Exploring the mechanism of Wogonin in the attenuation of LPS-induced inflammation in BV-2 cells and the protective effect of wogonin on SH-SY5Y cells

Exploring the mechanism of Wogonin in the attenuation of LPS-induced inflammation in BV-2 cells and the protective effect of wogonin on SH-SY5Y cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Exploring the mechanism of Wogonin in the attenuation of LPS-induced inflammation in BV-2 cells and the protective effect of wogonin on SH-SY5Y cells Ziqi Zhang, Mengfei Sun, Jingfeng Ouyang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7532665/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Parkinson's disease (PD) is a neurodegenerative disease that affects mainly middle-aged and elderly people, and its pathogenesis has not been clarified. To model neuroinflammatory components of PD in vitro, lipopolysaccharide (LPS)-induced inflammatory injury in BV-2 microglial cells was employed. Wogonin is a natural bioflavonoid extracted from the rhizome of the Chinese herb Scutellaria baicalensis . It has a neuroprotective effect and can play a role in alleviating the symptoms of neurodegenerative diseases. The aim of this study was to investigate the mitigating effect of wogonin on lipopolysaccharide-induced inflammation in BV-2 cells and the protective effect on SH-SY5Y cells by an experimentally validated method. The CCK-8 assay was used to detect the cell viability of each group. Enzyme-linked immunosorbent assay (ELISA), immunohistochemical staining (IHC), immunofluorescence staining (IF) and Western blot methods (WB) were used to detect the cell pathway indicators and inflammatory factors in each group. The results showed that LPS (1 µg/mL) induced polarization and activation of BV-2 cells and significantly increased the release of pro-inflammatory factors IL-6, TNF-α, and IL-1β, while decreasing the expression of tyrosine hydroxylase (TH) and promoting the aberrant aggregation of α-synaptic nucleoprotein (α-Syn) in SH-SY5Y neurons. After intervention with wogonin (16 µM), the above pathological processes were effectively reversed: inhibition of inflammatory factor secretion in BV-2 cells, restoration of TH expression in SH-SY5Y neurons, reduction of α-Syn deposition, and reduction of NF-κB p65 nuclear translocation and activation of the TLR4/MyD88 pathway. The effect was comparable to that of a TLR4 inhibitor (TAK-242), but the combination of the two did not show a synergistic effect. The study suggests that wogonin may inhibit LPS-induced release of inflammatory factors from BV-2 microglia and protect SH-SY5Y cells by regulating the TLR4/MyD88/NF-κB signaling pathway. Biological sciences/Cell biology Health sciences/Diseases Biological sciences/Immunology Biological sciences/Neuroscience Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Parkinson's disease (PD), the second most prevalent neurodegenerative disease worldwide[ 1 , 2 ], is characterized by progressive loss of midbrain substantia nigra dopaminergic neurons and abnormal aggregation of α-synuclein (α-Syn)[ 3 – 5 ]. Its pathogenesis is complex and has not been fully elucidated. It has been suggested that microglia-mediated neuroinflammation within the central nervous system is a key factor in several neurodegenerative diseases[ 6 , 7 ]. The central role of neuroinflammation in the pathological process of PD has also received much attention in recent years. Microglia, as the main immune defense of the Central Nervous System, are over-activated in response to stimulation by pathogens or injury signals, and activation of Toll-like receptor 4 (TLR4) on the cell membrane triggers the activation of a series of intracellular adaptor proteins, which leads to activation of proteins associated with the NF-κB pathway, resulting in the secretion of inflammatory factors and the release of interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and other pro-inflammatory factors, exacerbating neuronal oxidative damage and apoptosis[ 8 – 10 ]. Wogonin, a natural flavonoid active ingredient extracted from the traditional Chinese medicine Scutellaria baicalensis , possesses multiple pharmacological properties including antioxidant, anti-inflammatory and neuroprotective properties[ 11 ]. Some studies have shown that wogonin can inhibit the production of reactive oxygen species (ROS) thereby suppressing inflammatory responses and oxidative stress[ 12 – 14 ]. In an ischemic brain injury model, wogonin reduces neuronal damage by inhibiting pro-inflammatory microglia polarization and decreasing the secretion of neuroinflammatory factors through pathways[ 15 ]. In addition, wogonin has shown the ability to increase neuronal synaptic density in epilepsy disorders and to inhibit pro-inflammatory cytokine release and microglial synaptic phagocytosis through activation of the AKT/FoxO1 pathway[ 16 ]. The above studies suggest that wogonin has certain effects on inhibiting neuroinflammatory and protecting neurons from damage, and may improve neurodegenerative lesions by modulating apoptotic pathways and inhibiting oxidative stress, but its specific regulatory mechanism on microglia-mediated neuroinflammation is not clear. In this study, we constructed an in vitro neuroinflammation model with lipopolysaccharide (LPS)-activated BV-2 microglia, combined with the SH-SY5Y neuronal co-culture system, to systematically investigate the protective effect of wogonin on inflammatory cascade response and neuronal injury, and to explore its association with the TLR4/MyD88/NF-κB signaling pathway. Materials and Methods Materials The murine microglial BV-2 cell line was sourced from the Cell Resource Center of the Chinese Academy of Medical Sciences (Beijing, China). The human neuroblastoma SH-SY5Y cell line was sourced from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). TAK-242 (CAS: T125887) was purchased from Aladdin Biochemical Technology (Shanghai, China). Wogonin (CAS: B0002970) was acquired from Bepure (China). LPS (CAS: 028M4094V) was purchased from Sigma. DMEM (CAS: 8123098), penicillin-streptomycin solution (CAS: 15140-122), and FBS (CAS: 2350404RP) were obtained from Gibco. L-glutamine solution (CAS: 20212128) was purchased by Solarbio (Beijing, China). The Cell Counting Kit-8 was purchased from Dojindo Molecular Technologies (Japan). Mouse interleukin-6, tumor necrosis factor-alpha, and interleukin-1 beta enzyme-linked immunosorbent assay kits were provided by ABclonal Biotechnology (Wuhan, China). Rabbit monoclonal antibodies against NF-κB p65 (ab32536) and phosphorylated (p)-NF-κB p65 (ab76302), as well as goat polyclonal antibody targeting myeloid differentiation factor 88 (ab28763), were acquired from Abcam (UK). The BCA protein concentration assay kit, rabbit monoclonal β-actin antibody, and Alexa Fluor 488-conjugated goat anti-immunoglobulin G (H + L) were purchased from Beyotime Biotechnology (Shanghai, China). Mouse monoclonal α-synuclein (α-Syn) antibody, rabbit polyclonal tyrosine hydroxylase antibody, mouse monoclonal TLR4 antibody, horseradish peroxidase conjugated goat anti-mouse IgG (H + L), HRP-conjugated goat anti-rabbit IgG (H + L), HRP-conjugated rabbit anti-goat IgG (H + L), and DAB chromogenic kit were obtained from Sangon Biotech (Wuhan, China). Cell culture BV-2 and SH-SY5Y cells were cultured in DMEM supplemented with 10% FBS, 1% L-glutamine, and 1% penicillin-streptomycin solution. Cells were maintained in a humidified incubator at 37°C with 5% CO₂. When cell confluency reached 80–90%, the cells were passaged and seeded into new culture plates for subsequent experiments. BV-2 and SH-SY5Y cells were cultured and seeded into 96-well plates according to the aforementioned methods. After 24 h of incubation, the plates were removed, and supernatants from BV-2 cells in each group were collected separately. The collected supernatants were mixed with an equal volume of complete DMEM medium (1:1 ratio) to prepare conditioned medium. SH-SY5Y cells were then divided into experimental groups and treated with the respective conditioned medium. All plates were returned to the incubator for an additional 12 h of culture. Cell viability assay Cell viability was assessed using the CCK-8 following different interventions. Cells were seeded into 96-well plates at a density of 6 × 10⁴ cells/mL and cultured for 24 h until achieving 80% confluency in an adherent state. Cells were then treated with specified concentrations of compounds for designated durations. After treatment, the plates were incubated for an additional 24 h under standard culture conditions (37°C, 5% CO₂). Following incubation, the plates were removed, and cellular morphology across all groups was observed under the microscope and documented via photomicrographs. Absorbance (A) at 450 nm was measured using a microplate reader. Cell viability was calculated using the following formula: $$\:cell\:viability\left(\%\right)=\frac{\left(\text{A}\:\text{T}\text{r}\text{e}\text{a}\text{t}\text{m}\text{e}\text{n}\text{t}-\text{A}\:\text{S}\text{o}\text{l}\text{v}\text{e}\text{n}\text{t}\:\text{C}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\right)}{\left(\text{A}\:\text{C}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}-\text{A}\:\text{S}\text{o}\text{l}\text{v}\text{e}\text{n}\text{t}\:\text{C}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\right)}\times\:100\%$$ Determination the content of IL-6, TNF-α and IL-1β by ELISA The treated cells were cultured and seeded into 24-well plates according to the aforementioned protocol, followed by incubation for an additional 48 h. After the incubation period, supernatants from each well were collected and stored at − 80°C for subsequent analysis. The expression levels of IL-6, TNF-α, and IL-1β in the supernatants were quantified using ELISA kits in strict accordance with the manufacturer’s instructions. Immunohistochemistry SH-SY5Y cells were cultured according to the aforementioned protocol. After culture, cells were washed three times with PBS and fixed with 4% paraformaldehyde (PFA) for 30 min at room temperature. Following fixation, cells were permeabilized with 0.5% Triton X-100 for 5 min at room temperature and rinsed three times with PBS. Primary antibodies tyrosine hydroxylase (TH, 1:800 dilution) and α-synuclein (α-Syn, 1:1000 dilution) were applied to the cells and incubated on a shaker at 4°C for 24 h. After washing, species-matched secondary antibodies were added and incubated for 1 h at room temperature under light-protected conditions. DAB chromogen was applied for color development, followed by hematoxylin counterstaining of nuclei. Coverslips were mounted with neutral resin and observed under a microscope. Representative images were captured for analysis. Immunofluorescence SH-SY5Y cells were cultured as described above. After culture, cells were washed with PBS and fixed with PFA for 30 min at room temperature. Following three PBS washes, cells were permeabilized with 0.5% Triton X-100 for 10 min at RT. Primary antibody NF-κB p65 (1:800 dilution) was applied and incubated on a shaker at 4°C overnight. Cells were then treated with Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (1:250 dilution) under light-protected conditions at room temperature for 1 h. After washing with TBS, cell slides were counterstained with DAPI for 15 min to label nuclei. Slides were mounted with neutral resin and imaged using a fluorescence microscope. Representative images were captured with consistent exposure settings across all groups. Western blot SH-SY5Y cells were cultured as described above and washed with PBS. Cells were lysed in RIPA buffer supplemented with phosphatase and protease inhibitors for 15–30 min on ice. The lysates were centrifuged at 10,000 × g for 10 min at 4°C, and supernatants were collected in sterile tubes. Protein concentrations were quantified using a BCA assay kit. Equal amounts of protein were mixed with loading buffer, denatured by heating at 100°C for 5 min, and separated on 10% SDS-polyacrylamide gels. Proteins were then transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk in TBST for 90 min at room temperature. Primary antibodies β-actin (1:3000 dilution), TLR4 (1:5000 dilution), NF-κB p65, phosphorylated (p)-NF-κB p65, and MyD88 (1:1000 dilution) were applied and incubated at 4°C overnight. After three TBST washes, species-matched secondary antibodies were added and incubated for 1 h at room temperature. Membranes were washed again and developed using enhanced chemiluminescence reagent. Band densities were quantified using ImageJ software with β-actin as the loading control. Statistical analysis Measurement data are expressed as mean ± SD. Statistical analysis was performed using GraphPad Prism 9.1 software and IBM SPSS Statistics26. Comparisons between two groups were analyzed using Student’s t-test, and comparisons among multiple groups were assessed by one-way analysis of variance. A threshold of P < 0.05 was considered statistically significant. Results LPS inhibits BV-2 cell proliferation and induces pro-inflammatory cytokine release Accumulating evidence indicates that LPS promotes inflammatory responses both in vitro and in vivo, providing a model for studying neuroinflammatory mechanisms in PD. To determine the cytotoxic effects of LPS on BV-2 cells, BV-2 cells were pretreated with LPS at concentrations of 0, 0.1, 1, 10, or 100 μg/mL. BV-2 cell morphology was observed under a microscope, and images were captured. Cell viability was assessed using the CCK-8, and IL-6 levels in supernatants were quantified via ELISA. Fig.1A shows that in the group of LPS 0μg/ml, BV-2 cells exhibited typical semi-adherent and semi-suspension growth pattern. Suspended cells appeared uniformly round, while adherent cells displayed polymorphic morphologies (round, spindle, or polygonal) with smooth membrane edges, clear contours, and translucent cytoplasm accompanied by short protrusions. Compared to the LPS 0 μg/mL group: in the LPS 0.1 μg/mL group, the cell synapses were slightly longer, the cell volume was slightly enlarged, and there was no obvious morphological change; in the LPS 1 μg/mL group, the cell protrusions increased significantly, the cytosol was swollen and polarized, and the antennae were elongated, and some of the cells showed amebia-like changes; in the LPS 10 μg/mL group: the number of cells was reduced, and there were a lot of dead cells and fragments floating in the culture medium. LPS 10 μg/mL group: the number of cells was greatly reduced, with a large number of dead cells and fragments floating in the culture medium, and complete loss of cytoplasmic translucency; LPS 100 μg/mL group: the cells were almost completely dead, and only a few cell fragments remained. The results of CCK-8 showed (Fig.1B) that cell viability was significantly decreased in LPS 10μg/mL and LPS 100μg/mL groups compared with the group with LPS 0μg/ml (P<0.01). Whereas, no statistically significant difference in cell viability was observed in the LPS 0.1μg/mL and LPS 1μg/mL groups. The results of ELISA showed (Fig.1C) that IL-6 levels were increased in all other groups compared to the group with LPS 0μg/ml. Among them, IL-6 levels were significantly elevated in the group with LPS 1μg/mL, and no statistically significant differences were observed in the IL-6 levels of all other groups. Based on the above experimental results, we can conclude that LPS 10 μg/mL and LPS 100 μg/mL led to the loss of cell function or death. The cells in the LPS 1 μg/mL group showed a polarized morphology and a significant increase in the expression of inflammatory factors, but there was no significant change in the viability of the cells, which suggests that this concentration induces an activation phenotype without significantly affecting the survival rate, and it can be used as a modeling concentration for the subsequent experiments. Effects of TAK-242 at varying concentrations on BV-2 cell growth inhibition and inflammatory cytokine release TAK-242, a selective small-molecule inhibitor of the TLR4 signaling pathway, binds to the Cys747 residue within the TLR4 TIR domain, thereby suppressing LPS-induced production of pro-inflammatory cytokines and inhibiting downstream MyD88/TRIF signaling and autophagy. To evaluate the effects of TAK-242 on BV-2 cells, cells were treated with TAK-242 at concentrations of 0, 25, 50, 100, and 200 nM. Morphological changes were documented via microscopy. Cell viability was assessed by CCK-8. IL-6 levels in supernatants were quantified by ELISA. Fig.2A shows that the BV-2 cells in the TAK-242 0nM group grew adherently to the wall, were round or pike shaped, and showed a resting cell morphology. The cell boundaries were well defined with short tentacular extensions, most of the cells were in clusters, and a few were suspended and shiny. Compared with TAK-242 0nM group, the cells in each concentration group of TAK-242 did not show significant morphological changes. The results of CCK-8 showed (Fig.2B) that there was no significant difference in the cell viability of TAK-242 25nM, 50nM, and 100 nM groups compared with TAK-242 0nM group. The cell viability of TAK-242 200nM group was significantly reduced (P< 0.05). The results of ELISA showed (Fig.2C) that IL-6 levels were significantly lower in the TAK-242 50nM group, significantly higher in the TAK-242 200nM group, and there was no significant difference between the IL-6 levels in the TAK-242 25nM group and the TAK-242 100 nM group, as compared with those in the TAK-242 0nM group. Based on the above experimental results, we can conclude that TAK-242 200nM has a greater effect on cell viability and increases the expression of inflammatory factors. While there is no obvious morphological change in the cells of the TAK-242 50nM group, and there is no obvious change in the viability of the cells, but the expression of inflammatory factors is significantly reduced. This indicates that this concentration can inhibit the expression of inflammatory factors in BV-2 cells without significantly affecting the survival rate, and can be used as a subsequent experimental concentration. Effect of Wogonin at different concentrations on inflammatory cytokine secretion in BV-2 cells Previous studies suggest that wogonin exerts neuroprotective effects and ameliorates neuroinflammatory responses. In this study, we further investigated the impact of wogonin at varying concentrations on BV-2 cell morphology, viability, and inflammatory cytokine production. BV-2 cells were cultured with wogonin medium containing 0 μM, 4 μM, 8 μM, 16 μM, and 32 μM, respectively. Then the morphology of BV-2 cells in each group was observed under a microscope and photographed. Cellular morphology was documented via the microscopy, and viability was assessed using the CCK-8. IL-6 levels in supernatants were quantified by ELISA. Fig.3A shows that the cells in the wogonin 0 μM group were semi-suspended and semi-adherent, and the cells had the morphology of round, pike and polygonal. The cells had clear boundaries, high translucency and short tentacles. No significant morphological changes were observed in the cells of other wogonin concentration groups. Results of CCK-8 showed (Fig.3B) that no significant difference in cell viability was observed in the wogonin 4 μM, 8 μM, and 16 μM groups compared with the wogonin 0 μM group. Cell viability was significantly lower in the wogonin 32 μM group (P<0.01). The results of ELISA showed (Fig.3C) that IL-6 levels were significantly higher in the LPS group compared with the control group. Compared with the LPS group, there was no significant difference in the IL-6 level in the wogonin 4 μM group, and the IL-6 level was significantly lower in the wogonin 8 μM and wogonin 16 μM groups. Based on the above experimental results, we can conclude that cell viability decreased at 32 μM of wogonin, so this concentration was excluded. The IL-6 levels expressed by BV-2 cells was reduced to a greater extent at 32 μM of wogonin. Therefore, 16 μM was chosen as the optimal concentration of wogonin for subsequent experiments. Wogonin alleviates LPS-induced growth suppression and reduces inflammatory cytokine production in BV-2 cells Based on the experimental results above, three optimal concentrations were selected for subsequent studies: 1 μg/mL LPS, 50 nM TAK-242, and 16 μM Wogonin. Following the protocol described in the Materials and Methods section, BV-2 cells were divided into the following groups: Control group, LPS group (1 μg/mL LPS), Wogonin group (16 μM Wogonin + 1 μg/mL LPS), TAK-242 group (50 nM TAK-242 + 1 μg/mL LPS), TAK-242 + Wogonin group (50 nM TAK-242 + 16 μM Wogonin + 1 μg/mL LPS). Fig.4A shows that the BV-2 cells in the control group were half-adherent and half-suspended, the suspended cells were round, and the adherent cells were round, pike or polygonal, with smooth edges, clear boundaries, high transmittance, and short pseudopods could be seen to protrude. Compared with the control group, the cell protrusions in the LPS group increased significantly, the cell volume increased, the pseudopods were elongated, and some of the cells showed amoeba-like morphology. Compared with the LPS group, cell morphology was restored, the proportion of round cells increased, and the number of synapses was significantly reduced in the wogonin group, the TAK-242 group, and the TAK-242+ wogonin group. The results of CCK-8 showed (Fig.4B) that no significant difference in cell viability was observed in the LPS group compared with the control group. Compared with the LPS group, cell viability was more increased in the wogonin group, cell viability was significantly increased in the TAK-242 group (P < 0.01), and cell viability in the TAK-242+ wogonin group did not show any significant difference. The results of ELISA showed (Fig.4C-E) that the levels of IL-6, TNF-α, and IL-1β in the LPS group were significantly higher compared with the control group (P < 0.01). Compared with the LPS group, the levels of IL-6 were significantly lower in the wogonin group, the TAK-242 group, and the TAK-242 + wogonin group (P < 0.01), the levels of IL-1β were significantly lower in the wogonin group and the TAK-242 group (P < 0.01), and the levels of TNF-α were more reduced in the wogonin group, the TAK-242 group, and the TAK-242 + wogonin group. Based on the above results, we can conclude that both wogonin and TAK-242 were able to attenuate LPS-induced growth inhibition of BV-2 cells, increase the cell viability of BV-2 cells reduced by LPS, and reduce the expression of inflammatory factors, which had a certain protective effect on BV-2 cells. Wogonin mitigates LPS-induced inflammatory damage in SH-SY5Y microglial cells via the TLR4/MyD88/NF-κB signaling pathway Abnormal aggregation and deposition of α-syn is one of the characteristic pathological manifestations of PD. TH is a key enzyme in the dopamine synthesis pathway, responsible for the conversion of tyrosine to levodopa and then to dopamine. The expression levels of both can be used as biomarkers to assess the function of dopaminergic neurons and the progression of PD. IHC results showed (Fig.5A-C) that the intensity of α-syn protein expression was significantly enhanced (P<0.01) and the intensity of TH protein expression was significantly weakened (P<0.01) in SH-SY5Y cells of the LPS group, compared with the control group. Compared with the LPS group, the intensity of α-syn protein expression was significantly weakened (all P<0.01) and the intensity of TH protein expression was significantly enhanced (all P<0.01) in the SH-SY5Y cells of the wogonin group, the TAK-242 group, and the TAK-242 + wogonin group. The activation of NF-κB signaling pathway is closely related to neuroinflammation in PD. NF-κB p65 was activated and transferred to the nucleus, accelerating the release of inflammatory factors. IF results showed that (Fig.5D-E), compared with the control group, the expression of p65 protein in the SH-SY5Y cells of the LPS group was gradually shifted to the nucleus and was significantly enhanced (P < 0.01). Compared with the LPS group, p65 protein expression in the wogonin group, the TAK-242 group and the TAK-242 + wogonin group was dispersed to the outside of the nucleus, and the degree of expression was significantly reduced (P < 0.01). TLR4/MyD88/NF-κB pathway is one of the core signaling pathways in the inflammatory response, and microglial cells activate TLR4 when stimulated, which leads to the activation of MyD88 and the promotion of the NF-κB p65 is transferred to the nucleus and promotes the release of inflammatory factors. WB results showed (Fig.5F-G) that compared with the control group, SH-SY5Y cells in the LPS group showed a significant increase in the expression of p-p65 and MyD88 proteins (P < 0.01), a more increased expression of TLR4 protein, and no significant difference in the expression of p65 protein. Compared with the LPS group, the TLR4, p-p65 and MyD88 protein expression was significantly lower in the three groups of wogonin group, TAK-242 group and TAK-242 + wogonin group (P < 0.01), and there was no significant difference in p65 protein expression. Based on these results, we can conclude that wogonin and TAK-242 can protect SH-SY5Y cells, decrease α-syn and p65 protein expression, increase TH protein expression, and are related to the TLR4/MyD88/NF-κB pathway. Discussion PD, as the second most prevalent neurodegenerative disorder globally, has seen a rising incidence driven by population aging[ 17 , 18 ]. Despite advances in understanding its pathophysiology, the exact mechanisms underlying PD remain elusive. Current therapeutic strategies primarily alleviate symptoms but fail to halt pathological progression[ 19 , 20 ]. Neuroinflammation, particularly microglia-mediated inflammatory responses, is now recognized as a central pathogenic mechanism in PD[ 21 – 24 ]. Microglia, the primary immune cells of the central nervous system, undergo aberrant activation upon exposure to pathogen-associated molecular patterns (PAMPs) such as LPS. This activation triggers the TLR4/MyD88/NF-κB signaling cascade, leading to excessive release of pro-inflammatory cytokines such as IL-6, TNF-α, IL-1β and reactive oxygen species (ROS), which collectively drive dopaminergic neuronal degeneration[ 25 – 28 ]. While synthetic TLR4 inhibitors like TAK-242 exhibit anti-inflammatory potential, their clinical utility is limited by off-target toxicity and single-pathway targeting[ 29 , 30 ]. Consequently, natural compounds with multi-target efficacy and improved safety profiles have emerged as promising therapeutic candidates. Wogonin, a bioactive flavonoid derived from Scutellaria baicalensis , has garnered attention for its neuroprotective effects in PD models[ 31 – 33 ]. Mechanistic studies reveal that wogonin exerts multi-modal actions, including antioxidant, anti-inflammatory, regulation of apoptosis, and protection of dopaminergic neurons, and it has been found that wogonin can inhibit neuroinflammatory responses and improve neurological function, but the specific mechanisms need to be further explored[ 34 , 35 ]. The focus of this study was to investigate the neuroprotective effects of wogonin in an in vitro neuronal model and to reveal the underlying mechanisms behind these effects. BV-2 cells, commonly used as microglia in in vitro models, have a wide range of morphological, phenotypic, and functional characteristics of microglia[ 36 ].BV-2 cells are CNS immune macrophages that mediate inflammatory responses and play an important immunomodulatory role. LPS are strongly immunogenic particles located in the cell walls of Gram-negative bacteria, and are able to act as endotoxins in vivo to activate the innate immune system[ 37 ]. In the present study, we used LPS to stimulate the expression of inflammatory factors in BV-2 cells and administered SH-SY5Y cells with BV-2 cell inflammation model conditioned medium to establish a model of neuroinflammation for in vitro experiments with PD[ 38 ]. SH-SY5Y cells are a human neuroblastoma cell line widely used in neuroscience research with differentiation potential and neuron-like properties[ 39 , 40 ]. Microglia to develop a pro-inflammatory M1 phenotype, leading to the release of inflammatory factors that inhibit neuronal activity, which in turn causes abnormal neuronal function and loss[ 41 ]. This process is closely related to the TLR4/MyD88/NF-κB signaling pathway[ 42 ]. When LPS binds to TLR4 on the BV-2 cell membrane, the activated TLR4 recruits the downstream signaling molecule MyD88, which promotes the activation of NF-κB and transfers it to the nucleus of the cell, inducing the release of inflammatory factors, such as IL-6, TNF-α, and IL-1β, which can lead to the accumulation of neuronal function. The accumulation of these inflammatory factors and other harmful substances can lead to neuronal death, which in turn causes a series of clinical symptoms of PD[ 43 , 44 ]. In this study, BV-2 cells were firstly treated with different concentrations of LPS, and the cell viability and the content of inflammatory factor IL-6 in the supernatant of each group were detected, and the results showed that at a concentration of 1 µg/ml of LPS, the BV-2 cells were able to induce an activation phenotype as well as inflammation, and did not excessively affect the cell viability. Therefore, we concluded that 1 µg/ml was the most appropriate concentration of LPS to use in this experiment. We then administered different concentrations of the TLR4 inhibitor TAK-242 to BV-2 cells to verify its protective effects and screen for optimal concentrations. The results showed that 50 nM of TAK-242 was able to restore the viability of LPS-affected BV2 cells to the best extent and to reduce the expression of the inflammatory factor IL-6 to the most significant extent. It was verified that TAK-242 was able to inhibit TLR4 signaling and reduce the release of inflammatory mediators, thus reducing neuronal damage and having a protective effect on nerves. Then we administered different concentrations of wogonin to BV-2 cells and detected the cell morphology, cell viability and inflammatory factor IL-6 in each group. Since cell viability decreased at 32 µM of wogonin, this concentration was excluded to continue screening for the optimal concentration of baicalein. According to the results of ELISA for the detection of the IL-6 inflammatory factor, 16 µM of wogonin could reduce the expression of the inflammatory factor IL-6 to a lesser extent in the BV-2 cells that had been activated by LPS, and it could be used as the optimal concentration of wogonin for subsequent experiments. Inflammatory molecules produced by BV2 cells can lead to peripheral neuronal cell injury. In order to further verify the neuroprotective effects of wogonin from TAK-242 and the effect of their combination, an in vitro BV2-SH-SY5Y cell co-culture model was constructed. The results showed that wogonin could reduce the damage of SH-SY5Y cells in the co-culture system, and there was a statistically significant difference between the 16 µM wogonin group and the 50 nM TAK-242 and the model group, and both of them alone had a significant effect on the enhancement of cell viability and the reduction of inflammatory factors in BV-2 cells. However, the combination of the two did not result in a more significant protective effect. In summary, we found that LPS-induced BV-2 cells inhibited SH-SY5Y cell growth and induced neuronal inflammation. Moreover, both wogonin and TAK-242 were neuroprotective against LPS-induced inflammatory injury in SH-SY5Y cells. This protective effect may be related to the TLR4/MyD88/NF-κB signaling pathway. Wogonin may be a safe and promising drug candidate for the treatment of PD, which still needs to be validated in further animal models and clinical trials. Declarations Acknowledgments We are grateful to the Cell Resource Center of the Chinese Academy of Medical Sciences for providing the BV-2 cell line and to the Cell Bank of the Chinese Academy of Sciences for providing the SH-SY5Y cell line. Special thanks are extended to all laboratory members for their technical assistance and valuable discussions throughout the research. Author Contributions Data curation: Mengfei Sun, Ziqi Zhang. Formal analysis: Mengfei Sun, Ziqi Zhang. Investigation: Ziqi Zhang, Mengfei Sun. Methodology: Ziqi Zhang, Mengfei Sun. Supervision: Jingfeng Ouyang Writing – original draft: Ziqi Zhang. Writing – review & editing: Jingfeng Ouyang. Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Data Availability The datasets generated during this study are available from the corresponding author on reasonable request. References Global, regional, and national burden of neurological disorders, 1990-2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet Neurol. 2019;18. https://doi.org/10.1016/S1474-4422(18)30499-X Luo Y, Qiao L, Li M, Wen X, Zhang W, Li X. Global, regional, national epidemiology and trends of Parkinson’s disease from 1990 to 2021: findings from the Global Burden of Disease Study 2021. 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Naunyn Schmiedebergs Arch Pharmacol. 2025 [cited 18 Mar 2025]. https://doi.org/10.1007/s00210-025-03828-2 Fiebich BL, Batista CRA, Saliba SW, Yousif NM, de Oliveira ACP. Role of Microglia TLRs in Neurodegeneration. Front Cell Neurosci. 2018;12: 329. https://doi.org/10.3389/fncel.2018.00329 Chung LY-R, Lin Y-T, Liu C, Tai Y-C, Lin H-Y, Lin C-H, et al. Neuroinflammation Upregulated Neuronal Toll-Like Receptors 2 and 4 to Drive Synucleinopathy in Neurodegeneration. Front Pharmacol. 2022;13: 845930. https://doi.org/10.3389/fphar.2022.845930 Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.pdf Supplementary material S1 Fig. Effect of different concentrations of LPS on BV-2 cells.(A) Cell morphology of BV-2 cells at LPS 0-100 μg/ml;(B) BV-2 cell viability at different concentrations of LPS;(C) Inflammatory factor IL-6 in BV-2 cell supernatants at different concentrations of LPS; *P < 0.05; **P < 0.01. S2 Fig. Effect of different concentrations of TAK-242 on BV-2 cells.(A) Cell morphology of BV-2 cells at TAK-242 0-200ng/ml;(B) BV-2 cell viability at different concentrations of TAK-242;(C) Inflammatory factor IL-6 in BV-2 cell supernatants at different concentrations of TAK-242; *P < 0.05; **P < 0.01. S3 Fig. Effect of different concentrations of wogonin on BV-2 cells.(A) Cell morphology of BV-2 cells at wogonin 0-32μM;(B) BV-2 cell viability at different concentrations of wogonin;(C) Inflammatory factor IL-6 in BV-2 cell supernatants at different concentrations of wogonin; *P < 0.05; **P < 0.01. S4 Fig. Effects of wogonin and TAK-242 alone or in combination on BV-2 cells.(A) BV-2 cell morphology in different groups, groups a-e are control group, LPS group, LPS + wogonin group, LPS + TAK-242 group, LPS + wogonin + TAK-242 group, respectively (the same below);(B) BV-2 cell viability in a-e groups;#represents comparison with control group,*represents comparison with control group;## and ** indicate P < 0.01, # and * indicate P < 0.05 (the same below);（C-E）Inflammatory factor IL-6, TNF-α and IL-1β in BV-2 cell supernatants in a-e groups. S5 Fig. Effects of wogonin and TAK-242 alone or in combination on SH-SY5Y cells via the TLR4/MyD88/NF-κB signaling pathway.(A-C) IHC representative images of TH and α-syn contents in SH-SY5Y cells affected by BV-2 cells conditioned medium;(D-E) IF representative images of p65 fluorescent expression in SH-SY5Y cells affected by BV-2 cells conditioned medium; ( F-G) WB detection of changes in TLR-4, NF-κB p65, p-NF-κB p65 and MyD88 protein levels in SH-SY5Y cells. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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10:20:20","extension":"xml","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":109423,"visible":true,"origin":"","legend":"","description":"","filename":"d3ddca47a8b6464bae27a77dbed82d4e1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7532665/v1/aef12c060f0ca59246762644.xml"},{"id":94012016,"identity":"bcb56b54-dbec-403a-bfad-8bad5652320f","added_by":"auto","created_at":"2025-10-21 10:19:49","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":123101,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7532665/v1/5b30931a17d3326b09a5b4d8.html"},{"id":94011886,"identity":"3114b077-6a1f-4803-8cb5-c495ca7be435","added_by":"auto","created_at":"2025-10-21 10:19:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2639272,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of different concentrations of LPS on BV-2 cells.\u003c/strong\u003e (A) Cell morphology of BV-2 cells at LPS 0-100 μg/ml;(B) BV-2 cell viability at different concentrations of LPS;(C) Inflammatory factor IL-6 in BV-2 cell supernatants at different concentrations of LPS; *P \u0026lt; 0.05; **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7532665/v1/a2d3580e755e7188f4bdc10c.png"},{"id":94012098,"identity":"6e0f84ec-24ea-4a8f-8eb8-59f454ceb110","added_by":"auto","created_at":"2025-10-21 10:20:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":971870,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of different concentrations of TAK-242 on BV-2 cells.\u003c/strong\u003e (A) Cell morphology of BV-2 cells at TAK-242 0-200ng/ml;(B) BV-2 cell viability at different concentrations of TAK-242;(C) Inflammatory factor IL-6 in BV-2 cell supernatants at different concentrations of TAK-242; *P \u0026lt; 0.05; **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7532665/v1/61d0652e0d551fce121c0458.png"},{"id":94012055,"identity":"ef443480-264f-4933-9deb-90d2dd153aa2","added_by":"auto","created_at":"2025-10-21 10:20:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1201862,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of different concentrations of wogonin on BV-2 cells. \u003c/strong\u003e(A) Cell morphology of BV-2 cells at wogonin 0-32μM;(B) BV-2 cell viability at different concentrations of wogonin;(C) Inflammatory factor IL-6 in BV-2 cell supernatants at different concentrations of wogonin; *P \u0026lt; 0.05; **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7532665/v1/777e95d79a73402b4d424bc3.png"},{"id":94012012,"identity":"98d810ac-37d2-4c0e-a38e-eaab018b466c","added_by":"auto","created_at":"2025-10-21 10:19:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":836287,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of wogonin and TAK-242 alone or in combination on BV-2 cells.\u003c/strong\u003e(A) BV-2 cell morphology in different groups, groups a-e are control group, LPS group, LPS + wogonin group, LPS + TAK-242 group, LPS + wogonin + TAK-242 group, respectively (the same below);(B) BV-2 cell viability in a-e groups;#represents comparison with control group,*represents comparison with control group;## and ** indicate P \u0026lt; 0.01, # and * indicate P \u0026lt; 0.05 (the same below);（C-E）Inflammatory factor IL-6, TNF-α and IL-1β in BV-2 cell supernatants in a-e groups.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-7532665/v1/f0dd65ac6a6670ee9f21a80d.png"},{"id":94011884,"identity":"26c156b9-e6cc-4026-9240-ad9dfed4d24a","added_by":"auto","created_at":"2025-10-21 10:19:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3146140,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of wogonin and TAK-242 alone or in combination on SH-SY5Y cells via the TLR4/MyD88/NF-κB signaling pathway.\u003c/strong\u003e(A-C) IHC representative images of TH and α-syn contents in SH-SY5Y cells affected by BV-2 cells conditioned medium;(D-E) IF representative images of p65 fluorescent expression in SH-SY5Y cells affected by BV-2 cells conditioned medium; ( F-G) WB detection of changes in TLR-4, NF-κB p65, p-NF-κB p65 and MyD88 protein levels in SH-SY5Y cells. Groups: a, control group; b, LPS (1 μg/mL) treatment group; c, Wogonin (16 μM) + LPS (1 μg/mL) treatment group; d, TAK-242 (50 nM) + LPS (1 μg/mL) treatment group; e, Wogonin (16 μM) + TAK-242 (50 nM) + LPS (1 μg/mL) treatment group.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-7532665/v1/0d3a0487006bc4906d29a014.png"},{"id":96250016,"identity":"6df20ff2-3c53-471d-bef7-b9ccfa8c1325","added_by":"auto","created_at":"2025-11-19 07:37:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8780460,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7532665/v1/42a8df66-5be9-4de2-b2d5-5dc41854c874.pdf"},{"id":94012052,"identity":"f808e653-d91f-4787-9f1a-14dc262d6193","added_by":"auto","created_at":"2025-10-21 10:19:58","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":166547,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eS1 Fig.\u003c/strong\u003e \u003cstrong\u003eEffect of different concentrations of LPS on BV-2 cells.\u003c/strong\u003e(A) Cell morphology of BV-2 cells at LPS 0-100 μg/ml;(B) BV-2 cell viability at different concentrations of LPS;(C) Inflammatory factor IL-6 in BV-2 cell supernatants at different concentrations of LPS; *P \u0026lt; 0.05; **P \u0026lt; 0.01.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eS2 Fig.\u003c/strong\u003e \u003cstrong\u003eEffect of different concentrations of TAK-242 on BV-2 cells.\u003c/strong\u003e(A) Cell morphology of BV-2 cells at TAK-242 0-200ng/ml;(B) BV-2 cell viability at different concentrations of TAK-242;(C) Inflammatory factor IL-6 in BV-2 cell supernatants at different concentrations of TAK-242; *P \u0026lt; 0.05; **P \u0026lt; 0.01.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eS3 Fig. Effect of different concentrations of wogonin on BV-2 cells.\u003c/strong\u003e(A) Cell morphology of BV-2 cells at wogonin 0-32μM;(B) BV-2 cell viability at different concentrations of wogonin;(C) Inflammatory factor IL-6 in BV-2 cell supernatants at different concentrations of wogonin; *P \u0026lt; 0.05; **P \u0026lt; 0.01.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eS4 Fig. Effects of wogonin and TAK-242 alone or in combination on BV-2 cells.\u003c/strong\u003e(A) BV-2 cell morphology in different groups, groups a-e are control group, LPS group, LPS + wogonin group, LPS + TAK-242 group, LPS + wogonin + TAK-242 group, respectively (the same below);(B) BV-2 cell viability in a-e groups;#represents comparison with control group,*represents comparison with control group;## and ** indicate P \u0026lt; 0.01, # and * indicate P \u0026lt; 0.05 (the same below);（C-E）Inflammatory factor IL-6, TNF-α and IL-1β in BV-2 cell supernatants in a-e groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eS5 Fig. Effects of wogonin and TAK-242 alone or in combination on SH-SY5Y cells via the TLR4/MyD88/NF-κB signaling pathway.\u003c/strong\u003e(A-C) IHC representative images of TH and α-syn contents in SH-SY5Y cells affected by BV-2 cells conditioned medium;(D-E) IF representative images of p65 fluorescent expression in SH-SY5Y cells affected by BV-2 cells conditioned medium; ( F-G) WB detection of changes in TLR-4, NF-κB p65, p-NF-κB p65 and MyD88 protein levels in SH-SY5Y cells.\u003c/p\u003e","description":"","filename":"Supplementarymaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7532665/v1/cbd802915f4aaa55988a6c46.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exploring the mechanism of Wogonin in the attenuation of LPS-induced inflammation in BV-2 cells and the protective effect of wogonin on SH-SY5Y cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eParkinson's disease (PD), the second most prevalent neurodegenerative disease worldwide[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], is characterized by progressive loss of midbrain substantia nigra dopaminergic neurons and abnormal aggregation of α-synuclein (α-Syn)[\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Its pathogenesis is complex and has not been fully elucidated. It has been suggested that microglia-mediated neuroinflammation within the central nervous system is a key factor in several neurodegenerative diseases[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The central role of neuroinflammation in the pathological process of PD has also received much attention in recent years. Microglia, as the main immune defense of the Central Nervous System, are over-activated in response to stimulation by pathogens or injury signals, and activation of Toll-like receptor 4 (TLR4) on the cell membrane triggers the activation of a series of intracellular adaptor proteins, which leads to activation of proteins associated with the NF-κB pathway, resulting in the secretion of inflammatory factors and the release of interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and other pro-inflammatory factors, exacerbating neuronal oxidative damage and apoptosis[\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWogonin, a natural flavonoid active ingredient extracted from the traditional Chinese medicine \u003cem\u003eScutellaria baicalensis\u003c/em\u003e, possesses multiple pharmacological properties including antioxidant, anti-inflammatory and neuroprotective properties[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Some studies have shown that wogonin can inhibit the production of reactive oxygen species (ROS) thereby suppressing inflammatory responses and oxidative stress[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In an ischemic brain injury model, wogonin reduces neuronal damage by inhibiting pro-inflammatory microglia polarization and decreasing the secretion of neuroinflammatory factors through pathways[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In addition, wogonin has shown the ability to increase neuronal synaptic density in epilepsy disorders and to inhibit pro-inflammatory cytokine release and microglial synaptic phagocytosis through activation of the AKT/FoxO1 pathway[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The above studies suggest that wogonin has certain effects on inhibiting neuroinflammatory and protecting neurons from damage, and may improve neurodegenerative lesions by modulating apoptotic pathways and inhibiting oxidative stress, but its specific regulatory mechanism on microglia-mediated neuroinflammation is not clear. In this study, we constructed an in vitro neuroinflammation model with lipopolysaccharide (LPS)-activated BV-2 microglia, combined with the SH-SY5Y neuronal co-culture system, to systematically investigate the protective effect of wogonin on inflammatory cascade response and neuronal injury, and to explore its association with the TLR4/MyD88/NF-κB signaling pathway.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMaterials\u003c/h2\u003e\u003cp\u003eThe murine microglial BV-2 cell line was sourced from the Cell Resource Center of the Chinese Academy of Medical Sciences (Beijing, China). The human neuroblastoma SH-SY5Y cell line was sourced from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). TAK-242 (CAS: T125887) was purchased from Aladdin Biochemical Technology (Shanghai, China). Wogonin (CAS: B0002970) was acquired from Bepure (China). LPS (CAS: 028M4094V) was purchased from Sigma. DMEM (CAS: 8123098), penicillin-streptomycin solution (CAS: 15140-122), and FBS (CAS: 2350404RP) were obtained from Gibco. L-glutamine solution (CAS: 20212128) was purchased by Solarbio (Beijing, China). The Cell Counting Kit-8 was purchased from Dojindo Molecular Technologies (Japan). Mouse interleukin-6, tumor necrosis factor-alpha, and interleukin-1 beta enzyme-linked immunosorbent assay kits were provided by ABclonal Biotechnology (Wuhan, China). Rabbit monoclonal antibodies against NF-κB p65 (ab32536) and phosphorylated (p)-NF-κB p65 (ab76302), as well as goat polyclonal antibody targeting myeloid differentiation factor 88 (ab28763), were acquired from Abcam (UK). The BCA protein concentration assay kit, rabbit monoclonal β-actin antibody, and Alexa Fluor 488-conjugated goat anti-immunoglobulin G (H\u0026thinsp;+\u0026thinsp;L) were purchased from Beyotime Biotechnology (Shanghai, China). Mouse monoclonal α-synuclein (α-Syn) antibody, rabbit polyclonal tyrosine hydroxylase antibody, mouse monoclonal TLR4 antibody, horseradish peroxidase conjugated goat anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L), HRP-conjugated goat anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L), HRP-conjugated rabbit anti-goat IgG (H\u0026thinsp;+\u0026thinsp;L), and DAB chromogenic kit were obtained from Sangon Biotech (Wuhan, China).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eBV-2 and SH-SY5Y cells were cultured in DMEM supplemented with 10% FBS, 1% L-glutamine, and 1% penicillin-streptomycin solution. Cells were maintained in a humidified incubator at 37\u0026deg;C with 5% CO₂. When cell confluency reached 80\u0026ndash;90%, the cells were passaged and seeded into new culture plates for subsequent experiments.\u003c/p\u003e\u003cp\u003eBV-2 and SH-SY5Y cells were cultured and seeded into 96-well plates according to the aforementioned methods. After 24 h of incubation, the plates were removed, and supernatants from BV-2 cells in each group were collected separately. The collected supernatants were mixed with an equal volume of complete DMEM medium (1:1 ratio) to prepare conditioned medium. SH-SY5Y cells were then divided into experimental groups and treated with the respective conditioned medium. All plates were returned to the incubator for an additional 12 h of culture.\u003c/p\u003e\n\u003ch3\u003eCell viability assay\u003c/h3\u003e\n\u003cp\u003eCell viability was assessed using the CCK-8 following different interventions. Cells were seeded into 96-well plates at a density of 6 \u0026times; 10⁴ cells/mL and cultured for 24 h until achieving 80% confluency in an adherent state. Cells were then treated with specified concentrations of compounds for designated durations. After treatment, the plates were incubated for an additional 24 h under standard culture conditions (37\u0026deg;C, 5% CO₂). Following incubation, the plates were removed, and cellular morphology across all groups was observed under the microscope and documented via photomicrographs. Absorbance (A) at 450 nm was measured using a microplate reader. Cell viability was calculated using the following formula:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:cell\\:viability\\left(\\%\\right)=\\frac{\\left(\\text{A}\\:\\text{T}\\text{r}\\text{e}\\text{a}\\text{t}\\text{m}\\text{e}\\text{n}\\text{t}-\\text{A}\\:\\text{S}\\text{o}\\text{l}\\text{v}\\text{e}\\text{n}\\text{t}\\:\\text{C}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}\\right)}{\\left(\\text{A}\\:\\text{C}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}-\\text{A}\\:\\text{S}\\text{o}\\text{l}\\text{v}\\text{e}\\text{n}\\text{t}\\:\\text{C}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}\\right)}\\times\\:100\\%$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eDetermination the content of IL-6, TNF-α and IL-1β by ELISA\u003c/h3\u003e\n\u003cp\u003eThe treated cells were cultured and seeded into 24-well plates according to the aforementioned protocol, followed by incubation for an additional 48 h. After the incubation period, supernatants from each well were collected and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for subsequent analysis. The expression levels of IL-6, TNF-α, and IL-1β in the supernatants were quantified using ELISA kits in strict accordance with the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eSH-SY5Y cells were cultured according to the aforementioned protocol. After culture, cells were washed three times with PBS and fixed with 4% paraformaldehyde (PFA) for 30 min at room temperature. Following fixation, cells were permeabilized with 0.5% Triton X-100 for 5 min at room temperature and rinsed three times with PBS. Primary antibodies tyrosine hydroxylase (TH, 1:800 dilution) and α-synuclein (α-Syn, 1:1000 dilution) were applied to the cells and incubated on a shaker at 4\u0026deg;C for 24 h. After washing, species-matched secondary antibodies were added and incubated for 1 h at room temperature under light-protected conditions. DAB chromogen was applied for color development, followed by hematoxylin counterstaining of nuclei. Coverslips were mounted with neutral resin and observed under a microscope. Representative images were captured for analysis.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescence\u003c/h2\u003e\u003cp\u003eSH-SY5Y cells were cultured as described above. After culture, cells were washed with PBS and fixed with PFA for 30 min at room temperature. Following three PBS washes, cells were permeabilized with 0.5% Triton X-100 for 10 min at RT. Primary antibody NF-κB p65 (1:800 dilution) was applied and incubated on a shaker at 4\u0026deg;C overnight. Cells were then treated with Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (1:250 dilution) under light-protected conditions at room temperature for 1 h. After washing with TBS, cell slides were counterstained with DAPI for 15 min to label nuclei. Slides were mounted with neutral resin and imaged using a fluorescence microscope. Representative images were captured with consistent exposure settings across all groups.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eWestern blot\u003c/h3\u003e\n\u003cp\u003eSH-SY5Y cells were cultured as described above and washed with PBS. Cells were lysed in RIPA buffer supplemented with phosphatase and protease inhibitors for 15\u0026ndash;30 min on ice. The lysates were centrifuged at 10,000 \u0026times; g for 10 min at 4\u0026deg;C, and supernatants were collected in sterile tubes. Protein concentrations were quantified using a BCA assay kit. Equal amounts of protein were mixed with loading buffer, denatured by heating at 100\u0026deg;C for 5 min, and separated on 10% SDS-polyacrylamide gels. Proteins were then transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk in TBST for 90 min at room temperature. Primary antibodies β-actin (1:3000 dilution), TLR4 (1:5000 dilution), NF-κB p65, phosphorylated (p)-NF-κB p65, and MyD88 (1:1000 dilution) were applied and incubated at 4\u0026deg;C overnight. After three TBST washes, species-matched secondary antibodies were added and incubated for 1 h at room temperature. Membranes were washed again and developed using enhanced chemiluminescence reagent. Band densities were quantified using ImageJ software with β-actin as the loading control.\u003c/p\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eMeasurement data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Statistical analysis was performed using GraphPad Prism 9.1 software and IBM SPSS Statistics26. Comparisons between two groups were analyzed using Student\u0026rsquo;s t-test, and comparisons among multiple groups were assessed by one-way analysis of variance. A threshold of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eLPS inhibits BV-2 cell proliferation and induces pro-inflammatory cytokine release\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccumulating evidence indicates that LPS promotes inflammatory responses both in vitro and in vivo, providing a model for studying neuroinflammatory mechanisms in PD. To determine the cytotoxic effects of LPS on BV-2 cells, BV-2 cells were pretreated with LPS at concentrations of 0, 0.1, 1, 10, or 100 \u0026mu;g/mL. BV-2 cell morphology was observed under a microscope, and images were captured. Cell viability was assessed using the CCK-8, and IL-6 levels in supernatants were quantified via ELISA.\u003c/p\u003e\n\u003cp\u003eFig.1A shows that in the group of LPS 0\u0026mu;g/ml, BV-2 cells exhibited typical semi-adherent and semi-suspension growth pattern. Suspended cells appeared uniformly round, while adherent cells displayed polymorphic morphologies (round, spindle, or polygonal) with smooth membrane edges, clear contours, and translucent cytoplasm accompanied by short protrusions. Compared to the LPS 0 \u0026mu;g/mL group: in the LPS 0.1 \u0026mu;g/mL group, the cell synapses were slightly longer, the cell volume was slightly enlarged, and there was no obvious morphological change; in the LPS 1 \u0026mu;g/mL group, the cell protrusions increased significantly, the cytosol was swollen and polarized, and the antennae were elongated, and some of the cells showed amebia-like changes; in the LPS 10 \u0026mu;g/mL group: the number of cells was reduced, and there were a lot of dead cells and fragments floating in the culture medium. LPS 10 \u0026mu;g/mL group: the number of cells was greatly reduced, with a large number of dead cells and fragments floating in the culture medium, and complete loss of cytoplasmic translucency; LPS 100 \u0026mu;g/mL group: the cells were almost completely dead, and only a few cell fragments remained.\u003c/p\u003e\n\u003cp\u003eThe results of CCK-8 showed (Fig.1B) that cell viability was significantly decreased in LPS 10\u0026mu;g/mL and LPS 100\u0026mu;g/mL groups compared with the group with LPS 0\u0026mu;g/ml (P\u0026lt;0.01). Whereas, no statistically significant difference in cell viability was observed in the LPS 0.1\u0026mu;g/mL and LPS 1\u0026mu;g/mL groups.\u003c/p\u003e\n\u003cp\u003eThe results of ELISA showed (Fig.1C) that IL-6 levels were increased in all other groups compared to the group with LPS 0\u0026mu;g/ml. Among them, IL-6 levels were significantly elevated in the group with LPS 1\u0026mu;g/mL, and no statistically significant differences were observed in the IL-6 levels of all other groups. Based on the above experimental results, we can conclude that LPS 10 \u0026mu;g/mL and LPS 100 \u0026mu;g/mL led to the loss of cell function or death. The cells in the LPS 1 \u0026mu;g/mL group showed a polarized morphology and a significant increase in the expression of inflammatory factors, but there was no significant change in the viability of the cells, which suggests that this concentration induces an activation phenotype without significantly affecting the survival rate, and it can be used as a modeling concentration for the subsequent experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of TAK-242 at varying concentrations on BV-2 cell growth inhibition and inflammatory cytokine release\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTAK-242, a selective small-molecule inhibitor of the TLR4 signaling pathway, binds to the Cys747 residue within the TLR4 TIR domain, thereby suppressing LPS-induced production of pro-inflammatory cytokines and inhibiting downstream MyD88/TRIF signaling and autophagy. To evaluate the effects of TAK-242 on BV-2 cells, cells were treated with TAK-242 at concentrations of 0, 25, 50, 100, and 200 nM. Morphological changes were documented via microscopy. Cell viability was assessed by CCK-8. IL-6 levels in supernatants were quantified by ELISA.\u003c/p\u003e\n\u003cp\u003eFig.2A shows that the BV-2 cells in the TAK-242 0nM group grew adherently to the wall, were round or pike shaped, and showed a resting cell morphology. The cell boundaries were well defined with short tentacular extensions, most of the cells were in clusters, and a few were suspended and shiny. Compared with TAK-242 0nM group, the cells in each concentration group of TAK-242 did not show significant morphological changes.\u003c/p\u003e\n\u003cp\u003eThe results of CCK-8 showed (Fig.2B) that there was no significant difference in the cell viability of TAK-242 25nM, 50nM, and 100 nM groups compared with TAK-242 0nM group. The cell viability of TAK-242 200nM group was significantly reduced (P\u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003eThe results of ELISA showed (Fig.2C) that IL-6 levels were significantly lower in the TAK-242 50nM group, significantly higher in the TAK-242 200nM group, and there was no significant difference between the IL-6 levels in the TAK-242 25nM group and the TAK-242 100 nM group, as compared with those in the TAK-242 0nM group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBased on the above experimental results, we can conclude that TAK-242 200nM has a greater effect on cell viability and increases the expression of inflammatory factors. While there is no obvious morphological change in the cells of the TAK-242 50nM group, and there is no obvious change in the viability of the cells, but the expression of inflammatory factors is significantly reduced. This indicates that this concentration can inhibit the expression of inflammatory factors in BV-2 cells without significantly affecting the survival rate, and can be used as a subsequent experimental concentration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect\u003c/strong\u003e \u003cstrong\u003eof Wogonin at different concentrations on inflammatory cytokine secretion in BV-2 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious studies suggest that wogonin exerts neuroprotective effects and ameliorates neuroinflammatory responses. In this study, we further investigated the impact of wogonin at varying concentrations on BV-2 cell morphology, viability, and inflammatory cytokine production. \u0026nbsp; BV-2 cells were cultured with wogonin medium containing 0 \u0026mu;M, 4 \u0026mu;M, 8 \u0026mu;M, 16 \u0026mu;M, and 32 \u0026mu;M, respectively. Then the morphology of BV-2 cells in each group was observed under a microscope and photographed. Cellular morphology was documented via the microscopy, and viability was assessed using the CCK-8. IL-6 levels in supernatants were quantified by ELISA.\u003c/p\u003e\n\u003cp\u003eFig.3A shows that the cells in the wogonin 0 \u0026mu;M group were semi-suspended and semi-adherent, and the cells had the morphology of round, pike and polygonal. The cells had clear boundaries, high translucency and short tentacles. No significant morphological changes were observed in the cells of other wogonin concentration groups. Results of CCK-8 showed (Fig.3B) that no significant difference in cell viability was observed in the wogonin 4 \u0026mu;M, 8 \u0026mu;M, and 16 \u0026mu;M groups compared with the wogonin 0 \u0026mu;M group. Cell viability was significantly lower in the wogonin 32 \u0026mu;M group (P\u0026lt;0.01). The results of ELISA showed (Fig.3C) that IL-6 levels were significantly higher in the LPS group compared with the control group. Compared with the LPS group, there was no significant difference in the IL-6 level in the wogonin 4 \u0026mu;M group, and the IL-6 level was significantly lower in the wogonin 8 \u0026mu;M and wogonin 16 \u0026mu;M groups. Based on the above experimental results, we can conclude that cell viability decreased at 32 \u0026mu;M of wogonin, so this concentration was excluded. The IL-6 levels expressed by BV-2 cells was reduced to a greater extent at 32 \u0026mu;M of wogonin. Therefore, 16 \u0026mu;M was chosen as the optimal concentration of wogonin for subsequent experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWogonin alleviates LPS-induced growth suppression and reduces inflammatory cytokine production in BV-2 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the experimental results above, three optimal concentrations were selected for subsequent studies: 1 \u0026mu;g/mL LPS, 50 nM TAK-242, and 16 \u0026mu;M Wogonin. Following the protocol described in the Materials and Methods section, BV-2 cells were divided into the following groups: Control group, LPS group (1 \u0026mu;g/mL LPS), Wogonin group (16 \u0026mu;M Wogonin + 1 \u0026mu;g/mL LPS), TAK-242 group (50 nM TAK-242 + 1 \u0026mu;g/mL LPS), TAK-242 + Wogonin group (50 nM TAK-242 + 16 \u0026mu;M Wogonin + 1 \u0026mu;g/mL LPS).\u003c/p\u003e\n\u003cp\u003eFig.4A shows that the BV-2 cells in the control group were half-adherent and half-suspended, the suspended cells were round, and the adherent cells were round, pike or polygonal, with smooth edges, clear boundaries, high transmittance, and short pseudopods could be seen to protrude. Compared with the control group, the cell protrusions in the LPS group increased significantly, the cell volume increased, the pseudopods were elongated, and some of the cells showed amoeba-like morphology. Compared with the LPS group, cell morphology was restored, the proportion of round cells increased, and the number of synapses was significantly reduced in the wogonin group, the TAK-242 group, and the TAK-242+ wogonin group. The results of CCK-8 showed (Fig.4B) that no significant difference in cell viability was observed in the LPS group compared with the control group. Compared with the LPS group, cell viability was more increased in the wogonin group, cell viability was significantly increased in the TAK-242 group (P \u0026lt; 0.01), and cell viability in the TAK-242+ wogonin group did not show any significant difference. The results of ELISA showed (Fig.4C-E) that the levels of IL-6, TNF-\u0026alpha;, and IL-1\u0026beta; in the LPS group were significantly higher compared with the control group (P \u0026lt; 0.01). Compared with the LPS group, the levels of IL-6 were significantly lower in the wogonin group, the TAK-242 group, and the TAK-242 + wogonin group (P \u0026lt; 0.01), the levels of IL-1\u0026beta; were significantly lower in the wogonin group and the TAK-242 group (P \u0026lt; 0.01), and the levels of TNF-\u0026alpha; were more reduced in the wogonin group, the TAK-242 group, and the TAK-242 + wogonin group.\u003c/p\u003e\n\u003cp\u003eBased on the above results, we can conclude that both wogonin and TAK-242 were able to attenuate LPS-induced growth inhibition of BV-2 cells, increase the cell viability of BV-2 cells reduced by LPS, and reduce the expression of inflammatory factors, which had a certain protective effect on BV-2 cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWogonin mitigates LPS-induced inflammatory damage in SH-SY5Y microglial cells via the TLR4/MyD88/NF-\u0026kappa;B signaling pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAbnormal aggregation and deposition of \u0026alpha;-syn is one of the characteristic pathological manifestations of PD. TH is a key enzyme in the dopamine synthesis pathway, responsible for the conversion of tyrosine to levodopa and then to dopamine. The expression levels of both can be used as biomarkers to assess the function of dopaminergic neurons and the progression of PD. IHC results showed (Fig.5A-C) that the intensity of \u0026alpha;-syn protein expression was significantly enhanced (P\u0026lt;0.01) and the intensity of TH protein expression was significantly weakened (P\u0026lt;0.01) in SH-SY5Y cells of the LPS group, compared with the control group. Compared with the LPS group, the intensity of \u0026alpha;-syn protein expression was significantly weakened (all P\u0026lt;0.01) and the intensity of TH protein expression was significantly enhanced (all P\u0026lt;0.01) in the SH-SY5Y cells of the wogonin group, the TAK-242 group, and the TAK-242 + wogonin group.\u003c/p\u003e\n\u003cp\u003eThe activation of NF-\u0026kappa;B signaling pathway is closely related to neuroinflammation in PD. NF-\u0026kappa;B p65 was activated and transferred to the nucleus, accelerating the release of inflammatory factors. IF results showed that (Fig.5D-E), compared with the control group, the expression of p65 protein in the SH-SY5Y cells of the LPS group was gradually shifted to the nucleus and was significantly enhanced (P \u0026lt; 0.01). Compared with the LPS group, p65 protein expression in the wogonin group, the TAK-242 group and the TAK-242 + wogonin group was dispersed to the outside of the nucleus, and the degree of expression was significantly reduced (P \u0026lt; 0.01).\u003c/p\u003e\n\u003cp\u003eTLR4/MyD88/NF-\u0026kappa;B pathway is one of the core signaling pathways in the inflammatory response, and microglial cells activate TLR4 when stimulated, which leads to the activation of MyD88 and the promotion of the NF-\u0026kappa;B p65 is transferred to the nucleus and promotes the release of inflammatory factors. WB results showed (Fig.5F-G) that compared with the control group, SH-SY5Y cells in the LPS group showed a significant increase in the expression of p-p65 and MyD88 proteins (P \u0026lt; 0.01), a more increased expression of TLR4 protein, and no significant difference in the expression of p65 protein. Compared with the LPS group, the TLR4, p-p65 and MyD88 protein expression was significantly lower in the three groups of wogonin group, TAK-242 group and TAK-242 + wogonin group (P \u0026lt; 0.01), and there was no significant difference in p65 protein expression.\u003c/p\u003e\n\u003cp\u003eBased on these results, we can conclude that wogonin and TAK-242 can protect SH-SY5Y cells, decrease \u0026alpha;-syn and p65 protein expression, increase TH protein expression, and are related to the TLR4/MyD88/NF-\u0026kappa;B pathway.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePD, as the second most prevalent neurodegenerative disorder globally, has seen a rising incidence driven by population aging[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Despite advances in understanding its pathophysiology, the exact mechanisms underlying PD remain elusive. Current therapeutic strategies primarily alleviate symptoms but fail to halt pathological progression[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Neuroinflammation, particularly microglia-mediated inflammatory responses, is now recognized as a central pathogenic mechanism in PD[\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Microglia, the primary immune cells of the central nervous system, undergo aberrant activation upon exposure to pathogen-associated molecular patterns (PAMPs) such as LPS. This activation triggers the TLR4/MyD88/NF-κB signaling cascade, leading to excessive release of pro-inflammatory cytokines such as IL-6, TNF-α, IL-1β and reactive oxygen species (ROS), which collectively drive dopaminergic neuronal degeneration[\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. While synthetic TLR4 inhibitors like TAK-242 exhibit anti-inflammatory potential, their clinical utility is limited by off-target toxicity and single-pathway targeting[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Consequently, natural compounds with multi-target efficacy and improved safety profiles have emerged as promising therapeutic candidates.\u003c/p\u003e\u003cp\u003eWogonin, a bioactive flavonoid derived from \u003cem\u003eScutellaria baicalensis\u003c/em\u003e, has garnered attention for its neuroprotective effects in PD models[\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Mechanistic studies reveal that wogonin exerts multi-modal actions, including antioxidant, anti-inflammatory, regulation of apoptosis, and protection of dopaminergic neurons, and it has been found that wogonin can inhibit neuroinflammatory responses and improve neurological function, but the specific mechanisms need to be further explored[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The focus of this study was to investigate the neuroprotective effects of wogonin in an in vitro neuronal model and to reveal the underlying mechanisms behind these effects.\u003c/p\u003e\u003cp\u003eBV-2 cells, commonly used as microglia in in vitro models, have a wide range of morphological, phenotypic, and functional characteristics of microglia[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].BV-2 cells are CNS immune macrophages that mediate inflammatory responses and play an important immunomodulatory role. LPS are strongly immunogenic particles located in the cell walls of Gram-negative bacteria, and are able to act as endotoxins in vivo to activate the innate immune system[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In the present study, we used LPS to stimulate the expression of inflammatory factors in BV-2 cells and administered SH-SY5Y cells with BV-2 cell inflammation model conditioned medium to establish a model of neuroinflammation for in vitro experiments with PD[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. SH-SY5Y cells are a human neuroblastoma cell line widely used in neuroscience research with differentiation potential and neuron-like properties[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Microglia to develop a pro-inflammatory M1 phenotype, leading to the release of inflammatory factors that inhibit neuronal activity, which in turn causes abnormal neuronal function and loss[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. This process is closely related to the TLR4/MyD88/NF-κB signaling pathway[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. When LPS binds to TLR4 on the BV-2 cell membrane, the activated TLR4 recruits the downstream signaling molecule MyD88, which promotes the activation of NF-κB and transfers it to the nucleus of the cell, inducing the release of inflammatory factors, such as IL-6, TNF-α, and IL-1β, which can lead to the accumulation of neuronal function. The accumulation of these inflammatory factors and other harmful substances can lead to neuronal death, which in turn causes a series of clinical symptoms of PD[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, BV-2 cells were firstly treated with different concentrations of LPS, and the cell viability and the content of inflammatory factor IL-6 in the supernatant of each group were detected, and the results showed that at a concentration of 1 \u0026micro;g/ml of LPS, the BV-2 cells were able to induce an activation phenotype as well as inflammation, and did not excessively affect the cell viability. Therefore, we concluded that 1 \u0026micro;g/ml was the most appropriate concentration of LPS to use in this experiment. We then administered different concentrations of the TLR4 inhibitor TAK-242 to BV-2 cells to verify its protective effects and screen for optimal concentrations. The results showed that 50 nM of TAK-242 was able to restore the viability of LPS-affected BV2 cells to the best extent and to reduce the expression of the inflammatory factor IL-6 to the most significant extent. It was verified that TAK-242 was able to inhibit TLR4 signaling and reduce the release of inflammatory mediators, thus reducing neuronal damage and having a protective effect on nerves. Then we administered different concentrations of wogonin to BV-2 cells and detected the cell morphology, cell viability and inflammatory factor IL-6 in each group. Since cell viability decreased at 32 \u0026micro;M of wogonin, this concentration was excluded to continue screening for the optimal concentration of baicalein. According to the results of ELISA for the detection of the IL-6 inflammatory factor, 16 \u0026micro;M of wogonin could reduce the expression of the inflammatory factor IL-6 to a lesser extent in the BV-2 cells that had been activated by LPS, and it could be used as the optimal concentration of wogonin for subsequent experiments.\u003c/p\u003e\u003cp\u003eInflammatory molecules produced by BV2 cells can lead to peripheral neuronal cell injury. In order to further verify the neuroprotective effects of wogonin from TAK-242 and the effect of their combination, an in vitro BV2-SH-SY5Y cell co-culture model was constructed. The results showed that wogonin could reduce the damage of SH-SY5Y cells in the co-culture system, and there was a statistically significant difference between the 16 \u0026micro;M wogonin group and the 50 nM TAK-242 and the model group, and both of them alone had a significant effect on the enhancement of cell viability and the reduction of inflammatory factors in BV-2 cells. However, the combination of the two did not result in a more significant protective effect.\u003c/p\u003e\u003cp\u003eIn summary, we found that LPS-induced BV-2 cells inhibited SH-SY5Y cell growth and induced neuronal inflammation. Moreover, both wogonin and TAK-242 were neuroprotective against LPS-induced inflammatory injury in SH-SY5Y cells. This protective effect may be related to the TLR4/MyD88/NF-κB signaling pathway. Wogonin may be a safe and promising drug candidate for the treatment of PD, which still needs to be validated in further animal models and clinical trials.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to the Cell Resource Center of the Chinese Academy of Medical Sciences for providing the BV-2 cell line and to the Cell Bank of the Chinese Academy of Sciences for providing the SH-SY5Y cell line. Special thanks are extended to all laboratory members for their technical assistance and valuable discussions throughout the research.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData curation: Mengfei Sun, Ziqi Zhang.\u003c/p\u003e\n\u003cp\u003eFormal analysis: Mengfei Sun, Ziqi Zhang.\u003c/p\u003e\n\u003cp\u003eInvestigation: Ziqi Zhang, Mengfei Sun.\u003c/p\u003e\n\u003cp\u003eMethodology: Ziqi Zhang, Mengfei Sun.\u003c/p\u003e\n\u003cp\u003eSupervision: Jingfeng Ouyang\u003c/p\u003e\n\u003cp\u003eWriting – original draft: Ziqi Zhang.\u003c/p\u003e\n\u003cp\u003eWriting – review \u0026amp; editing: Jingfeng Ouyang.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during this study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGlobal, regional, and national burden of neurological disorders, 1990-2016: a systematic analysis for the Global Burden of Disease Study 2016. 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Front Cell Neurosci. 2021;15: 736310. https://doi.org/10.3389/fncel.2021.736310\u003c/li\u003e\n\u003cli\u003eXie Y, Qin Y, Wang J, Xu Z, Chen L, Kuang Y, et al. Tinosinenside A inhibits neuroinflammation and protects HT22 cells by suppressing the TLR4/NF-\u0026kappa;B/NLRP3 signaling pathway in BV2 cells. Naunyn Schmiedebergs Arch Pharmacol. 2025 [cited 18 Mar 2025]. https://doi.org/10.1007/s00210-025-03828-2\u003c/li\u003e\n\u003cli\u003eFiebich BL, Batista CRA, Saliba SW, Yousif NM, de Oliveira ACP. Role of Microglia TLRs in Neurodegeneration. Front Cell Neurosci. 2018;12: 329. https://doi.org/10.3389/fncel.2018.00329\u003c/li\u003e\n\u003cli\u003eChung LY-R, Lin Y-T, Liu C, Tai Y-C, Lin H-Y, Lin C-H, et al. Neuroinflammation Upregulated Neuronal Toll-Like Receptors 2 and 4 to Drive Synucleinopathy in Neurodegeneration. Front Pharmacol. 2022;13: 845930. https://doi.org/10.3389/fphar.2022.845930\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7532665/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7532665/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eParkinson's disease (PD) is a neurodegenerative disease that affects mainly middle-aged and elderly people, and its pathogenesis has not been clarified. To model neuroinflammatory components of PD in vitro, lipopolysaccharide (LPS)-induced inflammatory injury in BV-2 microglial cells was employed. Wogonin is a natural bioflavonoid extracted from the rhizome of the Chinese herb \u003cem\u003eScutellaria baicalensis\u003c/em\u003e. It has a neuroprotective effect and can play a role in alleviating the symptoms of neurodegenerative diseases. The aim of this study was to investigate the mitigating effect of wogonin on lipopolysaccharide-induced inflammation in BV-2 cells and the protective effect on SH-SY5Y cells by an experimentally validated method. The CCK-8 assay was used to detect the cell viability of each group. Enzyme-linked immunosorbent assay (ELISA), immunohistochemical staining (IHC), immunofluorescence staining (IF) and Western blot methods (WB) were used to detect the cell pathway indicators and inflammatory factors in each group. The results showed that LPS (1 \u0026micro;g/mL) induced polarization and activation of BV-2 cells and significantly increased the release of pro-inflammatory factors IL-6, TNF-α, and IL-1β, while decreasing the expression of tyrosine hydroxylase (TH) and promoting the aberrant aggregation of α-synaptic nucleoprotein (α-Syn) in SH-SY5Y neurons. After intervention with wogonin (16 \u0026micro;M), the above pathological processes were effectively reversed: inhibition of inflammatory factor secretion in BV-2 cells, restoration of TH expression in SH-SY5Y neurons, reduction of α-Syn deposition, and reduction of NF-κB p65 nuclear translocation and activation of the TLR4/MyD88 pathway. The effect was comparable to that of a TLR4 inhibitor (TAK-242), but the combination of the two did not show a synergistic effect. The study suggests that wogonin may inhibit LPS-induced release of inflammatory factors from BV-2 microglia and protect SH-SY5Y cells by regulating the TLR4/MyD88/NF-κB signaling pathway.\u003c/p\u003e","manuscriptTitle":"Exploring the mechanism of Wogonin in the attenuation of LPS-induced inflammation in BV-2 cells and the protective effect of wogonin on SH-SY5Y cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-21 10:15:45","doi":"10.21203/rs.3.rs-7532665/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8fd644be-2241-4ecb-88f0-78f6abd33dcf","owner":[],"postedDate":"October 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":56563694,"name":"Biological sciences/Cell biology"},{"id":56563695,"name":"Health sciences/Diseases"},{"id":56563696,"name":"Biological sciences/Immunology"},{"id":56563697,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2025-11-18T04:38:32+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-21 10:15:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7532665","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7532665","identity":"rs-7532665","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}