Protective effects of baicalin against L-glutamate-induced oxidative damage in HT-22 cells by inhibiting NLRP3 inflammasome activation via Nrf2/HO-1 signaling

Document Type : Original Article

Authors

1 Institute of Basic Medical Sciences, Xiyuan Hospital of China Academy of Chinese Medical Sciences, No.1 Xiyuan Caochang, Haidian District, Beijing, 100091, China

2 Key Laboratory of Pharmacology of Chinese Materia Medica, Beijing 100091, China

3 Hubei Provincial Hospital of Integrated Chinese and Western Medicine, Wuhan, Hubei 430015, China

Abstract

Objective(s): To explore the ability and underlying molecular mechanisms involved in the protective effects of Baicalin (BA) against L-Glutamate-induced mouse hippocampal neuron cell line HT-22.
Materials and Methods: The cell injury model of HT-22 cells was induced by L-glutamate, and cell viability and damage were detected by CCK-8 and LDH assays. Generation of intracellular reactive oxygen species (ROS) was measured by DCFH-DA in situ fluorescence method. The SOD activity and MDA concentration in the supernatants were determined by WST-8 and colorimetric method, respectively. Furthermore, Western blot and real-time qPCR analysis were utilized to detect the expression levels of the Nrf2/HO-1 signaling pathway and NLRP3 inflammasome proteins and genes.
Results: L-Glutamate exposure induced cell injuries in HT-22 cells, and the concentration of 5 mM L-Glutamate was chosen to be the modeling condition. Co-treatment with BA significantly promoted cell viability and reduced LDH release in a dose-dependent manner. In addition, BA attenuated the L-Glutamate-induced injuries by decreasing the ROS production and MDA concentration, while increasing the SOD activity. Moreover, we also found that BA treatment up-regulated the gene and protein expression of Nrf2 and HO-1, and then inhibited the expression of NLRP3.
Conclusion: Our study found that BA could relieve oxidative stress damage of HT-22 cells induced by L-Glutamate, and the mechanism might be related to the activation of Nrf2/HO-1 and inhibition of NLRP3 inflammasome.

Keywords


1. Jantas D, Greda A, Golda S, Korostynski M, Grygier B, Roman A, et al. Neuroprotective effects of metabotropic glutamate receptor group II and III activators against MPP(+)-induced cell death in human neuroblastoma SH-SY5Y cells: the impact of cell differentiation state. Neuropharmacology 2014;83:36-53.
2. Sims NR, Zaidan E. Biochemical changes associated with selective neuronal death following short-term cerebral ischaemia. Int J Biochem Cell Biol. 1995;27:531-550.
3. Dong XX, Wang Y, Qin ZH. Molecular mechanisms of excitotoxicity and their relevance to pathogenesis of neurodegenerative diseases. Acta Pharmacologica Sinica 2009;30:379-387.
4. Rossler OG, Bauer I, Chung HY, Thiel G. Glutamate-induced cell death of immortalized murine hippocampal neurons: neuroprotective activity of heme oxygenase-1, heat shock protein 70, and sodium selenite. Neurosci Lett 2004;362:253-257.
5. Ndountse LT, Chan HM. Role of N-methyl-D-aspartate receptors in polychlorinated biphenyl mediated neurotoxicity. Toxicol Lett 2009;184:50-55.
6. Murphy TH, Miyamoto M, Sastre A, Schnaar RL, Coyle JT. Glutamate toxicity in a neuronal cell line involves inhibition of cystine transport leading to oxidative stress. Neuron. 1989;2:1547-1558.
7. Tan S, Wood M, Maher P. Oxidative stress induces a form of programmed cell death with characteristics of both apoptosis and necrosis in neuronal cells. J Neurochem 1998;71:95-105.
8. Poljsak B, Suput D, Milisav I. Achieving the balance between ROS and anti-oxidants: when to use the synthetic anti-oxidants. Oxid Med Cell Longev 2013;2013:956792.
9.    Loboda A, Damulewicz M, Pyza E, Jozkowicz A, Dulak J. Role of Nrf2/HO-1 system in development, oxidative stress response and diseases: an evolutionarily conserved mechanism. Cell Mol Life Sci 2016;73:3221-3247.
10.    Zhu X, Guo F, Tang H, Huang C, Xie G, Huang T, et al. Islet transplantation attenuating testicular injury in type 1 diabetic rats is associated with suppression of oxidative stress and inflammation via Nrf-2/HO-1 and NF-kappaB pathways. J Diabetes Res 2019;2019:8712492.
11.    Karki R, Kanneganti TD. Diverging inflammasome signals in tumorigenesis and potential targeting. Nat Rev Cancer 2019;19:197-214.
12.    Tu L, Wu ZY, Yang XL, Zhang Q, Gu R, Wang Q, et al. Neuroprotective effect and mechanism of baicalin on Parkinson’s disease model induced by 6-OHDA. Neuropsychiatr Dis Treat 2019;15:3615-3625.
13.    Dai J, Qiu YM, Ma ZW, Yan GF, Zhou J, Li SQ, et al. Neuroprotective effect of baicalin on focal cerebral ischemia in rats. Neural Regen Res 2018;13:2129-2133.
14.    Zhang CY, Zeng MJ, Zhou LP, Li YQ, Zhao F, Shang ZY, et al. Baicalin exerts neuroprotective effects via inhibiting activation of GSK3beta/NF-kappaB/NLRP3 signal pathway in a rat model of depression. Int Immunopharmacol 2018;64:175-182.
15.    Cao Y, Mao X, Sun C, Zheng P, Gao J, Wang X, et al. Baicalin attenuates global cerebral ischemia/reperfusion injury in gerbils via anti-oxidative and anti-apoptotic pathways. Brain Res Bull 2011;85:396-402.
16. Cheng O, Li Z, Han Y, Jiang Q, Yan Y, Cheng K. Baicalin improved the spatial learning ability of global ischemia/reperfusion rats by reducing hippocampal apoptosis. Brain Res 2012;1470:111-118.
17. Liu Z, Zhang L, He Q, Liu X, Okeke CI, Tong L, et al. Effect of Baicalin-loaded PEGylated cationic solid lipid nanoparticles modified by OX26 antibody on regulating the levels of baicalin and amino acids during cerebral ischemia-reperfusion in rats. Int J Pharm. 2015;489(1-2):131-138.
18.    Hu H, Zhong X, Lin X, Yang J, Zhu X. Inhibitory effect of gualou guizhi decoction on microglial inflammation and neuron injury by promoting anti-inflammation via targeting mmu-miR-155. Evid Based Complement Alternat Med. 2021;2021:2549076.
19. Shi LY, Zhang L, Li H, Liu TL, Lai JC, Wu ZB, et al. Protective effects of curcumin on acrolein-induced neurotoxicity in HT22 mouse hippocampal cells. Pharmacol Rep 2018;70:1040-1046.
20. Lobner D. Comparison of the LDH and MTT assays for quantifying cell death: validity for neuronal apoptosis? J Neurosci Methods 2000;96:147-152.
21. Zelko IN, Mariani TJ, Folz RJ. Superoxide dismutase multigene family: A comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression. Free Radic Biol Med 2002;33:337-349.
22. Del Rio D, Stewart AJ, Pellegrini N. A review of recent studies on malondialdehyde as toxic molecule and biological marker of oxidative stress. Nutr Metab Cardiovasc Dis 2005;15:316-328.
23.    Li W, Khor TO, Xu C, Shen G, Jeong WS, Yu S, et al. Activation of Nrf2-anti-oxidant signaling attenuates NFkappaB-inflammatory response and elicits apoptosis. Biochem Pharmacol 2008;76:1485-1489.
24.    Park SY, Jung WJ, Kang JS, Kim CM, Park G, Choi YW. Neuroprotective effects of alpha-iso-cubebene against glutamate-induced damage in the HT22 hippocampal neuronal cell line. Int J Mol Med 2015;35:525-532.
25.    Ju XN, Mu WN, Liu YT, Wang MH, Kong F, Sun C, et al. Baicalin protects against thrombin induced cell injury in SH-SY5Y cells. Int J Clin Exp Pathol 2015;8:14021-14027.
26.    Jin X, Liu MY, Zhang DF, Zhong X, Du K, Qian P, et al. Baicalin mitigates cognitive impairment and protects neurons from microglia-mediated neuroinflammation via suppressing NLRP3 inflammasomes and TLR4/NF-kappaB signaling pathway. CNS Neurosci Ther 2019;25:575-590.
27.    Paudel KR, Kim DW. Microparticles-mediated vascular inflammation and its amelioration by anti-oxidant activity of baicalin. Anti-oxidants (Basel). 2020;9:890.
28.    Wang X, Yu JY, Sun Y, Wang H, Shan H, Wang S. Baicalin protects LPS-induced blood-brain barrier damage and activates Nrf2-mediated anti-oxidant stress pathway. Int Immunopharmacol 2021;96:107725.
29.    Lin D, Du Q, Wang H, Gao G, Zhou J, Ke L, et al. Antidiabetic micro-/nanoaggregates from ge-gen-qin-lian-tang decoction increase absorption of baicalin and cellular anti-oxidant activity in vitro. Biomed Res Int 2017;2017:9217912.
30.    Zhang L, Yang L, Xie X, Zheng H, Zheng H, Zhang L, et al. Baicalin magnesium salt attenuates lipopolysaccharide-induced acute lung injury via inhibiting of TLR4/NF-kappaB signaling pathway. J Immunol Res 2021;2021:6629531.
31.    Huang Z, Guo L, Huang L, Shi Y, Liang J, Zhao L. Baicalin-loaded macrophage-derived exosomes ameliorate ischemic brain injury via the anti-oxidative pathway. Mater Sci Eng C Mater Biol Appl 2021;126:112123.
32.    Duryee MJ, Klassen LW, Schaffert CS, Tuma DJ, Hunter CD, Garvin RP, et al. Malondialdehyde-acetaldehyde adduct is the dominant epitope after MDA modification of proteins in atherosclerosis. Free Radic Biol Med 2010;49:1480-1486.
33.    Bao L, Li J, Zha D, Zhang L, Gao P, Yao T, et al. Chlorogenic acid prevents diabetic nephropathy by inhibiting oxidative stress and inflammation through modulation of the Nrf2/HO-1 and NF-kB pathways. Int Immunopharmacol 2018;54:245-253.
34.    Kwon DH, Cha HJ, Choi EO, Leem SH, Kim GY, Moon SK, et al. Schisandrin a suppresses lipopolysaccharide-induced inflammation and oxidative stress in RAW 264.7 macrophages by suppressing the NF-kappaB, MAPKs and PI3K/Akt pathways and activating Nrf2/HO-1 signaling. Int J Mol Med 2018;41:264-274.
35.    Joshi G, Johnson JA. The Nrf2-ARE pathway: A valuable therapeutic target for the treatment of neurodegenerative diseases. Recent Pat CNS Drug Discov. 2012;7:218-229.
36.    Li S, Song Z, Liu T, Liang J, Yuan J, Xu Z, et al. Polysaccharide from ostrea rivularis attenuates reproductive oxidative stress damage via activating Keap1-Nrf2/ARE pathway. Carbohydr Polym 2018;186:321-31.
37.    Tang J, Jia X, Gao N, Wu Y, Liu Z, Lu X, et al. Role of the Nrf2-ARE pathway in perfluorooctanoic acid (PFOA)-induced hepatotoxicity in Rana nigromaculata. Environ Pollut 2018;238:1035-1043.
38.    Zu G, Zhou T, Che N, Zhang X. Salvianolic acid a protects against oxidative stress and apoptosis induced by intestinal ischemia-reperfusion injury through activation of Nrf2/HO-1 pathways. Cell Physiol Biochem 2018;49:2320-2332.
39.    Zeng Q, Lian W, Wang G, Qiu M, Lin L, Zeng R. Pterostilbene induces Nrf2/HO-1 and potentially regulates NF-kappaB and JNK-Akt/mTOR signaling in ischemic brain injury in neonatal rats. 3 Biotech 2020;10:192.
40.    Zhu X, Xi C, Thomas B, Pace BS. Loss of NRF2 function exacerbates the pathophysiology of sickle cell disease in a transgenic mouse model. Blood 2018;131:558-562.
41.    Kubo Y, Wruck CJ, Fragoulis A, Drescher W, Pape HC, Lichte P, et al. Role of Nrf2 in fracture healing: Clinical aspects of oxidative stress. Calcif Tissue Int 2019;105:341-352.
42.    Bao M, Liang M, Sun X, Mohyuddin SG, Chen S, Wen J, et al. Baicalin alleviates LPS-induced oxidative stress via NF-kappaB and Nrf2-HO1 signaling pathways in IPEC-J2 cells. Front Vet Sci 2021;8:808233.
43. Huang X, He Y, Chen Y, Wu P, Gui D, Cai H, et al. Baicalin attenuates bleomycin-induced pulmonary fibrosis via adenosine A2a receptor related TGF-beta1-induced ERK1/2 signaling pathway. BMC Pulm Med 2016;16:132.
44. Li Y, Liu T, Li Y, Han D, Hong J, Yang N, et al. Baicalin ameliorates cognitive impairment and protects microglia from lps-induced neuroinflammation via the SIRT1/HMGB1 pathway. Oxid Med Cell Longev 2020;2020:4751349.
45. Li J, Qiao Z, Hu W, Zhang W, Shah SWA, Ishfaq M. Baicalin mitigated Mycoplasma gallisepticum-induced structural damage and attenuated oxidative stress and apoptosis in chicken thymus through the Nrf2/HO-1 defence pathway. Vet Res 2019;50:83.
46.    Meng X, Hu L, Li W. Baicalin ameliorates lipopolysaccharide-induced acute lung injury in mice by suppressing oxidative stress and inflammation via the activation of the Nrf2-mediated HO-1 signaling pathway. Naunyn Schmiedebergs Arch Pharmacol 2019;392:1421-1433.
47. Lei L, Guo Y, Lin J, Lin X, He S, Qin Z, et al. Inhibition of endotoxin-induced acute lung injury in rats by bone marrow-derived mesenchymal stem cells: Role of Nrf2/HO-1 signal axis in inhibition of NLRP3 activation. Biochem Biophys Res Commun 2021;551:7-13.
48.    Du D, Lv W, Su R, Yu C, Jing X, Bai N, et al. Hydrolyzed camel whey protein alleviated heat stress-induced hepatocyte damage by activated Nrf2/HO-1 signaling pathway and inhibited NF-kappaB/NLRP3 axis. Cell Stress Chaperones 2021;26:387-401.
49.    Chen L, He PL, Yang J, Yang YF, Wang K, Amend B, et al. NLRP3/IL1beta inflammasome associated with the aging bladder triggers bladder dysfunction in female rats. Mol Med Rep 2019;19:2960-2968.
50. Xu M, Wang L, Wang M, Wang H, Zhang H, Chen Y, et al. Mitochondrial ROS and NLRP3 inflammasome in acute ozone-induced murine model of airway inflammation and bronchial hyperresponsiveness. Free Radic Res 2019;53:780-790.
51.    Li J, Ma C, Long F, Yang D, Liu X, Hu Y, et al. Parkin impairs antiviral immunity by suppressing the mitochondrial reactive oxygen species-Nlrp3 axis and antiviral inflammation. iScience 2019;16:468-484.
52.    Lin Q, Li S, Jiang N, Shao X, Zhang M, Jin H, et al. PINK1-parkin pathway of mitophagy protects against contrast-induced acute kidney injury via decreasing mitochondrial ROS and NLRP3 inflammasome activation. Redox Biol 2019;26:101254.
53.    Shen C, Liu J, Zhu F, Lei R, Cheng H, Zhang C, et al. The effects of cooking oil fumes-derived PM2.5 on blood vessel formation through ROS-mediated NLRP3 inflammasome pathway in human umbilical vein endothelial cells. Ecotoxicol Environ Saf 2019;174:690-698.
54.    Elliott EI, Sutterwala FS. Initiation and perpetuation of NLRP3 inflammasome activation and assembly. Immunol Rev 2015;265:35-52.
55.    Wang S, Zhao X, Yang S, Chen B, Shi J. Salidroside alleviates high glucose-induced oxidative stress and extracellular matrix accumulation in rat glomerular mesangial cells by the TXNIP-NLRP3 inflammasome pathway. Chem Biol Interact 2017;278:48-53.
56.    Li Y, Li J, Li S, Li Y, Wang X, Liu B, et al. Curcumin attenuates glutamate neurotoxicity in the hippocampus by suppression of ER stress-associated TXNIP/NLRP3 inflammasome activation in a manner dependent on AMPK. Toxicol Appl Pharmacol 2015;286:53-63.
57.    Zhao J, Wang Z, Yuan Z, Lv S, Su Q. Baicalin ameliorates atherosclerosis by inhibiting NLRP3 inflammasome in apolipoprotein E-deficient mice. Diab Vasc Dis Res 2020;17:6.
58.    Zhou QB, Jin YL, Jia Q, Zhang Y, Li LY, Liu P, et al. Baicalin attenuates brain edema in a rat model of intracerebral hemorrhage. Inflammation 2014;37:107-115.
59.    Zhang C, Yu P, Ma J, Zhu L, Xu A, Zhang J. Damage and phenotype change in PC12 cells induced by lipopolysaccharide can be inhibited by anti-oxidants through reduced cytoskeleton protein synthesis. Inflammation 2019;42:2246-2256.
60.    Xu WF, Liu F, Ma YC, Qian ZR, Shi L, Mu H, et al. Baicalin Regulates Proliferation, Apoptosis, Migration, and Invasion in Mesothelioma. Med Sci Monit 2019;25:8172-8180.
61.    Lee SG, Kim B, Yang Y, Pham TX, Park YK, Manatou J, et al. Berry anthocyanins suppress the expression and secretion of proinflammatory mediators in macrophages by inhibiting nuclear translocation of NF-kappaB independent of NRF2-mediated mechanism. J Nutr Biochem. 2014;25:404-411.
62.    Teles RBD, Diniz TC, Pinto TCC, de Oliveira RG, Silva MGE, de Lavor EM, et al. Flavonoids as therapeutic agents in alzheimer’s and parkinson’s diseases: A systematic review of preclinical evidences. Oxid Med Cell Longev 2018;2018: 7043213.
63.    Spagnuolo C, Moccia S, Russo GL. Anti-inflammatory effects of flavonoids in neurodegenerative disorders. Eur J Med Chem 2018;153:105-115.