Tetramethylpyrazine Derivative T-006 Ameliorates the Amyloid-β Plagues of Transgenic Alzhermer's Mice by Modulation of TLR4-mediated MyD88/NF-κB Signaling

Background Microglial activation mediated neuroinammation was considered as a vital trigger factor in the pathogenesis of Alzheimer’s disease (AD). T-006, a new tetramethylpyrazine derivative, has been recently found to alleviate cognitive decits via inhibition of Tau expression and phosphorylation in AD transgenic mouse models. Here, we hypothesized that T-006 may ameliorate AD-like pathology by suppressing the neuroinammation. Methods APP/PS1 transgenic AD mouse model was used here to evaluate the anti-inammatory effect of T-006 and its underlying mechanisms, as well as its potential protective effects against lipopolysaccharide (LPS)-activated microglial-induced neurotoxicity. Results Our results indicated that T-006 signicantly decreased the levels of total amyloid β peptide (Aβ) and glial brillary acidic protein (GFAP) as well as the ionized calcium binding adaptor molecule-1 (Ibα-1) expression in the APP/PS1 mice. Moreover, T-006 dramatically suppressed abnormal elevation of inammatory mediators and reduced the levels of Toll-like receptor 4 (TLR4), myeloid differential protein-88 (MyD88) and NF-κB signaling related proteins in lipopolysaccharide (LPS)-induced BV2 microglial cells. We also found that TAK242, a TLR4 inhibitor could abolish the down-regulation of T-006 on LPS-induced proinammatory mediators and reversed the downstream proteins expression containing MyD88 and NF-κB signaling. Importantly, T-006 prevented against neuroinammation induced neurotoxicity by mitigating reactive oxygen species (ROS) overproduction and mitochondrial membrane potential (MMP) dissipation. T-006 exerts in AD by suppressing the neuroinammation through modulation of TLR4-mediated MyD88/NF-κB signaling pathways. neurotoxicity by reversing mitochondrial impairment. Our results suggested that T-006 exerts neuroprotective effect in treating AD by suppressing the neuroinammation through modulation of TLR4-mediated MyD88/NF-κB signaling pathways.


Introduction
Alzheimer's disease (AD), a complex degenerative disease of central nervous system (CNS), is characterized by the decline of memory and cognitive ability. The extracellular deposits of amyloid β peptide (Aβ) in senile plaques (SP) and the formation of intracellular neuro brillary tangles (NFTs) caused by the hyperphosphorylation of Tau protein, are consisted of the most two important neuropathological features of AD, which contributed to the synapse loss and neurons death, and subsequently memory impairment [1]. Over 200 reagents targeting Aβ or Tao protein were researched in recent decades aimed to reverse or stop the degeneration progress of AD, but unfortunately, none is currently clinical available [2]. The underlying failure is that targeting the pathological features rather than the pathogenic factors seems an endless parade of the pharmaceutical anti-AD development [3]. Therefore, elucidating the precise mechanisms underlying the AD and seeking e cient agents with disease-modifying potential becomes the focus of current research.
Although the etiology of AD is not fully understood, increasing evidences con rmed that microglial activation mediated neuroin ammation was one of the vital trigger factors of AD [4,5].
Neuroin ammation is an important neuropathological process, which is triggered by microglia and astrocytes activation and closely related to the brain injury and neurodegenerative diseases including AD and Parkinson's disease [6,7]. Especially, microglia acted as the main immune-surveillance cells of CNS, of which over-activation increased pathological Aβ and Tau accumulation and synapse loss, and subsequently resulted in neuronal damage and death [8,9]. Therefore, inhibition of microglial activation and reduction of in ammatory mediators production is considered as bene cial strategy in AD therapy [10,11].
Tetramethylpyrazine (TMP) is the main active component of Chinese herb Ligusticum (Chuanxiong), which is widely used in clinical treatment of hypoxic-ischemic encephalopathy and cerebrocardiovascular diseases due to its ability to penetrate blood brain barrier (BBB) to generate therapeutic effects [12,13]. J147 is a multifunctional neuroprotectant with great potential to improve the degenerative process of AD, and its phase I clinical trial against AD was completed in 2020 (NCT03838185). In order to make full use of the advantages of TMP and improve its activity, we have synthesized a new TMP derivative named T-006 by replacing the methoxybenzene ring of J147 with TMP.
In previous studies, we found that T-006 alleviated cognitive de cits via inhibition of Tau and APP expression in AD transgenic mice models [14]. However, the speci c mechanism underlying the anti-AD effect of T-006 is unclear and whether this effect is regulated by the anti-neuroin ammation ability is still unknown. The present study thus aimed to investigate the anti-in ammatory effect of T-006 prophylactic treatment on a transgenic AD model and its underlying mechanisms, as well as its potential protective effects against lipopolysaccharide (LPS)-activated microglial-induced neurotoxicity.

Animals and treatment
The APP/PS1 mice were purchased from Jackson Laboratory and housed in a 12 h light/dark cycle under conditions of controlling humidity at 50 ± 10% and temperature at 22 ± 2°C. All experiments were performed in accordance with the guidelines from the Laboratory Animal Care and Use Ethics Committee of the Shenzhen Center for Disease Control and Prevention. Wide type (WT) and APP/PS1 mice were randomly divided into following groups (n=8-10/group): WT (Sham group), APP/PS1 (vehicle group), APP/PS1 + T-006 (3 mg/kg). Mice in T-006 group were administrated intragastrically once a day, while the sham group mice and vehicle group mice received equal volume of saline.

PC12 cells cultures
PC12 cells obtained from Jinan University (Guangzhou, China) were maintained in DMEM/F-12, supplemented with 12.5% FBS and 2.5% horse serum, as well as 1% penicillin/streptomycin mixture. Cells were seeded in 96-well plates (100 µL/well) at a concentration of 1.2×10 5 cells/mL in a humidi ed environment of 5% CO 2 at 37°C. Experiments were carried out 24 h after the cells were seeded.

MTT assay
The tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye (MTT) assay was used to evaluate neurotoxicity. The assay was performed as our described previously with minor modi cations (Chen et al., 2015). Brie y, BV2 cells were pre-treated with/without T-006 (1, 3, 10 µM) and minocycline (MINO, 5 µM) for 2 h before exposure to LPS (1 µg/mL) for 24 h. Control group denotes the cells were cultured in normal-medium. For neuroprotective experiment, the culture medium collected from BV2 cells exposed to 1 µg/mL LPS for 24 h was used as the activated microglia-conditioned medium (L-CM), which is performed as previous publication [15]. Then PC12 cells that treated with T-006 (1, 3, 10 µM) or MINO (5 µM), were cultured in L-CM for another 24 h. MTT (10 µL, 5 mg/mL) was added into the culture medium and cells were cultured at 37 o C for 4 h. After dissolving the formazan by replacing the medium with 100 µL of DMSO, the absorbance of the samples at 570 nm were read and recorded by a microplate reader (Molecular Service, USA).

Nitric oxide (NO) detection
The accumulation of NO in cell culture supernatant was detected by Griess reaction [16]. Concretely, BV2 microglial cells (3 × 10 4 cells/well) were seeded into 96-well plates and treated with T-006 (1, 3, 10 µM) for 2 h. After incubated with LPS (1 µg/mL) for 24 h, 50 µL of culture supernatant was mixed with equal volume of Griess reagent for 10 min at room temperature in the dark. Then, the absorbance was determined at 540 nm using a microplate reader. After the measurement of uorescence intensities, cell viability was measured by MTT assay. The extent of inhibition on ROS and RNS production was re ected by the mean uorescence intensities. The mean uorescence intensities were calculated by the formula: mean uorescence intensities (%) = detected uorescence intensities/cell viability × 100.

Cytokine enzyme-linked immunosorbent (ELISA) assays
BV2 cells were cultured in 96-well plate at a density of 5×10 4 cells/mL for 24 h. After treatment with 50 nM TAK242 (a TLR4 receptor inhibitor) for 30 min, cells were treated with T-006 (1, 3, 10 µM) or MINO (5 µM) for 2 h and then exposed to LPS (1 µg/mL) for 24 h. Control denotes normal-medium cultured cells. The concentrations of TNF-α and IL-6 in the culture supernatant were determined by ELISA assay kit according to the manufacturer's instructions (Elabscience Biotechnology, Wuhan, China). The absorbance of the sample at 450 nm was measured by a microplate reader.

Real-time reverse-transcription polymerase chain reaction (RT-PCR)
Total RNA was extracted from the cultured cells using Axygen total RNA miniprep kit (Corning, USA) and cDNA was obtained from the RNA using Hifair-II 1st Strand cDNA Synthesis SuperMix (Yeasen Biotechnology, China), according to the manufacturer's instructions. Real-time PCR (RT-PCR) program was executed as previous publication (Woo et al., 2014). In brief, Hieff® qPCR SYBR Green Master Mix and a Roche light cycler 96 sequence detection systems (Roche) were involved in the complement of RT-PCR. The primer sequences used were as follows: COX2: TGCACTATGGTTACAAAAGCTGG (forward) and TCAGGAAGCTCCTTATTTCCCTT (reverse); TNF-α: CTGAACTTCGGGGTGATCGG (forward) and GGCTTGTCACTCGAATTTTGAGA (reverse); IL-6: TCTATACCACTTCACAAGTCGGA (forward) and GAATTGCCATTGCACAACTCTTT (reverse); IL-1β: CTGTGACTCATGGGATGATGATG (forward) and CGGAGCCTGTAGTGCAGTTG (reverse); GAPDH: CATGTTCCAGTATGACTCCACTC (forward) and GGCCTCACCCCATTTGATGT (reverse). All data were expressed relative to GAPDH expression and quanti ed by Roche internal software.
2.12. Immuno uorescence staining BV2 cells were seeded on sterile coverslips in the 24-well plate at the density of 5×10 4 cells/mL for 24 h. After pretreated with T-006 (3, 10 µM) for 2 h, LPS (1 µg/mL) was added and incubated for 24 h, 12 h and 30 min, respectively. Control denotes normal-medium cultured cells. Cells were washed with ice-cold PBS and xed with 4% PFA in PBS for 30 min at room temperature. Subsequently, cells were blocked using 5% BSA blocking buffer for 1 h at room temperature before incubated with antibodies against TLR4 and NF-κB p65 overnight at 4°C. The cells were then washed with ice-cold PBS and incubated with secondary Alexa Fluor 488 and 594 labeled antibodies supplemented with DAPI (5 µM) for 2 h at room temperature in the dark. The coverslips were mounted and photos were taken under a uorescence microscope at 400× magni cation (Vert. A1, Zeiss, Germany).

Immunohistochemical staining
Mice were anesthetized with pentobarbital sodium (50 mg/kg; i.v.) and sacri ced after the behavioral experiment, and then xed with 4% paraformaldehyde. Subsequently, brain tissue was embedded in para n, and cut to make 5-µm thick serial coronal sections for Aβ, GFAP and Ibα-1 immunohistochemical staining, as our previous described [17]. The number of Aβ plaques and the GFAP-and Iba1-positive cells were identi ed using a uorescence microscope (Olympus, Japan), and then blindly counted via ImageJ (NIH, Bethesda, MD, USA).

Western blot assay
Western blot assay was performed as previously described [18]. Concretely, Brain tissues or cells were harvested using RIPA lysis buffer containing a cocktail of protease and phosphatase inhibitors. Subsequently, the total cytosol and nuclear proteins were isolated and their concentrations were determined by the BCA assay (Pierce, Rockford, IL, USA). The proteins (20-30 µg) were separated on a 10% SDS-polyacrylamide gel and then transferred to the polyvinyldi uoride membrane. After blocking with a 5% BSA blocking buffer, the polyvinyldi uoride membranes were co-incubated using primary antibodies overnight at 4 o C respectively against TLR4, MyD88, NF-κB p65, phospho-p65, iNOS, COX2, βactin and PCNA. Subsequently, the membranes were then washed with TBST and incubated with secondary antibodies at room temperature for 2 h. The signals were obtained using an ECL Plus kit using a detecting system (ProteinSimple, USA). Finally, the quantitative analysis was provided by ImageJ (NIH, Bethesda, MD, USA).

Data analysis
All data presented as means ± SEM were carried out at least three times independent experiments. Analysis of one-way ANOVA with Dunnett's test was used for multiple experimental statistical comparisons, with P < 0.05 being considered as statistical signi cance.

Results
3.1. T-006 alleviated the Aβ accumulation in the hippocampus of the APP/PS1 mice As reported, Tau tangles and Aβ plaques are two important hallmarks of AD. Previously, we found that T-006 improved cognitive ability after a long-term administration in AD transgenic mice of both APP/PS1-2xTg and APP/PS1/Tau-3xTg. Importantly, T-006 mitigated cognitive decline in 3xTg mice primarily via reducing the p-Tau and total Tau levels [14]. Thus, we wondered whether T-006 alleviated Aβ expression. Here, the Aβ expression levels in the brain of APP/PS1 mice, and quantify the Aβ plaques in the cortex and hippocampus were assessed. As illustrated in Fig. 1A-C, the Aβ deposition in APP/PS1 mice brain was obviously overexpressed both in the cortex and hippocampus compared to the WT mice. However, the T-006 treatment signi cant reduced Aβ deposition in the hippocampus, but not in the cortex. To further con rm which Aβ fractions were affected by T-006, Elisa analysis was used to test the soluble and insoluble forms of Aβ extracted from the hippocampus. The results showed that there was no signi cant difference in the soluble fractions of both Aβ1-42 and Aβ1-40 with or without T-006 treatment in APP/AS1 mice, while the insoluble fraction levels of both Aβ1-42 and Aβ1-40 were strikingly decreased in T-006 treatment group (Fig. 1D-G).

T-006 suppressed neuroin ammation in the APP/PS1 mice
Chronic neuroin ammation characterized with the abnormal activation of microglia and astrocytes has been observed in AD patients and AD animal models, respectively [19]. We next explored whether T-006 can attenuate neuroin ammation in AD mice. The immunostained assay of brain sections with antibodies against the GFAP and Ibα-1, which respectively represented a speci c marker of astrocyte and microglia, was performed. As shown in the Fig. 2A-C, compared with the WT mice, the activation of astrocytes and microglial were dramatically elevated in the hippocampus of the APP/PS1 mice. However, microglial activation, but not astrocytes activation, was signi cantly repressed by T-006 treatment in APP/PS1 mice. Western blot analysis also demonstrated that T-006 obviously down-regulated the Ibα-1 proteins expression of hippocampus in APP/PS1 mice (Fig. 2D and E).

T-006 diminished LPS-stimulated pro-in ammatory cytokine responses in BV2 microglial cells
Firstly, the cell cytotoxicity of T-006 on BV-2 cells was tested and results suggested that T-006 (1, 3 or 10 µM) had no cytotoxic effect on BV-2 cells for 24 h (Fig. 3A). Then the NO production of cells was detected. The result in Fig. 3B showed that LPS at the concentration of 1 µg/mL robustly stimulated the NO overproduction, while T-006 or MINO treatment signi cantly reversed the LPS-induced NO overproduction in BV2 cells. Importantly, the effect of T-006 at 10 µM was stronger than that of MINO. We next evaluated the effects of T-006 on the expression of IL-1β and TNF-α in LPS-stimulated BV-2 cells. The Elisa results revealed that T-006 signi cantly reversed the up-regulation of IL-1β and TNF-α proteins expression induced by LPS (Fig. 3C and D). Additionally, as illustrated in Fig. 1E-H, the mRNA levels of pro-in ammatory cytokines and mediators of COX2, TNF-α, IL-1β and IL-6 were markedly increased in LPS treated groups. Pretreatment with T-006 (1-10 µM) concentration dependently prevented LPS-induced increases in pro-in ammatory mediators, while T-006 treatment alone did not affect the levels of these proin ammatory cytokines compare with the control. Subsequently, we detected the protein expression of iNOS and COX2 through Western blot, and results revealed that LPS treated alone signi cantly upregulated the iNOS and COX2 proteins expression, which was also obviously attenuated by T-006 treatment (1-10 µM) in a concentration dependent manner (Fig. 3I-L). 3.4. T-006 suppressed the expression of TLR4 and its downregulator MyD88 in LPS-induced BV2 microglial cells TLR4 was considered as an important receptor of LPS, which could interact with a speci c adaptor molecular MyD88 and regulate in ammatory responses via activating the downstream signaling pathway including NF-κB [20]. Thus, whether the T-006 could modulate the TLR4 and its adaptor molecular MyD88 levels were investigated. As shown in Fig. 4A-C, pretreated with T-006 statistically inhibited the sharp increasement of TLR4 and MyD88 expression levels triggered by 1 µg/mL of LPS in BV2 cells. Consecutively, the immuno uorescence staining with TLR4-speci c antibody also demonstrated that T-006 at the concentrations of 3 and 10 µM down-regulated LPS-induced TLR4 overexpression in BV2 microglial cells (Fig. 4D). 3.5. T-006 inhibited the activation of NF-κB pathway in LPSactivated BV-2 microglial cells Increasing evidence indicated that NF-κB signaling pathway played a vital role in regulating microglialmediated in ammatory processes [21][22][23], and previous work reported that inhibition of NF-κB-driven gene transcriptional activity effected potent anti-in ammation against LPS-stimulated microglial cells activation [24]. Therefore, the effects of T-006 on NF-κB signaling pathway were also examined here. As shown in Fig. 5A and B, LPS markedly induced the expression of phosphorylation of NF-κB p65. In contrast, T-006 treatment concentration-dependently inhibited the phospho-NF-κB p65, which indicated that T-006 inhibited NF-κB signaling activation [25]. The ndings were also reinforced by immuno uorescence staining of the intracellular NF-κB p65 subunit. T-006 abrogated the LPS-evoked nuclear translocation of NF-κB p65 (Fig. 5C). These results suggested that T-006 alleviated LPS-induced neuroin ammation by inhibiting the activation of NF-κB signaling pathway.

T-006 diminished LPS-induced proin ammatory cytokines levels through suppression of TLR4-mediated MyD88/NF-κB pathway
To further con rm the inhibition of TLR4/MyD88 signaling pathway was associated with antiin ammatory effect of T-006 against the LPS-induced microglial activation, the speci c TLR4 inhibitor TAK242 was pretreated before BV2 cells exposure to T-006. The proteins expression of iNOS and COX2 were measured through Western blot. Expectedly, both T-006 (3 µM) and TAK242 (100 nM) administration robustly reversed the proteins expression increase of iNOS and COX2 induced by LPS, while no signi cance was observed between co-treatment with TAK242 and T-006 and TAK242 treated alone.
Additionally, we found that T-006 treatment alone had no effect on these two proteins expression, which was consistent with the above nding that T-006 did not affect the levels of these proin ammatory cytokines under basal conditions ( Fig. 6A-C). Since MyD88 acted as an important downstream targeting molecular adaptor of TLR4 [26], the effect of TAK242 on T-006's modulation of MyD88 expression was further investigated. As shown in Fig. 6D and E, T-006 signi cantly decreased the up-regulation of MyD88 protein levels in LPS-evoked BV2 microglial cells. However, treatment with combination of TAK242 (100 nM) and T-006 (3 µM) did not showed a stronger inhibitory effects compared to TAK242 or T-006. TLR4 depletion was reported to contribute to the inhibition of NF-κB signaling pathway [20]. Whether T-006's inactivation of the NF-κB signaling pathway was affected by TLR4 inhibitor TAK242 or not is unclear. As shown in Fig. 6F and G, T-006 considerably mitigated the LPS-induced phospho-NF-κB p65, whereas the addition of TAK242 did not affect this reversion effect of T-006. The immuno uorescent assay further con rmed the results that TLR4 inhibitor TAK242 did not affect the effect of T-006 on NF-κB signaling pathway. These data suggested that T-006 suppressed the pro-in ammatory responses in LPS-activated BV2 microglial cells via TLR4-mediated MyD88/NF-κB signaling pathway.

T-006 protected against LPS-activated BV2 microgliamediated neurotoxicity in PC12 cells
Since T-006 presented suppressive effects on neuroin ammation in LPS-activated microglia, the effects of T-006 against neuroin ammation-mediated neurotoxicity in PC12 cells under L-CM induction was also evaluated. As expected in the Fig. 7A, L-CM caused an obvious reduction (approximately 48.6%) in the viability of PC12 cells. Conversely, T-006 (1, 3, 10 µM) signi cantly attenuated the L-CM-caused cytotoxicity in a concentration-dependent manner. The cell viability of T-006 at 10 µM is higher than that of the positive control MINO (5 µM). In line with our previous nding [15], the vehicle-treated medium (vehicle group) did not affect the cell viability. As reported, the intracellular ROS overproduction is tightly involved in a cascade of harmful events, which ultimately induce neuronal damage and death [25]. Thus, intracellular ROS production was tested here and the result suggested that pretreatments with T-006 from 1 to 10 µM and MINO (5 µM) signi cantly reduced the ROS accumulation compared to the L-CM stimulation (Fig. 7B). To evaluate whether the neuroprotective effects of T-006 was related to maintenance of mitochondrial function, which acted as an important participants in in ammationmediated injury [27]. We tested the ATP release and the mitochondrial membrane potential (MMP) collapse under L-CM stimulation. As presented in the Fig. 7C, pretreatment with T-006 concentrationdependently and noticeably attenuated ATP decrease in L-CM co-treated PC12 cells. Additionally, the L-CM-induced MMP collapse was considerably reversed by T-006 treatment, indicating that the neuroprotective effect of T-006 was dependent on stabilizing MMP to preserve mitochondrial function ( Fig. 7D and E).

Discussion
Neuroin ammation, a self-defense reaction initiated by the central nervous system (CNS) in the brain injury, has been suggested as a causal factor in the pathogenesis and progression of many neurodegenerative diseases [7]. Although many anti-AD drugs targeting amyloid and Tau hypothesis failed to reach the clinical endpoints in past decades, a window was opened that neuroin ammation might be a new key pathogenic factor of AD, among which microglial-activated neuroin ammatiory responses by releasing a variety of proin ammatory cytokines could result in neurological injury [28]. Therefore, suppressing microglia-evoked neuroin ammation and reducing cellular injury may play an important role in AD therapy. In the current study, we showed that T-006 signi cantly decreased the Aβ accumulation by inhibiting neuroin ammation, and the anti-neuroin ammation effect of T-006 was tightly involved in the suppression of TLR4-meidated MyD88/NF-κB signaling. Importantly, T-006 provided neuroprotection against neuroin ammation-induced neuronal damage via attenuating ROS overproduction and subsequent mitochondrial dysfunction.
Although the etiology of AD is unclear, the brain Aβ pathology of SP has still be considered as the gold standard for AD diagnose since the decreased Aβ clearance is a common prelude to late-onset AD [29]. Here, we found that T-006 administration signi cantly reduced the deposition of Aβ in the hippocampus of APP/PS1 mice (Fig. 1). In addition, our previous results suggested that T-006 could considerably downregulated the expression levels of amyloid precursor protein (APP) and β-secretase (BACE-1) [14], which are key contributors of Aβ production [30]. Nevertheless, the failure of reducing the Aβ levels has achieved a consensus that only targeting the pathological products is far from enough for the strategy of anti-AD drugs development and there is an urgent need to understand the non-misfolded proteins pathogenesis in AD. As expected, in ammatory responses is closely linked with the activation of glia mainly including astrocytes and microglia in AD models, which are believed to initiate or aggravate the neurodegenerative processes by inducing Aβ oligomerization and tau hyperphosphorylation [1,31]. In the current study, we found that both the activation of astrocytes and microglia were signi cant augmented in the hippocampus of APP/PS1 mice, while T-006 obviously reversed this over-activation of microglia, but there is no signi cant difference in the activation of astrocytes compared with the vehicle group ( Fig. 2A-C). The Western blot assay further con rmed this inhibition of T-006 on the microglial activation by downregulating the Ibα-1 expression level (Fig. 2D and E). These results suggested that T-006 could attenuate the Aβ deposition via the inhibition of microglial activation and its underlying precise mechanism also needs deeper investigation.
Microglia are the rst responder to Aβ accumulation and its activation can initiate an in ammatory response by producing a large number of in ammatory molecules including TNF-α, IL-6, IL-1β, iNOS and COX2, which result in synaptic degeneration, neuronal cell death, and cognitive dysfunction [32]. Thus, the LPS was used here as stimulator of microglial activation to evaluate the anti-neuroin ammation activity of T-006. As shown in Fig. 3, LPS can increase TNF-α, IL-6 IL-1β, iNOS and COX2 expression levels. Not surprisingly, T-006 signi cantly and concentration-dependently reduced these proin ammatory molecules levels in LPS-induced BV2 microglia, implicating that T-006 attenuate aberrant microglial activation and subsequently over-activated microglia-induced in ammatory response. Accumulating evidence has shown that recognition of LPS by TLR4 plays a pivotal role in the neuroin ammation of AD, which can cause an overproduction of pro-in ammatory cytokines in response to neuronal dysfunction via activating MyD88/NF-κB downstream signaling [17,33]. Thus, inhibition of TLR4 to block the occurrence of these pro-in ammatory cytokines, exhibits potential therapeutic effects for the treatment of AD [34]. Interestingly, in the current study, treatment with T-006 statistically inhibited the increased effects triggered by LPS on TLR4 and MyD88 expression levels ( Fig. 4A-C). The immuno uorescence assay further con rmed that the down-regulation effect of T-006 on the TLR4 expression (Fig. 4D). Furthermore, we also evaluated the NF-κB expression affected by T-006. As illustrated in the Fig. 5, stimulation of LPS increased the phosphorylated NF-κB p65, resulting in a higher translocation of NF-κB p65 in nucleus, while T-006 effectively reversed this alteration. Furthermore, we assessed the action of TLR4 depletion in regulating T-006's anti-in ammatory effect using a TLR4 inhibitor TAK242. Consistent with the previous publication [35], TAK242 treatment signi cantly suppressed LPS-induced expression levels of iNOS and COX2, while co-treatment of T-006 with TAK242 did not abolish the down-regulation effect of TAK242 on these two pro-in ammatory factors (Fig. 6A-C). Subsequently, we examined whether TAK242 altered the effects of T-006 on TLR4 downstream signaling of MyD88/NF-κB. As expected, there is no signi cant difference between co-incubation of T-006 with TAK242 and TAK242 alone in the expression levels of MyD88 and phosphorylated NF-κB p65 (Fig. 6D-H). Based on these results, we presumed that TLR4mediated MyD88/NF-κB signaling pathway was tightly involved in the suppressive effect of T-006 on LPS-activated pro-in ammatory responses.
Several lines of studies have demonstrated that microglial activation caused overproduction of in ammatory molecules is a key contributor to neuronal injury [20,25]. Therefore, the inhibition of neuroin ammatory processes by blocking these pro-in ammatory molecules might be able to confer neuroprotection. In this study, we used the L-CM co-incubation with PC12 cells to test the neuroprotective effects of T-006 against in ammation-caused cell damage. In line with our previous publication [15], L-CM resulted in a signi cant reduction in the cell viability, while this L-CM-induced cell reduction could be counteracted by T-006 (Fig. 7A). Subsequently, we measured the intracellular ROS and found that T-006 signi cantly prevented the intracellular ROS aggregation stimulated by activated microglia (Fig. 7B). As reported, intracellular ROS overproduction is related to mitochondrial permeability transition pore opening, which in turn causes mitochondrial depolarization and consequently neuronal injury [36]. In this study, we also found that T-006 could both mitigate ATP production and MMP dissipation (Fig. 7C-E). Altogether, these results imply that T-006 exerted potent neuroprotection against neuroin ammation-induced neuronal damage.
In summary, our current study elucidated that T-006 signi cantly decreased the levels of total Aβ and GFAP as well as the Ibα-1 expression in the APP/PS1 mice. Additionally, T-006 effectively inhibited LPS-

Declarations
Ethics approval and consent to participate: All experiments were performed in accordance with the guidelines from the Laboratory Animal Care and Use Ethics Committee of the Shenzhen Center for Disease Control and Prevention. All efforts were made to ameliorate the suffering of animals. Deeply anesthetized mice were euthanized by perfusion followed by a physical method for tissue collection.
Consent for publication: Not applicable.
Availability of data and materials: All data generated or analyzed during this study are included in this published article.