Artemisinin protects against sepsis-associated encephalopathy by activating the AMPK axis in the microglia

Background and purpose: Artemisinin has been in use as an anti-malarial drug for almost half a century in the world. There is growing evidence that artemisinin also possesses potent anti-inflammatory and immunoregulatory properties. However, the efficacy of artemisinin treatment in sepsis-associated encephalopathy (SAE) remains unknown. Here, we evaluate the possible protective effects and explore the underlying mechanism of action of artemisinin on SAE. Methods: Male C57BL/6mice were pretreated with either vehicle or artemisinin, and then injected with LPS to establish an animal model of SAE. The cognitive function was then assessed using the Morris water maze. Neuronal damage and neuroinflammation in the hippocampus were evaluated by immunohistochemical analysis. Additionally, the protective mechanism of artemisinin was determined in vitro. Results: The results showed that artemisinin preconditioning attenuated LPS-induced cognitive impairment, neural damage, and microglial activation in the mouse brain. Luminex liquid chip revealed that artemisinin could inhibit the pro-inflammatory cytokines and chemokines induced by LPS in the BV2 microglia cells. Meanwhile, artemisinin suppressed the migratory ability of BV2 cells. Western blot demonstrated that artemisinin promoted adenosine monophosphate-activated protein kinaseα1 (AMPKα1) expression and suppressed nuclear translocation of NF-κB. Furthermore, knock-down of AMPKα1 markedly abolished the anti-inflammatory effects of artemisinin when exposed to LPS. Conclusion: Artemisinin is a potential therapeutic agent for SAE, and its effect was probably mediated by the activation of AMPKα1signalling pathway in microglia. the nuclei. Images were obtained using a fluorescence microscope. To analyse the ratio of nuclear and cytoplasmic immune fluorescence of NF-κB, the percentage of cells showing high fluorescence in different areas of a cell (either nucleus or cytoplasm) was counted. A total of 200 cells were counted per group. Densitometry analysis of the nuclear translocation of NF-κB was expressed as a percentage of total cells. Our results have shown that artemisinin could improve the cognitive function in SAE mice in the Morris water maze test. Artemisinin preconditioned mice exhibited better spatial learning-memory than the LPS-challenged group. It has been reported that


Introduction
Sepsis-associated encephalopathy (SAE) refers to cerebral nervous system dysfunction during sepsis [1]. Among the sepsis-induced systemic damage, SAE is one of the more severe clinical manifestation. It manifests as a rapid decline in cognitive functions and memory, even leading to coma [2].Anti-infection and supportive therapy are the major treatments used in the clinical treatment of SAE [3]. Until now the pathogenesis of SAE has not been fully elucidated, and there is no To evaluate cognitive disorder and the therapeutic effects of artemisinin in mice, the Morris water maze (MWM) test was conducted as described previously [14,16]. First, the mice were trained to find a hidden platform in the maze within 60s. If the mice failed to find the platform within 60s, it was guided to the platform and allowed to stay on it for 10s. The positioning navigation experiment was conducted after five days of LPS injection and the escape latency was recorded. On the 6th day of LPS injection, the space exploration experiment was performed where in the platform was removed from the pool. All mice were monitored for 60s to observe the average swimming speed, distance of swimming and the percentage of total time in the targeted quadrant. Data were analysed using SMART 3.0 software designed for the MWM test (Harvard Apparatus Technology Co, Ltd., USA).

Tissue preparation and histological analysis
After the MWM test, mice were euthanised and transcardially perfused with phosphate-buffered saline (PBS), followed by a solution containing 4% paraformaldehyde. Sections of hippocampal tissue from the brain were prepared and fixed in 4% paraformaldehyde solution overnight. After being dehydrated in ethanol, the tissues were embedded in paraffin and cut into 4 µm sections. Paraffinembedded sections of the brain tissue were deparaffinised in xylene and rehydrated through descending grades of ethanol (Tianjin Sheng Winton Chemical Co., Ltd., Tianjin, China). Sections were then stained with haematoxylin and examined under a light microscope (Nikon Technology Co, Ltd., Japan). The number of hippocampal Cornu Ammonis 1 (CA1) pyramidal neurons permm 2 was used to calculate the neuronal density.

Immunofluorescence histochemistry
After deparaffinization and rehydration, the slide was immersed in the EDTA antigen retrieval buffer and heated for antigen retrieval. The sections were blocked with 3% BSA for 30 min at room temperature, and then incubated with the primary antibody for ionised calcium binding adaptor molecule-1 (Iba-1, Abcam, Cambridge, UK. ab5076, 1:200), followed by fluorescent secondary antibodies. They were also incubated with DAPI solution at room temperature for 10 min to visualise the nucleus. The sections were imaged with a fluorescent microscope (Axio Observer Z1; Carl Zeiss AG, Germany). Red fluorescence was considered as an indicator of Iba-1 positivity.

Cell culture
BV2 microglial cells were cultured in DMEM (Thermo Fisher Scientific, MA, USA) supplemented with 10% FBS (Thermo Fisher Scientific, MA, USA), 0.5% penicillin, and 0.5% streptomycin (Thermo Fisher Scientific, MA, USA) at 37°C with a humidified atmosphere of 5% CO2 and 95% air. BV2 cells were then divided into three groups: control group, LPS group (treated with 100 ng/mLof LPS for 12 hours), and LPS+ART group (pretreated with 40 mΜ of artemisinin for 2 hours and then co-treated with 100 ng/mLof LPS for another 12 hours).

ELISA
The expression levels of IL-6 and TNF-α in the cell supernatants were measured using ELISA kits (Dakewei Bio-engineering Co., Ltd, Shenzhen, China) according to the manufacturer's instructions.
The optical density values were measured at 450 nm by the microplate reader within 5 min, and standard curves were plotted [17]. Both standards and samples were measured in triplicate.

Quantitative RT-PCR
The mRNA expression for IL-6 and TNF-α was determined by qRT-PCR as described previously [18].

Western blot analysis
Western blot analysis was performed on cell lysates according to standard protocols as described previously [11]. Briefly, the total protein of the cells was extracted with radio-immunoprecipitation

Wound healing assay
For the migration assay, BV2 microglial cells were seeded in 6-well plates and cultured until the formation of a 90% confluent monolayer. A linear wound was made by scratching the cell monolayer of BV2 cells with a sterile 200 μL pipette tip. After washing with PBS to remove the cell fragments, the cells were incubated in a medium without FBS. The areas of the wound and cell migration were photographed by a phase-contrast microscope at 0h and 12h after wounding. All images were analysed by IPP.

Transwell
A total of 100,000 BV2 cells were seeded in FBS-free DMEM media in the upper chamber of a transwell insert (Corning Incorporated, NY, USA). The lower chamber was filled with DMEM media supplemented with 10% FBS. After 12h of incubation, the cells in the upper chamber were removed, and the cells that had invaded the membrane were fixed with 4% paraformaldehyde and stained with crystal violet for 20 min. Five randomly selected fields were captured with an optical microscope and the number of invading cells was counted.

Small interfering RNA (siRNA) transfection
AMPKα1 targeting siRNA for use in the BV2 cell lines was synthesised by Gene Pharma Company (Gene Pharma, China). The sequence of the specific AMPKα1 siRNA was 5′-GCAUAUGCUGCAGGUAGAUTT-3′. The sequence of scrambled control siRNA was 5′-UUCUCCGAACGUGUCACGUTT-3′. Cells were transfected with AMPKα1 siRNA (si-AMPKα1) or scrambled control siRNA (si-NC) 8h prior to further experimentation using Lipofectamine iMAX (Invitrogen, USA) according to the manufacturer's instructions.

Immunocytochemistry staining
Cells were fixed in BD Cytofix/Cytoperm solution (BD Biosciences, NJ, USA) and permeabilised with 0.1% Triton-X. The cells were incubated overnight with primary antibody for nuclear factor kappa B (NF-κB. Cell Signaling Technology Inc., Danvers, USA. cst 3039s, 1:500) followed by labelling with FITC goat anti-rabbit IgG (Wuhan Servicebio Technology Co., Ltd, Wuhan, China. 1:400). Later the cells were incubated with 0.5 mg/mL DAPI for staining the nuclei. Images were obtained using a fluorescence microscope. To analyse the ratio of nuclear and cytoplasmic immune fluorescence of NF-κB, the percentage of cells showing high fluorescence in different areas of a cell (either nucleus or cytoplasm) was counted. A total of 200 cells were counted per group. Densitometry analysis of the nuclear translocation of NF-κB was expressed as a percentage of total cells.

Statistical analysis
The experimental results were expressed as mean±SD. Statistical analysis was performed with GraphPad Prism 7.0 software (San Diego, CA, USA) using one-way ANOVA followed by Tukey's posthoc analysis. A value of p less than 0.05 was considered as statistically significant.

Artemisinin ameliorated neuronal cell death and improved cognitive impairment in LPS-induced murine neuroinflammatory model
Morris water maze training was applied to assess the efficacy of artemisinin treatment in LPS-induced cognitive impairment. Five days after systemic injection of LPS, a positioning navigation experiment was performed to observe the cognitive function of mice. As shown in Fig.1A and B, compared to sham group, mice exposed to LPS exhibited a significantly prolonged escape latency to find the platform, while those pretreated with artemisinin exhibited a shorter escape latency (p<0.05). On the 6th day, the space exploration experiment was performed in the absence of the platform. Our data demonstrated that the percentage of total time in the targeted quadrant was significantly reduced in the LPS-challenged group compared with the sham group, while mice in the artemisinin-pretreated group spent significantly more time in the targeted quadrant than the LPS-challenged group (p<0.05).There was no significant difference between the three groups of mice with regard to the average speed nor the distance travelled in the space exploration test (p>0.05). These findings suggest that artemisinin could improve LPS-induced memory and cognitive impairment.
The hippocampus is closely related to cognitive and memory function. To determine whether artemisinin could affect neuronal survival in the hippocampal region and improve cognition, we performed haematoxylin staining of the hippocampus. It was found that LPS induced a significant reduction in the number of hippocampal CA1 neurons. Notably, artemisinin pretreatment could ameliorate the neuronal cell death in the CA1 region significantly (Fig.1C, D). Meanwhile, Iba-1 immunoreactive microglia was abundant in the hippocampus of the LPS group. Artemisinin administration induced a dramatic decrease in Iba-1 positive cells in the hippocampal region (Fig. 2).
This data demonstrated that artemisinin could ameliorate the hippocampal neuron cell death and improve cognitive impairment caused by LPS via suppressing microglial activation.

Artemisinin regulated the inflammatory profile and inhibited migratory ability of BV2 microglia
Microglial cell activation and proliferation precedes the onset of CNS injury [19]. Activated microglia produce a wide range of pro-inflammatory mediators. To investigate the effects of artemisinin on LPSactivated BV2 microglia, liquid chip was employed to determine the level of inflammatory cytokines in the supernatant of BV2 cells. We observed a marked release of pro-inflammation cytokines (TNF-α, IL-6) and chemokines (LIX, MCP-1, MIP-2) following 12 hours of LPS stimulation, which was however abolished by artemisinin treatment (Fig.3A). The reliability of the liquid chip results was validated by ELISA analysis of IL-6 and TNF-α (Fig.3D-E). Furthermore, mRNA and protein expression were confirmed with qRT-PCR and western blot, respectively. Our data showed significant increase in mRNA expression of IL-6 and TNF-αin the LPS-activated group, which was markedly inhibited by artemisinin ( Fig.4A, B). Similarly, western blot confirmed that artemisinin treatment significantly inhibited the protein expression of IL-6 and TNF-α after LPS stimulation (Fig.4 C, D). To observe the effect of artemisinin on microglial cell motility, both wound-healing assay and transwell chamber were employed. Compared with normal control, LPS stimulation efficiently enhanced the migratory ability of BV2 cells. However, such an effect was diminished in the presence of artemisinin (Fig.5). The above findings indicate that artemisinin could reduce the production of pro-inflammatory cytokines and microglial proliferation.

AMPKα1 pathway is involved in the anti-inflammatory effect of artemisinin
We next investigated whether the AMPKα1 pathway was involved in the anti-inflammatory effect of artemisinin. Preliminary western blot demonstrated that artemisinin could activate AMPKα1 in a dosedependent manner (Fig.6A). We further discovered that activation of AMPKα1 by artemisinin significantly attenuates LPS-induced inflammatory pathways in BV2 cells. Firstly, immunofluorescence analysis revealed that LPS stimulated nuclear translocation of NF-kB, whereas pretreatment with artemisinin markedly decreased the nuclear translocation of NF-κB ( Fig. 6C and D). Western blot analysis showed that the LPS-induced up-regulation of iNOS, IL-6, and TNF-α expression was also reversed by artemisinin pretreatment (Fig.6B). These results further suggest that activation of AMPKα1 by artemisinin significantly attenuated the activation of NF-κB inflammatory pathway induced by LPS.
The essential role of AMPKα1 activation in the anti-inflammatory effect of artemisinin was confirmed using si-RNA specific to AMPKα1. As shown in Fig.7A, immune blotting showed excellent knock-down efficiency of si-RNA specific to AMPKα1. We observed that artemisinin inhibited the LPS-induced increase in TNF-α production, but this effect was significantly reduced after transfection with AMPKα1 siRNA (Fig. 7B and C). Therefore, the results suggest that artemisinin attenuated the LPS-induced inflammation by activation of AMPKα1.

Discussion
In our study, artemisinin exhibited powerful anti-inflammatory and neuroprotective activities in the SAE model. This protection was mediated by the activation of AMPKα1 signalling in the microglia Pathological changes in the hippocampus were measured to detect neuronal survival and microglial activation in our study. Our results showed that artemisinin inhibited LPS-induced activation of microglia and neuronal death in the hippocampal CA1 region. Taken together, this study demonstrates that artemisinin could restore hippocampus-mediated cognition by inhibiting the microglial cell activation and neuronal death in the CA1 region.
To further evaluate the potential pharmacological mechanism of artemisinin in microglia, liquid chip technology was applied to detect the expression levels of cytokines in the BV2 cell supernatants. We The liquid chip also revealed that artemisinin inhibited the chemokines (LIX, MCP-1 and MIP-2) which were induced by LPS stimulation. Chemokines are crucial factors for an early inflammatory response that evoke migration of the microglial cells [32]. Microglial cells migrate to the site of injury or lesion, and play a pivotal role in the occurrence, and development of inflammation [33]. In this study, immunofluorescence of Iba-1, wound healing assay and transwell migration assay were performed to test whether artemisinin could reduce the migratory capacity of microglia cells. The immunofluorescence test for Iba-1 showed that artemisinin significantly inhibited LPS-induced migration of microglia in the brain. Wound healing assay and transwell migration assay also revealed that artemisinin inhibited the migratory ability of BV2 microglia. Our data suggests that the antiinflammatory effect of artemisinin might be exerted by suppressing microglial cell motility via inhibition of chemokine activity.
To further elucidate the mechanism of the anti-inflammatory and neuroprotective effects of artemisinin, we assessed its effect on the AMPK signalling pathway. AMPK is a highly conserved serine/threonine protein kinase present in eukaryotic cells [34,35]. AMPK participates in multiple biological processes by modulating cellular energy homeostasis. It has become an attractive drug target for diabetes, stroke, and tumour [36]. Recent studies found that AMPK is also involved in inflammatory response through regulation of NF-κB signalling pathway [37]. It has been reported that phosphorylated-AMPK inhibits the activation of NF-κB and decreases the production of IL-6, TNF-α and IL-1β [38][39][40][41]. These results suggest that AMPK phosphorylation may also serve as an important therapeutic target for inflammation [34,42]. However, whether artemisinin inhibits neuroinflammation through the AMPK/NF-κB pathway remains unexplored. Firstly, our study demonstrated that artemisinin could activate the AMPK in a dose-dependent manner in microglia cells. Furthermore, pretreatment with artemisinin markedly decreased the nuclear translocation of NF-κB in BV2 cells. PCR, western Blot and ELISA analysis showed that LPS-induced up-regulation ofIL-6, and TNF-α mRNA, and both proteins were also reversed by artemisinin pretreatment. Therefore, the present study suggests that artemisinin increased the expression of AMPK and blocked the nuclear translocation of NF-κB where it can initiate transcription by binding to gene promoter elements of IL-6 and TNF-α. These results suggest that the AMPK/NF-κB pathway is involved in the anti-inflammatory activity of artemisinin in neuroinflammation. Previous research has shown that artemisinin could regulate AMPK pathway, hence itis an attractive drug target for cancer and oxidative stress injury [43,44]. We demonstrate for the first time in the current study that AMPK pathway can be the therapeutic target of artemisinin for the treatment of neuroinflammation.

Conclusion
In summary, our data has proved for the first time that artemisinin inhibits hippocampal neuronal death and cognitive impairment by inhibiting microglial activation in the in-vivo model of SAE. In addition, it was found that artemisinin significantly increased the AMPKα1 to inhibit LPS-induced neuroinflammatory response in BV2 microglial cells. Our finding indicate that artemisinin may be a potential therapeutic agent in SAE.