Isoschaftoside Inhibits Lipopolysaccharide-Induced Inflammation in Microglia through Regulation of HIF-1α-Mediated Metabolic Reprogramming

Isoschaftoside is a C-glycosyl flavonoid extracted from the root exudates of Desmodium uncinatum and Abrus cantoniensis. Previous studies suggested that C-glycosyl flavonoid has neuroprotective effects with the property of reducing oxidative stress and inflammatory markers. Microglia are key cellular mediators of neuroinflammation in the central nervous system. The aim of this study was to investigate the effect of isoschaftoside on lipopolysaccharide-induced activation of BV-2 microglial cells. The BV-2 cells were exposed to 10 ng/ml lipopolysaccharide and isoschaftoside (0–1000 μM). Isoschaftoside effectively inhibited lipopolysaccharide-induced nitric oxide production and proinflammatory cytokines including iNOS, TNF-α, IL-1β, and COX2 expression. Isoschaftoside also significantly reduced lipopolysaccharide-induced HIF-1α, HK2, and PFKFB3 protein expression. Induction of HIF-1α accumulation by CoCl2 was inhibited by isoschaftoside, while the HIF-1α specific inhibitor Kc7f2 mitigated the metabolic reprogramming and anti-inflammatory effect of isoschaftoside. Furthermore, isoschaftoside attenuated lipopolysaccharide-induced phosphorylation of ERK1/2 and mTOR. These results suggest that isoschaftoside can suppress inflammatory responses in lipopolysaccharide-activated microglia, and the mechanism was partly due to inhibition of the HIF-1α-mediated metabolic reprogramming pathway.


Introduction
Microglial cells are the resident macrophages in the central nervous system, and they provide the frst line of defense against brain injury. Tey become activated in response to central nervous system injury or damage [1]. Te classic activation cascade of microglia can promote the synthesis and secretion of a large number of infammatory mediators including nitric oxide (NO), proinfammatory cytokines interleukin-1 (IL-1), IL-6, and tumor necrosis factor-α (TNF-α), leading to neuronal death and accelerating the pathological progress of neurodegenerative diseases [2,3]. Suppressing microglial overactivation is considered to be a promising therapeutic to a wide variety of CNS diseases.
Recent studies suggest that microglia activation can be regulated by their metabolism [4]. During the classic infammatory activation process, microglia cellular metabolism as well as energy production is reprogrammed from oxidative phosphorylation (OXPHOS) toward glycolysis even in the presence of oxygen, a phenomenon also known as the Warburg efect [5]. Inhibiting the glycolytic metabolism of activated microglia proved to be an efective therapeutic target for neuroinfammation and neurodegenerative diseases [6][7][8].
However, little is known about the efect of isoschaftoside on neuroinfammation in the central nervous system. Te objective of the present study was to investigate the efect of isoschaftoside on the infammatory response induced by lipopolysaccharide and its molecular mechanism in cultured BV2 microglial cells in vitro.

Materials and Methods
2.1. BV2 Microglial Cell Culture. Te murine BV2 microglial cells were purchased from China center for type culture collection (Wuhan, China) [11]. BV2 cells were cultivated in the DMEM medium supplemented with 10% FBS and 1% P/S in a humidifed 5% CO 2 atmosphere at 37°C.

Cell Viability
Assay. Cells were cultured in a 96-well plate. After experimental treatment, a cell count kit-8 was used to quantitatively evaluate the cell viability according to the manufacturer's instructions (Beyotime, China), and then the plates were incubated in the 5% CO 2 incubator for 2 h at 37°C. Te absorbance was determined at 450 nm using a microplate reader.

Measurement of Nitrite Production.
Te Griess reagent (Beyotime, China) was used to detect the concentration of NO in the cell culture supernatant. BV2 cells were seeded in 96-well plates and treated with 0-1000 μM isoschaftoside and 10 ng/mL lipopolysaccharide for 24 h. Te supernatant was collected and made to react with the Griess reagent at room temperature for 30 minutes. Te absorption of the mixture was measured at 540 nm using a microplate reader.
2.6. In Vitro Immunofuorescence. BV2 cells were cultured on gelatin-coated glass coverslips until fusion. Cells were induced with cobalt chloride for 24 h and then treated with isoschaftoside for 9 h. Te cells were washed twice in PBS and fxed with 4% paraformaldehyde for 10 minutes. Polyformaldehyde was removed by washing three times in PBS and then penetrated for 5 minutes with 0.2% Triton X-100 and 5% BSA in PBS for 30 minutes. Te slides were incubated with the primary antibody at 4°C overnight, followed by the goat IgG (1 : 200)-Alexa fuor488 secondary antibody, and they were then restained with DAPI (1 μg/ml). Te bx530 Olympus microscope with Olympus dp47 digital camera was used for photo taking, and the fuorescence intensity was analyzed by ImageJ.

Statistical
Analyses. Statistical analyses were determined by using GraphPad prism software 6 (La Jolla, CA), and the data are expressed as means ± SD. Two-way ANOVA was used for statistical comparisons followed by Tukey's post hoc test. Statistical signifcance was set at P < 0.05.

HIF-1α Is Required for Regulating the Anti-Infammatory
Efect of Isoschaftoside. To investigate whether the inhibitory efect of isoschaftoside on lipopolysaccharide-induced microglia activation depends on the inhibition of HIF-1α expression, BV2 cells were exposed to 10 μM Kc7f2 (a selective inhibitor of HIF-1α transcription) for 24 h, followed by incubation with lipopolysaccharide and isoschaftoside for 9 h. Te results showed that in the presence of Kc7f2, the inhibitory efect of isoschaftoside on lipopolysaccharideinduced neuroinfammation did no longer exist (Figures 3(e)-3(i)). Similarily, isoschaftoside had no further efect on HIF-1α, HK2, and PFKFB3 (Figures 3(a)-3(d)) expression when Kc7f2 was administered to BV2 cells. To further confrm that isoschaftoside can inhibit the upregulation of HIF-1α, BV2 cells were pretreated with CoCl 2 for 24 h followed by coadministration with isoschaftoside for 9 h. CoCl 2 causes accumulation of HIF-1α in microglia through inhibiting HIF-1α degradation [14]. As shown in Figures 4(a) and 4(b), the fuorescence intensity of HIF-1α increased signifcantly in CoCl 2 -treated cells, whereas isoschaftoside abolished this phenomenon. Consistent with the immunofuorescence assay, the protein levels of HIF-1α, HK2, and PFKFB3 in microglia were signifcantly increased after CoCl 2 treatment, while these upregulations were mitigated by isoschaftoside (Figures 4(c)-4(f )).

Discussion
Activated microglia play an important role in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease [16,17]. Te current study demonstrated that isoschaftoside, a C-glycosyl favonoid extracted from the root exudates of Desmodium uncinatum, prominently suppresses proinfammatory mediators' production in lipopolysaccharide-stimulated BV2 microglial cells.
Te regulatory efect of isoschaftoside on microglia activation may be related to its chemical structure ( Figure 6). Flavonoids are reported to have a wide range of pharmacological activities, including blood lipid lowering, hypoglycemia, antiviral, neuroprotective, antioxidant, and Evidence-Based Complementary and Alternative Medicine antitumor activities [18]. Extensive evidence supports the anti-infammatory features of numerous favonoids in both preclinical and clinical studies [19]. For example, favanone glycosides extracted from citrus fruits can reduce the levels of IL-1β and IL-6 in patients with neuritis [20]. In rat and mouse models of Parkinson's disease, the favonoids have been shown to reduce astrogliosis, microgliosis, and the levels of proinfammatory mediators in the brain [21]. Flavonoid-rich ethanol extract from the leaves of Diospyros kaki can alleviate microglia and astrocyte activation, inhibit neuroinfammation, and attenuate neuronal apoptosis and synaptic dysfunction in D-galactose-aged mice [22]. Flavonoid-agathisfavone derived from the Brazilian plant Poincianella pyramidalis is able to regulate microglia polarization and promote remyelination [23]. To the best of our knowledge, this is the frst report demonstrating that isoschaftoside is capable of regulating neuroinfammation in BV2 microglia. Te change of metabolic state recently has been suggested as an early inducement of neurodegenerative diseases [24]. Te HIF pathway is one of the key transcriptional regulators in immunity and infammation. Recent studies suggested that HIF-1α participates in the regulation of microglia metabolic reprogramming [24][25][26]. Tere are two key speed-limiting enzymes in the metabolic reprogramming pathway: PFKFB3 and HK2, which are closely related to the glycolysis transformation of lipopolysaccharide or hypoxia-activated microglia [27,28]. We explored whether isoschaftoside inhibits lipopolysaccharide-induced microglia activation by inhibiting the expression of HIF-1α, the upstream regulator of glycolysis. Our results showed that the HIF-1α expression was signifcantly reduced in the isoschaftoside-treated microglia compared with lipopolysaccharide-stimulated cells. In addition, induction of HIF-1α accumulation by CoCl 2 was also inhibited by isoschaftoside. Furthermore, the HIF-1α specifc inhibitor Kc7f2 mitigated the metabolic reprogramming and antiinfammatory efect of isoschaftoside. Tus, our results confrmed that HIF-1α is necessary for isoschaftoside to exert its anti-infammatory efect. ERK1/2 and mTOR have been suggested as important upstream pathways to induce HIF-1α transcription [29]. Terefore, it can be questioned whether the antiinfammatory property of isoschaftoside is associated with regulation of ERK1/2 and mTOR in activated microglial cells. Our results showed that lipopolysaccharide-induced phosphorylation of ERK1/2 and mTOR in microglia was inhibited by isoschaftoside. A recent study suggested that schaftoside reduced neuroinfammation in BV2 microglia cells through the TLR4/MyD88-mediated mitochondrial fssion pathway [30]. Terefore, it is possible that isoschaftoside regulates ERK1/2 and mTOR signaling pathways by binding to TLR4 in microglia as well.
Te fndings of this study have to be seen in light of possible limitation. Te immortalized murine microglial cell line BV2 was used to construct an infammatory model in our study. Although BV2 cells appear to be a valid substitute for primary microglia, however, the study indicated that BV2 cell responses to LPS and IL-4 were narrower and weaker than those of primary microglia [31]. Tus, the fndings of this study need to be validated in primary microglia in the future.
In summary, the present study found that isoschaftoside prevents lipopolysaccharide-induced activation of ERK1/2/ mTOR and HIF-1α mediated metabolic reprogramming, which leads to down-regulation of iNOS, COX-2, IL-1β, and TNF-α in BV2 microglial cells. Since isoschaftoside has very good bioavailability [32], our fndings suggest that isoschaftoside may be a good medication for prevention or treatment of infammation-related neurodegenerative diseases. Further preclinical and clinical studies are needed to better evaluate the potential therapeutic profle of isoschaftoside in neuroinfammation-mediated disorders.

Data Availability
Te data used to support the fndings of this study are included within the article.

Conflicts of Interest
Te authors declare no conficts of interest.