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 |