Glabridin Reduces Neuroinflammation by Modulating Inflammatory Signals in LPS-induced in Vitro and in Vivo Models

doi:10.21203/rs.3.rs-3334720/v1

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Research Article

Glabridin Reduces Neuroinflammation by Modulating Inflammatory Signals in LPS-induced in Vitro and in Vivo Models

https://doi.org/10.21203/rs.3.rs-3334720/v1

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published 19 Feb, 2024

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Objectives

Chronic neuroinflammation has become one of the important causes of common neurodegeneration disease. Therefore, the target of this study was to explore the protective action of glabridin on lipopolysaccharide (LPS)-induced neuroinflammation in vivo and in vitro and its mechanism.

Methods

The neuroinflammation model was established by LPS-induced BV2 cells. The cell viability with various concentrations of glabridin was determined by MTT assay, and the content of NO in each group was detected. A neuroinflammatory model was established in male C57BL/6J mice for water maze test. Subsequently, NF-κB and SOD indices were measured by ELISA, GFAP and IBA-1 indices were measured by immunofluorescence, and Nissl staining was used to explore the Nissl bodies in the hippocampus of mice.

Results

In vitro experiments, our results expressed that glabridin could markedly increase the cell activity of LPS-induced BV2 cells and reduce the NO expression in cells. It indicated that glabridin had a remarkable impact on the neuroinflammation of LPS-induced BV2 cells protection. In vivo neuroinflammation experiments, mice treated with different doses of glabridin showed significantly improved ability of memory compared with the LPS group in the Morris water maze test. The levels of NF-κB, GFAP, and the number of positive cells in Nissl staining were decreased. High-dose glabridin significantly increased the SOD content in the brain tissue and decreased the IBA-1 levels.

Conclusion

Glabridin can significantly reduce or even reverse LPS-induced neuroinflammation, which may be related with the fact that glabridin can reduce the NO expression, NF-κB, IBA-1, GFAP, and other inflammatory mediators, upregulate the expression of SOD to relieve oxidative stress of brain, and inhibit the activation of gliocyte in brain tissue.

Neuroinflammation

Glabridin

Lipopolysaccharide (LPS)

Cognitive impairment

BV2 cell

Neuroinflammation occurs more often in the central nervous system (CNS) and involves multistage physiological, complex responses triggered by nerve cell destruction in the brain, including infections, toxins, autoimmune activity, trauma, and processes that alter the neuronal activity (Carro et al., 2017; Tan et al., 2020). Studies have pointed out that neuroinflammation refers to the pathogenesis of Alzheimer's disease (AD), Parkinson's disease (PD), Depressive disorder (DD), and other common neurodegenerative diseases. The impairment of learning and memorial function and emotional dysfunction ascribed these neurodegenerative diseases have serious harm to patients and society. Neuroinflammation is caused mainly by active forms of microglia and astrocytes, both of which are attached to CNS immune glia with a dynamic biochemical cascade of inflammatory factors that alter the CNS microenvironment(Giovannoni & Quintana, 2020; Kwon & Koh, 2020; Silver, Schwab, & Popovich, 2014).

Patients with Alzheimer’s disease are possibilities that have higher endotoxin lipopolysaccharide (LPS) levels in the brains, which has been related to neuroinflammation and cognitive abilities decline (Leng & Edison, 2021; Y. Liu et al., 2018; Zhan, Stamova, & Sharp, 2018). The cholinergic drug galantamine is effective in mitigating the neuroinflammation and cognitive abilities decline induced by LPS (El-Saber Batiha, Magdy Beshbishy, El-Mleeh, Abdel-Daim, & Prasad Devkota, 2020; G. Li, Nikolic, & van Breemen, 2016). Additionally, cannabinoids may mediate some of the effects of anti-inflammatory by altering immunocyte metabolism ability and inducing autophagy (van Niekerk, Mabin, & Engelbrecht, 2019), and can also reduce inflammation by silencing small-diameter neurons (McKenna & McDougall, 2020; Rajesh, Mukhopadhyay, Hasko, & Pacher, 2008). Although studies have shown that non-steroidal anti-inflammatory drugs (NSAIDs) can remarkably mitigate the relative risk of dementia, long-term use of NSAIDs may lead to side effects such as gastrointestinal reactions and liver function damage. Therefore, the treatment of neuroinflammation is still a very challenging task.

Traditional herbal remedies are potential alternative therapies for treating a wide range of diseases (Yang et al., 2017). Some natural products are considered to have less side effects and better effects in the treatment of neuroinflammation than synthetic drugs. Consequently, medicinal plants have become increasingly widely accepted (Rao et al., 2019). Licorice glabridin (1,3-Benzenediol-,4-(3,4-dihydro-8,8-dimethyl-2h,8h-benzo[1,2-b:3,4-b'] dipyran-3-yl)-,(R)-), a licorice flavonoid, is a chief active substance extracted from licorice. Glabridin has been used on food and diet and demonstrated significant anti-inflammatory and anti-atherosclerotic activities, estrogen-like effects, and the ability of energy metabolism regulation (Markina et al., 2022; L. Wang, Yang, Yuan, Liu, & Liu, 2015).

Numerous studies have demonstrated that glabridin has neuroprotective and other beneficial effects on degenerative neurological diseases (Karthikkeyan, Pervaje, Pervaje, Prasad, & Modi, 2021; C. Li, Eom, & Jeong, 2015; Simmler, Pauli, & Chen, 2013). Kang JS et al. found that Glabridin inhibited inducible nitric oxide synthase (iNOS) expression and NO production in RAW 264.7 cells and peritoneal macrophages,and in an animal model, glabridin attenuated LPS-induced septic shock, resulting in decreased amounts of NO and TNF-α in plasma (Jong Soon Kang et al., 2005). Jee Youn Kim et al. found that DCs as inflammatory target cells of glabridin, which blocked NF-κB and MAPK signalings to inhibit dendritic cell maturation, and inhibited the production of pro-inflammatory cytokines, including IL-12p40, TNF-α, and IL-1β (Kim et al., 2010). Yehuda et al. found that glabridin inhibited LPS-induced NO production, iNOS gene expression, and nitrotyrosine formation in macrophages, which exhibited potential antiinflammatory effects under hyperglycemic conditions (Yehuda, Madar, Leikin-Frenkel, & Tamir, 2015). However, the bioavailability of glabridin is low, whether oral formulations can improve memory and the mechanism is still dimness. This study we used LPS-induced mouse microglial cell lines BV2 and C57BL/6J mice to construct in vitro and in vivo models of neuroinflammation to study the anti-neuroinflammatory effect and mechanism of different doses of glabridin. Our research results indicate the ability of glabridin to reduce neuroinflammation and contribute ideas into this mechanism of action, revealing the latent of glabridin as a therapeutic substances for neuroinflammation.

2. 1. Materials

Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), Penicillin-Streptomycin Solution, tryptase, phosphate-buffered saline (PBS) were purchased from Gibco (Carlsbad, CA, USA). Methyl-thiazolyl-tetrazolium (MTT) was purchased from Beijing Solarbio Science & Technology Company (Beijing, China). Dimethyl sulfoxide (DMSO) was purchased from Damao (Tianjin, China). LPS was purchased from Thermo Fisher Scientific (Suzhou, China). T75 cell culture flasks and 96-well plates were purchased from NEST (Wuxi, China). Glabridin powder was purchased from Aladdin (Shanghai, China). Azeliragon (TTP488) was purchased from InvivoChem. A nitric oxide (NO) kit was purchased from Beyotime (Shanghai, China). The ELISA kit was purchased from the Enzyme Label Biotechnology Company (Jiangsu, China). Adult (18–22 g) male C57BL/6J specific pathogen-free (SPF) mice were obtained from the Guangdong Medical Laboratory Animal Center from Sun Yat-Sen University (SCXK2011-0029, Guangzhou, China). The animal experimental protocol of this project adheres to the principles of animal protection, animal welfare and ethics, and all the test steps were in strict accordance with the Guidelines for the Care and Use of Laboratory Animals. (The 7th edition, USA). Tissue homogenizer was purchased from Tiangen Biotech Co., Ltd., Beijing, China.

2. 2. Preparation of glabridin

Glabridin (molecular weight 324.37) powders (C₂₀H₂₀O₄, purity ≥ 99%) was dissolved into 10 mM stock solution with Sodium Carboxymethyl Cellulose (CMC) solution, filtered using a microporous membrane with 0.22 µm, and kept aseptically in a freezer at − 20°C.

2. 3. Cell culture

Complete medium was obtained by adding 1% penicillin-streptomycin and 10% FBS to DMEM. Cells were grew in a T75 culture flask, cells were added to the 12 mL of complete culture medium and cultured in an cell incubator (Unity Lab Services, USA) at a stable temperature of 37°C in 5% CO₂ atmosphere. The nutrient solution was changed once a day.

The culture flask was observed under a microscope (Chongqing Optec Instrument Company, LTD), and BV2 cells were digested by 0.25% trypsin for counting after they were fully grown. Further, cells were added to 96-well plates with the density of 1 × 10⁵ cells/well. 100 µg/ml LPS powder was dissolved in PBS (Nam et al., 2018; L. Wang et al., 2021), and 100 ng/mL DMEM was treated into each well. The cells were then reinserted to incubator for an additional 24 h under the same conditions as previously stated.

2. 4. Cell Viability Assay

Methyl-thiazolyl-tetrazolium (MTT) was used as an target of cell activity (Pascua-Maestro et al., 2018). Inoculated BV2 cells were placed in 96-well plates with the density of 5×10³ cells in each well and classified into blank group, control group, and five administration groups for the cytotoxicity detection of glabridin at five concentrations. A 96-well plate was placed for 24 h in a constant-temperature incubator. Remove sterile glabridin from the 4°C and dilute with DMEM to concentrations gradient of 0.05, 0.1, 0.2, 0.4 and 0.8 µM. After culturing for 24 h, the culture medium was taking away from each well, drugs at five different concentrations (100 µL) were added to each well according to the groups. The equal amount of DMEM was treated into the control group; cells were not cultured and not reagents were added into the blank group.

The 96-well plate was placed in a constant-temperature incubator for further culturing for 24 h. Subsequent the cell supernatant was removed and each well was added with 100 µL 0.5 mg/mL MTT in DMEM without FBS under dark conditions. After the cell culture plate was incubated for 4 h at 37°C, then MTT solution was took away. The cell supernatant was removed, and 150 µL DMSO was treated to each well. After even shaking, each well of the absorbance was estimate at 570 nm using microplate analyzer (Thermo Scientific Company, USA) (Zhang, Naguro, & Herr, 2019).

2. 5. Measurement of nitric oxide production

The BV2 cells were cultured in 96-well plate (100 µl per well) at a concentration of 5×10⁵ cells/ml and classified into blank group, control group, LPS group, and four administration groups. The model and administration groups were treated with LPS, and the control group was cultured normally with DMEM. The 96-well plates were placed in a constant-temperature incubator for 24 h, and the medium was removed from the wells. After the sterile glabridin was diluted to 0.05, 0.1, 0.15 and 0.2 µM concentrations with DMEM, the solution was added to the four administration groups. DMEM was added to the model and control groups. The blank group did not plant cells, and no reagents were added. The plate was then put into an incubator for 24 h. The plates were removed from the incubator and, and 50 µL of the supernatant from each group was absorbed by a new 96-well plate and added to the corresponding position of the new plate. After culturing in an incubator for 10 min, the absorbancy of each group was measured by microplate analyzer (Thermo Scientific Company, USA) at 562 nm.

2. 6. Animals and experimental design

Twenty-five C57BL/6J mice without specific pathogen were separated into five groups according to mass: (1) no treatment control group, (2) LPS group, (3) TTP488 group, (4) high-dose glabridin, and (5) low-dose glabridin groups. The control group was injected with saline, and the LPS group was injected with LPS at 250 µg/kg. The TTP488 group were administered 5 mg/kg TTP488 and LPS, the high-dose glabridin group received 100 mg/kg glabridin and LPS, and the low-dose glabridin group 50 mg/kg and LPS. Treatments were administered intragastrically for 14 days. Based on the weight data recorded on the mice before intragastric administration, the daily volume of intragastric administration was estimated, and the standard was 0.2 mL per 10 g (weight of mice). The experimental protocol was approved by the Experimental Animal Center of Guangdong Province (approval documents: SCXK/20130002), and the source of purchase is Guangdong Experimental Animal Center.

2. 7. Morris water maze test

The Morris water maze test experiment lasted for seven days and included a learning and memory stage (D1-D5), directional navigation stage (D6), and spatial exploration stage (D7). We conducted the experiment as previously described (Fotakis & Timbrell, 2006). Briefly, mice during the learning and memory phases were lay in quadrants of the water maze test system, and the water escape time was recorded as the incubation period. For memory training, the mice were kept on a platform for 20 s. During the directional navigation phase, the mice were placed in the quadrant farthest from the platform, and both the incubation period and time to escape on the platform reflected their memory ability. In the spatial exploration phase, the survival platform on D7 was took away. The mice were lay up in the same quadrant of water, and over the next 60 seconds, the repetition of passing through the former platform area and their movement tracks were recorded (Dinel et al., 2020; Vorhees & Williams, 2006).

2. 8. Immunofluorescence assays

After the water maze experiment, the eyeballs of each group of mice were removed and their blood was separately collected. When the mice died, their brains were completely took away and part of the hippocampus was removed. Part of the whole brain and hippocampus of mice were fixed with 4% PFA tissue fixation solution, and then 4µm thick paraffin sections were cut into. Sections were immersed in xylene I for 20 min and xylene II for 20 min for dewaxing and rehydration. Put slices into the repair box that full of citrate antigen retrieval solution (PH6.0), heat it in a microwave for 8 minutes to boil, then paused heating for 8 minutes to hold it warm and then turn it to a medium low temperature for 7 minutes. After natural cooling at room temperature, place the slide into PBS (PH7.4), shake then wash it by using the decolorization shaker (3 times, 5 minutes each time). The tissue of mice were incubated at 4℃ overnight with GABAAR antibody diluted at 1:60 after staining. After douching with phosphate buffered saline (PBS) for 3 times, sections were incubated at 25℃ for 1 h with secondary antibody diluted at 1:800 and protected from light. IgG (H + L) (Cy3 conjugated Goat Anti-Rabbit IgG (H + L)) and IgG (H + L) (FITC conjugated Goat Anti-Rabbit IgG (H + L)) were added to stain for 10 minutes and then rinsed three times with PBS.

2. 9. Nissl standing

Brain tissue samples were washed with the PBS (twice, 5–10 min), paraffin embedded, prepared 5 µM thick sections, centrifuged at 12000rpm for 10min. The tissues were immersed completely in xylene I for 20 min and xylene II for 20 min for dewaxing, heated to 60℃ and stained with 1% toluidine blue for 40 min. After rinsing with distilled water, dehydrate with graded ethanol (70, 95, 100%). The sections were then washed with xylene for 5 minutes. Tissue sections were placed in a repair box that full of citrate antigen retrieval solution (PH 6.0) in a microwave for antigen repair, with a medium heat of 8min to boiling, a hold fire of 8min followed by a medium low temperature of 7 min. After natural lowering the temperature, the slides were situated in PBS (PH 7.4) and washed three times with a decolorizing shaking bed for 5 min each time.

Images of normal neurons were collected and observed under a microscope (magnification × 400). Three non-overlapping and intact areas were irregularly selected for each tissue section. Reckoned the number of normal neurons and took their average value.

2. 10. ELISA assay

The remaining mouse brain tissue was collected, and nine times the weight of the protein lysate was added to make the brain homogenate by RIPA buffer. After centrifugation at 5000 rpm for 3 min, supernatant was collected and stored at − 20℃ in a freezer. Manufacturer instructions were followed to determine sum protein content by using the Beyotime BCA Protein Concentration Assay Kit. Afterward, we ensured that protein contents of all samples were diluted with RIPA buffer to the same level. We determined superoxide dismutase (SOD) and NF-κB levels using an HRP-ELISA kit (Enzyme Label Biotechnology Company, Jiangsu) following the company’s protocol.

2. 11. Statistical analysis

One-way analysis of variance (ANOVA) was followed by Dennett's multiple comparison test. Zen (Carl Zeiss AG, Oberkochen, Baden-Württemberg, Germany) was used to quantify the dynamic change process of phenotypic parameters of microglia and astrocytes and add scales. GraphPad Prism 7 (GraphPad Software, La Jolla, CA, USA) was used to compare data. Image-J (National Institutes of Health, Bethesda, MD, USA) was used to measure the number of stained positive cells and calculate the integral optical density of the slice. Data are showed as mean ± standard error. P-values < 0.05 were considered to have statistics significance.

3. 1. Assessing glabridin cell viability

After testing to get the IC₅₀ value of glabridin was 0.399 µM. The cytotoxicity test of the drug could reflect whether the drug itself showed toxic effect on cells and magnitude of the effect, which was convenient to eliminate interference in the analysis of the drug's effect on the LPS model (Judson et al., 2016). The results showed that 0.2 µM glabridin had obvious toxicity to cells.

NO is a pro-inflammatory signaling molecule in the nervous system that is released in large quantities under the regulation of BV2 cells when producing neuroinflammation; therefore, it is used as an important indicator to detect the degree of neuroinflammation. The NO content in the LPS group was conspicuously more than that in the control group (p < 0.05), demonstrating this model was effective. All four doses of glabridin significantly inhibited NO activity when compared with the LPS group, and in the chosen concentration range, the higher the concentration, the more obvious was the inhibitory effect, indicating that glabridin in the selected concentration range has a good protective effect on LPS- induced neuroinflammation. Glabridin at 0.2 µM concentration reduced the level of NO to close to that of the control group, but because that concentration was highly toxic, little NO was produced (Han et al., 2017).

3. 2. Water Maze experiment

In the directional navigation stage, the mice in the control group could get onto the platform faster every day, whereas the mice in the LPS group were either slower or unable to mount the platform (p < 0.01). The implication of these results is that LPS had a notable effect on the memory ability of mice. In this model, the TTP488 group performed similarly to the control group when in contrasted to the LPS group (p < 0.01), indicating that the TTP488 almost completely prevented LPS-induced memory impairment. There was barely obvious difference between the date of high-dose and low-dose groups, but the mice in both groups were obviously faster than those in the LPS treatment control (p < 0.05) (Dinel et al., 2020; Vorhees & Williams, 2006).

3. 3. Glabridin reduces NF-κB content and protects nerve cells from oxidative stress

NF-κB is a fundamental cellular transcription factor that mediates the inflammatory response. It can activate the transcriptional function of genes and specifically bind to inflammatory factor genes. Therefore, NF-κB is a crucial index that reflects the extent of inflammation (Chawla, Roy, & Basak, 2021; Yu, Lin, Zhang, Zhang, & Hu, 2020), and the lower the content of NF-κB, the lower is the degree of inflammation. In contrast to the control group, NF-κB activity in the LPS group was obviously decreased (p < 0.05). Contrast with that in the LPS group, the activity of NF-κB in the TTP488 control and high-dose glabridin groups was decreased obviously (p < 0.05), and the decrease was similar (J. S. Kang et al., 2006). These results indicated that the inhibitory effect of glabridin at these two concentrations on NF-κB secretion was similar to that of the TTP488.

Neuroinflammation may lead to oxidative stress injury in nerve cells, and SOD can effectively protect nerve cells from oxidative stress injury during inflammation. Therefore, the SOD content can reflect the effectiveness of neuroinflammation in protecting nerve cells (Z. Wang et al., 2020). Within a certain range, the higher the SOD content, the better the protective effect. The SOD content in the brain tissue of the LPS group was significantly decreased (p < 0.05) compared to that in the brain tissue of the control group. SOD content in the TTP488 control and high- dose glabridin groups significantly increased (p < 0.05), which contrast with in the LPS group,. However, high dose glabridin had the same obvious effect on the increase in SOD content in mice brain tissue as the TTP488 had, indicating that high dose glabridin can play a good protective effect on nerve cells in neuroinflammation as the TTP488.

3. 4. Glabridin restored the number of Nissl bodies in nerve cells

There are many Nissl bodies in the body of neurons, which are composed of basophilic clumps and particles. They are mainly used for protein synthesis in neurons, and analyzing their number can reflect the functional activity of nerve cells. We focused on the neural cone cells found in the cortex and hippocampus to assess neuronal damage. After staining these cells, we used an inverted fluorescent microscope to note the staining depth, cell membrane integrity, and nuclear clarity. In the LPS group, the number of Nissl bodies was decreased, the color depth was shallow, the cell membrane was porous, the nucleus was poorly defined, and the conical cells were sparsely arranged. However, opposite results were observed in the control group. After glabridin and TTP488 treatment, the number of Nissl bodies of nerve cells was restored to a certain extent, the staining was deepened, the arrangement was increased (p < 0.05), and the nuclei were clearly visible. The implication of these results is that the glabridin and TTP488s could reduce the degree of neuronal damage by inhibiting neuroinflammation.

3. 5. Glabridin plays an anti-inflammatory role by inhibiting the activation of astrocytes and microglia

Fluorescein was labeled on the corresponding antibody. The antibody was directly bound to the antigen, and the content of the detected index was reflected by the fluorescence intensity. Two indices, GFAP and IBA-1, were detected. Elevated levels of both factors are markers of microglia and astrocytes activation (Lafrenaye et al., 2020). The darker the CY3 (IBA-1) or FITC (GFAP), the more severe is the inflammation (Norden, Trojanowski, Villanueva, Navarro, & Godbout, 2016). As shown in Fig. 5B and C, the proteins expressions of IBA-1 and GFAP in LPS group were significantly more than the control group (p < 0.05), while the expressions proteins of IBA-1 and GFAP in high-dose glabridin group were significantly decreased (p < 0.05). It is suggested that brain inflammation was more serious in the LPS group and glabridin may play an anti-inflammatory role by the inhibition of activated astrocytes and microglia, indicating that the TTP488 drug and high- and low-dose glabridin groups could effectively reverse neuroinflammation.

The anti-inflammatory and neuroprotective effects of glabridin have long been verified (K. Liu et al., 2017), but the protective effectiveness and mechanism of glabridin on neuroinflammation are not comprehensive (Simmler et al., 2013). In this study, LPS was used to induce a neuroinflammatory response in BV2 cells and was injected intraperitoneally to generate a model of learning and memory impairment, to explore the effective protective effects and the potential mechanism of different concentrations of glabridin on BV2 cells. Results showed that the production of NO was effectually inhibited and the surviving-rate of BV2 cells under the influence of LPS was increased by glabridin. In mice with learning and memory impairment, glabridin also significantly improved and reversed LPS induced memory impairment and restored memory ability (Cui, Ao, Li, & Yu, 2008). Simultaneously, the changes in SOD and NF-κB contents in the mouse brain tissue showed that glabridin's protective mechanism on neurocyte may be related to the regulation of SOD content to alleviate oxidative stress injury or of inflammatory factors such as NF-κB to restore the brain microenvironment (Griciuc et al., 2013). In summary, glabridin has a protective effect on LPS-induced neuroinflammation and has great potential in the development of therapeutic drugs for neuroinflammation (Park et al., 2010).

ACKNOWLEDGEMENTS

This work was funded by the Guangdong Provincial Key Laboratory of Plant Resources Biorefinery (No. 2021GDKLPRB02) and the Basic and Applied Basic Research Foundation of Guangdong Province (No. 2020A1515110715).

CONFLICTS OF INTEREST

The authors declare no conflicts of interest in the publication of this paper.

All animal experiments are conducted in accordance with relevant government and institutional regulations on the ethical use of animals and are approved by the Experimental Animal Center of Guangdong Province (approval documents: SCXK/20130002).

AUTHORS CONTRIBUTIONS

Jiyu Weng: Research, digitize and write the first draft.

Ying Wang: Research, digitize and write the first draft.

Zekai Tan: Digitize, investigation and verification.

Yanghe Yuan: Data collation.

Shiyuan Huang: Investigation.

Zexi Li: Formal analysis.

Yiming Li: Data collation and modification.

Lanyue Zhang: Conceptualization, supervision, resources and capital acquisition.

Zhiyun Du: Conceptualization, methodology, supervision, review and proofreading.

DATA AVAILABILITY STATEMENT

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

ORCID

Lanyue Zhang https://orcid.org/0000-0002-5153-5473

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Glabridin Reduces Neuroinflammation by Modulating Inflammatory Signals in LPS-induced in Vitro and in Vivo Models

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Journal Publication

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Abstract

Objectives

Methods

Results

Conclusion

Figures

1. Introduction

2. Material & Methods

2. 1. Materials

2. 2. Preparation of glabridin

2. 3. Cell culture

2. 4. Cell Viability Assay

2. 5. Measurement of nitric oxide production

2. 6. Animals and experimental design

2. 7. Morris water maze test

2. 8. Immunofluorescence assays

2. 9. Nissl standing

2. 10. ELISA assay

2. 11. Statistical analysis

3. Results

3. 1. Assessing glabridin cell viability

3. 2. Water Maze experiment

3. 3. Glabridin reduces NF-κB content and protects nerve cells from oxidative stress

3. 4. Glabridin restored the number of Nissl bodies in nerve cells

3. 5. Glabridin plays an anti-inflammatory role by inhibiting the activation of astrocytes and microglia

4. Discussion

Declarations

References

Status:

Journal Publication

Version 1