Mechanism of Yiqi Jiedu Prescriptions in Protection of Mitochondria in PC12 Cells Against Hypoxia Injury Based on Energy Metabolism

Yu-hao DAI; Li-ming LIU; Chen LIU; Wen-jie WU; Jian-ying SHEN; Shao-jing LI

doi:10.13422/j.cnki.syfjx.20220402

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Mechanism of Yiqi Jiedu Prescriptions in Protection of Mitochondria in PC12 Cells Against Hypoxia Injury Based on Energy Metabolism

更新时间：2022-01-13

- Mechanism of Yiqi Jiedu Prescriptions in Protection of Mitochondria in PC12 Cells Against Hypoxia Injury Based on Energy Metabolism
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 28, Issue 4, Pages: 34-41(2022)
- 作者机构：
  
  1.中国中医科学院中药研究所，北京 100700
  2.安徽中医药大学，合肥 230000
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20220402
  CLC： R2-0;R22;R285.5;R289;R33
- Received：17 September 2021，
  
  Published Online：06 December 2021，
  
  Published：20 February 2022
- 稿件说明：
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戴玉豪,刘黎明,刘陈等.基于能量代谢分析益气解毒方对缺氧损伤的PC12细胞线粒体保护作用的机制[J].中国实验方剂学杂志,2022,28(04):34-41.

DAI Yu-hao,LIU Li-ming,LIU Chen,et al.Mechanism of Yiqi Jiedu Prescriptions in Protection of Mitochondria in PC12 Cells Against Hypoxia Injury Based on Energy Metabolism[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(04):34-41.
戴玉豪,刘黎明,刘陈等.基于能量代谢分析益气解毒方对缺氧损伤的PC12细胞线粒体保护作用的机制[J].中国实验方剂学杂志,2022,28(04):34-41. DOI： 10.13422/j.cnki.syfjx.20220402.

DAI Yu-hao,LIU Li-ming,LIU Chen,et al.Mechanism of Yiqi Jiedu Prescriptions in Protection of Mitochondria in PC12 Cells Against Hypoxia Injury Based on Energy Metabolism[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(04):34-41. DOI： 10.13422/j.cnki.syfjx.20220402.

摘要

目的

采用Seahorse细胞能量代谢分析仪，通过缺氧损伤的高分化大鼠肾上腺嗜铬瘤细胞（PC12细胞）建立一种线粒体能量代谢评价方法并探究益气解毒方的线粒体保护作用。

方法

应用缺氧小室体外构建PC12细胞损伤模型，实验设置空白组，模型组（Model），益气解毒方高、中、低剂量组（25，5，1 µmol·L

-1

；YQ高、中、低）及阳性药曲美他嗪组（TMZ），每组3复孔，实验重复3次。采用建立的能量代谢分析方法对细胞线粒体复合物活性进行检测，筛选最佳给药浓度；随后设置益气解毒方全方及各拆方组，采用Seahorse细胞能量代谢分仪检测有氧呼吸和糖酵解功能；根据非线粒体呼吸值，质子漏值，基础呼吸值，最大呼吸值，ATP生成量，可提升呼吸能力检测值，通过主成份分析（PCA）和变量重要性指标（VIP），分析评价益气解毒方不同配伍组对缺氧损伤线粒体有氧呼吸作用效果；通过蛋白免疫印迹法（Western blot）检测细胞色素C（Cytochrome C），B细胞淋巴瘤-2（Bcl-2）和Bcl-2相关X蛋白（Bax）的表达。

结果

与其他浓度组比较，FCCP的最佳给药浓度为2 µmol·L

-1

；与模型组比较，5 µmol·L

-1

益气解毒方全方能提升线粒体复合物活性（

0.05）；与模型组比较，益气解毒方全方改善效果较好（

0.01）；在各拆方组中，人参皂苷和栀子苷配伍效果较好（

0.01）；VIP分析结果显示，对于线粒体呼吸功能改善，益气解毒方中栀子苷贡献度最大；与模型组比较，益气解毒方中、高剂量组线粒体细胞色素C的渗漏显著减少（

0.01），Bax蛋白的表达显著降低（

0.01），Bcl-2蛋白的表达明显升高（

0.05，

0.01）。

结论

建立了一种细胞水平、高通量线粒体能量代谢多指标定量评价方法，证明了益气解毒方全方能够显著改善缺氧损伤后PC12细胞线粒体能量代谢障碍，而发挥这一作用的机制可能与线粒体凋亡保护相关。

Abstract

Objective

To establish an evaluation method for mitochondrial energy metabolism with Seahorse analyzer and investigate the protective effect of Yiqi Jiedu prescriptions （YQ） on mitochondria in rat adrenal pheochromocytoma （PC12） cells against hypoxia injury.

Method

The PC12 cell injury model was induced

in vitro

using hypoxic chambers. Five groups were set up，

， a control group， a model group （model）， high- （25 µmol·L

-1

）， medium- （5 µmol·L

-1

） and low-dose （1 µmol·L

-1

） YQ groups， and a positive drug trimetazidine （TMZ） group， with three replicate wells in each group. The experiment was repeated three times. The established method for energy metabolism analysis was used to assay the activity of mitochondrial complex in cells and screen the optimal dosing concentration. Subsequently， the YQ group and modified YQ groups were set up， and the aerobic respiration and glycolysis function were assayed by the Seahorse analyzer. According to the non-mitochondrial oxygen consumption， proton leakage， basal respiration， maximum respiration， ATP production， and potentially improved respiration， the effects of modified YQ groups on the aerobic respiration of mitochondria damaged by hypoxia were evaluated by principal component analysis （PCA） and variable importance in projection （VIP）. The expression of cytochrome C， B-cell lymphoma-2 （Bcl-2）， and Bcl-2-associated X protein （Bax） was detected by Western blot.

Result

Compared with the groups of other concentrations， the optimal dosing concentration of carbonyl cyanide-4 （trifluoromethoxy）phenylhydrazone （FCCP） was 2 µmol·L

-1

. Compared with the model group， the medium-dose YQ group showed enhanced mitochondrial complex activity （

0.05）. The YQ groups were superior to the model group in improvement （

0.01）. The combination of ginsenoside and geniposide showed the optimal effect among the modified YQ groups （

0.01）. VIP analysis revealed that for the improvement of mitochondrial respiratory function， the contribution of geniposide in YQ was the greatest. Compared with the model group， the high-dose YQ group displayed reduced leakage of mitochondrial cytochrome C （

0.01）， decreased expression of Bax protein （

0.01）， and increased expression of Bcl-2 protein （

0.05，

0.01）.

Conclusion

A cellular， high-throughput quantitative evaluation method for mitochondrial energy metabolism was established， which demonstrated that YQ could significantly improve the impaired mitochondrial energy metabolism in PC12 cells damaged by hypoxia， and the underlying mechanism might be related to the protection against mitochondrial apoptosis.

关键词

Keywords

references

WANG W ， JIANG B ， SUN H ， et al . Prevalence，incidence，and mortality of stroke in China：results from a nationwide population-based survey of 480 687 adults ［J］. Circulation ， 2017 ， 135 （ 8 ）： 759 - 771 .

HOSSAIN M I ， ROULSTON C L ， STAPLETON D I . Molecular basis of impaired glycogen metabolism during ischemic stroke and hypoxia ［J］. PLoS One ， 2014 ， 9 （ 5 ）： e97570 .

HE Z ， NING N ， ZHOU Q ， et al . Mitochondria as a therapeutic target for ischemic stroke ［J］. Free Radic Biol Med ， 2020 ， 146 ： 45 - 58 .

SUCHADOLSKIENE O ， PRANSKUNAS A ， BALIUTYTE G ， et al . Microcirculatory，mitochondrial，and histological changes following cerebral ischemia in swine ［J］. BMC Neurosci ， 2014 ， 15 ： 2 .

GRASMICK K A ， HU H ， HONE E A ， et al . Uncoupling of the electron transport chain compromises mitochondrial oxidative phosphorylation and exacerbates stroke outcomes ［J］. J Neuroinfect Dis ， 2018 ， doi： 10.4172/2314-7326.1000283 http://dx.doi.org/10.4172/2314-7326.1000283 .

SIMS N R ， MUYDERMAN H . Mitochondria，oxidative metabolism and cell death in stroke ［J］. Biochim Biophys Acta ， 2010 ， 1802 （ 1 ）： 80 - 91 .

ANDRABI S S ， ALI M ， TABASSUM H ， et al . Pramipexole prevents ischemic cell death via mitochondrial pathways in ischemic stroke ［J］. Dis Model Mech ， 2019 ， 12 （ 8 ）： dmm033860 .

ANDRABI S S ， PARVEZ S ， TABASSUM H . Progesterone induces neuroprotection following reperfusion-promoted mitochondrial dysfunction after focal cerebral ischemia in rats ［J］. Dis Model Mech ， 2017 ， 10 （ 6 ）： 787 - 796 .

杜肖，路畅，贺晓丽，等 . 小续命汤有效成分组对脑缺血/再灌注大鼠恢复早期脑线粒体的保护作用研究［J］. 中国中药杂志， 2017 ， 42 （ 11 ）： 2139 - 2145 .

LI Z ， GRAHAM B H . Measurement of mitochondrial oxygen consumption using a Clark electrode ［J］. Methods Mol Biol ， 2012 ， 837 ： 63 - 72 .

SILVA A M ， OLIVEIRA P J . Evaluation of respiration with clark type electrode in isolated mitochondria and permeabilized animal cells ［J］. Methods Mol Biol ， 2012 ， 810 ： 7 - 24 .

IUSO A ， REPP B ， BIAGOSCH C ， et al . Assessing mitochondrial bioenergetics in isolated mitochondria from various mouse tissues using Seahorse XF96 analyzer ［J］. Methods Mol Biol ， 2017 ， 1567 ： 217 - 230 .

SURE V N ， SAKAMURI S ， SPERLING J A ， et al . A novel high-throughput assay for respiration in isolated brain microvessels reveals impaired mitochondrial function in the aged mice ［J］. Geroscience ， 2018 ， 40 （ 4 ）： 365 - 375 .

KOOPMAN M ， MICHELS H ， DANCY B M ， et al . A screening-based platform for the assessment of cellular respiration in Caenorhabditis elegans ［J］. Nat Protoc ， 2016 ， 11 （ 10 ）： 1798 - 1816 .

LI S ， WU C ， CHEN J ， et al . An effective solution to discover synergistic drugs for anti-cerebral ischemia from traditional Chinese medicinal prescriptionse ［J］. PLoS One ， 2013 ， 8 （ 11 ）： e78902 .

文丽梅，李韶菁，吴传鸿，等 . 益气解毒方对局灶性脑缺血大鼠线粒体损伤的保护作用［J］. 北京中医药大学学报， 2014 ， 37 （ 3 ）： 175 - 179 .

杨若聪，田朋朋，吴传鸿，等 . 益气解毒方基于线粒体保护的抗缺血性脑卒中研究［J］. 世界中医药， 2017 ，（ 11 ）： 200 - 204 .

LIU X ， ZHU X ， CHEN M ， et al . Resveratrol protects PC12 cells against OGD/ R-induced apoptosis via the mitochondrial-mediated signaling pathway ［J］. Acta Biochim Biophys Sin （Shanghai）， 2016 ， 48 （ 4 ）： 342 - 353 .

任民，徐革，彭伟，等 . 三七皂苷R1对缺氧诱导PC12细胞损伤的保护作用［J］. 中国临床药理学杂志， 36 （ 12 ）： 1657 - 1660 .

CLERC P ， POLSTER B M . Investigation of mitochondrial dysfunction by sequential microplate-based respiration measurements from intact and permeabilized neurons ［J］. PLoS One ， 2012 ， 7 （ 4 ）： e34465 .

WU Q ， YAN R ， SUN J . Probing the drug delivery strategies in ischemic stroke therapy ［J］. Drug Deliv ， 2020 ， 27 （ 1 ）： 1644 - 1655 .

SUCHER N J . Insights from molecular investigations of traditional Chinese herbal stroke medicines：implications for neuroprotective epilepsy therapy ［J］. Epilepsy Behav ， 2006 ， 8 （ 2 ）： 350 - 362 .

张锦，张允岭，郭蓉娟，等 . 从“毒损脑络”到“毒损络脉”的理论探讨［J］. 北京中医药， 2013 ， 32 （ 7 ）： 483 - 486 .

SILVA A M ， OLIVEIRA P J . Evaluation of respiration with clark-type electrode in isolated mitochondria and permeabilized animal cells ［J］. Methods Mol Biol ， 2018 ， 1782 ： 7 - 29 .

SEVERINGHAUS J W ， ASTRUP P B . History of blood gas analysis.IV.Leland Clark's oxygen electrode ［J］. J Clin Monit ， 1986 ， 2 （ 2 ）： 125 - 139 .

PLITZKO B ， LOESGEN S . Measurement of oxygen consumption rate （OCR） and extracellular acidification rate （ECAR） in culture cells for assessment of the energy metabolism ［J］. Bio Protoc ， 2018 ， 8 （ 10 ）： e2850 .

CUMMING B M ， REDDY V P ， STEYN A J C . The analysis of mycobacterium tuberculosis-induced bioenergetic changes in infected macrophages using an extracellular flux analyzer ［J］. Methods Mol Biol ， 2020 ， 2184 ： 161 - 184 .

ZHANG J ， ZHANG Q . Using seahorse machine to measure OCR and ECAR in cancer cells ［J］. Methods Mol Biol ， 2019 ， 1928 ： 353 - 363 .

AMIEL E ， EVERTS B ， FRITZ D ， et al . Mechanistic target of rapamycin inhibition extends cellular lifespan in dendritic cells by preserving mitochondrial function ［J］. J Immunol ， 2014 ， 193 （ 6 ）： 2821 - 2830 .

MILLMAN J R ， DOGGETT T ， THEBEAU C ， et al . Measurement of energy metabolism in explanted retinal tissue using extracellular flux analysis ［J］. J Vis Exp ， 2019 ， doi： 10.3791/58626 http://dx.doi.org/10.3791/58626 .

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