Isorhamnetin Attenuated the Release of Interleukin-6 from β-Amyloid-Activated Microglia and Mitigated Interleukin-6-Mediated Neurotoxicity

Alzheimer's disease (AD), characterized by the abnormal accumulation of β-amyloid (Aβ), is the most prevalent type of dementia, and it is associated with progressive cognitive decline and memory loss. Aβ accumulation activates microglia, which secrete proinflammatory factors associated with Aβ clearance impairment and cause neurotoxicity, generating a vicious cycle among Aβ accumulation, activated microglia, and proinflammatory factors. Blocking this cycle can be a therapeutic strategy for AD. Using Aβ-activated HMC3 microglial cells, we observed that isorhamnetin, a main constituent of Oenanthe javanica, reduced the Aβ-triggered secretion of interleukin- (IL-) 6 and downregulated the expression levels of the microglial activation markers ionized calcium binding adaptor molecule 1 (IBA1) and CD11b and the inflammatory marker nuclear factor-κB (NF-κB). Treatment of the SH-SY5Y-derived neuronal cells with the Aβ-activated HMC3-conditioned medium (HMC3-conditioned medium) or IL-6 increased reactive oxygen species production, upregulated cleaved caspase 3 expression, and reduced neurite outgrowth, whereas treatment with isorhamnetin counteracted these neurodegenerative presentations. In the SH-SY5Y-derived neuronal cells, IL-6 upregulated the phosphorylation of tyrosine kinase 2 (TYK2) and signal transducer and activator of transcription 1 (STAT1), whereas isorhamnetin normalized this abnormal phosphorylation. Overexpression of TYK2 attenuated the neuroprotective effect of isorhamnetin on IL-6-induced neurotoxicity. Our findings demonstrate that isorhamnetin exerts its neuroprotective effect by mediating the neuroinflammatory IL-6/TYK2 signaling pathway, suggesting its potential for treating AD.


Introduction
Alzheimer's disease (AD), the most common cause of dementia in older people, is an irreversible and a progressive neurodegenerative disease that slowly impairs cognitive function, memory and thinking, and eventually the ability to perform the simplest daily tasks [1]. With the rapid increase in the aging population in developing and developed countries, AD is becoming a major health problem globally [2]. However, the lack of effective treatment for preventing disease progression necessitates the development of new agents that can halt the pathogenesis of AD. Pathologically, AD is characterized by the presence of senile plaques in the brain [1]. Senile plaques are composed of the β-amyloid (Aβ) peptide, a fragment of the amyloid peptide precursor protein (APP) [3]. Aβ peptides aggregate to form oligomers and other high-order polymerized structures that cause neuronal death through various mechanisms including neuroinflammation, oxidative stress, excitotoxicity, energy depletion, and apoptosis [4].
Neuroinflammation is a prominent feature in AD pathology and a potential target for the treatment and prevention of AD [5]. Neuroinflammation links Aβ peptide deposition to neuronal death by activating microglia through Toll-like receptor 2 [6]. Activated microglia secrete proinflammatory factors including interleukin-(IL-) 1β [7], IL-6 [8], and tumor necrosis factor-(TNF-) α [7]. The expression levels of IL-1β, IL-6, and TNF-α have been reported to be upregulated in the brains of AD animal models [7,9] and patients with AD [1]. In addition, an increased risk of AD is associated with the genetic variants of IL-1β [10], IL-6 [11], and TNF-α [12]. Abnormal production of proinflammatory factors causes neurotoxicity and impairs Aβ peptide clearance, forming a vicious cycle among Aβ peptide accumulation, activated microglia, and proinflammatory factors. Blocking this cycle can be a vital strategy for halting neurodegeneration in AD.
Isorhamnetin, a small-molecular compound with an aromatic heterocyclic structure, is the main constituent of the Oenanthe javanica extract. Isorhamnetin exerts an antiinflammatory effect by modulating IκB kinase (IKK)α, IKKβ, and nuclear factor-(NF-) κB [13]. In a rat model, isorhamnetin demonstrated neuroprotection against Aβ peptides [14]. This study investigated the effects of isorhamnetin on microglial activation and neuroinflammation in the cell models of AD. Isorhamnetin inhibited Aβ peptide-mediated microglial activation and exerted a neuroprotective effect on IL-6-mediated neurotoxicity. These findings support the potential of isorhamnetin for use in AD treatment.
2.3. Cell Viability Assessment Using Lactate Dehydrogenase and 2,5-Diphenyl-2H-Tetrazolium Bromide Assays. According to the manufacturer's instructions (Roche), the cells were incubated with 100 μL of lactate dehydrogenase (LDH) reagent at room temperature for 20 min. The absorbance of the samples was measured at 490 nm by using a spectrophotometer. For the 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, the cells were cultivated with 20 μL of MTT (5 mg/mL in phosphate-buffered saline (PBS)) and incubated at 37°C for 2 hours. Subsequently, the formation of purple formazan crystals was determined by measuring absorbance at 570 nm by using a microplate spectrophotometer. [15]. Aβ42 peptides were dissolved in hexafluoro-2-propanol (HFIP) and incubated at room temperature for at least 1 hour. To obtain the peptide film, HFIP was removed through evaporation and the resulting peptides were stored at −20°C or −80°C. The resulting film was adjusted to a concentration of 5 mM by using dimethyl sulfoxide and then diluted to achieve appropriate concentrations for experiments.
2.6. Transfection with Plasmid DNA. The SH-SY5Y cells were transfected with the TYK2 expression vector (Sino Biological) by using the X-treme transfection reagent (Roche) according to the manufacturer's instructions. Subsequently, the expression of TYK2 was confirmed through Western blotting after 48 hours of transfection.

2.7.
Reactive Oxidative Species Analysis. The cells were seeded in six-well plates (10 5 cells/well) and differentiated into neuronal cells through treatment with retinoic acid (RA) (20 μM) for 5 days. Subsequently, the neuronal cells were treated with isorhamnetin or IL-6 for 48 hours. For reactive oxygen species (ROS) analysis, the fluorogenic Cell-ROX deep red reagent (Molecular Probes) was added to the live cells, followed by incubation at 37°C for 6 hours. Thereafter, the cells were washed with PBS and red fluorescence (indicating the presence of ROS) was examined using a Leica TCS confocal microscope at the excitation and emission wavelengths of 644 and 665 nm, respectively.

Neurite
Outgrowth Evaluation Using Immunofluorescence Staining. Neurons were cultured on a coverslip and washed with PBS. Subsequently, they were fixed with 4% paraformaldehyde for 20 min at room temperature. After the residual fixation buffer was removed using PBS, the cells were blocked in PBS containing 5% BSA for 20 min at room temperature and then hybridized with the primary antibody tubulin class III (TUBB3) (1 : 5000 dilution, BioLegend) overnight at 4°C. After being washed with PBS, the cells were incubated with an Alexa 488conjugated secondary antibody in the dark for 1 hour. For cell counting, the cells were stained with 4 ′ ,6-diamidino-2phenylindole (1 : 1000 dilution). Subsequently, the mounted coverslips were examined using a Leica TCS confocal microscope. Neurite outgrowth on the SH-SY5Y-derived neurons (>200 cells) was analyzed using MetaMorph software (Molecular Devices).
2.9. Preparation of HMC3-Conditional Medium. The HMC3 cells were treated by Aꞵ oligomers (200 nM) for 48 hours, and the medium was replaced with fresh culture medium for 24 hours. The supernatant free from Aꞵ oligomers was collected for further experiments.
2.10. ELISA. The conditional medium (2 mL) was collected from a 3.5 cm dish seeded with 10 5 HMC3 cells/well after treatment with Aꞵ oligomers (200 nM) for 24 or 48 hours, followed by isorhamnetin (10 μM) for 48 hours. The collected medium was centrifuged at 1500 rpm for 5 min at room temperature. We used 100 μL of the supernatant for the assessment. The concentrations of the proinflammatory cytokines/factors TNF-α, IL-1β, and IL-6 were analyzed using commercially available ELISA kits according to the manufacturer's protocol (IL-1β: Thermo Fisher; IL-6: R&D; TNF-α: R&D).
2.11. Statistical Analyses. All statistical analyses were performed using Student's t-test or one-way analysis of variance (ANOVA) with Bonferroni's post hoc test by using SPSS 18.0 (SPSS Inc., Chicago, IL, USA).

Activation of Human Microglia by the Synthetic Aβ
Oligomer. Studies have revealed that Aβ oligomers activate microglia to secrete proinflammatory factors [7,8]. Therefore, we treated the human microglial HMC3 cells with Aβ oligomers [15,16] (Figure 1(a)). Aβ oligomers demonstrated higher fluorescent signals in the thioflavin T assay than did monomeric Aβ (fold change: 2.13, P < 0:001, Figure 1 Figure 1(g)). By contrast, the secretions of IL-1β (treatment vs no treatment: 0.42 pg/mL vs 0.34 pg/mL, Supplementary Figure 1A) and TNF-α (treatment vs no treatment: 6.73 pg/mL vs 11.21 pg/mL, Supplementary Figure 1A) were not altered by treatment with Aβ oligomers. Twenty-four-hour treatment with Aβ oligomers to HMC3 cells demonstrated consistent results of cytokine releases (Supplementary Figure 1A). These results demonstrated that Aβ oligomers can activate microglia and increase IL-6 secretion from microglia. Given 48 hours treatment with Aβ oligomers to HMC3 cells, it generated relatively pronounced IL-6 secretion compared with 24 hours treatment. Therefore, we chose this condition for further experiments.

Discussion
Elucidating interactions between neuroinflammation and neurodegeneration can facilitate the development of new treatment strategies for AD. In this study, we established cell models to mimic neurodegenerative interactions among Aβactivated microglia, inflammatory factors, and neurons. The results reveal that the Aβ-activated NF-κB inflammatory signaling pathway upregulated the expression of the microglial 11 Oxidative Medicine and Cellular Longevity activation markers CD11b, CD68, and IBA1 and increased the secretion of IL-6 from the HMC3 microglial cells. Both the HMC3-conditioned medium and IL-6 reduced cell viability, impaired neurite outgrowth, and increased ROS production and cleaved caspase 3 expression in the SH-SY5Yderived neuronal cells. We observed that isorhamnetin attenuated the neuroinflammation and neurodegeneration induced by the HMC3-conditioned medium and IL-6. Furthermore, the results indicate that isorhamnetin suppressed neuroinflammation by deactivating the TYK2/STAT1 signaling pathway. These findings indicate the role of IL-6 in the nonautonomous interaction between microglia and neurons in AD. Furthermore, the findings demonstrate that the neuroprotective effect of isorhamnetin involves mediation of the noncanonical IL-6 signaling pathway, thus indicating the potential of isorhamnetin for use in AD treatment ( Figure 8).
Our study demonstrates that IL-6 was the main constituent in the HMC3 conditional medium that regulated the neuroinflammatory interaction between microglia and neurons for neurodegeneration (Figures 2 and 4). Isorhamnetin suppressed the activation of the NF-κB pathway, downregulated the expression of the microglial activation markers, and reduced the secretion of IL-6 from the Aβ-activated microglia ( Figure 2). In lipopolysaccharide-induced BV2 mouse microglial cells, isorhamnetin reduced oxidative stress by reactivating TLR4 and the NF-κB pathway [23]. Moreover, isorhamnetin was reported to attenuate oxidative stress and neurotoxicity by upregulating NRF2/HO-1 [24]. Similar to isorhamnetin, Gx-50, a natural compound derived from Zanthoxylum bungeanum, suppressed the activation of TLR4 and its downstream MyD88 and TRAF6 and reduced the expression levels of TNF-α, IL-1β, nitric oxide (NO), prostaglandin E2, inducible NO synthase, and cyclooxygenase-2 in Aβ-activated primary rat microglia and APP + transgenic mice [7]. In addition, KHG26792, a novel azetidine derivative, attenuated the activation of the NF-κB signaling pathway and reduced the expression levels of IL-6, IL-1β, TNF-α, NO, ROS, and NADHP oxidase in Aβ-treated rat primary microglia [25]. These compounds exert neuroprotective effects possibly by mediating neuroinflammation, and the neuroprotective potential of these compounds should be further explored in neuronal and animal models for AD.
The canonical downstream pathway of IL-6 in inflammatory cells and microglia involves the activation of JAK2 and STAT3. IL-6 upregulated the expression of STAT3 in rodent neurons [26,27]. However, the downstream signaling of IL-6 in human neurons remains contentious. The phosphorylation of STAT3 was upregulated on SH-SY5Y cells exposed to a high IL-6 concentration (10 ng/mL) [27]. In this study, we observed that HMC3-conditioned medium and a low IL-6 concentration (350 pg/mL; this concentration was equal to that in the HMC3-conditioned medium) caused neurotoxicity without upregulating the JAK2/STAT3 signaling pathway in the SH-SY5Y-derived neuronal cells ( Figure 5). However, the noncanonical TYK2/STAT1 phosphorylation was upregulated following treatment with a low IL-6 concentration. TYK2, a member of the JAK family, is associated with the intracellular domain of various cytokines that affect inflammatory responses [28]. In addition, the activation of the TYK2/STAT signaling pathway is associated with inflammatory responses, apoptosis, and ROS production [21,22,29]. Knockout of TYK2 mitigated neuronal death in APP/PS1 transgenic mice [23]. Our results indicate that isorhamnetin downregulated TYK2/STAT1 phosphorylation to attenuate the neurotoxic effects of IL-6, whereas TYK2 overexpression attenuated the neuroprotective effect of isorhamnetin (Figures 6 and 7). These results highlight the role of TYK2/STAT1 signaling in AD pathogenesis and isorhamnetin in protection against neuroinflammation.
Isorhamnetin, the main constituent of flavonoids, possesses antioxidative properties [30,31]. In addition to protection against neuroinflammation, isorhamnetin exerts antioxidative and neurotropic effects. Isorhamnetin potentiated the effect of the nerve growth factor on the neurite outgrowth of PC12 cells [32]. In mice treated with scopolamine, isorhamnetin reduced oxidative stress and suppressed cholinergic signaling pathways, reduced cholinesterase activity [33], and improved impairment in spatial and nonspatial learning and memory [34]. Furthermore, treatment with isorhamnetin in stroke mice improved the integrity of the blood-brain barrier and reduced the levels of IL-1β, IL-6, and TNF-α in the ischemic cortex [35]. Together with our findings, these results suggest the pleiotropic effects of isorhamnetin for neuroprotection. Isorhamnetin can effectively penetrate the blood-brain barrier [36] and thus can be used for treating neurological diseases. Furthermore, our results reveal that isorhamnetin exhibite neuroprotection in the SH-SY5Y-derived neuronal cells following IL-6 treatment, suggesting that isorhamnetin can reduce neuroinflammationmediated neurodegeneration.  Figure 8: The anti-inflammatory and neuroprotective effects of isorhamnetin on Alzheimer's disease. Aβ oligomers activate microglia to secrete IL-6, which further upregulates the TYK2/ STAT1 pathway to increase production of reactive oxygen species (ROS), impair neurite outgrowth, and upregulate apoptosis on neurons (neuroinflammation). Isorhamnetin attenuates microglial activation and IL-6 secretion and downregulates the TYK2/ STAT1 pathway to reduce IL-6-mediated neuroinflammation.
In conclusion, our study indicates the pivotal role of IL-6 in mediating nonautonomous interactions between microglia and neurons in AD and that the TYK2/STAT1 signaling pathway can be a target for treating neuroinflammation in AD. In addition, our results demonstrate that isorhamnetin alleviates neuroinflammation by deactivating the TYK2/ STAT1 signaling pathway. These findings provide new insights into neuroinflammatory pathogenesis as well as a novel therapeutic target for AD. Additional animal studies should be conducted to validate our findings.

Data Availability
The datasets generated during the current study are available from the corresponding author upon reasonable request.