【摘要】 目的:基于p53/p21通路探讨左归丸对帕金森病（Parkinson’s disease,PD）小鼠的脑保护作用机制。方法:采用随机数字表法将60只雄性C57BL/6小鼠分为对照组、模型组、左归丸低剂量组(1.8 g·kg^-1·d^-1)、左归丸中剂量组(3.6 g·kg^-1·d^-1)、左归丸高剂量组(7.2 g·kg^-1·d^-1)、美多芭组(50 mg·kg^-1·d^-1)。除对照组外，其余各组小鼠均通过腹腔注射1-甲基-4-苯基-1,2,3,6-四氢吡啶（1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP）(30 mg·kg^-1·d^-1)建立PD模型，连续5 d。从造模第1天起，各给药组同步开始灌胃给药，每日1次，持续10 d。采用行为学评估、免疫组化、Western Blot、Elisa、β-半乳糖苷酶（β-galactosidase,SA-β-gal）染色、免疫荧光染色法观察左归丸对PD小鼠细胞衰老的影响。结果:旷场实验结果显示，与对照组比较，模型组小鼠旷场运动总距离缩短（P<0.01）;与模型组比较，左归丸中剂量组、左归丸高剂量组及美多芭组小鼠运动总距离增加（P<0.01）。爬杆实验结果显示，与对照组比较，模型组小鼠爬杆时间延长（P<0.05）;与模型组比较，左归丸高剂量组爬杆时间缩短（P<0.05）。免疫组织化学染色及Western Blot法检测酪氨酸羟化酶（tyrosine hydroxylase,TH）蛋白结果显示，与对照组比较，模型组TH蛋白表达降低（P<0.01）;与模型组比较，左归丸中剂量组、左归丸高剂量组及美多芭组TH蛋白表达升高（P<0.05或P<0.01）。Elisa结果显示，与对照组比较，模型组黑质组织和纹状体组织多巴胺（dopamine,DA）、二羟基苯基乙酸（dihydroxy-phenyl acetic acid,DOPAC）、高香草酸（homovanillic acid,HVA）水平降低（P<0.01）;与模型组比较，左归丸低剂量组、左归丸中剂量组、左归丸高剂量组、美多芭组黑质组织和纹状体组织DA、DOPAC、HVA水平均升高（P<0.05或P<0.01）。免疫荧光双染检测结果显示，与对照组比较，模型组核纤层蛋白B1（nuclear lamina protein B1,Lamin B1）阳性细胞数减少（P<0.01），胶质纤维酸性蛋白（glial fibrillary acidic protein,GFAP）阳性细胞数增加（P<0.01）;与模型组比较，左归丸低剂量组、左归丸中剂量组、左归丸高剂量组、美多芭组Lamin B1阳性细胞数增加（P<0.05或P<0.01）,GFAP阳性细胞数减少（P<0.01）。Western blot检测结果显示，与对照组比较，模型组黑质组织p53（tumour protein 53,TP53）、细胞周期蛋白依赖性激酶抑制剂1A（Cyclin-dependent kinase inhibitor 1A,CDKN1A,p21）、细胞周期蛋白依赖性激酶抑制剂2A（Cyclin-dependent kinase inhibitor 2A,CDKN2A,p16）蛋白表达均升高（P<0.01）;与模型组相比，左归丸低剂量组、左归丸中剂量组、左归丸高剂量组、美多芭组黑质组织p53、p21、p16蛋白表达均降低（P<0.01）。SA-β-gal染色法结果显示，与对照组比较，模型组黑质组织SA-β-gal蛋白阳性表达面积增加（P<0.01）;与模型组相比，左归丸中剂量组、左归丸高剂量组、美多芭组黑质组织SA-β-gal蛋白阳性表达面积降低（P<0.05或P<0.01）。Elisa检测结果显示，与对照组比较，模型组黑质组织IL-6、IL-8水平均升高（P<0.01）;与模型组相比，左归丸低剂量组、左归丸中剂量组、左归丸高剂量组、美多芭组黑质组织IL-6、IL-8水平均降低（P<0.05或P<0.01）。结论:左归丸可能通过抑制p53/p21通路，减轻细胞衰老，从而对PD模型小鼠发挥保护作用。更多还原

【Abstract】 Objective: To explore the neuroprotective mechanism of Zuogui Pills in Parkinson’s disease（PD） mice based on the p53/p21 pathway. Methods: Sixty male C57 BL/6 mice were randomly divided into a control group,model group,low-dose Zuogui Pills group(1. 8 g·kg^-1·d^-1),medium-dose Zuogui Pills group(3. 6 g·kg^-1·d^-1),high-dose Zuogui Pills group(7. 2 g·kg^-1·d^-1),and Madopar group(50 mg·kg^-1·d^-1) using a random number table method. Except for the control group,all other groups were modeled by intraperitoneal injection with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine（MPTP）(30 mg·kg^-1·d^-1) for five consecutive days. From the first day of modeling,the treatment groups were administered orally once daily for 10 days. Behavioral assessment,immunohistochemistry,Western Blot,ELISA,β-galactosidase（SA-β-gal） staining,and immunofluorescence staining were used to observe the effect of Zuogui Pills on cellular senescence in PD mice. Results: The open field test results showed that,compared with the control group,the total distance traveled by mice in the model group was reduced（P < 0. 01）; compared with the model group,the total distance traveled by mice in the medium-dose and high-dose Zuogui Pill groups,as well as the Madopar group,was increased（P < 0. 01）. The pole-climbing test results showed that,compared with the control group,the time for mice in the model group to climb the pole was prolonged（P < 0. 05）; compared with the model group,the pole-climbing time was shortened in the high-dose Zuogui Pill group（P < 0. 05）. Immunohistochemistry and Western Blot detection of tyrosine hydroxylase（TH） protein showed that,compared with the control group,TH protein expression in the model group was decreased（P < 0. 01）; compared with the model group,TH protein expression was increased in the medium-dose and high-dose Zuogui Pill groups,as well as in the Madopar group（P < 0. 05 or P < 0. 01）. ELISA results showed that,compared with the control group,the levels of dopamine（DA）,dihydroxyphenyl acetic acid（DOPAC）,and homovanillic acid（HVA） in the substantia nigra and striatum tissues of the model group were decreased（P < 0. 01）; compared with the model group,the levels of DA,DOPAC,and HVA in the substantia nigra and striatum tissues were increased in the low-dose,medium-dose,and high-dose Zuogui Pill groups,as well as in the Madopar group（P < 0. 05 or P <0. 01）. Immunofluorescence double staining results showed that compared with the control group,the number of Lamin B1（nuclear lamina protein B1） positive cells in the model group was reduced（P < 0. 01）,and the number of GFAP（glial fibrillary acidic protein）positive cells was increased（P < 0. 01）. Compared with the model group,the numbers of Lamin B1 positive cells in the low-dose,medium-dose,and high-dose Zuogui pills groups,as well as in the Madopar group,were increased（P < 0. 01）,while the numbers of GFAP positive cells were decreased（P < 0. 05 or P < 0. 01）. Western blot results showed that compared with the control group,the protein levels of p53（tumor protein 53,TP53）,CDKN1A（Cyclin-dependent kinase inhibitor 1A,p21）,and CDKN2A（Cyclin-dependent kinase inhibitor 2A,p16） in the substantia nigra tissue of the model group were all elevated（P < 0. 01）. Compared with the model group,the protein levels of p53,p21,and p16 in the substantia nigra tissue were reduced in the low-dose,medium-dose,and high-dose Zuogui pills groups,as well as in the Madopar group（P < 0. 01）. SA-β-gal staining results showed that compared with the control group,the area of SA-β-gal positive expression in the substantia nigra tissue of the model group was increased（P <0. 01）; compared with the model group,the area of SA-β-gal positive expression was decreased in the medium-dose and highdose Zuogui pills groups,as well as in the Madopar group（P < 0. 05 or P < 0. 01）. ELISA results showed that compared with the control group,the levels of IL-6 and IL-8 in the substantia nigra tissue of the model group were increased（P < 0. 01）; compared with the model group,the levels of IL-6 and IL-8 in the substantia nigra tissue were reduced in the low-,medium-,and high-dose Zuogui Pill groups,as well as in the Madopar group（P < 0. 05 or P < 0. 01）. Conclusion: Zuogui Pill may exert a protective effect on PD model mice by alleviating cellular senescence through inhibition of the p53/p21 pathway.更多还原