HSYA inhibits Aβ1-42 -induced neuroinammation by promoting microglial M2 polarization via TREM2/TLR4/NF- κB pathway in BV-2 cells.

Hydroxysaor yellow A (HSYA), an extract from Carthamus tinctorius L. Dry owers (Compositae). HSYA has been shown to have neuroprotective effects in several AD models. However, the exact mechanisms of HSYA regulate neuroinammation have still not been claried. In this study, we investigated the mechanism by which HSYA regulates microglia activation and neuroinammation via TREM2, and further claried its underlying molecular mechanism. We silenced TREM2 in BV-2 cells and evaluated the expression of inammatory markers (TNF-α, IL-1β, IL-4, IL-6, IL-10, and IL-13). The results showed that HSYA could up-regulate cell activity and improve the morphology of BV-2 cells injured by Aβ 1−42 . HSYA upregulated expression of M1 markers (iNOS, IL-1β, IL-6) and down-regulated expression of M2 markers (Arg-1, IL-4, IL-10, IL-13) by TREM2, and changed microglia from M1 pro-inammatory phenotype to M2 anti-inammatory phenotype. HSYA inhibited the activation of TLR4/ NF-κB transduction pathway induced by Aβ 1−42 by up-regulating TREM2, and regulated the transcription of inammatory cytokines by downstream transcription factors NF-κB p65 and IκB-α. In conclusion, HSYA regulated microglial inammatory phenotype by regulating microglial (M1/M2) polarization in Aβ 1−42 -induced BV-2 cells which may be mediated through TREM2/TLR4/NF-κB pathway. signicantly by Aβ 1-42 inducation. on the of of IκB-α, when these results indicate that HSYA eliminates 1-42 induced TLR4 and through TREM2, which contribute to the over- activation of microglia. et al. 2011). ELISA and qPCR results showed that after induction by Aβ 1-42 , the expression of pro-inammatory cytokines IL-1β and IL-6 in BV-2 cells was up-regulated, while the anti-inammatory factors IL-4 and IL-13 were down-regulated. After HSYA intervention, the expression of IL-1β and IL-6 was signicantly down-regulated, and the expression of IL-4, IL-10, and IL-13 was up-regulated. It is suggested that HSYA exerts a neuroprotective effect by up-regulating the expression of anti-inammatory factors and down-regulating the expression of proinammatory factors that mediate inammation. TREM2 is a major anti-inammatory receptor in vivo, which transmits activation signals by binding to DAP12 and inhibits the release of inammatory mediators(Daws et al. 2001; Ford and McVicar 2009). In this study, using Aβ 1-42 inductions in the presence of TREM2 silence, the up-regulation of pro-inammatory cytokine expression and the down-regulation of anti-inammatory factor expression in BV-2 cells were more signicant. The therapeutic effect of HSYA is useless in the absence of TREM2. The results indicate that TREM2 plays an indispensable role in HSYA regulating the expression of microglia inammatory factors.


Introduction
Alzheimer's disease (AD) is a degenerative disease of the central nervous system. Its main clinical manifestations are progressive cognitive impairment and memory impairment (Reitz and Mayeux 2014). AD is characterized by brain atrophy and enlargement of the ventricles, accumulation of protein amyloid-beta plaques in the brain, and the presence of neuro brillary tangles (Nestor et al. 2008;Bloom 2014). Numerous studies have shown that Aβ plays a dominant role in the occurrence and development of AD. According to the amyloid cascade hypothesis, the neuro brillary tangles and neuroin ammation observed in AD are caused by Aβ accumulation (Kepp 2016).
Neurotoxic Aβ can activate astrocytes and microglia (Rogers et al. 1988). Microglia are the resident immune cells of the central nervous system and the rst line of defense of the central nervous system(CNS) (Schafer et al. 2012). Once exogenous stimulation or microenvironment changes is felt, microglia can be activated to have the ability to deformation and phagocytosis. Microglia mediate multiple facets of neuroinflammation, which plays a double-edged role in various brain diseases via distinct microglial phenotypes. In different microenvironments, activated microglia can be polarized in M1 phenotype with host defense and pro-in ammatory functions and the M2 phenotype with neuroprotection, nerve repair, or neural ring remodeling functions (Zhou et al. 2014; Colton et al. 2006). Activated M1 microglia can release a large number of in ammatory cytotoxic mediators, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and oxidative stressrelated indicators such as reactive oxygen species (ROS), reactive nitrogen, and nitric oxide (NO). By contrast, the activated M2 microglia can increase the expression of anti-inflammatory mediators and neurotrophic factors, such as transforming growth factor-β, interleukin10 (IL-10), arginase-1 (Arg-1), and CD206(Dong et al. 2019; Subhramanyam et al. 2019). Therefore, inhibiting the overactivated inflammatory microglia M1 phenotype by switching to the protective M2 phenotype appears to be a potential therapeutic strategy in neuroinflammatory disorders.
The activation process of Aβ on microglia is inseparable from the large number of Aβ receptors on the microglia membrane, such as Triggering Receptor Expressed on Myeloid Cells-2 (TREM2) and Toll-like receptors (TLR) (Beach et al. 2010). Several genetic variants of the TREM2 have been demonstrated to increase the risk of Alzheimer's disease (AD), thereby supporting the role of microglia and immune cells in AD (Guerreiro et al. 2013). TREM2 can regulate the function of microglia and transform its phenotype from M1 to M2. It can inhibit the release of in ammatory cytotoxic mediators and up-regulate the expression of anti-in ammatory factors, thereby reducing neuroin ammation damage to brain cells (Rohn 2013;Jonsson et al. 2013). TLR4/NF-κB and other signal molecules are the same as TREM2, which are critical to the modulation of microglial activation and neuroin ammation. TLR4 is mainly expressed in cerebral microglia. Stimulated by appropriate ligands, the NF-κB pathway is further activated to induce the transcription of pro-in ammatory cytokines, which play an important role in the activation of M1 microglia (Shi et al. 2019;Wan et al. 2016). Studies were con rmed that the TREM2 pathway and TLR4 pathway are mutually inhibited, that is, anti-in ammatory effect of TREM2 can antagonize the proin ammatory effect of TLR4 (Rosciszewski et al. 2017).
Hydroxy sa ower yellow A (HSYA), is the main active component of sa ower. In recent years, HSYA has been found that a neuroprotective effect is related to various mechanisms, such as reducing in ammation, scavenging oxygen free radicals, and inhibiting apoptosis after ischemia-reperfusion (Keiko et al. 2010). Gene chips showed that HSYA can signi cantly inhibit the expression of in ammatory factors after cerebral ischemia (Liu et al. 2013). Other studies have reported that HSYA can inhibit the in ammatory response of microglia after oxygen-glucose deprivation and have neuroprotective effects(J. Li et al. 2013). HSYA can also inhibit the in ammatory induced by Aβ 1-42 , play a neuroprotective effect and improve the learning, and memory ability of AD mice (Hou et al. 2020). More and more studies have been conducted on the pharmacological mechanism of HSYA, indicating that HSYA may be a multitarget drug candidate for the treatment of AD. However, whether HSYA inhibits the polarization of Aβ 1-42induced microglial phenotype and promotes microglial polarization to M2 phenotype remains unclear. Moreover, the molecular mechanisms underlying HSYA-mediated anti-neuroinflammatory effects remain unknown.
In the current study, we aim to investigate whether the imbalance between TLR4 and TREM2 of microglia in Aβ 1-42 -induced BV-2 cells mediates microglia polarization and neuroin ammation. We also analyzed the potential effects of HSYA on the polarization of microglia M1/M2. Our results may provide in-depth theoretical support for HSYA as a drug for the treatment of LOAD.

Preparation of Aβ 1-42 solutions
To generate soluble oligomers, Aβ 1-42 peptide was dissolved in 1,1,1,3,3,3-hexa uoro-2-propanol (HFIP; Sigma-Aldrich) at a concentration of 1 mM and then incubated for 24 hours under a fume hood(T. Jiang et al. 2014). The residual peptide lm was dissolved to a concentration of 5 mM in dry dimethyl sulfoxide (DMSO). For oligomeric conditions, the peptide was dissolved in the peptide in a serumfree DMEM high glucose medium to a nal concentration of 100 uM and kept at 4 °C for 24 hours.

Cell Culture and Treatment
Immortalized mouse BV-2 microglia were cultured in DMEM (Gibco, USA)high glucose medium with 0.1% penicillin-streptomycin (Gibco, USA) and 10% fetal bovine serum (Zhejiang Tianhang Biotechnology Co., Ltd., China) in a humidified atmosphere containing 5% CO 2 and 95% air at 37℃. The cells were subcultured for further passages when they reached 80% confluence and the culture medium were changed every two days. The cells in the logarithmic growth phase can be used in the experiment. Cells were pretreated with or without Aβ 1-42 for 24 hours and treated with various concentrations of HSYA (1, 2.5, 5, 10, and 20 μM) for 24 hours.

Cell Viability Assay and Morphological Analysis
BV-2 cell viability was assessed by the MTT assay using a 96-well culture plate. BV-2 cells were inoculated with Aβ 1-42 or treated with different concentrations of HSYA for 24 hours, and the appropriate concentration was selected for the follow-up experiment. Brie y, BV2 cells were seeded and pretreated with Aβ 1-42 for 24 hours and treated with different concentrations of HSYA.MTT (Soleibao, China) solution (20 μL) was added to each well. After incubation at 37 °C for four hours. The liquid was discarded and 200 μL DMSO were added to shake for 10 minutes. The absorbance at 570 nm was read on the enzyme meter (Thermo, USA). For morphological analysis, the cells were imaged with the Zeiss inverted microscope (Axio observer A1, Zeiss, Germany) at 100× magni cation.

Lentivirus Transduction
The lentivirus encoded the TREM2 shRNA sequence 5′-AGCGGAATGGGAGCACAGTCA-3′. Lentivirus containing TREM2 shRNA (LV-shTREM2) at 1 × 10 8 TU/ml were purchased from Genechem (Shanghai, China). BV-2 cells were plated into a 6-well culture plate(5 × 10 4 cells/well) and incubated overnight. The TREM2 lentiviral particles were used to infect the cells at an MOI of 10. After 12 hours of lentiviral adsorption and infection, the transfected cells were screened by the complete culture medium of 2.5 μg/mL puromycin (Soleibao, China). The lentivirus transduction e ciency was observed by a uorescence microscope and the expression levels of TREM2 were validated using Western blot. were inoculated into a 6-well culture plate(1× 10 6 cells/well). The cells were incubated with Aβ 1-42 (1 μM) for 24 hours and treated with HSYA (5 μM) for 24 hours. The supernatant was collected and the concentrations were measured by ELISA according to the manufacturer's instructions. Optical density (OD) was measured at 450nm using a microplate reader (Thermo, USA)

Quantitative PCR (qPCR) Assay
Total RNA was extracted using the UNIQ-10 Column Trizol Total RNA Isolation kit (Sangon Biotech, China). According to the standard protocol, the isolated RNA was treated with PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa Bio INC, China) to eliminate genomic DNA and reverse-transcribed into singlestranded cDNA. Speci c primers (Sangon Biotech, China) were used to amplify target genes by using QuantiNova TM SYBR Green PCR kit (Qiagen, Germany). The Rotor-Gene Q system (QIAGEN, Malaysia) for qPCR analysis. Reaction conditions: pre-denaturation 95 °C, 10 min; 95 °C, 10s, 60°C, 45s, 40 cycles. Each sample was analyzed in triplicate, and calculate the relative expression of mRNA after normalizing IL-6 and IL-10. By comparing the CT value of the target gene with that of GAPDH, the relative change of gene expression level was 2 −ΔΔCt . All primer sequences used are listed in Table 1. Table 1 Primers sequences used for qPCR.

Gene
Forward primer Reverse primer minutes in the dark and mounted with 50% glycerol (Solarbio, China). Fluorescence images were acquired using a confocal laser(LSM510, Zeiss, Germany). The quanti cation of the uorescence intensity was performed by analyzing the uorescence images using the ImageJ software.

Statistical analysis
Statistical analysis was performed using SPSS software 22.0(IBM, Inc., Armonk, NY, USA). One-way analysis of variance (ANOVA) followed by the Tukey test was used to assess the statistical signi cance of differences between groups. The results are expressed as the mean ± standard error (SEM). P<0.05 was considered a signi cant difference. P<0.05 is considered statistically signi cant.

Effects of HSYA on the viability of BV-2 cells induced by Aβ 1-42 .
To determine whether HSYA influences the viability of BV-2 cells, an MTT assay was performed 24 hours after treatment with various concentrations of HSYA ranging from 1 μM to 20 μM. Results (Fig. 1a) showed that HSYA concentrations less than 10 μM did not induce any group, the morphology of microglia in the HSYA treatment group was improved, that is, the increase of cell protrusions and the improvement of adherence (Fig. 1f). These results suggest that HSYA could reverse the decreased cell viability and morphological changes induced by Aβ 1-42 .
3.2 HSYA has no obvious effect on phagocytosis of Aβ 1-42 by BV-2 cells Abnormal accumulation of Aβ can activate microglia, and acute activation can promote the phagocytosis and clearance of Aβ by microglia, thereby exerting neuroprotective effects (Colton et al. 2006). Flow cytometry was used to detect the phagocytosis of Aβ 1-42 of BV-2 cells treated with different concentrations of HSYA. Compared with the control group, the phagocytosis of Aβ 1-42 of BV-2 cells in the HSYA treatment group was enhanced, but it was no signi cant change. (Fig. 2 a-b ) 3.3 The effects of HSYA on the expression of TREM2 in BV-2 cells TREM2 is a β-amyloid receptor that regulates the function of microglia. As showed in Fig. 3a  Ren wrote the manuscript; Chunhui Wang and Yanli Hu revised the manuscript. All authors read and approved the manuscript for publication. The authors declare that all data were generated in-house and that no paper mill was used.

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