Anti-inflammatory and DNA Repair Effects of Astragaloside IV on PC12 Cells Damaged by Lipopolysaccharide

Abstract

Objective

This study aimed to establish a neural cell injury model in vitro by stimulating PC12 cells with lipopolysaccharide (LPS) and to examine the effects of astragaloside IV on key targets using high-throughput sequence technology and bioinformatics analyses.

Methods

PC12 cells in the logarithmic growth phase were treated with LPS at final concentrations of 0.25, 0.5, 0.75, 1, and 1.25 mg/mL for 24 h. Cell morphology was evaluated, and cell survival rates were calculated. A neurocyte inflammatory model was established with LPS treatment, which reached a 50% cell survival rate. PC12 cells were treated with 0.01, 0.1, 1, 10, or 100 µmol/L astragaloside IV for 24 h. The concentration of astragaloside IV that did not affect the cell survival rate was selected as the treatment group for subsequent experiments. NOS activity was detected by colorimetry; the expression levels of ERCC2, XRCC4, XRCC2, TNF-α, IL-1β, TLR4, NOS and COX-2 mRNA and protein were detected by RT-qPCR and Western blotting. The differentially expressed genes (DEGs) between the groups were screened using a second-generation sequence (fold change>2, P<0.05) with the following KEGG enrichment analysis, RT-qPCR and Western blotting were used to detect the mRNA and protein expression of DEGs related to the IL-17 pathway in different groups of PC12 cells.

Results

The viability of PC12 cells was not altered by treatment with 0.01, 0.1, or 1 µmol/L astragaloside IV for 24 h (P>0.05). However, after treatment with 0.5, 0.75, 1, or 1.25 mg/mL LPS for 24 h, the viability steadily decreased (P<0.01). The mRNA and protein expression levels of ERCC2, XRCC4, XRCC2, TNF-α, IL-1β, TLR4, NOS, and COX-2 were significantly increased after PC12 cells were treated with 1 mg/mL LPS for 24 h (P<0.01); however, these changes were reversed when PC12 cells were pretreated with 0.01, 0.1, or 1 µmol/L astragaloside IV in PC12 cells and then treated with 1 mg/mL LPS for 24 h (P<0.05). Second-generation sequencing revealed that 1026 genes were upregulated, while 1287 genes were downregulated. The DEGs were associated with autophagy, TNF-α, interleukin-17, MAPK, P53, Toll-like receptor, and NOD-like receptor signaling pathways. Furthermore, PC12 cells treated with a 1 mg/mL LPS for 24 h exhibited increased mRNA and protein expression of CCL2, CCL11, CCL7, MMP3, and MMP10, which are associated with the IL-17 pathway. RT-qPCR and Western blotting analyses confirmed that the DEGs listed above corresponded to the sequence assay results.

Conclusion

LPS can damage PC12 cells and cause inflammatory reactions in nerve cells and DNA damage. astragaloside IV plays an anti-inflammatory and DNA damage protective role and inhibits the IL-17 signaling pathway to exert a neuroprotective effect in vitro.

References

[1] Hopfner F, Höglinger GU, Kuhlenbäumer G, et al.. β-adrenoreceptors and the risk of Parkinson’s disease. Lancet Neurol, 2020, 19(3): 247-254

[2] Mackenzie F, Ruhrberg C. Diverse roles for VEGF-A in the nervous system. Development, 2012, 139(8): 1371-1380

[3] Leonard BE, Myint A. Inflammation and depression: Is there a causal connection with dementia?. Neurotox Res, 2006, 10(2): 149-160

[4] Wuwongse S, Chang RC, Law AC. The putative neurodegenerative links between depression and Alzheimer’s disease. Prog Neurobiol, 2010, 91(4): 362-375

[5] Kumar RA, Damanpreet S. Targeting Glycogen synthase kinase-3 for oxidative stress and neuroinflammation: Opportunities, challenges and future directions for cerebral stroke management. Neuropharmacology, 2018, 139: 124-136

[6] Wang P, Sun L, Shen A, et al.. Involvement of Src-Suppressed C Kinase Substrate in Neuronal Death Caused by the Lipopolysaccharide-Induced Reactive Astrogliosis. Inflammation, 2010, 33(6): 359-373

[7] Togbe D, Schnyder-Candrian S, Schnyder B, et al.. Toll-like receptor and tumor necrosis factor dependent endotoxin-induced acute lung injury. Int J Exp Pathol, 2007, 88(6): 387-391

[8] Monga S, Denora N, Laquintana V, et al.. The Neuro-Protective Effects of the TSPO Ligands CB86 and CB204 on 6-OHDA-Induced PC12 Cell Death as an In Vitro Model for Parkinson’s Disease. Biology (Basel), 2021, 10(11): 1183

[9] Bournival J, Quessy P, Martinoli MG. Protective effects of resveratrol and quercetin against MPP+-induced oxidative stress act by modulating markers of apoptotic death in dopaminergic neurons. Cell Mol Neurobiol, 2009, 29(8): 1169-1180

[10] Envelope SY, Envelope WP, Envelope F Q, et al.. Research progress of astragaloside IV in the treatment of atopic diseases. Biomed Pharmacother, 2022, 156: 113989

[11] Streit WJ, Mrak RE, Griffin WST. Microglia and neuroinflammation: a pathological perspective. J Neuroinflammation, 2004, 1(1): 14

[12] Honig LS. Inflammation in Neurodegenerative Disease. Arch Neurol, 2000, 57(6): 786

[13] Glass CK, Saijo K, Winner B, et al.. Mechanisms underlying inflammation in neurodegeneration. Cell, 2010, 140(6): 918-934

[14] Brown GC. The endotoxin hypothesis of neurodegeneration. J Neuroinflammation, 2019, 16(1): 180

[15] Miklossy J. Chronic Inflammation and Amyloidogenesis in Alzheimer’s Disease-Role of Spirochetes. J Alzheimers Dis, 2008, 13(4): 381-391

[16] Yuan Q, Wu Y, Wang G, et al.. Preventive effects of arctigenin from Arctium lappa L against LPS-induced neuroinflammation and cognitive impairments in mice. Metab Brain Dis, 2022, 37(6): 2039-2052

[17] Wang LM, Wu Q, Kirk RA, et al.. Lipopolysaccharide endotoxemia induces amyloid-β and p-tau formation in the rat brain. Am J Nucl Med Mol Imaging, 2018, 8(2): 86-99

[18] Zhan X, Stamova B, Jin LW, et al.. Gram-negative bacterial molecules associate with Alzheimer disease pathology. Neurology, 2016, 22: 2324-2332

[19] Yuhai Z, Lin C, Vivian J, et al.. Microbiome-Derived Lipopolysaccharide Enriched in the Perinuclear Region of Alzheimer’s Disease Brain. Front Immunol, 2017, 8: 1064

[20] Wakulik K, Wiatrak B, Szczukowski L, et al.. Effect of Novel Pyrrolo[3,4-d]pyridazinone Derivatives on Lipopolysaccharide-Induced Neuroinflammation. Int J Mol Sci, 2020, 21(7): 2575

[21] Buchanan MM, Hutchinson M, Watkins LR, et al.. Toll-like receptor 4 in CNS pathologies. J Neurochem, 2010, 114(1): 13-27

[22] Wadachi R, Hargreaves KM. Trigeminal nociceptors express TLR-4 and CD14: a mechanism for pain due to infection. J Dent Res, 2006, 85(1): 49-53

[23] Duan T, Du Y, Xing C, et al.. Toll-Like Receptor Signaling and Its Role in Cell-Mediated Immunity. Front Immunol, 2022, 13: 812774

[24] Gioannini TL, Zhang D, Teghanemt A, et al.. An essential role for albumin in the interaction of endotoxin with lipopolysaccharide-binding protein and sCD14 and resultant cell activation. J Biol Chem, 2002, 277(49): 47818-47825

[25] Kim JI, Lee CJ, Jin MS, et al.. Crystal structure of CD14 and its implications for lipopolysaccharide signaling. J Biol Chem, 2005, 280(12): 11347-11351

[26] Kelley SL, Lukk T, Nair SK, et al.. The crystal structure of human soluble CD14 reveals a bent solenoid with a hydrophobic amino-terminal pocket. J Immunol, 2013, 190(3): 1304-1311

[27] Iyama T, Wilson DM. DNA repair mechanisms in dividing and nondividing cells. DNA Repair (Amst), 2013, 12(8): 620-636

[28] Wu T, Hu E, Xu S, et al.. clusterProfiler 4.0: A universal enrichment tool for interpreting omics data. Innovation (Camb), 2021, 2(3): 284-287

[29] Laura N, Eleonora P, Mauro, et al.. The Response to Oxidative DNA Damage in Neurons: Mechanisms and Disease. Neural Plast, 2016, 2016: 3619274

[30] Xiao S, Cui S, Lu X, et al.. The ERCC2/XPD Lys751Gln polymorphism affects DNA repair of benzo[a]pyrene induced damage, tested in an in vitro model. Toxicol In Vitro, 2016, 34: 300-308

[31] Bayer FE, Deichsel S, Mahl P, et al.. Drosophila Xrcc2 regulates DNA double-strand repair in somatic cells. DNA Repair (Amst), 2020, 88: 102807

[32] Toh MR, Ngeow J. Homologous Recombination Deficiency: Cancer Predispositions and Treatment Implications. Oncologist, 2021, 26(9): e1526-e1537

[33] Ely LK, Fischer S, Garcia KC, et al.. Structural basis of receptor sharing by interleukin 17 cytokines. Nat Immunol, 2009, 10(12): 1245-1251

[34] Waisman A, Hauptmann J, Regen T. The role of IL-17 in CNS diseases. Acta Neuropathol, 2015, 129(5): 625-637

[35] Garg AV, Ahmed M, Vallejo AN, et al.. The deubiquitinase A20 mediates feedback inhibition of interleukin-17 receptor signaling. Sci Signal, 2013, 6(278): ra44

[36] Cruz JA, Childs E, Amatya N, et al.. IL-17 Signaling Triggers Degradation of the Constitutive NF-κB Inhibitor ABIN-1. Immunohorizons, 2017, 1(7): 133-141

[37] Hammond TR, Marsh SE, Stevens B. Immune Signaling in Neurodegeneration. Immunity, 2019, 50(4): 955-974

[38] Chen JM, Jiang GX, Li QW, et al.. Increased serum levels of interleukin-18, −23 and −17 in chinese patients with Alzheimer’s disease. Dement Geriatr Cogn Disord, 2014, 38(5–6): 321-329

[39] Behairi N, Belkhelfa M, Mesbah-Amroun H, et al.. All-trans-Retinoic Acid Modulates Nitric Oxide and Interleukin-17A Production by Peripheral Blood Mononuclear Cells from Patients with Alzheimer’s Disease. Neuroimmunomodulation, 2015, 22(6): 385-393

[40] Saresella M, Calabrese E, Marventano I, et al.. Increased activity of Th-17 and Th-9 lymphocytes and a skewing of the postthymic differentiation pathway are seen in Alzheimer’s disease. Brain Behav Immun, 2011, 25(3): 539-547

[41] Moustafa AA, Chakravarthy S, Phillips JR, et al.. Motor symptoms in Parkinson’s disease: A unified framework. Neurosci Biobehav Rev, 2016, 68: 727-740

[42] Liu Z, Qiu AW, Huang Y, et al.. IL-17A exacerbates neuroinflammation and neurodegeneration by activating microglia in rodent models of Parkinson’s disease. Brain Behav Immun, 2019, 81: 630-645

[43] Dai HY, Ji D, Tan C, et al.. Research progress on the role and regulatory mechanism of pathogenic Th17 cells in neuroinflammation. Yi Chuan (Chinese), 2022, 44(4): 289-299