Fig 2: TS treatment promoted Nrf2 to inhibit the expression of BACE1 in the brain of APP/PS1 mice. (A) The effect of TS on the survival rate of Aß-induced PC12 cells for 24 h was evaluated by MTT assay (#### p < 0.0001, **** p < 0.0001). (B) The effect of TS on Nrf2 and BACE1 proteins in the brain of APP/PS1 mice were detected by Western blot. (C) Quantitative analysis of the relative expression level of Nrf2 protein in each group (# p = 0.0195, * p = 0.0151). (D) Quantitative analysis of the relative expression level of BACE1 protein in each group (# p = 0.0343, *** p = 0.0008, *** p = 0.001). (E) The effect of TS on Nrf2 and BACE1 protein in Aß-induced PC12 cells were detected by Western blot. (F) Quantitative analysis of the relative expression concertation of Nrf2 protein in each group (# p = 0.0119, ** p = 0.0027, *** p = 0.0003). (G) Quantitative analysis of the relative expression concertation of BACE1 protein in each group (### p = 0.0003, ** p = 0.0016, *** p = 0.0003, *** p = 0.0002, *** p = 0.0001). (H) PC12 cells were transfected with Nrf2-siRNA and transfection reagent at 1:1 for 36h, and the expression of Nrf2 was detected by Western blotting. (I) Quantitative analysis of the relative expression level of Nrf2 protein in Nrf2-siRNA and ns-siRNA groups (* p = 0.0199). (J) The effect of TS on transcription the level of BACE1 in Nrf2-/- PC12 cells was investigated by qPCR (## p = 0.0093, #### p < 0.0001, * p = 0.0234, ++ p = 0.0030). (K) The effect of TS on the level of Nrf2 and BACE1 promoter binding was investigated by CHIP experiment (*** p = 0.0008). (L) The effect of TS on the level of Aß aggregation was detected by ThT fluorescence method (** p = 0.0058, *** p = 0.0003, *** p = 0.0002). #: Comparing with WT group; *: Compared with model group; +: Compared with ns siRNA group. Data are presented as mean ± SEM (n = 3).

Fig 3: DOR inhibition or MOR activation enhanced BACE1 activities in normal conditions, while DOR activation attenuated BACE1-mediated cleavage activities in Aß1–42 injury. BACE1 activities were examined at 30 min and 48 h after the drug treatment (N = 3 in each group). C: control. C + U: DOR was activated by UFP-512. C + U + N: PC12 cells were treated with UFP-512 plus naltrindole at the same time under normal conditions. C + N: DOR was inhibited by naltrindole. A: PC12 cells were treated with 20 µM Aß1–42 oligomer to mimic the AD injury. A + U: DOR was activated by UFP-512 under AD injury. A + U + N: PC12 cells were treated with UFP-512 plus naltrindole together under AD injury. A + N: DOR was inhibited by naltrindole under AD injury. (A) DOR-mediated regulation of BACE1 activities in the physiological condition. *p < 0.05, **p<0.01 vs. (C). Note that 30-min treatment with DOR agonist UFP-512 significantly decreased BACE1 activities, whereas the application of DOR antagonist naltrindole enhanced BACE1 activities at both 30 min and 48 h after the treatment. (B) Effects of DADLE and DAMGO on BACE1 activity in normal conditions. *p < 0.05 vs. (C). Note that DADLE did not induce any significant change in BACE1 activity, while MOR agonist DAMGO significantly upregulated BACE1 activities. (C) Effects of DOR agonist UFP-512 and antagonist naltrindole on BACE1 activity under AD injury. The PC12 cells were treated with 20 µM Aß1–42 oligomer for 48 h to mimic AD injury. UFP-512 and naltrindole were simultaneously or separately applied to these cells for 48 h or at the last 30 min of Aß1–42 oligomer exposure. *p < 0.05, **p < 0.01 vs. (A). Note that DOR activation by UFP-512 significantly reduced BACE1 activities, whereas its inhibition with naltrindole reversed such effect. (D) Effects of MOR activation on BACE1 activity under AD injury. The cells with AD injury were exposed to DADLE or DAMGO at 1–5 µM. *p < 0.05, **p < 0.01 vs. (A). Note that the BACE1 cleavage activity was significantly reduced by DADLE at 1 µM after 48-h treatment, but largely enhanced by the application of DAMGO at 1 or 5 µM.

Fig 4: Knocking-down DOR greatly enhanced BACE1 activity and expression for abnormal APP cleavage and contributed to the Aß42 production. The PC12 cells transfected with DOR siRNA or negative control siRNA were cultured in the conditions of normal control and Aß1–42 oligomer for 48 h. Similarly, APPswe SH-SY5Y cells were also transfected with human DOR siRNA or negative control siRNA for Aß42 measurement. N = 3 in each group. NS: not significant, *p < 0.05 vs. (C) or (A); **p < 0.01 vs. (C) or (A); ***p < 0.001 vs. (C) or (A). (A) BACE1 activity before and after DOR knockdown. Note that DOR knockdown significantly increased BACE1 activities in both normal and AD conditions. (B) DOR knockdown induced effects on Aß42 production and release in PC12 and APPswe SH-SY5Y cell models. DOR knockdown caused a sharp increase in the Aß42 level production in the cell lysate, but not in the medium shed in PC12 cells and the same was true in APPswe SH-SY5Y cell model. (C) BACE1 expression was largely increased by DOR knockdown in physiological condition but not under Aß1–42 injury. Note that DOR knockdown up-regulated BACE1 protein in physiological condition, but did not further increase BACE1 protein in the cells exposed to Aß1–42 olimoger that already had an increased BACE1 protein. (D) DOR knockdown was associated with a rise in APP processing. Note that along with an increase in BACE1 expression after DOR knockdown, the transfection with DOR siRNA also significantly increased APP cleavage in the physiological condition. Aß1–42 olimoger caused a quick cleavage of APP, which was not further enhanced by DOR knockdown.

Fig 5: DOR activation down-regulated, but MOR activation up-regulated BACE1 expression for APP processing. BACE1 mRNA and proteins were measured in the PC12 cells in the conditions of normal control and Aß1–42 injury (48 h). APP processing was evaluated by measuring APP full length protein and AICD. N = 3 in each group. *p < 0.05, **p<0.01 vs. (C) or (A). (A) DAMGO-induced alternations in BACE1 mRNA in physiological condition and AD injury. (B) DAMGO-induced upregulation of BACE1 protein in both physiological condition and AD injury, while DOR activation specifically attenuated BACE1 expression under AD injury. (C) APP cleavage was associated with DOR activation and inhibition, while MOR activation significantly accelerated APP processing. Note that DOR activation had no significant effect on BACE1 mRNA expression though it tended to induce a slight reduction in both physiological and Aß1–42 oligomer conditions. However, it significantly reduced the level of BACE1 protein in AD condition (Aß1–42 oligomer exposure), which could be largely reversed by DOR antagonist naltrindole. In contrast, MOR activation with DAMGO significantly increased the expression of both BACE1 mRNA and protein in both physiological and Aß1–42 oligomer conditions. Consistently, the APP cleavage was seriously interfered by DOR activation in Aß1–42 oligomer conditions, while DAMGO accelerated APP processing in both physiological and Aß1–42 oligomer conditions with a significant decrease in APP full length protein and a large increase in AICD, the APP cleavage product.