Anti-inflammatory effects of B vitamins protect against tau hyperphosphorylation and cognitive impairment induced by 1,2 diacetyl benzene: An in vitro and in silico study

https://doi.org/10.1016/j.intimp.2022.108736Get rights and content

Highlights

Abstract

1,2 diacetyl benzene (DAB) penetrates the blood–brain barrier, causing neuroinflammation, tau hyperphosphorylation, and cognitive impairment. Converging evidence supports the anti-inflammatory effects of B vitamins on cognitive impairment, but the effects of B vitamins on cognitive impairment induced by DAB remain unclear. Here, we investigated the anti-inflammatory properties of B vitamins in DAB-stimulated human neuroblastoma SH-SY5Y cells. In this in-silico analysis, we investigated the genes, transcription factors, miRNAs, and sponges linked with DAB, B vitamins and the pathogenesis of cognitive impairment. We found vitamins B1, B2, and B3 had anti-inflammatory properties in DAB-stimulated SH-SY5Y cells, possibly via inhibiting NF-κB activation. Furthermore, vitamins B1, B2, and B3 inhibited GSK-3β, β-amyloid, and tau hyperphosphorylation in SH-SY5Y cells. These vitamins can also modulate genes induced by DAB (IL1B, IL6, IL10, iNOS, COX2, NFκB, GSK3B, TNF, and APP) in SH-SY5Y cells. In silico analyses, inflammatory response related pathways, “Alzheimer’s disease”, “pathways of neurodegeneration-multiple disease”, and “prolactin signaling pathway”, were highlighted. Additionally, we explored a network-based approach to identify key genes, transcription factors, miRNAs, and pathways in cognitive impairment. The transcription factors NFKB2 and BATF3 were shown to be the most important in regulating genes. We also found eight significant miRNAs related to cognitive impairment, and these miRNAs were also validated by qPCR. Finally, we developed and tested in silico miRNA sponge sequences for these miRNAs.

Introduction

Cognitive impairment occurs when a person has difficulty concentrating, remembering, making decisions, or learning new things that affect their daily life. Cognitive impairment can range from mild to severe [1]. Mild cognitive impairment has been known as a transitional stage between normal cognitive aging and overt dementia [2]. Mild cognitive impairment is a neurological disorder that affects the elderly and is characterized by minor but noticeable memory and/or thinking ability deficits with little impact on daily life functions [3]. Severe cognitive impairment can result in the loss of the capacity to comprehend the meaning or significance of something, as well as the ability to speak or write, leading to an inability to live independently [1]. Cognitive impairment affects patients' psychological, physical, economic, and social well-being, as well as caregivers, families, and communities [1], [4].

There are many risk factors that contribute to the pathogenesis of cognitive impairment (e.g., genetics, sex, age, family history, low educational attainment, unhealthy diets, physical inactivity, smoking, harmful use of alcohol, hypertension, diabetes, depression, etc.) [1], [4]. It has been known that cognitive impairment is not a normal aspect of growing older, despite the fact that age is the strongest recognized risk factor [5]. However, these factors have not been enough to explain the prevalence of cognitive impairment. Converging evidence suggests that organic solvents such as 1,2 diacetyl benzene (DAB) also play an important role in the etiology of cognitive impairment [6], [7], [8]. In the context of urbanization and industrialization, humans are more likely to be exposed to organic solvents such as gasoline, cosmetics, printing inks, pharmaceuticals, household cleaners, and paints [7], [9], [10], [11], [12], [13], [14]. DAB, a small component of gasoline and fuel oils, is a neurotoxic metabolite of the colorless liquid 1,2-diethylbenzene [6]. DAB can penetrate across the blood–brain barrier, interact with proteins, and produce polymers [15]. DAB has been shown to cause activated microglia, inflammatory response, oxidative stress, impaired hippocampus neurogenesis, and cognitive impairment in animals [7], [8]. Therefore, it is important to control organic solvents, especially DAB, to lessen the burden of cognitive impairment and enhance the quality of life.

Chronic neuroinflammation and tau hyperphosphorylation are linked to a variety of neurodegenerative disorders, including cognitive impairment [16]. Therefore, it has been proposed that regulating neuroinflammation and neurotoxicity is a potential therapeutic method for treating cognitive impairment. B vitamins are a group of water-soluble vitamins that act as cofactors or coenzymes in neuron excitability, axonal transport, neurotransmitter synthesis, and cellular metabolism [17]. Increasing evidence suggests that B vitamins, including B1, B2, and B3, are implicated in the pathogenesis of cognitive impairment by regulating inflammation, tau hyperphosphorylation, glycogen synthase kinase-3β (GSK-3β), and NF-κB activation [18], [19], [20], [21], [22], [23]. However, little is known about the anti-inflammatory effect of vitamins B1, B2, and B3 on tau hyperphosphorylation induced by DAB. Thus, we hypothesized that vitamins B1, B2, and B3 can protect against tau hyperphosphorylation caused by DAB in the human neuroblastoma SHSY5Y cell line. We demonstrated that vitamins B1, B2, and B3 show anti-inflammatory effects on DAB-stimulated human neuroblastoma through suppression of inflammation, tau hyperphosphorylation, GSK-3β, and NF-κB activation. Next, we investigated the genes (gene interactions, networks, biological processes, pathways, and diseases) and miRNAs (gene, transcription factors, miRNA interactions, networks, pathways, and diseases) linked with DAB, B vitamins and the pathogenesis of cognitive impairment. Using the microRNA sponge generator and tester, we also developed and tested in silico miRNA sponge sequences for miRNA-induced cognitive impairment.

Section snippets

Materials

DAB (99% pure), thiamine hydrochloride (99% pure), riboflavin (98% pure), and nicotinamide (99.5% pure) were purchased from Sigma-Aldrich Chemical Co. (Madison, WI, USA). Anti-tau, and anti-p-tau (Ser396) were obtained from Cell Signaling Technology Inc. (MA, USA). p-Tau 46, glycogen synthase kinase (GSK)-3β, p-GSK-3β Ser9, and p-GSK-3β Tyr 216, anti-Cox2, anti-β-actin, and secondary antibodies were purchased from Santa Cruz Biotechnology. Anti-inducible nitric oxide synthase (iNOS) was

Cell viability was not affected by vitamins B1, B2 and B3

After culturing SH-SY5Y cells with various doses of DAB for 24 h, we used the MTT assay to identify the effects of DAB and B vitamins on cell viability. As shown in Fig. 1A, the viability of SH-SY5Y cells was not affected by 0–5 µM DAB. When the concentration of DAB was increased, however, it significantly decreased the viability of SH-SY5Y cells. Next, we assessed whether vitamin B1, B2, and B3 concentrations had an effect on the viability of SH-SY5Y cells. As shown in Fig. 1B-D, vitamins in

Discussion

Inflammation and tau hyperphosphorylation are thought to be implicated in the pathogenesis of cognitive impairment [7]. Inflammation is normally a protective response that aids in the healing process. However, chronic inflammation can cause tissue damage, neurodegeneration, and impaired neurogenesis [29]. Over phosphorylation of the microtubule-related protein tau can increase the release of insoluble tau aggregation, which has been linked with cognitive impairment [30]. DAB has been known as a

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This study supported by grants National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (grant nos. NRF2013R1A1A3008851 and 2018R1D1A1B07049610).

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