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中华脑科疾病与康复杂志(电子版) ›› 2022, Vol. 12 ›› Issue (06) : 324 -331. doi: 10.3877/cma.j.issn.2095-123X.2022.06.002

基础研究

山茱萸环烯醚萜苷改善6-OHDA诱导帕金森病细胞模型的损伤
郑丽华1, 钱一菁1, 黄崇甄2,(), 周春娜1   
  1. 1. 200433 上海,长海医院药剂科
    2. 200031 上海,上海图书馆
  • 收稿日期:2022-05-09 出版日期:2022-12-15
  • 通信作者: 黄崇甄

Cornel iridoid glycoside ameliorates 6-OHDA-induced damage in cell model of Parkinson's disease

Lihua Zheng1, Yijing Qian1, Chongzhen Huang2,(), Chunna Zhou1   

  1. 1. Department of Pharmacy, Changhai Hospital, Shanghai 200433, China
    2. Shanghai Library, Shanghai 200031, China
  • Received:2022-05-09 Published:2022-12-15
  • Corresponding author: Chongzhen Huang
引用本文:

郑丽华, 钱一菁, 黄崇甄, 周春娜. 山茱萸环烯醚萜苷改善6-OHDA诱导帕金森病细胞模型的损伤[J/OL]. 中华脑科疾病与康复杂志(电子版), 2022, 12(06): 324-331.

Lihua Zheng, Yijing Qian, Chongzhen Huang, Chunna Zhou. Cornel iridoid glycoside ameliorates 6-OHDA-induced damage in cell model of Parkinson's disease[J/OL]. Chinese Journal of Brain Diseases and Rehabilitation(Electronic Edition), 2022, 12(06): 324-331.

目的

探索山茱萸环烯醚萜苷(CIG)对6-羟基多巴(6-OHDA)诱导的帕金森病(PD)细胞模型损伤的作用。

方法

将PC12细胞分为空白对照组(未作处理)、PD模型组(给予6-OHDA诱导PD细胞模型)、PD模型+CIG 20 μmol/L组(给予6-OHDA和20 μmol/L CIG)、PD模型+CIG 40 μmol/L组(给予6-OHDA和40 μmol/L CIG)、PD模型+CIG 80 μmol/L组(给予6-OHDA和80 μmol/L CIG)。采用细胞计数试剂盒8法检测细胞存活情况,DCFH-DA荧光探针评估活性氧(ROS)含量,流式细胞术测定细胞凋亡,比色法分析超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量,酶联免疫吸附测定法分析炎症因子肿瘤坏死因子-α(TNF-α)和白细胞介素-1β(IL-1β)分泌,Western blot实验测定B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、天冬氨酸特异性半胱氨酸蛋白酶3(Caspase3)、Cleaved Caspase3、P47phox、酪氨酸羟化酶(TH)、血红素加氧酶-1(HO-1)、转录因子NF-E2相关因子2(Nrf2)和醌氧化还原酶1(NQO1)蛋白表达。

结果

与空白对照组比较,PD模型组PC12细胞活力、SOD活性及Bcl-2、TH、HO-1、Nrf2和NQO1蛋白表达水平明显降低,细胞凋亡率、ROS含量、MDA含量、Bax、Caspase3、Cleaved Caspase3、P47phox蛋白表达水平、TNF-α、IL-1β含量显著升高,差异具有统计学意义(P<0.05)。与PD模型组比较,PD模型+CIG 20 μmol/L组、PD模型+CIG 40 μmol/L组、PD模型+CIG 80 μmol/L组PC12细胞活力、SOD活性及Bcl-2、TH、HO-1、Nrf2和NQO1蛋白表达水平明显升高,细胞凋亡率、ROS含量、MDA含量、Bax、Caspase3、Cleaved Caspase3、P47phox蛋白表达水平、TNF-α、IL-1β含量显著降低,均呈浓度依赖性,差异具有统计学意义(P<0.05)。

结论

CIG可以改善6-OHDA诱导的PC12细胞增殖,抑制细胞凋亡,减轻氧化应激损伤和改善炎症反应,从而保护PD细胞模型损伤。

Objective

To explore the protective effect of cornel iridoid glycoside (CIG) on 6-hydroxydopa (6-OHDA)-induced damage to Parkinson's disease (PD) cell model.

Methods

PC12 cells were divided into blank control group (untreated), PD model group (with 6-OHDA induced PD cell model), PD model+CIG 20 μmol/L group (with 6-OHDA and 20 μmol/L CIG), PD model+CIG 40 μmol/L group (administered 6-OHDA and 40 μmol/L CIG), PD model+CIG 80 μmol/L group (administered 6-OHDA and 80 μmol/L CIG). Cell counting kit 8 method was used to detect cell survival, DCFH-DA fluorescent probe was applied to assess ROS content, flow cytometry was performed to determine cell apoptosis, and colorimetric analysis of superoxide dismutase (SOD) activity and malondialdehyde (MDA) content, enzyme-linked immunosorbent assay to investigate the secretion of inflammatory factors tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), and Western blot to determine Bcl-2, Bax, Caspase3, Cleaved Caspase3, P47phox, TH, HO-1, Nrf2 and NQO1 protein expression.

Results

Compared with the blank control group, cell viability, SOD activity, Bcl-2, TH, HO-1, Nrf2 and NQO1 protein expression levels of PC12 cells in the PD model group were significantly reduced, and the apoptosis rate, ROS content, MDA content, Bax, Caspase3, Cleaved Caspase3, P47phox protein expression levels, TNF-α, IL-1β content were significantly increased (P<0.05). Compared with the PD model group, the PD model+CIG 20 μmol/L group, PD model+CIG 40 μmol/L group, PD model+CIG 80 μmol/L group PC12 cell viability, SOD Activity, Bcl-2, TH, HO-1, Nrf2 and NQO1 protein expression levels were remarkably increased, apoptosis rate, ROS content, MDA content, Bax, Caspase3, Cleaved Caspase3, P47phox protein expression levels, TNF-α, IL-1β content obviously reduced, all showed concentration dependence (P<0.05).

Conclusion

CIG can promote 6-OHDA-induced PC12 cell proliferation, inhibit apoptosis, reduce oxidative stress damage and improve inflammatory response, thereby protecting the PD cell model damage.

表1 CCK-8法检测不同浓度CIG下PC12细胞的吸光度A值(Mean±SD,n=9)
表2 CIG对6-OHDA诱导的PC12细胞ROS含量的影响(Mean±SD,n=9)
图1 5组PC12细胞流式细胞术检测结果及Bcl-2、Bax、Caspase3和Cleaved Caspase3蛋白表达比较A:流式细胞仪检测结果;B:Western blot检测结果;PD:帕金森病;CIG:山茱萸环烯醚萜苷;Bcl-2:B细胞淋巴瘤/白血病-2;Bax:Bcl-2相关X蛋白;Caspase3:天冬氨酸特异性半胱氨酸蛋白酶3;GAPDH:甘油醛-3-磷酸脱氢酶
表3 CIG对6-OHDA诱导的PC12细胞凋亡及凋亡蛋白表达的影响(Mean±SD,n=9)
图2 Western blot检测PC12细胞中P47phox和TH蛋白表达PD:帕金森病;CIG:山茱萸环烯醚萜苷;TH:酪氨酸羟化酶;GAPDH:甘油醛-3-磷酸脱氢酶
表4 5组PC12细胞P47phox和TH蛋白表达量比较(Mean±SD,n=9)
表5 CIG对6-OHDA诱导的PC12细胞内SOD活性和MDA含量的影响(Mean±SD,n=9)
表6 CIG对6-OHDA诱导的PC12细胞TNF-α和IL-1β含量的影响(ng/L,Mean±SD,n=9)
图3 Western blot检测PC12细胞中HO-1、Nrf2和NQO1蛋白表达PD:帕金森病;CIG:山茱萸环烯醚萜苷;HO-1:血红素加氧酶-1;Nrf2:转录因子NF-E2相关因子2;NQO1:醌氧化还原酶1;GAPDH:甘油醛-3-磷酸脱氢酶
表7 CIG对6-OHDA诱导的PC12的HO-1、Nrf2和NQO1蛋白表达影响(Mean±SD,n=9)
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