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

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.

BER can pass through the blood-brain barrier and reach the brain parenchyma in a dose-and time-dependent manner [24], and has multiple neuropharmacological properties including neuroprotective and neurotrophic effects. It also stimulates anti-neuronal apoptosis, improves cerebral microcirculation, reduces depression, and inhibits acetylcholinesterase [25][26][27]. Notably, one study [28] has reported that BER can decrease the production of Aβ 40/42 , but the mechanism remains unclear. Further investigation of how BER inhibits the expression of BACE may have significant impact on the treatment of AD. In this study, we therefore focused on the mechanism of BER on BACE and Aβ 40/42 inhibition, using HEK293 cells stably transfected with APP695 containing the Swedish mutation.

Results
Effects of BER and U0126 on the proliferation and cytotoxicity of HEK293 cells The MTT assay was used to detect the treatments on the proliferation of HEK293 cells. Relative to the vehicle group, no significant declines were observed in the cells receiving treatments (P > 0.05) ( Figure 1A, B and 1C). The LDH release of cultured medium was used to assay the treatments for the cytotoxicity of HEK293 cells. Compared with vehicle treatment, BER and U0126 showed no significant effects on the release of LDH in the culture medium (P > 0.05) ( Figure 1D, E and 1F), but 3% H 2 O 2 significantly increased the release of LDH in the culture medium (P < 0.01).

Effects of BER and U0126 on the expression of BACE
We assayed the expression of BACE in HEK293 cells by WB. BER (1 μM, 5 μM, 10 μM, and 20 μM) was found to significantly reduce the expression of BACE for 48 hours of incubation ( Figure 3A). BER (5 μM) was found to significantly reduce the expression of BACE for 8 hours, 24 hours, 48 hours, and 72 hours of incubation ( Figure 3B). However, U0126 (0.5 μM) was found to significantly increase the expression of BACE and alleviate the inhibition of BER (5 μM) on the expression of BACE (Figure 3C).

Discussion
In this study, we observed that BER significantly decreased the production of Aβ 40/42 and the expression of BACE via activation of the ERK1/2 pathway in a dose-and time-dependent manner. We also found that U0126, an antagonist of ERK1/2 pathway, abolished the effects of BER on both Aβ 40/42 and BACE. BER had previously been demonstrated to be able to reduce cancerous conditions by inhibiting the proliferation of tumor cells [29,30], but we did not find that BER could inhibit the proliferation and show cytotoxicity toward HEK 293 cells by MTT and LDH assays. From this, it can be concluded that the inhibition of BER on the production of Aβ 40/42 is not associated with the anti-proliferative or cytotoxic qualities of BER.
The enzyme BACE is crucial to the production of Aβ 40/42 and the expression of BACE increases in the brains of AD patients [5]. For this reason, BACE has been considered as a therapeutic target for AD treatments. On the other hand, the expression and activity of BACE is regulated by the ERK1/2 pathway in a doseand time-dependent manner [31], and BER increases the expression of LDLR and glucose uptake by activating the ERK1/2 pathway [10,15]. So berberine-induced reduction of BACE1 protein levels is related to ERK1 activation. Furthermore, though BER has been shown unable to inhibit the activity of BACE in vitro [25], the ERK1/2 pathway negatively modulates BACE1 activity in vivo [31]. Thus, we think that BER might also decrease the production of Aβ 40/42 by inhibit BACE1 activity via activating ERK1/2 pathway, and it need to be studied in the next study.
At the same time, BER may decrease the production of Aβ 40/42 by affecting the activity of α-secretase and γsecretase. It has been reported that ERK1/2 is an endogenous negative regulator of γ-secretase activity, and NSAIDs can inhibit γ-secretase activity by inhibiting the Rho-ROCK pathway [6,32,33]. BER inhibits tumor cell migration by inhibiting the Rho-ROCK pathway in HONE1 cells [34], so it is possible that BER inhibits the activity of γ-secretase by activating the ERK1/2 pathway and inhibiting the Rho-ROCK pathway. Moreover, BER, an acetylcholinesterase inhibitor, may be able to upregulate α-secretase activity by promoting the translocation of α-secretase to the cell surface [35]. All these possibilities require further study.

Conclusion
In this study, we demonstrated that BER can decrease the production of Aβ 40/42 by inhibiting the expression of BACE via activation of the ERK1/2 pathway. In previous studies, we demonstrated that BER improved impaired spatial memory and increased both the activation of microglia and the expression of insulin degrading enzyme (IDE) in the rat model of AD [36][37][38]. Other researchers have demonstrated other pharmacological effects of BER in HEK293 cells, e.g., inhibiting Aβ 42 aggregation and attenuating the Tau hyperphosphorylation induced by calyculin A [39,40]. Together, we consider BER to be a very promising drug for use in AD patients.

Methods
Cell culture and treatments

MTT analysis
After the cells were treated in the manner described above, 10 μl of 1 mg/ml MTT stock (Sigma, St. Louis, MO, U.S.) were added to each well and the incubation continued for another 4 hours. One hundred microliters of a solution containing 20% SDS and 50% dimethylformamide (pH 4.8) were then added to each well. After overnight incubation, absorption values at a wavelength of 570 nm were determined by spectrophotometer.

Cellular toxicity analysis
HEK293 cells were plated at a density of approximately 1 × 10 4 cells per well on 24-well plates. After 24 hours of incubation, the conditioned media were replaced with new media containing BER, U0126, and BER with U0126 at the final concentrations and the final times indicated. Lactate dehydrogenase (LDH) activity was determined to evaluate the cell toxicity of BER, U0126, and BER with U0126 by using cytotoxicity detection kits (Njjcbio Institute, China) according to the manufacturer's instructions. Hydrogen peroxide (3%) was used as a positive control and added to the conditioned media during the last hour of incubation. The baseline was determined in control wells containing no cells and the values obtained there were subtracted from those obtained from experimental wells.

Sandwich ELISA
HEK293 cells were plated at a density of approximately 4 × 10 4 cells per well on 6-well plates. After 24 hours of incubation, the conditioned media were replaced by new media containing BER, U0126, and BER with U0126 at the final concentrations and final times indicated. The cultured media were harvested and extra cellular Aβ levels were determined by using the Human Aβ 40/42 Assay Kit (Cusabiao Biotech Co., Ltd., U.S.) according to the manufacturer's instructions.

Statistical analysis
All of the data were expressed as mean ± SD and the analysis was carried out using the one way analysis of variance (ANOVA). Values of P < 0.05 were considered statistically significant.