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BMC Complementary Medicine and Therapies

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Open Access 01-12-2020 | Research article

Germacrone protects against oxygen-glucose deprivation/reperfusion injury by inhibiting autophagy processes in PC12 cells

Authors: Jianxing Zhang, Li Yuan, Sujie Wang, Jiang Liu, Huiqin Bi, Guojuan Chen, Jingjing Li, Lili Chen

Published in: BMC Complementary Medicine and Therapies | Issue 1/2020

Abstract

Background

Germacrone is an anti-inflammatory ingredient in the Chinese medicine zedoary turmeric. The purpose of this study was to explore the protective mechanism of germacrone against PC12 cells injury caused by oxygen-glucose deprivation/reperfusion (OGD/R).

Methods

OGD/R injury model of PC12 cells was established by using OGD/R (2 h/24 h). The cell viability was assessed by MTT assay and LDH release. The ultrastructure of cells was observed by transmission electron microscopy (TEM). The expression of autophagy related proteins in cells was determined by Western Blot.

Results

The results of ultrastructural observation showed that PC12 cells damaged by OGD/R showed typical autophagy characteristics. In addition, OGD/R observably up-regulated the expression of autophagy related proteins: the class III type phosphoinositide 3-kinase (PI3K III), light chain 3(LC3), and Beclin-1 in PC12 cells, and inhibited the expression of the class I type phosphoinositide 3-kinase (PI3K I), Protein kinase B (Akt), the mammalian target of rapamycin (mTOR), and B-cell lymphoma 2(Bcl-2) proteins. Furthermore, germacrone increased the cell viability of OGD/R-damaged PC12 cells by down-regulating the expression of LC3 protein in cells in a concentration-dependent manner. More importantly, germacrone significantly inhibited the expression of PI3K III, LC3, and Beclin-1 in OGD/R-injured PC12 cells, and up-regulated the expressionof PI3K I, Akt, mTOR, and Bcl-2 proteins in cells, and this inhibited or up-regulated effect was reversed by PI3K I inhibitor (ZSTK474).

Conclusion

The above results indicated that germacrone could inhibit the autophagy effect in OGD/R injury model of PC12 cells, the mechanism of inhibition was regulated by PI3K III/Beclin-1/Bcl-2 and PI3K I/Akt/mTOR pathways, thereby improving the cell viability of PC12 cells and playing a neuroprotective role, which provided a new drug for the treatment of OGD/R.

White E. The role for autophagy in cancer. J Clin Invest. 2015;125(1):42–6.PubMedPubMedCentralCrossRef

Gatica D, Lahiri V, Klionsky DJ. Cargo recognition and degradation by selective autophagy. Nat Cell Biol. 2018;20(3):233–42.PubMedPubMedCentralCrossRef

Levy JMM, Towers CG, Thorburn A. Targeting autophagy in cancer. Nat Rev Cancer. 2017;17(9):528–42.PubMedCrossRef

Luo C, Ouyang M-W, Fang Y-Y, Li S-J, Zhou Q, Fan J, Qin Z-S, Tao T. Dexmedetomidine protects mouse brain from ischemia-reperfusion injury via inhibiting neuronal autophagy through up-regulating HIF-1α. Front Cell Neurosci. 2017;11:197.PubMedPubMedCentralCrossRef

Zhao Y, Huang G, Chen S, Gou Y, Dong Z, Zhang X. Homocysteine aggravates cortical neural cell injury through neuronal autophagy overactivation following rat cerebral ischemia-reperfusion. Int J Mol Sci. 2016;17(8):1196.PubMedCentralCrossRef

Zhao Y-N, Guo X-F, Li J-M, Chen C-X, Li S-X, Xu C-J. mTOR/autophagy pathway in the hippocampus of rats suffering intermittent hypoxia preconditioning and global cerebral ischemia-reperfusion. Oncotarget. 2017;8(14):23353–9.PubMedPubMedCentral

Schaaf MB, Keulers TG, Vooijs MA. Rouschop KM.LC3/GABARAP family proteins: autophagy-(un) related functions. FASEB J. 2016;30(12):3961–78.PubMedCrossRef

Frost LS, Lopes VS, Bragin A, Reyes-Reveles J, Brancato J, Cohen A, Mitchell CH, Williams DS, Boesze-Battaglia K. The contribution of melanoregulin to microtubule-associated protein 1 light chain 3 (LC3) associated phagocytosis in retinal pigment epithelium. Mol Neurobiol. 2015;52(3):1135–51.PubMedCrossRef

do Nascimento-Neto LG, Cabral MG, Carneiro RF, Silva Z, Arruda FV, Nagano CS, Fernandes AR, Sampaio AH, Teixeira EH, Videira PA. Halilectin-3, a lectin from the marine sponge haliclona caerulea, induces apoptosis and autophagy in human breast cancer mcf7 cells through caspase-9 pathway and lc3-ii protein expression. Anticancer Agents Med Chem. 2018;18(4):521–8.PubMedCrossRef

10.

Song B-Q, Chi Y, Li X, Du W-J, Han Z-B, Tian J-J, Li J-J, Chen F, Wu H-H, Han L-X. Inhibition of notch signaling promotes the adipogenic differentiation of mesenchymal stem cells through autophagy activation and PTEN-PI3K/AKT/mTOR pathway. Cell Physiol Biochem. 2015;36(5):1991–2002.PubMedCrossRef

11.

Maejima Y, Kyoi S, Zhai P, Liu T, Li H, Ivessa A, Sciarretta S, Del Re DP, Zablocki DK, Hsu C-P. Mst1 inhibits autophagy by promoting the interaction between Beclin1 and Bcl-2. Nat Med. 2013;19(11):1478–88.PubMedPubMedCentralCrossRef

12.

Heras-Sandoval D, Pérez-Rojas JM, Hernández-Damián J, Pedraza-Chaverri J. The role of PI3K/AKT/mTOR pathway in the modulation of autophagy and the clearance of protein aggregates in neurodegeneration. Cell Signal. 2014;26(12):2694–701.PubMedCrossRef

13.

Kumar D, Shankar S, Srivastava RK. Rottlerin induces autophagy and apoptosis in prostate cancer stem cells via PI3K/Akt/mTOR signaling pathway. Cancer Lett. 2014;343(2):179–89.PubMedCrossRef

14.

Tsai J-P, Lee C-H, Ying T-H, Lin C-L, Lin C-L, Hsueh J-T, Hsieh Y-H. Licochalcone a induces autophagy through PI3K/Akt/mTOR inactivation and autophagy suppression enhances Licochalcone A-induced apoptosis of human cervical cancer cells. Oncotarget. 2015;6(30):28851–66.PubMedPubMedCentralCrossRef

15.

Liu B, Gao Y-Q, Wang X-M, Wang Y-C, Fu L-Q. Germacrone inhibits the proliferation of glioma cells by promoting apoptosis and inducing cell cycle arrest. Mol Med Rep. 2014;10(2):1046–50.PubMedCrossRef

16.

Chen Q, Wang G, Tang L, Yu X, Li Z, Yang X. Effect of germacrone in alleviating HUVECs damaged by H2O2-induced oxidative stress. Zhongguo Zhong Yao Za Zhi. 2017;42(18):3564–71.PubMed

17.

Feng J, Bai X, Cui T, Zhou H, Chen Y, Xie J, Shi Q, Wang H, Zhang G. In vitro antiviral activity of germacrone against porcine reproductive and respiratory syndrome virus. Curr Microbiol. 2016;73(3):317–23.PubMedCrossRef

18.

Xie XH, Zhao H, Hu YY, Gu XD. Germacrone reverses Adriamycin resistance through cell apoptosis in multidrug-resistant breast cancer cells. Exp Ther Med. 2014;8(5):1611–5.PubMedPubMedCentralCrossRef

19.

Kong Q, Ma Y, Yu J, Chen X. Predicted molecular targets and pathways for germacrone, curdione, and furanodiene in the treatment of breast cancer using a bioinformatics approach. Sci Rep. 2017;7(1):15543.PubMedPubMedCentralCrossRef

20.

Ye L, Wu J, Chen W, Feng Y, Shen Z. Novel anti-cancer agents based on germacrone: design, synthesis, biological activity, docking studies and MD simulations. RSC Adv. 2017;7(7):3760–7.CrossRef

21.

Lim MS, Choung SY, Jeong KW. Germacrone inhibits estrogen receptor α-mediated transcription in MCF-7 breast cancer cells. Phytother Res. 2016;30(12):2036–43.PubMedCrossRef

22.

Zhao Q, Wang Q, Wang J, Tang M, Huang S, Peng K, Han Y, Zhang J, Liu G, Fang Q, You Z. Maternal immune activation-induced PPARγ-dependent dysfunction of microglia associated with neurogenic impairment and aberrant postnatal behaviors in offspring. Neurobiol Dis. 2019;125:1–13.PubMedCrossRef

23.

Zhang L, Zhang J, You Z. Switching of the Microglial activation phenotype is a possible treatment for depression disorder. Front Cell Neurosci. 2018;12:306.PubMedPubMedCentralCrossRef

24.

Wu T, Yin F, Kong H. Germacrone attenuates cerebral ischemia/reperfusion injury in rats via antioxidative and antiapoptotic mechanisms. J Cell Biochem. 2019;120:18901–9.PubMedCrossRef

25.

Yang G, Wang N, Seto SW, Chang D, Liang H. Hydroxysafflor yellow a protects brain microvascular endothelial cells against oxygen glucose deprivation/reoxygenation injury: involvement of inhibiting autophagy via class I PI3K/Akt/mTOR signaling pathway. Brain Res Bull. 2018;140:243–57.PubMedCrossRef

26.

Van Meerloo J, Kaspers GJ, Cloos J. Cell sensitivity assays: the MTT assay. In: Cancer cell culture: Springer; 2011. p. 237–45.

27.

Welinder C, Ekblad L. Coomassie staining as loading control in Western blot analysis. J Proteome Res. 2011;10(3):1416–9.PubMedCrossRef

28.

Masuda G, Yashiro M, Kitayama K, Miki Y, Kasashima H, Kinoshita H, Morisaki T, Fukuoka T, Hasegawa T, Sakurai K. Clinicopathological correlations of autophagy-related proteins LC3, Beclin 1 and p62 in gastric cancer. Anticancer Res. 2016;36(1):129–36.PubMed

29.

Kang Y, Liu J, Wu J, Yin Q, Liang H, Chen A, Shao L. Graphene oxide and reduced graphene oxide induced neural pheochromocytoma-derived PC12 cell lines apoptosis and cell cycle alterations via the ERK signaling pathways. Int J Nanomed. 2017;12:5501–10.CrossRef

30.

Greene LA. NGF-responsive clonal PC12 pheochromocytoma cells as tools for neuropharmacologic investigations. In: Chemotherapy: Proceedings of the 7th International Congress of Pharmacology, vol.10. Paris: Elsevier;1978; 2016. p. 197.

31.

Liu Y, Levine B. Autosis and autophagic cell death: the dark side of autophagy. Cell Death Differ. 2015;22(3):367–76.PubMedCrossRef

32.

Wang M, Li Y-J, Ding Y, Zhang H-N, Sun T, Zhang K, Yang L, Guo Y-Y, Liu S-B, Zhao M-G. Silibinin prevents autophagic cell death upon oxidative stress in cortical neurons and cerebral ischemia-reperfusion injury. Mol Neurobiol. 2016;53(2):932–43.PubMedCrossRef

33.

Lin S-P, Ye S, Long Y, Fan Y, Mao H-F, Chen M-T, Ma Q-J. Circular RNA expression alterations are involved in OGD/R-induced neuron injury. Biochem Biophys Res Commun. 2016;471(1):52–6.PubMedCrossRef

34.

Hu Z, Yang B, Mo X, Zhou F. HspB8 mediates neuroprotection against OGD/R in N2A cells through the phosphoinositide 3-kinase/Akt pathway. Brain Res. 2016;1644:15–21.PubMedCrossRef

35.

Gaucher S, Jarraya M. Comparison of the PrestoBlue and LDH release assays with the MTT assay for skin viability assessment. Cell Tissue Bank. 2015;16(3):325–9.PubMedCrossRef

36.

Lazari MM, Orhon I, Codogno P, Dupont N. Monitoring of autophagy and cell volume regulation in kidney epithelial cells in response to fluid shear stress. In: Autophagy: Springer; 2019. p. 331–40.

37.

Z-t M, W-n L, Y-r Z, Gong Q-H. Icariin attenuates OGD/R-induced autophagy via Bcl-2-dependent cross talk between apoptosis and autophagy in PC12 cells. Evid Based Complement Alternat Med. 2016:4343084.

38.

Li T-T, Zhu D, Mou T, Guo Z, Pu J-L, Chen Q-S, Wei X-F, Wu Z-J. IL-37 induces autophagy in hepatocellular carcinoma cells by inhibiting the PI3K/AKT/mTOR pathway. Mol Immunol. 2017;87:132–40.PubMedCrossRef

39.

Jian J, Xuan F, Qin F, Huang R. Bauhinia championii flavone inhibits apoptosis and autophagy via the PI3K/Akt pathway in myocardial ischemia/reperfusion injury in rats. Drug Des Devel Ther. 2015;9:5933–45.PubMedPubMedCentral

40.

Liu Y, Wang W, Fang B, Ma F, Zheng Q, Deng P, Zhao S, Chen M, Yang G, He G. Anti-tumor effect of germacrone on human hepatoma cell lines through inducing G2/M cell cycle arrest and promoting apoptosis. Eur J Pharmacol. 2013;698(1–3):95–102.PubMedCrossRef

41.

Liao Q, Qian Z, Liu R, An L, Chen X. Germacrone inhibits early stages of influenza virus infection. Antiviral Res. 2013;100(3):578–88.PubMedCrossRef

42.

Liu Y-Y, Zheng Q, Fang B, Wang W, Ma F-Y, Roshan S, Banafa A, Chen M-J, Chang J-L, Deng X-M. Germacrone induces apoptosis in human hepatoma HepG2 cells through inhibition of the JAK2/STAT3 signalling pathway. J Huazhong Univ Sci Technolog Med Sci. 2013;33(3):339–45.PubMedCrossRef

43.

Guo M-L, Liao K, Periyasamy P, Yang L, Cai Y, Callen SE, Buch S. Cocaine-mediated microglial activation involves the ER stress-autophagy axis. Autophagy. 2015;11(7):995–1009.PubMedPubMedCentralCrossRef

44.

Chang Y, Kong R. Ganoderic acid a alleviates hypoxia-induced apoptosis, autophagy, and inflammation in rat neural stem cells through the PI3K/AKT/mTOR pathways. Phytother Res. 2019;33:1448–56.PubMedCrossRef

45.

Huang X-P, Ding H, Yang X-Q, Li J-X, Tang B, Liu X-D, Tang Y-H, Deng C-Q. Synergism and mechanism of Astragaloside IV combined with Ginsenoside Rg1 against autophagic injury of PC12 cells induced by oxygen glucose deprivation/reoxygenation. Biomed Pharmacother. 2017;89:124–34.PubMedCrossRef

Title: Germacrone protects against oxygen-glucose deprivation/reperfusion injury by inhibiting autophagy processes in PC12 cells
Authors: Jianxing Zhang
Li Yuan
Sujie Wang
Jiang Liu
Huiqin Bi
Guojuan Chen
Jingjing Li
Lili Chen
Publication date: 01-12-2020
Publisher: BioMed Central
Published in: BMC Complementary Medicine and Therapies / Issue 1/2020
Electronic ISSN: 2662-7671
DOI: https://doi.org/10.1186/s12906-020-2865-1

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Germacrone protects against oxygen-glucose deprivation/reperfusion injury by inhibiting autophagy processes in PC12 cells

Abstract

Background

Methods

Results

Conclusion

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Abstract

Background

Methods

Results

Conclusion

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