Hyperoside Attenuate Inflammation in HT22 Cells Via Upregulating SIRT1 To Activities Wnt/β-Catenin And Sonic Hedgehog Pathways Part 1

Please contact oscar.xiao@wecistanche.com for more information

Neuroinflammation plays important role in the pathogenesis and progression of altered neurodevelopment, sensorineural hearing loss, and certain neurodegenerative diseases. Hyperoside (quercetin-3-O-β-D-galactoside) is an active compound isolated from Hypericum plants. In this study, we investigate the protective effect of peroxide on neuroinflammation and its possible molecular mechanism. Lipopolysaccharide(LPS) and hyperoside were used to treat HT22 cells. The cell viability was measured by MTT assay. The cell apoptosis rate was measured by flow cytometry assay. The mRNA expression levels of interleukin-1β(IL-1β), interleukin-6(IL-6), interleukin-8(IL-8), and tumor necrosis factor-α(TNF-a) were determined by quantitative reverse transcription-polymerase chain reaction. The levels of oxidative stress indices superoxide dismutase (SOD), reactive oxygen species (ROS), catalase(CAT), glutathione(GSH), and malondialdehyde(MDA) were measured by the kits. The expression of neurotrophic factor and the relationship among hyperoside, silent mating type information regulation 2 homolog-1(SIRT1) and Wnt/β-catenin, and the sonic hedgehog was examined by western blotting. In the LPS-induced HT22 cells, hyperoside promotes cell survival; alleviates the level of IL-1β, IL-6, IL-8, TNF-α, ROS, MDA, Bax, and caspase-3; and increases the expression of CAT, SOD, GSH, Bcl-2, BDNF, TrkB, and NGF. In addition,hvperoside upregulated the expression of SIRT1. Further mechanistic investigation showed that hyperoside alleviated LPS-induced inflammation, oxidative stress, and apoptosis by upregulating SIRTl to activate Wnt/β-catenin and sonic hedgehog pathways. Taken together, our data suggested that hyperoside acts as a protector in neuroinflammation.

Please click here to know more

1. Introduction

Neuroinflammation is a chronic inflammation of the brain tissue, which plays an important role in the pathogenesis and progression of altered neurodevelopment, sensorineural hearing loss, and certain neurodegenerative diseases[1-5]. In the early stages of the central auditory pathway, noise-induced hearing loss and conductive hearing loss are related to neuroinflammation [6]. In addition, neuroinflammation contributes to neuronal death and neurological deterioration by increasing the production of proinflammatory factors and oxidative stress[7]. Numerous studies have shown that hippocampal neurons are susceptible to neuroinflammatory and cause neurological complications [8]. However, there are still no effective agents or methods to restore and prevent neuronal damage caused by neuroinflammation. Thus, the identification of effective inflammatory protective candidate agents is crucial.

Hyperoside(quercetin3-O-β-D-galactoside) is an active compound isolated from Hypericum plants. It has antioxidant and anti-inflammatory activities, decreasing calcium overload and inhibiting apoptosis [9, 10]. Previous studies have confirmed that hyperoside effectively prevents neurological complications caused by neuroinflammation. In the hyperglycemia-induced oxidative stress and inflammation acute diabetes model, the administration of hyperoside prevented cognitive dysfunction, neuroinflammation, and oxidative stress caused by DM through the TNF-α/NF-Bx/caspase-3 signaling pathway [11].In diseases such as Parkinson's disease, hyperoside acts as a protective agent by attenuating LPS-induced activation of microglia [12]. So far, there are few studies on the protective effect of hyperoside on neuroinflammation, and the mechanism has not been fully elucidated.

Lipopolysaccharide(LPS) is widely used to activate the innate immune system. Previous studies have shown that LPS is usually used to prepare neuroinflammation models induced by inflammatory responses [13,14]. In this study, we exposed HT22 cells to LPS to mimic a cellular model of neuroinflammation. Simultaneously, the protective effect of hyperoside on neuroinflammation and its possible molecular mechanism was studied through this model. We found that hyperoside protects HT22 cells from LPS-induced inflammation; oxidative stress and apoptosis are closely related to SIRT1levels. Further analysis showed that hyperoside alleviated LPS-induced inflammation, oxidative stress, and apoptosis by upregulating SIRT1 to activate Wnt/β-catenin and sonic hedgehog pathways.

2. Materials and Methods

2.1.Reagents and Drugs. LPS, hyperoside, and 3-(4,5- bromide dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium

(MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's modified Eagle medium containing 10%fetal bovine serum(FBS)was purchased from Gibco(Carls-bad, USA).100U/ml penicillin and 100mg/ml streptomycin were purchased from Sigma-Aldrich(St. Louis, MO, USA). LiCl and sonic hedgehog agonist SAg were purchased from Merck(San Diego, CA, USA). Primary antibodies against Bcl-2,Bax, caspase-3,BDNF,NGF, SIRT1,Wnt1,β-catenin, Shh, Patch, and GAPDH and secondary antibodies were all purchased from Cell Signaling (Boston, MA, USA). 2.2. Cell Culture and Treatments. The HT22 murine neuronal cell line was purchased from Kunming Cell Bank of the Chi-nese Academy of Sciences(Kunming, China). HT22 cells were seeded at2×104 cells/well in a 6-well plate and cultured in the DMEM with 10% fetal bovine serum, 100U/ml penicillin， and 100μg/ml streptomycin at 37C in a 5% CO， humidified incubator. When HT22 reached 70% confluency after 24h, cell transfection pretreated with different concentrations of hyperoside and LPS（1μg/ml） was performed.

Cistanche can improve immunity

2.3.Cell Viability. Cell viability was determined by MTT assay.

Briefly, the HT22 cells were seeded into 24-well plates at a density of 2×104 cells/ml for 24h. Cells were starved overnight and then pretreated with different concentrations of hyperoside for 24h followed by incubation with LPS （1μg/ml） for another 24h. The medium was refreshed and incubated with 50μl of MTT （5 mg/ml prepared in phosphate-buffered saline)for 4h at 37C. Next, the solution was removed and added DMSO to the plates. The absorbance at 570nm was measured using a plate reader.

2.4.Western Blotting.

RIPA buffer was added to HT22 cells(Invitrogen; USA) to collect the total protein; then the protein concentrations were determined assessed using the BCA method (Invitrogen, USA).

The protein samples were mixed with a 5x loading buffer and denatured at the boil. The proteins were separated by 15% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. Next, the membranes were incubated with primary antibodies(Bcl-2, Bax, caspase-3, BDNF, NGF, SIRT1, Wntl,β-catenin, Shh, Patch, and GAPDH)at 4Covernight. Then, the next day, the membranes were washed with PBS and incubated with secondary antibodies for 1 h. Finally, the protein bands were measured using the ImageJ software. The data were collected from at least three independent experiments.

2.5.qRT-PCR. Total RNA of HT22 cells was isolated using the TRIzol RNA Extraction Kit (Invitrogen, Grand Island, NY, USA), and the isolated total RNA was reverse-transcribed to cDNA using a reverse transcription kit (Takara, Kyoto,Japan).Next, SYBR Premix Ex Taq II (Takara, Kyoto, Japan) was used to perform qRT-PCR amplification. The IL-1β, I-6,and TNF-α amplification primers were as follows:IL-1β,5'-GATGGTCGCATTAGCTCC-3'and 5'-GGCTGTAGCTGTAGCGTC-3';IL-6,5'-ATTG CGGCGGCTGACGCGTAG-3'and 5'-GTCTGTTGCGC GAGCTGGTA-3';IL-8,5'-GTCGAGCTGCCGCGTAGCG T-3'and 5'-CGCGATGCGTGCAGC-3'; and TNF-a,5'-CGTCAGCCGATTTGCTATCT-3'and5'-CGGACTCCG CAAAGTCTAAG-3'.The relative expression of mRNA was analyzed using the 2-Act method.

2.6.SOD, GSH, and MDA Assay. We measured oxygen species(ROS), catalase(CAT), superoxide dismutase(SOD), glutathione(GSH), and malondialdehyde (MDA) levels activity using the corresponding assay kits (Nanjing Jian-cheng Bio Company, China).

2.7.Flow Cytometry.

Flow cytometry assay was used to measure the cell apoptosis rate according to a previous report [15]. HT22 cells in each group were harvested and resuspended. The apoptotic cells were double-labeled with annexin V-FITC and PI using an annexin V-FITC/PI apoptosis detection kit (Beyotime Biotechnology, China) for 30 min at room temperature in the dark. Then, the fluorescence intensity of the cells was quantified by flow cytometry.

2.8. Statistical Analysis.

In this study, the difference between two groups was compared by using a t-test, and that among groups was analyzed by one-way analysis of variance (ANOVA).All data were presented as the mean values±

figure 1:Hyperoside alleviated apoptosis and inflammation in the LPS-induced HT22 cells. HT22 cell viability was measured by the MTT assay. Cell viability was observed by a microscope (100x)(a, b). The expression of Bcl-2, Bax, and caspase-3 in HT22 cells was measured by western blotting(c). The HT22 cell apoptosis rate was measured by flow cytometry assay(d). The levels of IL-1β,IL-6. IL-8 and TNF-αmRNA in HT22 cells were measured by art-PCR(e);* was considered significant compared to control (*P<0.05);# was considered significant compared to LPS ("P <0.05).

This article is extracted from Hindawi Neural Plasticity Volume 2021, Article ID 8706400, 10 pages https://doi.org/10.1155/2021/8706400