Decrease in the production of β-amyloid by berberine inhibition of the expression of β-secretase in HEK293 cells

Abstract

Background: Berberine (BER), the major alkaloidal component of Rhizoma coptidis, has multiple pharmacological effects including inhibition of acetylcholinesterase, reduction of cholesterol and glucose levels, anti-inflammatory, neuroprotective and neurotrophic effects. It has also been demonstrated that BER can reduce the production of beta-amyloid40/42, which plays a critical and primary role in the pathogenesis of Alzheimer's disease. However, the mechanism by which it accomplishes this remains unclear.

Results: Here, we report that BER could not only significantly decrease the production of beta-amyloid40/42 and the expression of beta-secretase (BACE), but was also able to activate the extracellular signal-regulated kinase1/2 (ERK1/2) pathway in a dose- and time-dependent manner in HEK293 cells stably transfected with APP695 containing the Swedish mutation. We also find that U0126, an antagonist of the ERK1/2 pathway, could abolish (1) the activation activity of BER on the ERK1/2 pathway and (2) the inhibition activity of BER on the production of beta-amyloid40/42 and the expression of BACE.

Conclusion: Our data indicate that BER decreases the production of beta-amyloid40/42 by inhibiting the expression of BACE via activation of the ERK1/2 pathway.

Figure 1

Evaluation of the treatments on the proliferation and the cytotoxicity of HEK293 cells by MTT assay and LDH assay. (A) Effects of BER (1 μM, 5 μM,10 μM, and 20 μM) on the proliferation of HEK293 for 48 hours of incubation, P > 0.05 compared with vehicle-treated group (n = 5). (B) Effects of BER (5 μM) on the proliferation of HEK293 for 8, 24, 48, and 72 hours of incubation, P > 0.05 compared with vehicle-treated group (n = 5). (C). Effects of BER (5 μM), U0126 (0.5 μM), and U0126 with BER (0.5 μM+5 μM) on the proliferation of HEK293 cells for 48 hours of incubation, P > 0.05 compared with vehicle-treated group (n = 5). (D) Effects of BER (1 μM, 5 μM, 10 μM, and 20 μM) on the cytotoxicity of HEK293 for 48 hours of incubation, *P < 0.05 when compared with vehicle-treated group (n = 5). (E) Effects of BER (5 μM) on the cytotoxicity of HEK293 for 8, 24, 48, and 72 hours of incubation, *P < 0.05 when compared with vehicle-treated group (n = 5). (F). Effects of BER (5 μM), U0126 (0.5 μM), and U0126 with BER (0.5 μM+5 μM) on the cytotoxicity of HEK293 cell for 48 hours of incubation, *P < 0.05 when compared with vehicle-treated group (n = 5).

Figure 2

Evaluation of the treatments on the production of Aβ40/42 in HEK293 cell by ELISA. (A) BER (1 μM, 5 μM, 10 μM, and 20 μM) can inhibit the production of Aβ40 for 48 hours of incubation,*P < 0.01 compared with vehicle-treated group, #P < 0.05 compared with BER (1 μM) groups (n = 5). (B) BER (5 μM) can inhibit the production of Aβ40 from 8 to 72 hours of incubation, *P < 0.01 compared with vehicle-treated group, #P > 0.05 compared with BER (8 h) groups (n = 5). (C) U0126 (0.5 μM) can abolish the inhibition of BER (5 μM) on the production of Aβ40, *P < 0.01 compared with vehicle-treated group, #P < 0.05 compared with vehicle-treated group (n = 5). (D) BER (1 μM, 5 μM, 10 μM, and 20 μM) can inhibit the production of Aβ42 for 48 hours of incubation,*P < 0.01 compared with the vehicle-treated group, #P < 0.05 compared with BER (1 μM and 5 μM) groups (n = 5). (E) BER (5 μM) can inhibit the production of Aβ42 from 8 to 72 hours of incubation, *P < 0.01 compared with the vehicle-treated group, #P < 0.05 compared with BER (8- and 24-hour) groups (n = 5). (F) U0126 (0.5 μM) can abolish the inhibition of BER (5 μM) on the production of Aβ42, *P < 0.01 compared with vehicle-treated group, #P < 0.05 compared with vehicle-treated group (n = 5).

Figure 3

Evaluation of the treatments on the expression of BACE by Western blot. (A) BER (1 μM, 5 μM, 10 μM, and 20 μM) can significantly decrease the expression of BACE in a dose-dependent manner for 48 hours of incubation, *P < 0.05 compared with vehicle-treated group, #P < 0.01 compared with vehicle-treated group (n = 3). (B) BER (5 μM) can decrease the expression of BACE from 8 to 72 hours of incubation, *P < 0.05 compared with vehicle-treated group, #P < 0.01 compared with the vehicle-treated group (n = 3). (C) BER (5 μM) can decrease the expression of BACE for 48 hours of incubation, U0126 (0.5 μM), and BER with U0126 (5 μM +0.5 μM) can increase the expression of BACE for 48 hours of incubation, *P < 0.05 compared with vehicle-treated group (n = 3).

Figure 4

Evaluation of the treatments on the expression of the activation of ERK1/2 pathway by Western blot. BER (1 μM, 5 μM, 10 μM, and 20 μM) can significantly increase phosphorylation-ERK1/2 for 48 hours of incubation, *P < 0.05 compared with vehicle-treated group, #P < 0.01 compared with vehicle-treated group (n = 3). (B) BER (5 μM) can increase phosphorylation-ERK1/2 from 8 to 72 hours of incubation, *P < 0.05 compared with vehicle group, #P < 0.01 compared with vehicle-treated group (n = 3). (C) U0126 (0.5 μM) can completely abolish the activation of BER on ERK1/2, *P < 0.05 compared with the vehicle-treated group, #P < 0.01 compared with vehicle-treated group (n = 3).