摘要
目的:探讨毛蕊花苷(Verbascoside,VB)对帕金森(Parkinson′s disease,PD)模型小鼠的改善作用及明确可能的机制。方法:将60只C57BL/6雄性小鼠随机分为正常组、模型组、VB低、中、高剂量组(分别为30、60、120 mg/kg)。适应性培养一周以后,除空白组外,各组连续5 d腹腔注射1-甲基-4-苯基-1,2,3,6-四氢吡啶盐酸盐(MPTP)建立PD亚急性模型,造模后药物组连续灌胃给药7 d,正常组小鼠注射等量生理盐水。通过旷场实验、爬杆实验、抓力测试观察小鼠行为学改变;HE染色和Nissl染色观察黑质神经元组织病理结构;透射电镜观察神经元超微结构变化;免疫组织化学检测酪氨酸羟化酶(TH)、α-突触核蛋白(α-Syn)以及Toll样受体4(TLR4)蛋白阳性表达;Western blot检测小鼠黑质TH、α-Syn及TLR4、NF-κB、PNF-κB蛋白水平变化;Elisa法检测血清中炎症因子IL-1β、IL-6、肿瘤坏死因子-α(TNF-α)水平;利用分子对接和分子动力学验证VB与TLR4/NF-κB信号通路及相关因子的结合能力及稳定性。结果:与正常组相比,模型组小鼠行走总路程降低、爬杆时间显著延长,前肢抓力减弱(P<0.05);透射电镜观察神经元超微结构改变,细胞核溶解、变形,线粒体损伤;H&E染色和Nissl染色显示神经元数量减少,形态结构改变,神经元尼氏体阳性细胞数减少(P<0.05);免疫组织化学和Western blot结果显示α-syn、TLR4、NF-κB p65表达水平升高,TH水平明显降低(P<0.05),血清中IL-1β、IL-6、TNF-α表达显著升高(P<0.05);与模型组比较,VB治疗组明显改善小鼠活动能力(P<0.05),神经元细胞形态逐渐恢复,神经元尼氏体阳性细胞增多(P<0.05);α-syn、TLR4、NF-κB p65蛋白表达降低,TH蛋白表达升高(P<0.05),血清中IL-1β、IL-6、TNF-α水平明显降低(P<0.05)。分子对接及分子动力学模拟结果显示VB与蛋白结合优良,性质较稳定。结论:VB对PD小鼠具有保护作用,其作用机制可能与TLR4/NF-κB通路有关,进而调节神经免疫。
Objective:To explore the improving effect of verbascoside on Parkinson's disease(PD)model mice and to clarify the possible mechanism.Methods:60 C57BL/6 male mice were randomly divided into normal group,model group,low,middle and high dose groups(30,60,120 mg/kg,respectively).After a week of adaptive culture,except for the blank group,the subacute model of PD was established by intraperitoneal injection of 1-methyl-4-phenyl-1(MPTP)in each group for 5 consecutive days.After a week of adaptive culture,except for the blank group,the subacute model of PD was established by intraperitoneal injection of 1-methyl-4-phenyl-1(MPTP)in each group for 5 consecutive days.The establishment of the model,the drug group was intragastrically administered continuously for 7 days,and the mice in the normal group were injected with the same amount of saline.The behavioral changes of mice were observed by open field test,pole climbing test and grip test;the morphology,apoptosis and Nissl bodies of substantia nigra neurons were observed by HE staining and Nissl staining;the ultrastructural changes of neurons were observed by transmission electron microscope;the positive expressions of tyrosine hydroxylase(TH),α-synuclein(α-Syn)and toll-like receptor 4(TLR4)were detected by immunohistochemistry.The protein levels of TH,α-Syn,TLR4,NF-κB and P-NF-κB in substantia nigra of mice were detected by Western blot,and the levels of inflammatory factors IL-1β,IL-6 and tumor necrosis factor-α(TNF-α)in serum were detected by Elisa.Molecular docking was used to verify the binding ability of verbascoside to TLR4/NF-κB signal pathway and related factors,and molecular dynamics was used to verify the binding mode and stability of verbascoside with compounds.Results:Compared with the normal group,the total walking distance,average speed,free activity time,pole climbing time and forelimb grip in the model group were significantly longer than those in the normal group(P<0.05).The ultrastructural changes of neurons,nuclear lysis,deformation and mitochondrial damage were observed under transmission electron microscope,and the number and morphological changes of HE staining substantia nigra neurons were decreased(P<0.05).Nissl staining showed that the number of Nissl positive cells decreased(P<0.05),the positive expression of TH decreased and the positive expression ofα-syn and TLR4 increased(P<0.05).Western blotting showed that the expression of TH protein decreased,the expression ofα-syn,TLR4,NF-κB p65 protein increased(P<0.05),and the expression of IL-1β,IL-6 and TNF-αin serum increased significantly(P<0.05).Compared with the model group,the activity ability of mice in the treatment group was significantly improved(P<0.05),the morphology of neurons gradually recovered and the number of neurons increased(P<0.05).The number of Nissl positive cells increased(P<0.05).The expression of TH protein increased,while the expression ofα-syn,TLR4,NF-κB p65 protein decreased(P<0.05),and the expression of IL-1β,IL-6,TNF-αin serum decreased significantly(P<0.05).The results of molecular docking and molecular dynamics simulation show that the combination of mulberry glycoside with the compound is good and the property is stable.Conclusion:Verbascoside has protective effect on PD mice,and its mechanism may be related to TLR4/NF-κB pathway,and then regulate neuroimmunity.
作者
阿迪莱·艾比布力
克力比奴儿·赛地尔丁
逯冉冉
杨新玲
ADILAI Aibibuli;KELIBINUER Saidierding;LU Ran-ran;YANG Xin-ling(The Second Affiliated Hospital of Xinjiang Medical University,Urumqi 841100,China)
出处
《海南医学院学报》
2023年第24期1855-1862,1869,共9页
Journal of Hainan Medical University
基金
中央引导地方科技发展专项资金项目(ZYD2022C17)
国家自然科学基金(82160232)。
