牵正散水煎剂对PSI诱导的体外帕金森病模型的影响
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摘要
目的:建立PC12细胞帕金森病模型;探讨牵正散水煎剂对PSI诱导PC12细胞的体外帕金森病模型的增殖的影响。方法:利用MTT法检测牵正散水煎剂对PC12细胞的细胞毒性作用;采用DAPI染色及伊红染色相结合的方法,利用倒置荧光显微镜观察确定PSI诱导PC12细胞形成的帕金森病细胞模型的合适剂量;利用HE染色观察PSI诱导PC12细胞的体外帕金森病模型的嗜酸性小体;利用MTT法确定牵正散水煎剂对PSI诱导PC12细胞的体外帕金森病模型增殖的影响。结果:MTT法测定牵正散水煎剂对PC12细胞的毒性作用,计算得到IC_0,IC_(25)及IC_(50),分别为3.038 6 mg/mL、8.403 4 mg/mL、13.768 2 mg/mL;DAPI及伊红染色显示PSI在浓度为6μmol/L时,PC12细胞的形态改变及胞浆中嗜酸性小体表现最为典型;HE染色后,倒置显微镜下观察结果显示PSI作用下PC12形态及数量均发生改变;HE染色显示细胞数量减少,有聚集倾向,细胞体积增大,细胞胞浆内出现帕金森特征性病理改变路易小体的类似结构嗜酸性小体;MTT结果显示,不同浓度的牵正散水煎剂可以改善PSI对PC12细胞增殖的抑制作用。结论:获得PC12细胞帕金森病体外培养细胞模型;确定一定浓度的牵正散水煎剂可以不同程度地改善体外帕金森病细胞模型的增殖情况;为牵正散的临床应用提供实验依据,为临床药物治疗帕金森病提供新的思路。
Objective: To study the effect of Qianzheng San Decoction on Parkinson's disease(PD) model in vitro of PC12 cell induced by Proteasome Inhibitor I(PSI). Methods: MTT method was used to detect the cytotoxic effect of Qianzheng San Decoction on PC12 cells; DAPI staining and eosin staining were used to determine the appropriate concentration of PSI inducing PC12 cell leading to successful PD model; Eosinophilic body was observed with HE staining; MTT method was used to determine the effect of Qianzheng San Decoction on cell proliferation in the model. Results: In terms of toxic effect of Qianzheng San Decoction to PC12 by MTT method, IC_0, IC_(25) and IC_(50) were 3.038 6 mg/mL, 8.403 4 mg/mL and 13.768 2 mg/mL respectively. DAPI and eosin staining showed that the morphological changes and eosinophilic bodies in the cytoplasm of PC12 cells were most typical when PSI concentration was 6 μmol/L. After HE staining, inverted microscope observation showed that the morphology and quantity of PC12 changed under the action of PSI. HE staining showed a decrease in the number of cells, a tendency to aggregate, an increase in cell volume, and a similar eosinophilic body with Parkinson's characteristic pathological changes in the cytoplasm, suggesting successful modeling. MTT results showed that different concentrations of Qianzheng San Decoction could improve the inhibitory effect of PSI on the proliferation of PC12 cells. Conclusion: PC12 cell model of PD in vitro was successfully established; and the proliferation of PC12 could be improved by a certain concentration of the decoction. This study provided experimental basis for clinical application of Qianzheng San, and provided new ideas for the medication of PD.
Objective: To study the effect of Qianzheng San Decoction on Parkinson's disease(PD) model in vitro of PC12 cell induced by Proteasome Inhibitor I(PSI). Methods: MTT method was used to detect the cytotoxic effect of Qianzheng San Decoction on PC12 cells; DAPI staining and eosin staining were used to determine the appropriate concentration of PSI inducing PC12 cell leading to successful PD model; Eosinophilic body was observed with HE staining; MTT method was used to determine the effect of Qianzheng San Decoction on cell proliferation in the model. Results: In terms of toxic effect of Qianzheng San Decoction to PC12 by MTT method, IC_0, IC_(25) and IC_(50) were 3.038 6 mg/mL, 8.403 4 mg/mL and 13.768 2 mg/mL respectively. DAPI and eosin staining showed that the morphological changes and eosinophilic bodies in the cytoplasm of PC12 cells were most typical when PSI concentration was 6 μmol/L. After HE staining, inverted microscope observation showed that the morphology and quantity of PC12 changed under the action of PSI. HE staining showed a decrease in the number of cells, a tendency to aggregate, an increase in cell volume, and a similar eosinophilic body with Parkinson's characteristic pathological changes in the cytoplasm, suggesting successful modeling. MTT results showed that different concentrations of Qianzheng San Decoction could improve the inhibitory effect of PSI on the proliferation of PC12 cells. Conclusion: PC12 cell model of PD in vitro was successfully established; and the proliferation of PC12 could be improved by a certain concentration of the decoction. This study provided experimental basis for clinical application of Qianzheng San, and provided new ideas for the medication of PD.
引文
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[18] Zhang L,Chang M,Li H,et al.Proteomic changes of PC12 cells treated with proteasomal inhibitor PSI[J].Brain Res,2007,115(3):196-203.
[2] 刘宇翔,尹邦良,刘振华.中国帕金森病患者的经济负担及相关因素调查研究[J].中国现代医学杂志,2016,26(8):105-108.
[3] Alves G,Forsaa EB,Pedersen KF,et al.Epidemiology of Parkinson's disease[J].J Neurol,2008,255(Suppl 5):18-32.
[4] Lonneke ML de Lau,Monique MB Breteler.Epidemiology of Parkinson's disease[J].Lancet Neurology,2006,5(6):525-535.
[5] 陈军虎,刘远新,王新亭.帕金森病中医病因病机的研究进展[J].新疆中医药,2016,34(6):74-76.
[6] 陈笑丹,张丽萍.帕金森病的中医治疗进展[J].长春中医药大学学报,2016,32(1):213-215.
[7] Liu Y,GAO J,Peng M,et al.A Review on Central Nervous System Effects of Gastrodin[J].Frontiers in Pharmacology,2018,9:24.
[8] Wei W,Chen HY,Fan W,et al.Chinese medicine for idiopathic Parkinson's disease:A meta analysis of randomized controlled trials[J].Chinese Journal of Integrative Medicine,2017,23(1):55-61.
[9] Liu CT,Wu BY,Hung YC,et al.Decreased risk of dementia in migraine patients with traditional Chinese medicine use:a population-based cohort study[J].Oncotarget,2017,8(45):79680-79692.
[10] 李晓秀,王俊平,汲坤,等.牵正散提取物对帕金森病模型小鼠震颤、运动障碍、黑质神经元超微结构的影响[J].中国实验方剂学杂志,2015,21(21):130-133.
[11] 史亚楠,刘岑.帕金森病中医治疗进展[J].辽宁中医药大学学报,2015,17(11):105-108.
[12] 熊成成,蔡婉萍,林嘉娜,等.白附子不同炮制品药理作用评价研究[J].中药材,2016,39(8):1763-1766.
[13] 李晶峰,孙佳明,张辉.僵蚕的化学成分及药理活性研究[J].吉林中医药,2015,35(2):175-177.
[14] 史磊,张天锡,杜聪颖,等.中药全蝎活性成分、药理作用及临床应用研究进展[J].辽宁中医药大学学报,2015,17(4):89-91.
[15] 陈璐,张丽慧,张家凤,等.帕金森病细胞模型与实验研究[J].健康研究,2013,33(3):179-183.
[16] Guo DJ,Li F,Yu PH,et al.Neuroprotective effects of luteolin against apoptosis induced by 6-hydroxydopamine on rat pheochromocytoma PC12 cells[J].Pharm Biot,2013,51(2):190-196.
[17] 范振群,刘树民,周世慧,等.帕金森病体外细胞模型的研究进展[J].实验动物与比较医学,2013,33(1):74-78.
[18] Zhang L,Chang M,Li H,et al.Proteomic changes of PC12 cells treated with proteasomal inhibitor PSI[J].Brain Res,2007,115(3):196-203.
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