PL201, a Reported Rhamnoside Against Alzheimer's Disease Pathology, Alleviates Neuroinflammation and Stimulates Nrf2 Signaling

Neuroinflammation induced by overactivated glia cells is believed to be a major hallmark of Alzheimer's disease (AD) and a hopeful target against AD. A rhamnoside PL201 was previously reported to promote neurogenesis and ameliorate AD, and in this study, we revealed that PL201 also significantly reduced accumulation of the activated microglia and proinflammatory cytokines in APP/PS1 mice. In vitro, PL201 consistently suppressed the microglia induction of proinflammatory cytokines after stimulation with lipopolysaccharides and Aβ42. Further mechanistic studies demonstrated that PL201 considerably enhanced the expression level and the nuclear translocation of Nrf2, a key regulator of neuroinflammation. Moreover, PL201 effectively stimulated Nrf2 signaling cascade, including upregulation of HO-1 and downregulation of NF-κB pathway. Thus, our findings indicated the anti-neuroinflammatory effect by PL201 in vivo and suggested that PL201 or the like, with multiple functions such as neurogenesis, mitochondria maintenance, and anti-neuroinflammation, could be a promising candidate in AD treatment.


Animals
All animal studies were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Both male and female mice were randomized in different experiment groups in this study. Nine-month-old APP/PS1 mice and wild-type (WT) littermates were used in this study. APP/PS1 transgenic mice expressing the mutated human app (Swedish mutations K595N M596L) and the human presenilin1 with a deletion of exon 9 were obtained from The Jackson Laboratory (stock no.004462) (36). All mice were bred and maintained in a temperature-controlled room and maintained on a 12-h light/dark cycle, with ad libitum access to food and water.

Drug Administration
PL201 was synthesized as previously described (35). PL201 was dissolved in water. Nine-month-old APP/PS1 mice were orally administered with 30 mg/kg PL201 daily for 60 days. Control mice were treated with water as a vehicle. After drug treatment, mice of each group were euthanized, and the whole brains were removed. Brains were collected for measurement of proinflammatory and anti-inflammatory cytokines.

Cell Culture and Drug Treatment
BV2 cells were cultured in DMEM (Invitrogen, Camarillo, CA); HMC3 cells were cultured in MEM (Invitrogen, Camarillo, CA). All medium was supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin, and all cells were cultured at 37 • C in an atmosphere of 95% air and 5% CO 2 . In all in vitro assays, BV2 and HMC3 cells were starved for 12 h in serum-free culture medium before PL201 treatment. To detect the expression of proinflammatory cytokines, cells were pretreated with PL201 at various concentrations (10-300 µM). After the pretreatment, 300 ng/ml LPS (Sigma, O55:B5) or 3 µM oligomer Aβ42 (GL Biochem, Shanghai) was added for further 24 h stimulation. To detect Nrf2 ′ s downstream genes, cells were treated with PL201 at various concentrations (10-300 µM) for 6 h. To identify the nuclear translocation and expression of Nrf2, cells were treated with PL201 at various concentrations (10-300 µM) for 1 h. To assess the activation of NF-κB pathway, cells were pretreated with PL201 for 2 h followed by 1 h of LPS or oligomer Aβ42 stimulation.

Preparation of Aβ42 Oligomers
Aβ42 peptide was prepared as previously described (37). In brief, 1 mg Aβ42 was dissolved in 1 ml cold hexafluoroisopropanol (Sigma, 52517), and the solution was aliquoted into Protein LoBind tubes (Eppendorf, 030108094); each tube containing 50 µg Aβ42 was dried overnight at RT. The residue was dissolved in 2.5 µl dimethyl sulfoxide (DMSO) followed by 117 µl cold phenol-free DMEM/F-12 medium (Thermo Fisher, 21041025) to obtain a 100-µM stock solution. After vortex for 60 s and incubated at 4 • C for 24 h, the solution was centrifuged at 16,000 × g for 10 min, and the supernatant containing Aβ42 oligomers was used for further experiments. The content of Aβ42 oligomers was confirmed by Western blot using anti-Aβ antibody (6E10, BioLegend, 803002; 1:1,000).

Preparation of Mouse Brain Homogenate
Mice cortex extract was obtained as reported previously (38,39). In brief, cortex tissues were homogenized and sonicated 30 × 15 s with a 15-s interval between each sonication in Trisbuffered saline (TBS, pH 8.0) containing 0.1% Igepal. Samples were centrifuged at 16,000 × g at 4 • C for 30 min to remove any remaining insoluble debris in the solution. Samples were then detected by ELISA kits. The results were normalized to total protein.

Quantitative Real-Time Polymerase Chain Reaction
Total RNA was extracted from BV2 cells and HMC3 cells using TRIZOL Reagent according to the manufacturer's protocols. Equal amounts of RNA were reverse transcribed using a cDNA synthesis kit (Takara). qRT-PCR experiments were performed using synthetic primers and a 2 × HotStart SYBR Green qRT-PCR Master Mix kit (ExCell). Primers used in this study are listed in Table 1.

Immunostaining and Confocal Microscopy
Immunostaining was performed as previously described (28). In detail, mice were killed, and dissected brains were fixed in 4% paraformaldehyde (PFA) (Sigma) at 4 • C for 24 h. After fixation, tissues were washed in cold PBS for three times and then dehydrated in 30% sucrose overnight at 4 • C and embedded in optimal cutting temperature (OCT) (Thermo Fisher), followed by continuous serial cryostat sectioning at 30 µm. Sections were then washed with PBS (pH 7.4) and blocked with a blocking buffer (PBS supplemented with 0.5% Triton X-100 and 5% normal horse serum) for 1 h at room temperature. Afterwards, the sections were incubated with the following primary antibodies overnight at 4 • C: anti-Iba1 (Wako, 019-19741; 1:500), and anti-6E10 (BioLegend, 803002, 1:300). These sections were washed with PBS and incubated with Alexa fluorescence-conjugated secondary antibodies (Invitrogen, 1:1,000) for 1 h at room temperature followed by 10-min nuclei staining using 4 ′ 6diamidino-2-phenylindole (DAPI Beyotime Biotechnology, 1:5,000). The images were acquired using a Zeiss confocal laser scanning microscope (Axio Scan.Z1). The instrument settings, including numeric gain, laser power, and magnification, were kept constant between animals to avoid potential technical artifacts. Cultured BV2 cells and HMC3 cells were fixed in 4% PFA in PBS at room temperature for 15 min, and the immunostaining was carried out following the same procedure as brain sections. The following primary antibodies were used: anti-p65 (Santa Cruz, sc-372; 1:300) and anti-Nrf2 (Cell Signaling, 12721; 1:200). The images were captured using a Zeiss confocal laser scanning microscope (Zeiss 880 Airyscan). The instrument settings, including numeric gain, laser power, and magnification, were kept constant between animals to avoid potential technical artifacts.
As previously reported, ImageJ software was applied for quantitative analysis for Iba1 staining (39). First, red/green/blue (RGB) images were converted to 8-bit grayscale, and the threshold levels were adjusted to highlight positive signals. We quantified at least four sections per mouse. The Iba1 intensity from the cortex and hippocampus were qualified via Image J. Statistical analysis based on a blind review of data.
ImageJ software was applied for quantitative analysis for Nrf2 and p65 in cultured BV2 cells and HMC3 cells. The quantitative method was used following the same procedure as brain sections.

Nuclear and Cytoplasmic Extraction
BV2 cells and HMC3 cells were cultured in six-well plates and grown for 24 h and then treated with various concentrations of PL201 for indicated time. Then, these cells were washed with PBS and lysed for 10 min in buffer A (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl 2 , and 1% NP-40) containing phosphatase inhibitor and protease inhibitor. After centrifugation at 3,000 × g for 5 min at 4 • C, the supernatant was collected to a new microfuge tube as the cytoplasmic fraction. The pellet was washed in buffer A for three times and resuspended in buffer B (20 mM HEPES, pH 7.9, 20% glycerol, 1.5 mM MgCl 2 , 0.2 mM EDTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride). The suspension was then sonicated for 30 s, and centrifuged at 14,000 × g for 10 min at 4 • C to obtain the nuclear fraction.

Cell Viability
Cell viability was detected by CellTiter-Glo Luminescent assay. One hundred microliters CellTiter-Glo Reagent was added to each well of 96-well plate and mixed for 2 min on an orbital shaker to lyse cells. The cell viability was judged according to the luminescence.

Statistical Analysis
GraphPad Prism 7 (GraphPad Software, La Jolla, CA, United States) was used to draw graphs and perform data analysis. All values were represented as the mean ± SEM. One-way ANOVA followed by the Tukey multiple comparisons test were used to compare values from multiple groups. Twotailed unpaired t-test was applied for comparisons between two groups. Values were considered to have significant difference when p < 0.05.

PL201 Treatment Decreases Microglia
Reactivity and Proinflammatory Cytokine Production in APP/PS1 Mice Accumulating evidence suggest that the pathogenesis of Alzheimer's disease is strongly associated with immunological alterations in the brain. In AD patients and mouse models, neuroinflammation has often been observed accompanied by excessive accumulation of microglia. To investigate whether PL201 treatment affected neuroinflammation in AD mice, we evaluated the microglial reactivity in the hippocampus and cortex by staining with ionized calcium binding adaptor molecule 1 (Iba1) antibody, which is a specific marker of microglia in the brain. We found that the microglia accumulating around the amyloid plagues significantly increased in the hippocampus and cortex of APP/PS1 mice when compared with the WT controls (Figures 1A,B). Moreover, reactive microgliosis was reduced in PL201-treated AD mice as well (Figures 1A-D). Since the activated microglia produce cytokines and chemokines, which contribute to AD pathology, we further detected the level of several cytokines and chemokines in brain samples from WT and AD mice. The results showed that, in AD mice, the levels of proinflammatory cytokines and chemokines including IL-6, TNFα, MIP-1α, and IL-1β were elevated, while the level of antiinflammatory cytokine IL-10 was decreased (Figures 1E-I). After PL201 treatments, the levels of these proinflammatory cytokines were reduced in AD mice, but the levels of IL-10 remained unchanged. Collectively, these data demonstrated that PL201 treatment could suppress proinflammatory cytokine secretion and decrease neuroninflammation in AD mice. Emerging findings suggest that a significant increase in inflammatory response in AD is associated with Nrf2. We examined Nrf2 expression in mouse brains and found that PL201 had a tendency to increase Nrf2 expression in AD mice (Figures 1J,K).

PL201 Activates Nrf2 Signaling
Since Nrf2 signaling plays an important role in modulating neuroinflammation, we next tested whether PL201 could activate Nrf2 signaling in both BV2 and HMC3 cells. The results revealed that PL201 activated Nrf2 nuclear translocation in a dosedependent manner. Meanwhile, Nrf2 levels in the cytoplasm were correspondingly reduced after PL201 treatment (Figures 3A-D).
To detect the specificity of Nrf2 antibody (41), we used tbhQ (15 µM) as a positive control and showed the whole extension of lanes in the Western blot. (Figures S3A,B). Similarly, using immunostaining, we observed that nuclear accumulation of Nrf2 was markedly increased after 1 h treatment with PL201 in BV2 ( Figure 3E) and HMC3 cells (Figure 3F). We also determined gene level of Nrf2 after PL201 stimulation; the results indicated that PL201 effectively activated the mRNA expression of Nrf2, with a significant effect at the concentration of 300 µM in BV2 and HMC3 cells (Figures S3C,D). HO-1 is a major Nrf2 target gene; therefore, we tested the level of HO-1. The expression of the HO-1 significantly increased after PL201 treatment for 6 h in BV2 cells (Figures 3G-I). Similar result was also observed in HMC3 cells (Figures 3J-L). Moreover, the function of Nrf2 in the regulation of HO-1 induced by PL201 was studied using Nrf2 special inhibitor-brusatol. BV2 cells were pretreated with brusatol followed by PL201 for 6 h; brusatol completely abolished PL201-induced HO-1 expression (Figures S3E,F). Interestingly, we tested HO-2 in parallel to HO-1 and found that the protein level of HO-2 was not altered after PL201 treatment (Figures S4A-F). Besides HO-1, 6 h treatment  The mRNA expression of IL-6, TNFα, and IL-1β were measured by qRT-PCR. Quantifications were expressed as mean ± SEM (compared with control, # p < 0.05, ## p < 0.01, ### p < 0.005; compared with LPS-stimulated condition, *p < 0.05, **p < 0.01, ***p < 0.005).
of PL201 also dose-dependently increased the levels of GCLM and NQO1, which are both NRF2-target genes (Figures S5A-F). Taken together, these results indicated that PL201 treatment activates Nrf2 signaling pathway.

Nrf2 Signaling Is Required for the Anti-inflammatory Effects of PL201
We next investigated whether the anti-inflammatory effects of PL201 were mediated by Nrf2 signaling. To test this, we pretreated BV2 cells with an Nrf2-specific inhibitor brusatol before adding PL201 and found that brusatol attenuated the inhibitory effect of PL201 on the release of proinflammatory cytokines in LPS-treated BV2 cells (Figures 4A-E). We next blocked HO-1, which is a critical downstream mediator of Nrf2, by its inhibitor SNPP. BV2 cells were pretreated in the absence or presence of SNPP or PL201, followed by LPS stimulation for 24 h. Similar to that of Nrf2 inhibition, SNPP also attenuated the inhibitory effects of PL201 on proinflammatory cytokines such as IL-6, TNFα, and IL-1β (Figures 4F-J). We investigated the effect of PL201, brusatol, and Snpp on cytokines expression alone. They did not have effect on the expression of proinflammatory cytokines (Figures S6A-C). In summary, these data suggested that Nrf2 signaling is required for the anti-inflammatory effects of PL201.

PL201 Suppresses NF-κB Pathway
Emerging evidence suggest that the cross-talk between Nrf2 and NF-κB signaling is important for the regulation of inflammation. To explore the effect of PL201 on NF-κB pathway, BV2 cells were pretreated with PL201 for 2 h followed by LPS stimulation for another 1 h. After that, nuclear protein was extracted as previously reported. The nuclear accumulation of p50 and p65 markedly increased after LPS treatment, which is reduced by PL201 treatment (Figures 5A-C). Using immunofluorescence microscopy, we also observed that PL201 significantly inhibited the nuclear translocation of the NF-κB p65 subunit in LPS or oligomer Aβ42-activated microglia cells (Figures 5D,E). IκBα is a negative regulator of NF-κB. Previous reports showed that elevating the expression of Nrf2 inhibited NF-κB activity. We hypothesized that PL201 might suppress NF-κB activity by inducing Nrf2. To test this, we pretreated BV2 cells in the absence or presence of brusatol or PL201, followed by LPS stimulation for 1 h, and found that brusatol reversed the inhibitory effect of PL201 on IκB-α phosphorylation, while the basal protein levels of Nrf2 and IκB-α have no changes after brusatol stimulation (Figures 5F-H). These results demonstrated that Nrf2 mediates PL201-dependent downregulation of NF-κB activity.

DISCUSSION
In AD, microglia accumulate and secrete proinflammatory cytokines, which accelerate neuronal injury (42,43). Our results demonstrated that PL201 treatment significantly reduced the levels of microglia activation and proinflammatory cytokines in the brains of APP/PS1 mice. Furthermore, we found that PL201 inhibited proinflammatory cytokine production in microglia cells after LPS or Aβ stimulation, which indicated that PL201 may affect microglia directly. In summary, these data demonstrated that PL201 may suppress the inflammatory response mediated by microglia both in vivo and in vitro.  (30 nM) and then exposed to LPS for 1 h; the protein levels of phosphorylated IkBα and total IkBα were analyzed by Western blot, the expression of Nrf2 was measured by Western blot. Quantifications were expressed as mean ± SEM (compared with vehicle, # p < 0.05, ## p < 0.01, ### p < 0.005; compared with LPS-stimulated condition, *p < 0.05, **p < 0.01, ***p < 0.005; compared with PL201 under brusatol treatment, $ p < 0.05, $$ p < 0.01, $$$ p < 0.005).
Frontiers in Immunology | www.frontiersin.org Nrf2 has been reported to regulate the inflammatory response of microglia, and involved in the pathogenesis of AD (18,44). Thus, we evaluated whether the effect of PL201 was mediated by Nrf2. The results indicated that PL201 could activate Nrf2 signaling by promoting Nrf2 metastasis into the nuclei of microglia cells. PL201 also elevated the mRNA expression of Nrf2 in a dose-dependent manner. Meanwhile, the expression of Nrf2 target genes concluding HO-1, NQO1, and GCLM could be induced by PL201. Interestingly, the antineuroinflammatory effect of PL201 can be blocked by Nrf2 inhibitor. PL201 could inhibit NF-κB signal dependent on Nrf2 activation. Collectively, we proposed that PL201 is a Nrf2 signaling activator. Emerging evidence indicated that Nrf2 may play an important role in tau pathology (27,45,46). Maybe PL201 could affect tau phosphorylation. Nrf2 also acts as a regulator of autophagy and takes an important role in regulating ADrelated protein (47). Future work will be needed to detect if pharmacological induction of Nrf2 may be a valid strategy to facilitate degradation of toxic proteins such as Aβ or tau in the brain.
A large number of anti-inflammatory drugs, including non-steroidal anti-inflammatory drug (NSAID), peroxisome proliferator-activated receptor gamma (PPAR-γ) activators, and TNFα inhibitors, have been assessed in clinical trials for AD, but the results are not yet conclusive (2,4). Since the complicated etiology of AD, developing multitarget drugs might be a promising strategy to treat this disease. We previously reported that PL201 treatment improved mitochondrial function of neural stem cells and promoted neurogenesis in AD mice. In this study, we further demonstrated that PL201 stimulates Nrf2 signaling in microglia and significantly suppresses neuroinflammation. Taken together, our results indicated that PL201 may serve as a potential multitarget treatment for AD.

CONCLUSIONS
In summary, we found that PL201 significantly reduced neuroinflammation in microglia via Nrf2/HO-1/NF-κB signaling pathway. Our findings indicated that PL201 might serve as a promising therapeutic agent for AD.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/Supplementary Material.

ETHICS STATEMENT
The animal study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

AUTHOR CONTRIBUTIONS
YA designed this research and performed the cellular experiments and wrote the manuscript. YA and HZ conducted the animal experiments. GP and SH revised the manuscript. All authors read and approved the final manuscript.