Effects of inhibiting PI3K/Akt/mTOR signaling pathway on autophagy, apoptosis and PD characteristic expression proteins in MPP+-treated SH-SY5Y cells

doi:10.11958/20221595

Abstract

Abstract:

Objective To investigate effects of inhibiting phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway on autophagy, apoptosis, the expression of Parkinson's disease (PD) characteristic protein α-synuclein (α-syn) and tyrosine hydroxylase (TH) in SH-SY5Y cells treated with 1-methyl-4-phenylpyridine (MPP⁺). Methods SH-SY5Y cells were treated with MPP⁺, PI3K/Akt activator insulin-like growth factor-1 (IGF-1) and PI3K/Akt inhibitor LY294002. CCK-8 was performed to measure the cell viability. Flow cytometry was used to detect cell apoptosis rate. Acridine orange (AO) staining was used to detect autophagic vacuoles. Western blot assay was used to measure protein levels of α-syn, TH, p62, microtubule-associated protein 1 light chain 3B (LC3B), p-mTOR, mTOR, p-PI3K, PI3K, p-Akt and Akt. Results MPP⁺ intervention significantly reduced the survival rate of SH-SY5Y cells in a dose-dependent manner (P<0.05). The survival rate of SH-SY5Y cells decreased and the apoptosis rate increased after MPP⁺ and IGF-1 treatment (P<0.05). The autophagic vacuoles in cells decreased, the ratio of LC3BⅡ/Ⅰ decreased, and the level of p62 protein increased (P<0.05). The level of TH protein decreased and the level of α-syn protein increased (P<0.05). The ratios of p-PI3K/PI3K, p-Akt/Akt and p-mTOR/mTOR were increased (P<0.05). The effect of LY294002 on SH-SY5Y cells was opposite to that of MPP⁺ and IGF-1, and LY294002 was able to reverse the effect of MPP⁺ on SH-SY5Y cells to a certain extent (P<0.05). Conclusion Inhibition of PI3K/Akt/mTOR signaling pathway has a protective effect on MPP⁺-induced SH-SY5Y cytotoxicity, which provides a new theoretical basis for the treatment of PD.

Key words: Parkinson disease, phosphatidylinositol 3-kinases, proto-oncogene proteins c-akt, TOR serine-threonine kinases, autophagy, apoptosis, 1-methyl-4-phenylpyridinium, SH-SY5Y cells

CLC Number:

R742

Figures/Tables 8

References 24

[1]	CAI L J, TU L, HUANG X M, et al. LncRNA MALAT1 facilitates inflammasome activation via epigenetic suppression of Nrf2 in Parkinson's disease[J]. Mol Brain, 2020, 13(1):130. doi:10.1186/s13041-020-00656-8.
[2]	MERGHANI M M, ARDAH M T, AL SHAMSI M, et al. Dose-related biphasic effect of the Parkinson's disease neurotoxin MPTP,on the spread, accumulation, and toxicity of α-synuclein[J]. Neurotoxicology, 2021, 84:41-52. doi:10.1016/j.neuro.2021.02.001.
[3]	ITOKAZU Y, FUCHIGAMI T, MORGAN J C, et al. Intranasal infusion of GD3 and GM1 gangliosides downregulates alpha-synuclein and controls tyrosine hydroxylase gene in a PD model mouse[J]. Mol Ther, 2021, 29(10):3059-3071. doi:10.1016/j.ymthe.2021.06.005.
[4]	SIMON D K, TANNER C M, BRUNDIN P. Parkinson disease epidemiology, pathology, genetics, and pathophysiology[J]. Clin Geriatr Med, 2020, 36(1):1-12. doi:10.1016/j.cger.2019.08.002.
[5]	DJAJADIKERTA A, KESHRI S, PAVEL M, et al. Autophagy induction as a therapeutic strategy for neurodegenerative diseases[J]. J Mol Biol, 2020, 432(8):2799-2821. doi:10.1016/j.jmb.2019.12.035.
[6]	GUO Y L, DUAN W J, LU D H, et al. Autophagy-dependent removal of α-synuclein: a novel mechanism of GM1 ganglioside neuroprotection against Parkinson's disease[J]. Acta Pharmacol Sin, 2021, 42(4):518-528. doi:10.1038/s41401-020-0454-y.
[7]	CONG L H, LI T, WANG H, et al. IL-17A-producing T cells exacerbate fine particulate matter-induced lung inflammation and fibrosis by inhibiting PI3K/Akt/mTOR-mediated autophagy[J]. J Cell Mol Med, 2020, 24(15):8532-8544. doi:10.1111/jcmm.15475.
[8]	JIANG D, PENG Y. The protective effect of decoction of Rehmanniae via PI3K/Akt/mTOR pathway in MPP⁺-induced Parkinson's disease model cells[J]. J Recept Signal Transduct Res, 2021, 41(1):74-84. doi:10.1080/10799893.2020.1787445.
[9]	ZHANG D, YUAN Y, ZHU J, et al. Insulin-like growth factor 1 promotes neurological functional recovery after spinal cord injury through inhibition of autophagy via the PI3K/Akt/mTOR signaling pathway[J]. Exp Ther Med, 2021, 22(5):1265. doi:10.3892/etm.2021.10700.
[10]	GBD 2017 US NEUROLOGICAL DISORDERS COLLABORATORS, FEIGIN V L, VOS T, et al. Burden of neurological disorders across the US from 1990-2017:A global burden of disease study[J]. JAMA Neurol, 2021, 78(2):165-176. doi:10.1001/jamaneurol.2020.4152.
[11]	JANG W, KIM H J, LI H, et al. The neuroprotective effect of erythropoietin on rotenone-induced neurotoxicity in SH-SY5Y cells through the induction of autophagy[J]. Mol Neurobiol, 2016, 53(6):3812-3821. doi:10.1007/s12035-015-9316-x.
[12]	KOGAN M, MCGUIRE M, RILEY J. Deep brain stimulation for Parkinson disease[J]. Neurosurg Clin N Am, 2019, 30(2):137-146. doi:10.1016/j.nec.2019.01.001.
[13]	JANKOVIC J. Parkinson's disease: clinical features and diagnosis[J]. J Neurol Neurosurg Psychiatry, 2008, 79(4):368-376. doi:10.1136/jnnp.2007.131045.
[14]	BRUNDIN P, DAVE K D, KORDOWER J H. Therapeutic approaches to target alpha-synuclein pathology[J]. Exp Neurol, 2017, 298(Pt B):225-235. doi:10.1016/j.expneurol.2017.10.003.
[15]	NASCIMENTO A C, ERUSTES A G, RECKZIEGEL P, et al. α-Synuclein overexpression induces lysosomal dysfunction and autophagy impairment in human neuroblastoma SH-SY5Y[J]. Neurochem Res, 2020, 45(11):2749-2761. doi:10.1007/s11064-020-03126-8.
[16]	CHEN L, HUANG Y, YU X, et al. Corynoxine protects dopaminergic neurons through inducing autophagy and diminishing neuroinflammation in rotenone-induced animal models of Parkinson's disease[J]. Front Pharmacol, 2021, 12:642900. doi:10.3389/fphar.2021.642900.
[17]	SONG Q, PENG S, ZHU X. Baicalein protects against MPP⁺/MPTP^- induced neurotoxicity by ameliorating oxidative stress in SH-SY5Y cells and mouse model of Parkinson's disease[J]. Neurotoxicology, 2021, 87:188-194. doi:10.1016/j.neuro.2021.10.003.
[18]	FAKHRI S, IRANPANAH A, GRAVANDI M M, et al. Natural products attenuate PI3K/Akt/mTOR signaling pathway:A promising strategy in regulating neurodegeneration[J]. Phytomedicine, 2021, 91:153664. doi:10.1016/j.phymed.2021.153664.
[19]	BU L L, LIU Y Q, SHEN Y, et al. Neuroprotection of exendin-4 by enhanced autophagy in a Parkinsonian rat model of α-synucleinopathy[J]. Neurotherapeutics, 2021, 18(2):962-978. doi:10.1007/s13311-021-01018-5.
[20]	YAN D, MA Z, LIU C, et al. Corynoxine B ameliorates HMGB1-dependent autophagy dysfunction during manganese exposure in SH-SY5Y human neuroblastoma cells[J]. Food Chem Toxicol, 2019, 124:336-348. doi:10.1016/j.fct.2018.12.027.
[21]	GIACOPPO S, BRAMANTI P, MAZZON E. Triggering of inflammasome by impaired autophagy in response to acute experimental Parkinson's disease: involvement of the PI3K/Akt/mTOR pathway[J]. Neuroreport, 2017, 28(15):996-1007. doi:10.1097/WNR.0000000000000871.
[22]	WANG S J, WANG Q, MA J, et al. Effect of moxibustion on mTOR-mediated autophagy in rotenone-induced Parkinson's disease model rats[J]. Neural Regen Res, 2018, 13(1):112-118. doi:10.4103/1673-5374.224380.
[23]	ENOGIERU A B, HAYLETT W, HISS D C, et al. Regulation of AKT/AMPK signaling, autophagy and mitigation of apoptosis in Rutin-pretreated SH-SY5Y cells exposed to MPP[J]. Metab Brain Dis, 2021, 36(2):315-326. doi:10.1007/s11011-020-00641-z.
[24]	ZHOU J, ZHAO Y, LI Z, et al. miR-103a-3p regulates mitophagy in Parkinson's disease through Parkin/Ambra1 signaling[J]. Pharmacol Res, 2020, 160:105197. doi:10.1016/j.phrs.2020.105197.

组别	细胞存活率	细胞凋亡率
Control组	100.00±0.00	9.36±0.98
MPP⁺组	52.64±3.25^a	23.48±3.07^a
IGF-1组	58.69±4.15^a	19.67±3.24^a
LY294002组	136.29±8.34^ab	4.19±0.45^ab
MPP⁺+LY294002组	78.24±7.52^bc	16.35±2.18^bc
F	226.163^＊＊	70.666^＊＊

组别	细胞存活率	细胞凋亡率
Control组	100.00±0.00	9.36±0.98
MPP⁺组	52.64±3.25^a	23.48±3.07^a
IGF-1组	58.69±4.15^a	19.67±3.24^a
LY294002组	136.29±8.34^ab	4.19±0.45^ab
MPP⁺+LY294002组	78.24±7.52^bc	16.35±2.18^bc
F	226.163^＊＊	70.666^＊＊

组别	自噬空泡比例（%）	p62	LC3BⅡ/Ⅰ
Control组	75.36±9.36	0.45±0.03	0.89±0.09
MPP⁺组	24.36±2.58^a	1.28±0.09^a	0.24±0.03^a
IGF-1组	26.47±2.64^a	1.15±0.10^a	0.17±0.02^a
LY294002组	89.89±9.08^ab	0.24±0.02^ab	1.28±0.11^ab
MPP⁺+LY294002组	61.51±5.59^bc	0.79±0.06^bc	0.57±0.05^bc
F	119.538^＊＊	256.787^＊＊	267.938^＊＊

组别	自噬空泡比例（%）	p62	LC3BⅡ/Ⅰ
Control组	75.36±9.36	0.45±0.03	0.89±0.09
MPP⁺组	24.36±2.58^a	1.28±0.09^a	0.24±0.03^a
IGF-1组	26.47±2.64^a	1.15±0.10^a	0.17±0.02^a
LY294002组	89.89±9.08^ab	0.24±0.02^ab	1.28±0.11^ab
MPP⁺+LY294002组	61.51±5.59^bc	0.79±0.06^bc	0.57±0.05^bc
F	119.538^＊＊	256.787^＊＊	267.938^＊＊

组别	TH	α-syn
Control组	0.73±0.07	0.34±0.03
MPP⁺组	0.22±0.03^a	0.78±0.08^a
IGF-1组	0.24±0.04^a	0.75±0.06^a
LY294002组	0.98±0.10^ab	0.16±0.02^ab
MPP⁺+LY294002组	0.48±0.04^bc	0.56±0.05^bc
F	167.842^＊＊	154.391^＊＊