD發(fā)病機(jī)制復(fù)雜,一般認(rèn)為可能與氧化應(yīng)激、線粒體損傷、神經(jīng)免疫炎癥和神經(jīng)興奮性毒性有關(guān)。 人參皂苷Rg3(ginsenoside Rg3,以下簡(jiǎn)稱Rg3)是中國(guó)傳統(tǒng)中藥人參的有效成分,Rg3能預(yù)防和治療癌癥、改善心臟血管功能、抗血小板凝集、保護(hù)腦神經(jīng)細(xì)胞和提高機(jī)體免疫力。本課題組前期研究表明,Rg3能減輕魚藤酮納米脂質(zhì)載體(R-NLC)誘導(dǎo)的PD模型大鼠行為運(yùn)動(dòng)障礙,并能有效抑制黑質(zhì)DA神經(jīng)元凋亡。進(jìn)一步研究發(fā)現(xiàn),這一作用與Rg3減弱氧化應(yīng)激反應(yīng)、上調(diào)Bcl-2和Bax的比值以及抑制‘caspase通路激活有關(guān)。 作為上述工作的繼續(xù),本實(shí)驗(yàn)采用魚藤酮誘導(dǎo)PC12細(xì)胞損傷模型,用R-NLC制備PD大鼠模型,從體內(nèi)、體外兩條途徑,進(jìn)一步確證人參皂苷Rg3對(duì)魚藤酮誘導(dǎo)的多巴胺能神經(jīng)元損傷的保護(hù)作用,著重探討p38MAPK-iNOS-NO通路在其中的作用,以進(jìn)一步探討Rg3保護(hù)DA能神經(jīng)元的分子生物學(xué)機(jī)制。主要結(jié)果如下： 1.人參皂苷Rg3對(duì)魚藤酮誘導(dǎo)的PD模型DA能神經(jīng)元的保護(hù)作用 方法：PCl2細(xì)胞分組：(1)對(duì)照組：0.1%DMSO;(2)模型組：1μM魚藤酮；(3)1μM魚藤酮+0.3125μRg3；(4)1μM魚藤酮+0.625μM Rg3;(5)1μM魚藤酮+1.25μM Rg3；(6)1μM魚藤酮+2.5μM Rg3；(7)1μM魚藤酮+5μM Rg3。Rg3溶液在魚藤酮前4小時(shí)加入,用MTT法測(cè)定24h各組細(xì)胞OD值,計(jì)算細(xì)胞存活率,流式細(xì)胞術(shù)測(cè)定細(xì)胞凋亡率。 動(dòng)物分組：(1)對(duì)照組：sc空白NLC+ig0.5%CMC-Na；(2)模型組：sc R-NLC+ig0.5%CMC-Na；(3)陽性藥物組：sc R-NLC+ig司來吉蘭(11mg/kg)；(4)Rg3低劑量組：sc R-NLC+igRg3(3mg/kg)；(5)Rg3中劑量組：sc R-NLC+igRg3(6mg/kg)；(6)Rg3高劑量組：sc R-NLC+igRg3(12mg/kg).大鼠皮下注射(sc)R-NLC,首劑量0.5mg/kg,第二次0.8mg/kg,此后每次1mg/kg,全程2天一次,共28天。其它藥物提前3天灌胃給藥,每天一次,共31天。末次注射給藥后24h,分離大鼠黑質(zhì)和紋狀體。以評(píng)分法考察大鼠行為學(xué)表現(xiàn)、HE染色觀察黑質(zhì)細(xì)胞形態(tài),高效液相-電化學(xué)法測(cè)定紋狀體DA含量。 結(jié)果：(1)對(duì)PC12細(xì)胞損傷的影響：模型組OD值為0.340±0.007,細(xì)胞存活率為69.5%,0.3125、0.625、1.25、2.5、5μMRg3組OD值分別為0.354±0.011(P0.05),0.388±0.018(P0.01),0.423±0.009(P0.01),0.441±0.007(P0.01)和0.416±0.007(P0.01),細(xì)胞存活率分別為72.4%,79.3%,86.5%,90.3%和85.1%,與模型組比較有顯著升高。 (2)對(duì)PC12細(xì)胞凋亡的影響：魚藤酮使PC12細(xì)胞出現(xiàn)早期和晚期凋亡,總凋亡率達(dá)71.1%(P0.01)。1.25,2.5μMRg3預(yù)處理后,細(xì)胞凋亡率下降為47.9%和28.1%(P0.01)。 (3)對(duì)PD大鼠外觀行為和DA能神經(jīng)元的影響：①行為學(xué)評(píng)分：與模型組(3.83±3.13)評(píng)分比較,低、中、高劑量Rg3干預(yù)后,評(píng)分分別為1.57±0.98、1.57±0.98和1.43±1.13,呈顯著性降低(P0.05)。②黑質(zhì)HE染色：與對(duì)照組相比,模型組大鼠黑質(zhì)神經(jīng)元出現(xiàn)核固縮,胞體縮小,胞內(nèi)尼氏體明顯減少；Rg3干預(yù)后能明顯改善上述變化。③紋狀體DA含量：模型組紋狀體DA含量為0.57±0.11nmol/g,較對(duì)照組(14.15±3.68nmol/g)顯著降低(P0.01)。低、中、高劑量Rg3組DA含量分別為1.38±0.79nmol/g (P0.05)、1.15±0.34nmol/g (P0.01)和9.49±6.93nmol/g (P0.01),與模型組比較,呈顯著升高。 結(jié)論：人參皂苷Rg3可抑制體內(nèi)、外魚藤酮誘導(dǎo)的PD模型DA能神經(jīng)元損傷,并能抑制魚藤酮誘導(dǎo)的PC12細(xì)胞凋亡。 2.人參皂苷Rg3對(duì)魚藤酮誘導(dǎo)的PD模型DA能神經(jīng)元iNOS活力和NO含量的影響 方法：PC12細(xì)胞分組：(1)對(duì)照組：0.1%DMSO;(2)模型組：1μM魚藤酮；(3)1μM魚藤酮+1.25μM Rg3；(4)1μM魚藤酮+2.5μM Rg3；大鼠分組和給藥方法同第一部分。比色法測(cè)定細(xì)胞和大鼠黑質(zhì)iNOS活力和NO含量。 結(jié)果：(1)細(xì)胞iNOS活力和NO含量：模型組iNOS活力和NO含量分別為0.692±0.030U/mL和7.67±1.45μM,與對(duì)照組(0.431±0.051U/mL和2.90±0.53μM)比較,呈顯著升高(P0.01)；1.25、2.51μM Rg3組iNOS活力分別為0.636±0.031U/mL和0.643±0.009U/mL,NO含量分別為4.84±1.20μM和4.91±1.72μM,與模型組比較,呈顯著下降(P0.05或0.01)。 (2)大鼠黑質(zhì)iNOS活力和NO含量：模型組黑質(zhì)iNOS活力和NO含量分別為0.223±0.044U/mgprot和8.06±2.35μmol/gprot,與對(duì)照組(0.142±0.016U/mgprot和4.57±1.30μmol/gprot)比較,呈顯著升高(P0.01)；低、中、高劑量Rg3組黑質(zhì)iNOS活力分別為0.140±0.033U/mgprot,0.132±0.023U/mgprot和0.145±0.035U/mgprot,NO含量分別為5.26±1.18μmol/gprot,4.41±1.12μmol/gprot和5.40±1.03μmol/gprot,與模型組比較,呈顯著下降(低、高劑量組P0.05,中劑量組P0.01)。 結(jié)論：人參皂苷Rg3能顯著抑制體內(nèi)、外魚藤酮誘導(dǎo)的iNOS活力增加和NO產(chǎn)生,提示Rg3可能通過抑制NO的生成來保護(hù)DA能神經(jīng)元。 3.人參皂苷Rg3對(duì)魚藤酮誘導(dǎo)的PD模型DA能神經(jīng)元p38MAPK與NO的關(guān)系研究 方法：(1)體外實(shí)驗(yàn)：分組：(1)對(duì)照組：0.1%DMSO；(2)模型組：1μM魚藤酮；(3)1μM魚藤酮+10μM SB203580; MTT法測(cè)定細(xì)胞存活率,比色法測(cè)定iNOS活力和NO含量。 (2)體內(nèi)實(shí)驗(yàn)：大鼠分組和給藥方法同第一部分。末次給藥后24h,Western blot法測(cè)定黑質(zhì)中p-p38MAPK蛋白表達(dá)；免疫組化法測(cè)定p-p38MAPK和iNOS的表達(dá),并對(duì)其進(jìn)行非線性相關(guān)分析。 結(jié)果：(1)p38MAPK抑制劑SB203580對(duì)細(xì)胞存活率、iNOS活力和NO含量的影響：SB203580組細(xì)胞存活率為81.0%,較模型組(69.0%)顯著升高(P0.05)。模型組iNOS活力和NO含量分別為0.692±0.030U/mL和7.67±1.45μM, SB203580干預(yù)后,分別下降為0.634±0.014U/mL和5.27±1.66μM (P0.05)。提示魚藤酮引起PD損傷的機(jī)制與p38MAPK有關(guān),p38MAPK通路可能調(diào)控了iNOS活力和NO水平。 (2)對(duì)p-p38MAPK表達(dá)的影響：與對(duì)照組相比,模型組黑質(zhì)中p-p38MAPK表達(dá)水平顯著增強(qiáng),Rg3預(yù)處理后,p-p38MAPK表達(dá)水平顯著降低,提示Rg3抗PD大鼠黑質(zhì)DA能神經(jīng)元凋亡的作用機(jī)制可能與下調(diào)p-p38MAPK水平有關(guān)。 (3)p-p38MAPK和iNOS表達(dá)量的相關(guān)性分析：與對(duì)照組相比,模型組黑質(zhì)中p-p38MAPK和iNOS陽性細(xì)胞百分?jǐn)?shù)均有顯著升高；Rg3預(yù)處理后,兩種蛋白陽性細(xì)胞百分?jǐn)?shù)都呈明顯降低。對(duì)兩種蛋白陽性細(xì)胞百分?jǐn)?shù)進(jìn)行相關(guān)性分析,表明p-p38MAPK與iNOS呈正相關(guān)。提示Rg3可能是通過抑制p38MAPK的磷酸化,從而減少iNOS的活化。 結(jié)論：Rg3可能是通過抑制p38MAPK的磷酸化,繼而降低iNOS活化水平、減少NO生成,而產(chǎn)生抗PD作用的。
[Abstract]:Parkinson's disease (Parkinson's disease PD) is one of the most common in the elderly neurodegenerative disease, the main pathological features of substantia nigra (Substantia Nigra, SN) compacta dopamine (dopamine, DA) neurons degeneration and death of Lewis corpuscles (Lewy body, LB) formed by reduce the level of DA in the nigrostriatal pathway, the basal ganglia circuits function change. Most patients showed muscle rigidity, bradykinesia, tremor and gait instability.PD pathogenesis is complex, generally considered possible mitochondrial oxidative stress, injury, inflammation and immune related neural excitotoxicity.
Ginsenoside Rg3 (ginsenoside Rg3, hereinafter referred to as Rg3) is the active ingredient of traditional Chinese medicine China ginseng, Rg3 can prevent and treat cancer, improve cardiovascular function, platelet aggregation, protect brain cells and improve immunity. Ourprevious studies showed that Rg3 can reduce the rotenone nanostructured lipid carrier (R-NLC) behavior of PD model rats induced dyskinesia, and can inhibit the apoptosis of substantia nigra DA neurons. Further studies showed that the effect of Rg3 and decreased oxidative stress, upregulation of Bcl-2 and the ratio of Bax and inhibit the activation of caspase. "
As a continuation of the above work, this experiment used rotenone induced PC12 cell injury model, model, PD rats were prepared with R-NLC from the body, in two ways, further confirmed the protective effect of ginsenoside Rg3 on neuronal injury induced by rotenone dopamine, focused on the p38MAPK-iNOS-NO pathway in the role in molecular biology mechanism to further explore the Rg3 protect DA neurons. The main results are as follows:
Protective effect of 1. ginsenoside Rg3 on DA neurons of rotenone induced PD model
Methods: PCl2 cell groups: (1) control group: 0.1%DMSO (2); model group: 1 M rotenone; (3) 1 M rotenone +0.3125 Rg3; (4) 1 M +0.625 M Rg3 rotenone; (5) 1 M +1.25 M Rg3 rotenone; (6) 1 M +2.5 M Rg3 rotenone; (7) 1 M +5 M rotenone solution Rg3.Rg3 to 4 hours before the determination of 24h in rotenone, each cell OD value by MTT method, the cell survival rate, cell apoptosis was detected by flow cytometry.
Animal grouping: (1) blank control group: SC NLC+ig0.5%CMC-Na (2); model group: SC R-NLC+ig0.5%CMC-Na; (3) positive drug group: SC R-NLC+ig selegiline (11mg/kg); (4) low dose Rg3 group: SC R-NLC+igRg3 (3mg/kg); (5) Rg3 middle dose group: SC R-NLC (+igRg3 6mg/kg (6) Rg3); high dose group: SC R-NLC+igRg3 (12mg/kg). Rats by subcutaneous injection of R-NLC (SC), the first dose of 0.5mg/kg, second 0.8mg/kg, then 1mg/kg every time, the entire 2 days time, a total of 28 days to 3 days in advance. Other drugs administered orally, once a day, a total of 31 day. The last injection after administration of 24h, the separation of the substantia nigra and striatum in rats. To score method to study the behavior of rats, to observe the morphology of nigral cells stained with HE, DA content in striatum was determined by HPLC.
Results: (1) effect on PC12 cell injury model: od group is 0.340 + 0.007, the survival rate of cells was 69.5% 0.3125,0.625,1.25,2.5,5, the OD value of MRg3 group were 0.354 + 0.011 (P0.05), 0.388 + 0.018, 0.423 + 0.009 (P0.01) (P0.01), 0.441 + 0.007 and 0.416 + 0.007 (P0.01) (P0.01), cell survival rates were 72.4%, 79.3%, 86.5%, 90.3% and 85.1%, compared with the model group increased significantly.
(2) the effect of rotenone on the apoptosis of PC12 cells: rotenone induced early and late apoptosis of PC12 cells, and the total apoptosis rate reached 71.1% (P0.01). After pretreatment with.1.25,2.5 MRg3, the apoptosis rate decreased to 47.9% and 28.1% (P0.01), respectively.
(3) influence on the appearance and behavior of PD rats and DA neurons: scores of praxiology: model group (3.83 + 3.13) score, low, high dose intervention of Rg3 scores were 1.57 + 0.98,1.57 + 0.98 and 1.43 + 1.13, was significantly decreased (P0.05). The substantia nigra HE staining: compared with the control group, the model group of rat substantia nigra neurons karyopyknosis, cell body shrinkage, cytoplasmic Nissl body reduced obviously; after Rg3 intervention can significantly improve the change. The content of striatal DA: model group DA in striatum was 0.57 + 0.11nmol/g, 14.15 + 3.68nmol/g (compared with the control group) decreased significantly (P0.01). The low and high dose of Rg3 group DA was 1.38 + 0.79nmol/g (P0.05), 0.34nmol/g (P0.01) 1.15 + and 9.49 + 6.93nmol/g (P0.01), compared with the model group was significantly increased.
Conclusion: ginsenoside Rg3 can inhibit the injury of DA neurons in PD model induced by rotenone and inhibit the apoptosis of PC12 cells induced by rotenone.
Effect of 2. ginsenoside Rg3 on iNOS activity and NO content of DA neurons in PD model induced by rotenone
Methods: PC12 cells were divided into: (1) control group: 0.1%DMSO; (2) model group: 1 M M rotenone; (3) 1 micron M rotenone +1.25 M M; (4) 1, M, rotenone +2.5, small M Rg3; rats were divided into groups and administrated with the first part.
Results: (1) iNOS activity and NO content of cells: iNOS activity and NO content in model group were 0.692 + 0.030U/mL and 7.67 + 1.45 M, and the control group (0.431 + 0.051U/mL and 2.90 + 0.53 M), was significantly increased (P0.01); 1.25,2.51 M group Rg3 iNOS activity were 0.636 + 0.031U/mL and 0.643 + 0.009U/mL, the content of NO was 4.84 + 1.20 M and 4.91 + 1.72 M, compared with the model group was significantly decreased (P0.05 or 0.01).
(2) the substantia nigra iNOS activity and NO content in rats: the substantia nigra iNOS activity and NO content were 0.223 + 0.044U/mgprot and 8.06 + 2.35 mol/gprot, and the control group (0.142 + 0.016U/mgprot and 4.57 + 1.30 mol/gprot), was significantly increased (P0.01); low, high dose Rg3 group of substantia nigra iNOS activity were 0.140 + 0.033U/mgprot, 0.132 + 0.023U/mgprot and 0.145 + 0.035U/mgprot, the content of NO was 5.26 + 1.18 mol/gprot 4.41 + 1.12 mol/gprot and 5.40 + 1.03 mol/gprot, compared with the model group was significantly decreased (low, high dose group, middle dose group P0.05, P0.01).
Conclusion: ginsenoside Rg3 can significantly inhibit rotenone induced iNOS activity and NO production, suggesting that Rg3 may protect DA neurons by inhibiting the generation of NO.
3. ginsenoside Rg3 on the relationship between p38MAPK and NO in DA neurons of rotenone induced PD model
Methods: (1) in vitro experiments: grouped: (1) control group: 0.1%DMSO; (2) model group: 1 M rotenone; (3) 1 M M rotenone +10 M SB203580; MTT method was used to measure cell viability, and iNOS activity and NO content were determined by colorimetry.
(2) in vivo experiments: the rats were divided into groups. The rats were divided into groups. The same method was given in the first part. After the last administration, the expression of p-p38MAPK protein in substantia nigra was detected by 24h Western blot method. The expression of p-p38MAPK and iNOS was detected by immunohistochemistry, and the correlation analysis between them was made.
Results: (1) p38MAPK inhibitor SB203580 on cell viability, iNOS activity and NO content: SB203580 group cell survival rate was 81%, compared with the model group (69%) was significantly increased (P0.05). INOS activity and NO content in model group were 0.692 + 0.030U/mL and 7.67 + 1.45 M and the intervention of SB203580 decreased to 0.634 + 0.014U/mL and 5.27 + 1.66 M (P0.05). It is suggested that rotenone induced PD damage mechanism associated with p38MAPK, p38MAPK pathway may regulate the activity of iNOS and NO levels.
(2) effects on the expression of p-p38MAPK: compared with the control group, model group, the expression level of p-p38MAPK in the substantia nigra was significantly enhanced after pretreatment with Rg3, the expression level of p-p38MAPK was significantly decreased, suggesting that Rg3 anti PD rat nigral DA neuron apoptosis mechanism may be related to the down-regulation of p-p38MAPK level.
(3) analysis the correlation between the expression of p-p38MAPK and iNOS: compared with the control group, the model group in the substantia nigra of p-p38MAPK and iNOS positive cell percentage increased significantly; after Rg3 pretreatment, two protein positive cell percentage were significantly reduced. The correlation analysis of two protein positive cells showed a positive percentage, p-p38MAPK iNOS. Suggesting that Rg3 may be through inhibiting the phosphorylation of p38MAPK, thereby reducing the activation of iNOS.
Conclusion: Rg3 may inhibit the phosphorylation of p38MAPK, then reduce the activation level of iNOS, reduce the formation of NO, and produce anti PD effect.

【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R-332;R285.5

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