Fasudil inhibits lipopolysaccharide-induced astrocytic injury by regulating Nrf2/HO-1 signaling pathway

doi:10.12307/2021.144

Abstract

Abstract: BACKGROUND: More and more evidences show that ROCK signaling pathway is involved in inflammation and oxidative stress. Fasudil is an effective Rho kinase inhibitor. Our previous series of studies have proved that fasudil has neuroprotective effects. However, it is still unclear that whether fasudil has protective effect on oxidative damage to astrocytes and its possible mechanisms.
OBJECTIVE: To explore the protective effect and mechanism of fasudil on lipopolysaccharide-induced oxidative injury in astrocytes.
METHODS: Primary C57BL/6 mouse astrocytes were cultured and divided into PBS control group, lipopolysaccharide stimulation group (1 mg/L), lipopolysaccharide (1 mg/L) combined with fasudil (15 mg/L) treatment group. After 24 hours of treatment, MTT assay was used to detect the viability of astrocytes. The content of malondialdehyde and the activity of superoxide dismutase in astrocytes were measured using the kit to evaluate the level of oxidative stress. Immunofluorescence staining was used to detect the expression of cleaved-caspase-3, the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and the expression of heme oxygenase-1 (HO-1) in astrocytes. Western blot assay was used to detect the total Nrf2, nuclear Nrf2 and HO-1 protein levels.
RESULTS AND CONCLUSION: (1) Compared with the PBS control group, the astrocyte viability in the lipopolysaccharide stimulation group was reduced, and fasudil treatment improved the cell damage induced by lipopolysaccharide. (2) Compared with the PBS control group, lipopolysaccharide resulted in a significant decrease in the activity of superoxide dismutase and a significant increase in the level of malondialdehyde in astrocytes (P < 0.01). Fasudil significantly increased the activity of superoxide dismutase (P < 0.01) and reduced the level of malondialdehyde (P < 0.01). (3) Compared with the PBS control group, the expression of cleaved-caspase-3 was significantly increased in the lipopolysaccharide stimulation group. Compared with the lipopolysaccharide stimulation group, the expression of cleaved-caspase-3 was decreased in the lipopolysaccharide combined with fasudil treatment group. (4) Fasudil could promote the nuclear translocation of Nrf2 and significantly increase the expression of total Nrf2, nucleus Nrf2 and HO-1 after lipopolysaccharide stimulation. (5) The results indicate that fasudil can inhibit lipopolysaccharide-induced astrocytic injury by regulating the Nrf2/HO-1 signaling pathway.

Key words: astrocyte, lipopolysaccharide, fasudil, nuclear factor erythroid 2-related factor 2, oxidative stress, apoptosis, experiment

CLC Number:

Guo Minfang, Zhang Huiyu, Zhang Peijun, Bai Zhenjun, Yu Jingwen, Wang Yuyin, Wei Wenyue, Song Lijuan, Chai Zhi, Yu Jiezhong, Ma Cungen. Fasudil inhibits lipopolysaccharide-induced astrocytic injury by regulating Nrf2/HO-1 signaling pathway[J]. Chinese Journal of Tissue Engineering Research, 2021, 25(31): 5012-5017.

Figures/Tables 6

References

[1] LIGUORI I, RUSSO G, CURCIO F, et al. Oxidative stress, aging, and diseases. Clin Interv Aging. 2018;13:757-772.
[2] TOBORE TO. On the central role of mitochondria dysfunction and oxidative stress in Alzheimer’s disease. Neurol Sci. 2019;40(8):1527-1540.
[3] POHANKA M. Oxidative stress in Alzheimer disease as a target for therapy. Bratisl Lek Listy. 2018;119(9):535-543.
[4] MATSCHKE V, THEISS C, MATSCHKE J. Oxidative stress: the lowest common denominator of multiple diseases. Neural Regen Res. 2019;14(2):238-241.
[5] FRAUNBERGER EA, SCOLA G, LALIBERTÉ VL, et al. Redox Modulations, Antioxidants, and Neuropsychiatric Disorders. Oxid Med Cell Longev. 2016; 2016:4729192.
[6] MCBEAN GJ. Cysteine, Glutathione, and Thiol Redox Balance in Astrocytes. Antioxidants (Basel). 2017;6(3):62.
[7] CABEZAS R, BAEZ-JURADO E, HIDALGO-LANUSSA O, et al. Growth Factors and Neuroglobin in Astrocyte Protection Against Neurodegeneration and Oxidative Stress. Mol Neurobiol. 2019;56(4):2339-2351.
[8] JOE EH, CHOI DJ, AN J, et al. Astrocytes, Microglia, and Parkinson’s Disease. Exp Neurobiol. 2018;27(2):77-87.
[9] GONZÁLEZ-REYES RE, NAVA-MESA MO, VARGAS-SÁNCHEZ K, et al. Involvement of Astrocytes in Alzheimer’s Disease from a Neuroinflammatory and Oxidative Stress Perspective. Front Mol Neurosci. 2017;10:427.
[10] RIZOR A, PAJARILLO E, JOHNSON J, et al. Astrocytic Oxidative/Nitrosative Stress Contributes to Parkinson’s Disease Pathogenesis: The Dual Role of Reactive Astrocytes. Antioxidants (Basel). 2019;8(8):265.
[11] MOLINARI C, MORSANUTO V, GHIRLANDA S, et al. Role of Combined Lipoic Acid and Vitamin D3 on Astrocytes as a Way to Prevent Brain Ageing by Induced Oxidative Stress and Iron Accumulation. Oxid Med Cell Longev. 2019;2019:2843121.
[12] SHEFA U, JEONG NY, SONG IO, et al. Mitophagy links oxidative stress conditions and neurodegenerative diseases. Neural Regen Res. 2019;14(5):749-756.
[13] HAMDI Y, KADDOUR H, VAUDRY D, et al. The octadecaneuropeptide ODN protects astrocytes against hydrogen peroxide-induced apoptosis via a PKA/MAPK-dependent mechanism. PLoS One. 2012;7(8):e42498.
[14] SANDBERG M, PATIL J, D’ANGELO B, et al. NRF2-regulation in brain health and disease: implication of cerebral inflammation. Neuropharmacology. 2014;79:298-306.
[15] CAO S, CHAO D, ZHOU H, et al. A novel mechanism for cytoprotection against hypoxic injury: δ-opioid receptor-mediated increase in Nrf2 translocation. Br J Pharmacol. 2015;172(7):1869-1881.
[16] NITURE SK, KASPAR JW, SHEN J, et al. Nrf2 signaling and cell survival. Toxicol Appl Pharmacol. 2010;244(1):37-42.
[17] SHU L, WANG C, WANG J, et al. The neuroprotection of hypoxic preconditioning on rat brain against traumatic brain injury by up-regulated transcription factor Nrf2 and HO-1 expression. Neurosci Lett. 2016;611:74-80.
[18] FU PC, TANG RH, YU ZY, et al. The Rho-associated kinase inhibitors Y27632 and fasudil promote microglial migration in the spinal cord via the ERK signaling pathway. Neural Regen Res. 2018;13(4):677-683.
[19] YAN Y, YU J, GAO Y, et al. Therapeutic potentials of the Rho kinase inhibitor Fasudil in experimental autoimmune encephalomyelitis and the related mechanisms. Metab Brain Dis. 2019;34(2):377-384.
[20] WANG J, SUI RX, MIAO Q, et al. Retraction: Effect of Fasudil on remyelination following cuprizone-induced demyelination. CNS Neurosci Ther. 2020;26(7):778.
[21] 张慧宇,郭敏芳,于婧文,等.法舒地尔(Fasudil)抑制脂多糖诱导的小鼠星形胶质细胞活化和炎性反应及其机制[J].细胞与分子免疫学杂志, 2018,34(6):505-510.
[22] WANG Y, ZHAO CS. Sigma-1 receptor activation ameliorates LPS-induced NO production and ROS formation through the Nrf2/HO-1 signaling pathway in cultured astrocytes. Neurosci Lett. 2019;711:134387.
[23] POURHANIFEH MH, SHAFABAKHSH R, REITER RJ, et al. The Effect of Resveratrol on Neurodegenerative Disorders: Possible Protective Actions Against Autophagy, Apoptosis, Inflammation and Oxidative Stress. Curr Pharm Des. 2019;25(19):2178-2191.
[24] CHERBUIN N, WALSH E, BAUNE BT, et al. Oxidative stress, inflammation and risk of neurodegeneration in a population sample. Eur J Neurol. 2019; 26(11):1347-1354.
[25] SOFRONIEW MV, VINTERS HV. Astrocytes: biology and pathology. Acta Neuropathol. 2010;119(1):7-35.
[26] PACHECO SM, AZAMBUJA JH, DE CARVALHO TR, et al. Glioprotective Effects of Lingonberry Extract Against Altered Cellular Viability, Acetylcholinesterase Activity, and Oxidative Stress in Lipopolysaccharide-Treated Astrocytes. Cell Mol Neurobiol. 2018;38(5):1107-1121.
[27] SHARMA A, PATRO N, PATRO IK. Lipopolysaccharide-Induced Apoptosis of Astrocytes: Therapeutic Intervention by Minocycline. Cell Mol Neurobiol. 2016;36(4):577-592.
[28] PORTER AG, JÄNICKE RU. Emerging roles of caspase-3 in apoptosis. Cell Death Differ. 1999;6(2):99-104.
[29] SHU Q, FAN H, LI SJ, et al. Protective effects of Progranulin against focal cerebral ischemia-reperfusion injury in rats by suppressing endoplasmic reticulum stress and NF-κB activation in reactive astrocytes. J Cell Biochem. 2018;119(8):6584-6597.
[30] OTTERBEIN LE, FORESTI R, MOTTERLINI R. Heme Oxygenase-1 and Carbon Monoxide in the Heart: The Balancing Act Between Danger Signaling and Pro-Survival. Circ Res. 2016;118(12):1940-1959.
[31] LOBODA A, DAMULEWICZ M, PYZA E, et al. Role of Nrf2/HO-1 system in development, oxidative stress response and diseases: an evolutionarily conserved mechanism. Cell Mol Life Sci. 2016;73(17):3221-3247.
[32] ALI T, KIM T, REHMAN SU, et al. Natural Dietary Supplementation of Anthocyanins via PI3K/Akt/Nrf2/HO-1 Pathways Mitigate Oxidative Stress, Neurodegeneration, and Memory Impairment in a Mouse Model of Alzheimer’s Disease. Mol Neurobiol. 2018;55(7):6076-6093.
[33] JO MG, IKRAM M, JO MH, et al. Gintonin Mitigates MPTP-Induced Loss of Nigrostriatal Dopaminergic Neurons and Accumulation of α-Synuclein via the Nrf2/HO-1 Pathway. Mol Neurobiol. 2019;56(1):39-55.
[34] HUNG SY, LIOU HC, FU WM. The mechanism of heme oxygenase-1 action involved in the enhancement of neurotrophic factor expression. Neuropharmacology. 2010;58(2):321-329.