Aβ1-42 promotes microglial activation and apoptosis in the progression of AD by binding to TLR4

doi:10.21203/rs.3.rs-4894942/v1

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Aβ1-42 promotes microglial activation and apoptosis in the progression of AD by binding to TLR4

https://doi.org/10.21203/rs.3.rs-4894942/v1

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Alzheimer's disease (AD) is one of the most common age-related neurodegenerative diseases and the most devastating form of senile dementia. It has a complex mechanism and no effective treatment. Exploring the pathogenesis of AD and providing ideas for treatment can effectively improve the prognosis of AD. Microglia were incubated with β-amyloid protein 1–42 (Aβ1–42) to construct an AD cell model. After microglia were activated, cell morphology changed, the expression level of inflammatory factors increased, cell apoptosis was promoted, and the expression of Tau protein and related proteins increased. By up-regulating and down-regulating Toll-like receptor 4 (TLR4), the cells were divided into Lv-NC group, Lv-TLR4 group, Sh-NC group, and Sh-TLR4 group. The expression of inflammatory factors was detected again. It was found that compared with the Lv-NC group, the expression of various inflammatory factors in the Lv-TLR4 group decreased, cell apoptosis was inhibited, and the expression of Tau protein and related proteins decreased. Compared with the Sh-NC group, the expression of inflammatory factors in the Sh-TLR4 group increased, cell apoptosis was promoted, and the expression of Tau protein and related proteins increased. These results indicate that Aβ1–42 may promote microglial activation and apoptosis by binding to TLR4. Reducing the expression of TLR4 can reduce the occurrence of inflammatory response in AD cells and slow down cell apoptosis. Therefore, TLR4 is expected to become a new target for the prevention and treatment of AD.

Biological sciences/Neuroscience/Cell death in the nervous system

Biological sciences/Neuroscience/Diseases of the nervous system

Biological sciences/Neuroscience/Synaptic transmission

Currently, the three major pathological features of Alzheimer's disease (AD) are: β-amyloid protein (Aβ) deposition, microtubule-associated protein (Tau protein) phosphorylation, and neurodegeneration[1]. In recent years, the correlation between neuroinflammatory response and AD has attracted the attention of the medical science community. Studies have shown that neuroinflammation depends on microglia in the innate immune system and participates in the entire development process of AD[2]. Among them, TLR4 (Toll-like receptor 4) is an important mediator for regulating inflammatory factors[3], but its specific mechanism of action on the activation and apoptosis of microglia is still unclear.

Many anti-AD drugs in clinical practice have a narrow spectrum of action and large toxic side effects. They can only relieve symptoms to a certain extent and cannot affect the course of AD. They are also expensive[4]. Therefore, by constructing an AD cell model, it is more helpful to explore new drugs for treatment while exploring the pathogenesis of AD. Among them, amyloid β-protein 1–42 (Aβ1–42) has unique characteristics such as solubility, high molecular weight, and helicity[5], which has been recognized by many scholars. Studies have shown that the mouse model constructed by Aβ1–42 can better simulate the pathological phenomena of AD patients[6]. Studies have shown that Aβ aggregation activates microglia, leading to the activation of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasomes, and subsequently promoting the expression of inflammatory factors such as IL-1β and TNF-α[7]. In addition, aggregated Aβ can activate complement, leading to synaptic modification and loss of microglial phagocytosis[8]. Innate immune responses can activate microglia in the brain, leading to increased expression of TLR4, NLRP3 inflammasomes, and pro-apoptosis-related proteins, resulting in neuroinflammation, Aβ accumulation, synaptic loss, and neurodegeneration[9]. The interaction between TLR4, complement, and inflammasome signaling pathways can lead to the occurrence of autoimmune responses in brain cells of AD patients[10]. Therefore, inhibiting the expression of TLR4 is expected to be a preventive and therapeutic approach for AD.

In this study, at the cellular level, an AD cell model was constructed by culturing BV2 cells with Aβ1–42. The expression levels of related inflammatory factors and proteins were verified by knocking down and overexpressing the TLR4 gene, thereby exploring the mechanism by which Aβ1–42 promotes microglial activation and apoptosis in AD by binding to TLR4, providing new therapeutic ideas for the prevention and clinical treatment of AD.

Detection of related indicators after Aβ1–42 induced BV2 cells

Effects of Aβ1–42 on cell morphology and cell viability

Microscopic observation: After incubating BV2 cells with Aβ1–42 for 24 hours, the cells became ameba-like as the concentration increased, showing enlarged cell bodies, shortened or even disappeared protrusions, and rod-shaped or round cell morphology (Fig. 1). As the action time prolonged, the cell morphology became worse. After 48 hours of Aβ1–42 action, the cell fragments increased, the cells died in pieces, and the formed cells were less than 50% (Fig. 2). Therefore, the subsequent experiments used 24-hour related detection. Compared with the control group, the cell viability decreased significantly and was dose- and time-dependent. After Aβ1–42 acted on BV2 cells for 24 hours, the difference in cell viability was statistically significant from the Aβ1–42 concentration of 1µmol/L (P < 0.05, Fig. 3A), and gradually decreased with the increase of concentration. When the concentration of Aβ1–42 was 5 µmol/L, the difference in cell viability was statistically significant from 16 h after Aβ1–42 acted on BV2 cells (P < 0.05, Fig. 3B), and gradually decreased with time.

Effect of Aβ1–42 on apoptosis of BV2 cells

Compared with the blank control group, the early apoptosis rate of cells in each Aβ1–42 (0, 1, 2, 2.5, 5, 10) µmol/L group increased, and increased with the increase of Aβ1–42 concentration (P < 0.05,Fig. 4)

Effects of Aβ1–42 on the expression of inflammatory factors in BV2 cells

ELISA results showed that as the concentration of Aβ1–42 increased, the levels of IL-1β, TNF-α, IL-6, and IL-18 in BV2 cells increased significantly. When the concentration of Aβ1–42 was < 5 µmol/L, inflammation The expression of factors was dose-dependent, and compared with the control group, the expression of each inflammatory factor at a concentration > 1 µmol/L was significantly increased (P < 0.05, Fig. 5).

The effect of A β 1–42 on TLR4 and related inflammatory factors in BV2 cells

RT-PCR results showed that compared with the Aβ1–42 concentration of 0 µmol/L, the mRNA expression of NLRP3, TLR4, caspase1, Bcl-2, and Bax significantly increased when the Aβ1–42 concentration was 5 µmol/L (P< 0.05, Fig. 6), the mRNA expression of Bcl-2 was significantly reduced (P < 0.05, Fig. 6). .( The primer sequences are shown in Table 3, and the fluorescence quantification program is shown in Table 4)

Effects of Aβ1–42 on Tau protein-related indices in BV2 cells

The Western blotting results showed that compared with the Control group, the expression of AT8, Tau, P-Tau, and GSK-3βproteins was significantly enhanced in the Aβ1–42 group(P < 0.05,Fig. 7). This indicates that A β 1–42 can promote an increase in the expression levels of Tau protein and phosphorylated protein (Table 1)

Table 1 Differences in protein expression between blank group and model group (X ± S)

group	AT8	Tau	P-Tau	GSK-3β
blank group	0.86 ± 0.01	0.88 ± 0.01	0.97 ± 0.01	0.86 ± 0.12
model group	1.08 ± 0.02	1.04 ± 0.01	1.09 ± 0.01	1.26 ± 0.01
t	10.631	8.762	11.013	12.215
p	<0.001	<0.05	<0.001	<0.001

Aβ1–42 induced BV2 cells to construct an AD cell model, and the detection of related indicators after constructing TLR4 knockdown and overexpression cell models by lentiviral transfection

Lentiviral model construction and validation

According to the results shown in Fig. 8(a.), MOI = 150 and HitransGP infection enhancement solution (25x) were finally used as infection conditions for subsequent experiments. Lentivirus was used to construct TLR4 knockdown and overexpression BV2 stably transfected cell lines induced by Aβ1–42, and RT-PCR and Westernbloting techniques were used to verify the transfection efficiency of TLR4 knockdown and overexpression lentivirus at the mRNA and protein levels. After knockdown/overexpression, the mRNA and protein expression levels of TLR4 were significantly down-regulated/increased [P < 0.05, Fig. 8(b→c)].

Effects of lentiviral transfection on cell apoptosis

Compared with NC-Lv, the early apoptosis rate of cells in the Lv-TLR4 group was significantly decreased, and compared with the NC-Sh group, the early apoptosis rate of cells in the Lv-TLR4 group was significantly increased (Fig. 9,P < 0.05).

Effects of lentiviral transfection on inflammatory factors in each group of cells

The ELISA detection results showed that compared with NC Lv, the levels of IL-1 β, TNF - α, IL-6, and IL-18 in the Lv-TLR4 group were significantly decreased (P < 0.05, Fig. 12); Compared with the NC-Sh group, the levels of IL-1 β, TNF - α, IL-6, and IL-18 were significantly increased in the Sh-TLR4 group (P < 0.05, Fig. 10).

The effect of lentiviral knockdown on TLR4 and related inflammatory factors in AD cells

The RT-PCR results showed that compared with NC Lv, the mRNA expression levels of NLRP3, TLR4, caspase-1, and Bax in the Lv-TLR4 group were significantly decreased (P < 0.05), while the mRNA expression level of Bcl-2 was significantly increased (P < 0.05); Compared with the NC-Sh group, the mRNA expression levels of NLRP3, TLR4, caspase-1and Bax in the Sh-TLR4 group were significantly increased (P < 0.05), while the mRNA expression level of Bcl-2 was significantly decreased (P < 0.05, Fig. 11).(The primer sequences are shown in Table 3, and the fluorescence quantification program is shown in Table 4)

Effects of lentiviral transfection on Tau protein-related indicators in AD cells

The WB results showed that compared with NC Lv, the expression of ASC, Caspase-1, AT8, Tau, P-Tau, PP2A, and GSK-3β proteins was significantly decreased in the Lv-TLR4 group (P < 0.05); Compared with the NC-Sh group, the Sh-TLR4 group showed significantly increased expression of ASC, Caspase-1, AT8, Tau, P-Tau, PP2A, and GSK-3 β proteins (P < 0.05, Fig. 12). This suggests that A β 1–42 may cause Tau protein phosphorylation by binding to TLR4 (Table 2)

Table 2

Differences in target protein expression between negative control group and transfection group(x̄± S)
group	AT8	Tau	P-Tau	GSK-3β
NC-TLR4	0.81 ± 0.01	0.82 ± 0.01	0.77 ± 0.02	0.94 ± 0.02
Lv-TLR4	0.44 ± 0.01	0.26 ± 0.01	0.57 ± 0.02	0.37 ± 0.01
t	10.829	13.192	9.762	12.615
p	<0.01	<0.01	<0.01	<0.001
NC-Sh	0.74 ± 0.01	0.72 ± 0.01	0.87 ± 0.01	0.85 ± 0.01
Sh-TLR4	1.00 ± 0.06	0.88 ± 0.02	1.25 ± 0.01	1.15 ± 0.01
t	11.197	7.542	13.122	10.384
p	<0.001	<0.05	<0.001	<0.001

AD is a progressive, irreversible neurodegenerative disease with cognitive impairment as its main clinical manifestation. Its etiology and pathogenic mechanism are complex and unclear. There is currently no effective treatment. It not only seriously affects the quality of life and survival of patients[11], but also imposes a heavy economic burden on society and families[12]. Therefore, research on the pathogenic mechanism of AD is of great significance. At present, there is still controversy about the pathogenic mechanism of AD, but most people still tend to favor the "β-amyloid protein cascade hypothesis"[13]. Studies have shown that neuroinflammation plays an important role in the occurrence and development of AD[14], among which neuroinflammation caused by the activation of microglia in the central nervous system may play a dominant role[15].

Microglia are innate immune cells in the central nervous system. After brain tissue is stimulated, microglia will show two extreme phenotypes of polarization, the pro-inflammatory phenotype M1 and the anti-inflammatory M2 phenotype. This in turn causes a series of inflammatory reactions, leading to damage to nerve cell function and even nerve cell apoptosis [16]. The activation of microglia is a continuously progressing process, first manifested by an increase in the number of cells, followed by changes in morphology. Zhu M[17] et al. found in experiments that after BV2 cells were induced by Aβ1–42 incubation, the cells appeared similar to In the amoeba-like morphology, the cell body enlarges and the processes become shorter or even disappear, which is consistent with the results of this experimental study. Experiments have found that injecting Aβ1–42 into the hippocampus of mice can induce microglia activation and polarization toward M1 type, leading to a series of inflammatory reactions and significant effects on the central nervous system [18][19]. In this study, after using Aβ1–42 to induce BV2 cells to construct an AD cell model, the expression of IL-1β, IL-18, TNF-α, IL-6 inflammatory factors and the expression of NLRP3, TLR4, caspase- 1. The results show that the mRNA levels of Bcl2, Bax and other indicators are positively correlated with the concentration of Aβ1–42, suggesting that Aβ1–42 induces BV2 cells to polarize towards the M1 type and leads to an increase in the expression of inflammatory factors. At the same time, WB was used to detect the expression of apoptosis-related proteins, Tau protein and Tau protein phosphorylation-related proteins. The results showed that the above indicators increased to varying degrees. Combined with the flow cytometry results, it was suggested that the activation of microglia is through binding to TLR4 receptors. body, leading to the production of inflammatory factors such as NLRP3, and at the same time promoting the production of pro-apoptotic genes, Tau protein aggregation, and Tau protein hyperphosphorylation, thereby damaging nerve cells and leading to cell apoptosis.

Studies have shown that the TLR4/NF-κB signaling pathway mediates Aβ1-42-induced microglial activation[20], This is consistent with the results of this study. The use of TLR4 inhibitors can reduce the expression of the M1 marker inducible nitric oxide synthase (iNOS) [20]. In this study, TLR4 was knocked down by lentiviral transfection and it was found that compared with the model group, the apoptosis rate and inflammatory factor expression of the knockdown group were significantly decreased, and the expression of inflammatory factors such as NLRP3, Tau protein and Tau protein phosphorylation-related proteins were significantly decreased. In the subsequent reverse verification of TLR4 overexpression, the detection of the above indicators once again confirmed that Aβ1–42 induced microglial activation by binding to TLR4 receptors, leading to NLRP3 inflammasome activation and the production of proinflammatory factors, thereby affecting the progression of Alzheimer's disease.

In summary, inhibiting the expression of TLR4 can inhibit the activation of microglia induced by Aβ1–42, thereby inhibiting the activation of NLRP3 inflammasome and the production of inflammatory factors, inhibiting the apoptosis of neuronal cells, abnormal aggregation and hyperphosphorylation of Tau protein, and helping to alleviate neurofibrillary tangles, thereby achieving the effect of delaying and improving the progression of AD, providing new targets and ideas for the treatment of AD.

Cells, reagents and instruments

BV2 cells (Procell), Aβ1–42 (chinapeptidesCO., Ltd), lentivirus (Jikai Gene), DMEM high glucose medium, fetal bovine serum, BV2 cell-specific medium, 0.25% trypsin solution (containing EDTA, phenol red dissolved in PBS), PBS buffer, cell freezing solution (Procell); TBgreen (Takara); ELISA kits (IL-1β, IL-18, IL-6, TNF-α), color rapid gel preparation kit 10%, Western-specific primary and secondary antibody dilution solution, ultrasensitive ECL chemiluminescence kit, protein loading buffer, protein-free rapid blocking solution, mouse anti-GAPDH monoclonal antibody, BCA protein quantification kit (Boster); RNA extraction kit, protein electrophoresis maker (Polymer); AnnexinⅤ-FITC apoptosis detection kit purchased from (Elabscience); Methyl sulfone (DMSO) (Solaibao); hexafluoroisopropanol (HFIP) (Beijing Mairuida); reverse transcription kit, RIPA lysis buffer, phosphorylation protease inhibitor (Service-Bio); enhanced CCK-8 detection kit (Biyuntian); ECL ultra-sensitive luminescent liquid (Pumei Biotechnology); glycogen synthase kinase (GSK-3β), PTau, Tau and GAPDH antibodies (Abcam); AT8 antibody (Wuhan Sanying); HRP-labeled goat anti-rabbit secondary antibody (Abmart); RT-PCR detection primers (Sangon Biotechnology, high-speed refrigerated centrifuge, electrophoresis instrument, transfer instrument, microplate reader, PCR instrument, WB exposure instrument (BioRad); CO2 cell culture incubator (Likang Biotechnology); flow cytometer (BD); cell clean bench (Sujing Antai), -80℃ refrigerator (ThermoScientific), -20℃ refrigerator (Siemens), 4℃ refrigerator (Haier), ice maker (SANYO)

Cell grouping and processing

BV2 cells were incubated with different concentrations of Aβ1–42 (0, 1, 2, 2.5, 5, 10) µmol/L to construct an AD cell model. After that, the cells seeded in a T25 culture flask were gently washed with pre-cooled PBS, digested with trypsin, and centrifuged at 1000×r/min for 5 min to collect the cells for subsequent detection.

CCK-8 method

BV2 cells in the logarithmic growth phase were collected and inoculated in a 96-well plate at a standard of 3×103/well. After conventional culture for 24 hours, the cells were divided into groups for drug treatment. The concentrations of Aβ1–42 were 0, 1, 2, 2.5, 5, and 10 µmol/L, and conventional culture was continued for 24 hours and 48 hours. The cell morphology was observed under a microscope and photographed. After that, the old culture medium was removed in the clean bench, and new culture medium containing 10% (volume fraction) CCK8 solution was added. The cells were incubated in a conventional incubator in the dark for 2 hours. The absorbance value (A) at 450nm was detected by an enzyme marker, and the average value of each duplicate well was taken. The cell survival rate was calculated according to the following formula.

Cell viability =\(\:\frac{\text{A}-\text{d}\text{o}\text{p}\text{e}\text{d}\:-\:\text{A}-\text{b}\text{l}\text{a}\text{n}\text{k}}{\text{A}-\text{d}\text{o}\text{p}\text{e}\text{d}\:-\:\text{A}-\text{b}\text{l}\text{a}\text{n}\text{k}}\)×100%

Flow cytometry

BV2 cells in the logarithmic growth phase were collected and seeded in six-well plates at a density of 1×105 cells/2 ml per well. After the cells adhered to the wall, different concentrations of Aβ1–42 (0, 1, 2, 2.5, 5, and 10 µmol/L) were added and routine culture was continued for 24 h. After that, the cells were collected and FITC/PI staining was performed according to the operating instructions of the apoptosis detection kit, and cell apoptosis was detected by flow cytometry.

ELISA method

The cells inoculated in the 6-well plate were gently washed with pre-cooled PBS, then digested with trypsin, centrifuged at 1000 × r/min for 5 min, and the cells were collected. The operation was strictly followed the instructions of the ELISA kit.

RT-PCR technology

Cells were collected and the RNA extraction kit was strictly followed. The purity of the extracted RNA samples was determined. The samples were diluted in proportion and then reverse transcribed. The samples were added to the eight-tube strip according to the reaction system of 20 µL. The primer sequences are shown in Table 3 and the fluorescence quantitative program is shown in Table 4. The target gene and internal reference Ct values were obtained by RT-PCR, and then the relative expression was calculated by the 2-△△Ct method.

Table 3

.Primer sequence information
Target gene	Primer sequences
TLR4	F-TGAGGACTGGGTGAGAAATGAGC
TLR4	R-CTGCCATGTTTGAGCAATCTCAT
Caspapse-1	F-AAAGACAAGCCCAAGGTGATC
Caspapse-1	R-CCAAGTCACAAGACCAGGCATA
ASC	F-CAGCACAGGCAAGCACTCATT
ASC	R-TCATCTTGTCTTGGCTGGTGG
NLRP3	F-ATTACCCGCCCGAGAAAGG
NLRP3	R-TCGCAGCAAAGATCCACACAG
GAPDH	F-CCTCGTCCCGTAGACAAAATG
GAPDH	R-TGAGGTCAATGAAGGGGTCGT
Note: TLR4, Toll-like receptor 4; Caspase-1, cysteine protease-1; NLRP3, NOD-like receptor pyrin domain-associated protein 3; Bcl-2, Bcl-2; Bax, Bcl-associated X protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Table 4

Fluorescence quantification program
Two-step PCR amplification standard procedure
Stage 1: Pre-denaturation Reps: 1 95℃ 30s Stage 2: PCR reaction Reps: 40 95℃ 5s 60℃ 34s

WB method

BV2 cells in the logarithmic growth phase were inoculated into 96-well plates at 3×103 cells per well. After culturing for 24 hours under normal conditions, 5 µmol/L Aβ1–42 was added to the cells for 24 hours after the cells adhered to the wall to construct the AD cell model. The cells were collected, and then 350 µL of RIPA lysis buffer and phosphorylation protease inhibitors (1 ml RIPA lysis buffer: 10 µL phosphorylation protease inhibitors) were added to each well. The cells were lysed on ice for 30 minutes, and centrifuged at 13000×r/min in a precooled 4°C low-temperature ultracentrifuge for 15 minutes. After centrifugation, the supernatant was aspirated as a protein sample. The protein concentration was determined by the BCA method. After the sample was diluted with ultrapure water, an appropriate proportion of loading buffer was added and boiled in a 100°C metal bath for 10 minutes. The boiled samples can be stored in a -80°C refrigerator for a long time. The expression level of the target protein was then detected by WB.

Lentivirus infection pilot test

BV2 cells in the logarithmic growth phase were inoculated into a 96-well plate at 3×103 cells per well and cultured under normal conditions for 24 hours. After the cells adhered to the wall, 5 µmol/L Aβ1–42 was added and incubated for 24 hours to construct an AD cell model. A lentiviral titer gradient was set to explore the optimal cell infection conditions and the appropriate MOI value, MOI=(virus titer×virus volume)/cell number.

Statistical methods

FlowJo10.8.1 software was used to analyze the flow cytometry data, ImageJ software was used to analyze the grayscale of WB bands, and GraphpadPrism10 statistical software was used to analyze the data. One-way analysis of variance was used for comparison between groups, and Tukey-t test was used for comparison between two groups.

Competing interests

The authors declare no competing interests.

Funding

This research was funded by the Science and Technology Innovation 2030-"Brain Science and Brain-like Research" major project and the Gansu Provincial Science and Technology Plan Project.

Author Contribution

Yi Zhang and Cheng Gu designed the study. Rui-xia Dou completed the experiments. Rui-xia Dou, Lu-lu Zhang, and Yun-hua Liang completed the data processing, icon production, paper writing, and revised the manuscript. Yi Zhang, Cheng Gu, and Rui-xia Dou revised the manuscript for important intellectual content. All authors contributed to and approved the final version of the manuscript.

Data Availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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No competing interests reported.

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Aβ1-42 promotes microglial activation and apoptosis in the progression of AD by binding to TLR4

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Version 1

Abstract

Figures

Introduction

Results

Detection of related indicators after Aβ1–42 induced BV2 cells

Effects of Aβ1–42 on cell morphology and cell viability

Effect of Aβ1–42 on apoptosis of BV2 cells

Effects of Aβ1–42 on the expression of inflammatory factors in BV2 cells

The effect of A β 1–42 on TLR4 and related inflammatory factors in BV2 cells

Effects of Aβ1–42 on Tau protein-related indices in BV2 cells

Lentiviral model construction and validation

Effects of lentiviral transfection on cell apoptosis

Effects of lentiviral transfection on inflammatory factors in each group of cells

The effect of lentiviral knockdown on TLR4 and related inflammatory factors in AD cells

Effects of lentiviral transfection on Tau protein-related indicators in AD cells

Discussion

Conclusions

Materials and methods

Cells, reagents and instruments

Cell grouping and processing

CCK-8 method

Flow cytometry

ELISA method

RT-PCR technology

WB method

Lentivirus infection pilot test

Statistical methods

Declarations

Competing interests

Funding

Author Contribution

Data Availability

References

Additional Declarations

Supplementary Files

Status:

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