Decrease in the production of beta-amyloid by berberine inhibition of the expression of beta-secretase in HEK293 cells
© Zhu et al; licensee BioMed Central Ltd. 2011
Received: 20 October 2011
Accepted: 12 December 2011
Published: 12 December 2011
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.
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.
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.
KeywordsAlzheimer's disease berberine beta-amyloid40/42 beta-secretase extracellular signal-regulated kinase1/2
Alzheimer's disease (AD) is the most prominent form of senile dementia. In the pathogenesis of AD, amyloid-β peptide (Aβ) plays a critical and primary role . The aggregation and accumulation of extracellular and intracellular Aβ40/42 impairs synaptic plasticity and memory [2, 3]. Aβ40/42 is generated by β-secreatase- (beta-site amyloid precursor protein cleaving enzyme, BACE) and γ-secretase-mediated sequential cleavages of amyloid precursor protein (APP). Inhibition of the production of Aβ40/42 can be expected to delay the development of AD . In fact, some nonsteroidal anti-inflammatory drugs (NSAIDs), including sulindac sulfide, S-ibuprofen, R-ibuprofen and indomethacin, have been shown to inhibit the production of Aβ40/42 by inhibiting the expression of BACE and the activity of γ-secretase via activating peroxisome proliferator-activated receptor γ (PPAR γ) and inhibiting Rho-Rho associated kinase (Rho-ROCK) pathway [5, 6]. Additionally, some statins, including sinvastatin, rosuvastatin, and lovastatin, the cholesterol-lowering drugs, have been found to reduce levels of Aβ40/42 by promoting the expression of α-secretase and inhibiting BACE activity [7–9].
Berberine (BER), an isoquinoline alkaloid existing in Cortex phellodendri (Huangbai) and Rhizoma coptidis (Huanglian), has a long history in China as a non-prescription drug for the treatment of diarrhea and gastrointestinal disorders. In recent years, many studies have indicated that BER has multiple pharmacological effects. BER is a novel cholesterol-lowering drug distinct from the statin family. It works by increasing the expression of low-density lipoprotein receptors (LDLR) and inhibiting lipid synthesis [10, 11]. BER can also improve insulin resistance and exerts an insulin-independent glucose-lowering effect, stimulating insulin secretion and sensitizing insulin activity, inducing glycolysis, and increasing glucose transport and uptake activity [12–17]. At the same time, some studies have found that BER exerts anti-inflammatory effects by inhibiting arachidonic acid metabolism and the production of some inflammatory factors including cyclooxygenase-2 (COX-2), interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), Interleukin-1 (IL-6) and inductible nitric oxide synthase (iNOS)[18–23].
BER can pass through the blood-brain barrier and reach the brain parenchyma in a dose- and time-dependent manner , and has multiple neuropharmacological properties including neuroprotective and neurotrophic effects. It also stimulates anti-neuronal apoptosis, improves cerebral microcirculation, reduces depression, and inhibits acetylcholinesterase [25–27]. Notably, one study  has reported that BER can decrease the production of Aβ40/42, 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 Aβ40/42 inhibition, using HEK293 cells stably transfected with APP695 containing the Swedish mutation.
Effects of BER and U0126 on the proliferation and cytotoxicity of HEK293 cells
Effects of BER and U0126 on the production of Aβ40/42
Effects of BER and U0126 on the expression of BACE
Effects of BER and U0126 on the activation of ERK1/2 pathway
In this study, we observed that BER significantly decreased the production of Aβ40/42 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 Aβ40/42 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 Aβ40/42 is not associated with the anti-proliferative or cytotoxic qualities of BER.
The enzyme BACE is crucial to the production of Aβ40/42 and the expression of BACE increases in the brains of AD patients . 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 dose- and time-dependent manner , 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 , the ERK1/2 pathway negatively modulates BACE1 activity in vivo . Thus, we think that BER might also decrease the production of Aβ40/42 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 Aβ40/42 by affecting the activity of α-secretase and γ-secretase. 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 , 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 . All these possibilities require further study.
In this study, we demonstrated that BER can decrease the production of Aβ40/42 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 [36–38]. Other researchers have demonstrated other pharmacological effects of BER in HEK293 cells, e.g., inhibiting Aβ42 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.
Cell culture and treatments
HEK293 cells stably transfected with APP695 containing the Swedish mutation were maintained in Dulbecco's modified Eagle's medium (DMEM), supplemented with 5% fetal bovine serum and G418 (Sigma, St. Louis, MO, U.S.) (100 μg/mL) in a humidified atmosphere at 37°C with 5% CO2. HEK293 cells were given BER (Sigma, St. Louis, MO, U.S.) (1 μM, 5 μM, 10 μM, and 20 μM), U0126 (Sigma, St. Louis, MO, U.S.) (0.5 μM), and BER with U0126 (5 μM+0.5 μM) for 48 hours, and HEK293 cells were also given BER (5 μM) for 8 hours, 24 hours, 48 hours and 72 hours.
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 × 104 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.
HEK293 cells were plated at a density of approximately 4 × 104 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 Aβ40/42 Assay Kit (Cusabiao Biotech Co., Ltd., U.S.) according to the manufacturer's instructions.
Western blotting (WB) analysis
HEK293 cells were plated at a density of 4 × 104 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. Cells were lysed in a cell and tissue protein extraction reagent and protease inhibitor cocktail and phosphotase inhibitor cocktail (Kangchen Bio-tech, China), phenyl-methyl-sulfonyl-fluoride-proteomics grade kit (Kangchen Bio-tech, China). Protein extracts (protein 50 μg) were subjected to SDS-PAGE. The levels of BACE, p-ERK1/2, ERK1/2, and GAPDH in the cell lysates were quantified by WB analysis using polyclonal antibody anti-BACE (487-501, 1:10000 dilution, EMD Bioscience, Germany), monoclonal antibody anti-phospho-ERK1/2 and ERK1/2 (137F5 and 197G2, 1:1000 dilution, Cell Signaling Technology, U.S.), and polyclonal antibody anti-GAPDH (IBN9003L, 1:3000 dilution, KB Biotech, China), respectively. This was followed by application of peroxidase-conjugated secondary antibodies (GGHL-15P,1:5000 dilution, ICL Lab, U.S.). Immunoreactive signals were detected by enhanced chemiluminescence using ECL Plus WB detection reagents (Pierce); signal intensity was determined with a densitometer, LAS-3000 (Fuji Photo Film Co., Ltd., Tokyo, Japan). The amounts of immunoreactive BACE on internal control GAPDH and p-ERK1/2 on internal control ERK1/2 in each sample were calculated by using Quality One software (Bio-Red, U.S.).
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.
enzyme-linked immunosorbent assay
extracellular signal-regulated kinase1/2
This study was supported by the National Natural Science Foundation of China (30800359), and was partly accomplished in the laboratory of neurology department, the first affiliated hospital of Sun Yat-Sen University.
- Goedert M, Spillantini MG: A century of Alzheimer's disease. Science. 2006, 314 (5800): 777-81. 10.1126/science.1132814.View ArticlePubMedGoogle Scholar
- Lesné S, Koh MT, Kotilinek L, Kayed R, Glabe CG, Yang A, Gallagher M, Ashe KH: A specific amyloid-beta protein assembly in the brain impairs memory. Nature. 2006, 440 (7082): 352-7. 10.1038/nature04533.View ArticlePubMedGoogle Scholar
- Cleary JP, Walsh DM, Hofmeister JJ, Shankar GM, Kuskowski MA, Selkoe DJ, Ashe KH: Natural oligomers of the amyloid-beta protein specifically disrupt cognitive function. Nat Neurosci. 2005, 8 (1): 79-84. 10.1038/nn1372.View ArticlePubMedGoogle Scholar
- Roberson ED, Mucke L: 100 years and counting: prospects for defeating Alzheimer's disease. Science. 2006, 314 (5800): 781-4. 10.1126/science.1132813.PubMed CentralView ArticlePubMedGoogle Scholar
- Sastre M, Dewachter I, Rossner S, Bogdanovic N, Rosen E, Borghgraef P, Evert BO, Dumitrescu-Ozimek L, Thal DR, Landreth G, Walter J, Klockgether T, van Leuven F, Heneka MT: Nonsteroidal anti-inflammatory drugs repress beta-secretase gene promoter activity by the activation of PPARgamma. Proc Natl Acad Sci USA. 2006, 103 (2): 443-8. 10.1073/pnas.0503839103.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhou Y, Su Y, Li B, Liu F, Ryder JW, Wu X, Gonzalez-DeWhitt PA, Gelfanova V, Hale JE, May PC, Paul SM, Ni B: Nonsteroidal anti-inflammatory drugs can lower amyloidogenic Abeta42 by inhibiting Rho. Science. 2003, 302 (5648): 1215-7. 10.1126/science.1090154.View ArticlePubMedGoogle Scholar
- Fassbender K, Simons M, Bergmann C, Stroick M, Lutjohann D, Keller P, Runz H, Kuhl S, Bertsch T, von Bergmann K, Hennerici M, Beyreuther K, Hartmann T: Simvastatin strongly reduces levels of Alzheimer's disease beta -amyloid peptides Abeta 42 and Abeta 40 in vitro and in vivo. Proc Natl Acad Sci USA. 2001, 98 (10): 5856-61. 10.1073/pnas.081620098.PubMed CentralView ArticlePubMedGoogle Scholar
- Xiu J, Nordberg A, Qi X, Guan ZZ: Influence of cholesterol and lovastatin on alpha-form of secreted amyloid precursor protein and expression of alpha7 nicotinic receptor on astrocytes. Neurochem Int. 2006, 49 (5): 459-65. 10.1016/j.neuint.2006.03.007.View ArticlePubMedGoogle Scholar
- Famer D, Crisby M: Rosuvastatin reduces caspase-3 activity and up-regulates alpha-secretase in human neuroblastoma SH-SY5Y cells exposed to A beta. Neurosci Lett. 2004, 371 (2-3): 209-14. 10.1016/j.neulet.2004.08.069.View ArticlePubMedGoogle Scholar
- Kong W, Wei J, Abidi P, Lin M, Inaba S, Li C, Wang Y, Wang Z, Si S, Pan H, Wang S, Wu J, Wang Y, Li Z, Liu J, Jiang JD: Berberine is a novel cholesterol-lowering drug working through a unique mechanism distinct from statins. Nat Med. 2004, 10 (12): 1344-51. 10.1038/nm1135.View ArticlePubMedGoogle Scholar
- Brusq JM, Ancellin N, Grondin P, Guillard R, Martin S, Saintillan Y, Issandou M: Inhibition of lipid synthesis through activation of AMP kinase: an additional mechanism for the hypolipidemic effects of berberine. J Lipid Res. 2006, 47 (6): 1281-8. 10.1194/jlr.M600020-JLR200.View ArticlePubMedGoogle Scholar
- Yin J, Hu R, Chen M, Tang J, Li F, Yang Y, Chen J: Effects of berberine on glucose metabolism in vitro. Metabolism. 2002, 51 (11): 1439-43. 10.1053/meta.2002.34715.View ArticlePubMedGoogle Scholar
- Leng SH, Lu FE, Xu LJ: Therapeutic effects of berberine in impaired glucose tolerance rats and its influence on insulin secretion. Acta Pharmacol Sin. 2004, 25 (4): 496-502.PubMedGoogle Scholar
- Ko BS, Choi SB, Park SK, Jang JS, Kim YE, Park S: Insulin sensitizing and insulinotropic action of berberine from Cortidis rhizoma. Biol Pharm Bull. 2005, 28 (8): 1431-7. 10.1248/bpb.28.1431.View ArticlePubMedGoogle Scholar
- Kim SH, Shin EJ, Kim ED, Bayaraa T, Frost SC, Hyun CK: Berberine activates GLUT1-mediated glucose uptake in 3T3-L1 adipocytes. Biol Pharm Bull. 2007, 30 (11): 2120-5. 10.1248/bpb.30.2120.View ArticlePubMedGoogle Scholar
- Yin J, Gao Z, Liu D, Liu Z, Ye J: Berberine improves glucose metabolism through induction of glycolysis. Am J Physiol Endocrinol Metab. 2008, 294 (1): E148-56.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhou L, Yang Y, Wang X, Liu S, Shang W, Yuan G, Li F, Tang J, Chen M, Chen J: Berberine stimulates glucose transport through a mechanism distinct from insulin. Metabolism. 2007, 56 (3): 405-12. 10.1016/j.metabol.2006.10.025.View ArticlePubMedGoogle Scholar
- Huang CG, Chu ZL, Wei SJ, Jiang H, Jiao BH: Effect of berberine on arachidonic acid metabolism in rabbit platelets and endothelial cells. Thromb Res. 2002, 106 (4-5): 223-7. 10.1016/S0049-3848(02)00133-0.View ArticlePubMedGoogle Scholar
- Fukuda K, Hibiya Y, Mutoh M, Koshiji M, Akao S, Fujiwara H: Inhibition by berberine of cyclooxygenase-2 transcriptional activity in human colon cancer cells. J Ethnopharmacol. 1999, 66 (2): 227-33. 10.1016/S0378-8741(98)00162-7.View ArticlePubMedGoogle Scholar
- Kuo CL, Chi CW, Liu TY: The anti-inflammatory potential of berberine in vitro and in vivo. Cancer Lett. 2004, 203 (2): 127-37. 10.1016/j.canlet.2003.09.002.View ArticlePubMedGoogle Scholar
- Hsiang CY, Wu SL, Cheng SE, Ho TY: Acetaldehyde-induced interleukin-1beta and tumor necrosis factor-alpha production is inhibited by berberine through nuclear factor-kappaB signaling pathway in HepG2 cells. J Biomed Sci. 2005, 12 (5): 791-801. 10.1007/s11373-005-9003-4.View ArticlePubMedGoogle Scholar
- Iizuka N, Miyamoto K, Hazama S, Yoshino S, Yoshimura K, Okita K, Fukumoto T, Yamamoto S, Tangoku A, Oka M: Anticachectic effects of Coptidis rhizoma, an anti-inflammatory herb, on esophageal cancer cells that produce interleukin 6. Cancer Lett. 2000, 158 (1): 35-41. 10.1016/S0304-3835(00)00496-1.View ArticlePubMedGoogle Scholar
- Pan LR, Tang Q, Fu Q, Hu BR, Xiang JZ, Qian JQ: Roles of nitric oxide in protective effect of berberine in ethanol-induced gastric ulcer mice. Acta Pharmacol Sin. 2005, 26 (11): 1334-8. 10.1111/j.1745-7254.2005.00186.x.View ArticlePubMedGoogle Scholar
- Wang X, Xing D, Wang W, Lei F, Su H, Du L: The uptake and transport behavior of berberine in Coptidis Rhizoma extract through rat primary cultured cortical neurons. Neurosci Lett. 2005, 379 (2): 132-7. 10.1016/j.neulet.2004.12.050.View ArticlePubMedGoogle Scholar
- Jung HA, Min BS, Yokozawa T, Lee JH, Kim YS, Choi JS: Anti-Alzheimer and antioxidant activities of coptidis rhizoma alkaloids. Biol Pharm Bull. 2009, 32 (8): 1433-8. 10.1248/bpb.32.1433.View ArticlePubMedGoogle Scholar
- Ye M, Fu S, Pi R, He F: Neuropharmacological and pharmacokinetic properties of berberine: a review of recent research. J Pharm Pharmacol. 2009, 61 (7): 831-7.View ArticlePubMedGoogle Scholar
- Kulkarni SK, Dhir A: Berberine: a plant alkaloid with therapeutic potential for central nervous system disorders. Phytother Res. 2010, 24 (3): 317-24. 10.1002/ptr.2968.View ArticlePubMedGoogle Scholar
- Asai M, Iwata N, Yoshikawa A, Aizaki Y, Ishiura S, Saido TC, Maruyama K: Berberine alters the processing of Alzheimer's amyloid precursor protein to decrease Abeta secretion. Biochem Biophys Res Commun. 2007, 352 (2): 498-502. 10.1016/j.bbrc.2006.11.043.View ArticlePubMedGoogle Scholar
- Kettmann V, Kosfalova D, Jantova S, Cernakova M, Drimal J: In vitro cytotoxicity of berberine against HeLa and L1210 cancer cell lines. Pharmazie. 2004, 59 (7): 548-51.PubMedGoogle Scholar
- Letasiova S, Jantova S, Muckova M, Theiszova M: Antiproliferative activity of berberine in vitro and in vivo. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2005, 149 (2): 461-3.View ArticlePubMedGoogle Scholar
- Tamagno E, Guglielmotto M, Giliberto L, Vitali A, Borghi R, Autelli R, Danni O, Tabaton M: JNK and ERK1/2 pathways have a dual opposite effect on the expression of BACE1. Neurobiol Aging. 2009, 30 (10): 1563-73. 10.1016/j.neurobiolaging.2007.12.015.View ArticlePubMedGoogle Scholar
- Kim SK, Park HJ, Hong HS, Baik EJ, Jung MW, Mook-Jung I: ERK1/2 is an endogenous negative regulator of the gamma-secretase activity. FASEB J. 2006, 20 (1): 157-9.PubMedGoogle Scholar
- Tung YT, Hsu WM, Wang BJ, Wu SY, Yen CT, Hu MK, Liao YF: Sodium selenite inhibits gamma-secretase activity through activation of ERK. Neurosci Lett. 2008, 440 (1): 38-43. 10.1016/j.neulet.2008.05.048.View ArticlePubMedGoogle Scholar
- Tsang CM, Lau EP, Di K, Cheung PY, Hau PM, Ching YP, Wong YC, Cheung AL, Wan TS, Tong Y, Tsao SW, Feng Y: Berberine inhibits Rho GTPases and cell migration at low doses but induces G2 arrest and apoptosis at high doses in human cancer cells. Int J Mol Med. 2009, 24 (1): 131-8.PubMedGoogle Scholar
- Zimmermann M, Gardoni F, Marcello E, Colciaghi F, Borroni B, Padovani A, Cattabeni F, Di Luca M: Acetylcholinesterase inhibitors increase ADAM10 activity by promoting its trafficking in neuroblastoma cell lines. J Neurochem. 2004, 90 (6): 1489-99. 10.1111/j.1471-4159.2004.02680.x.View ArticlePubMedGoogle Scholar
- Zhu F, Qian C: Berberine chloride can ameliorate the spatial memory impairment and increase the expression of interleukin-1beta and inducible nitric oxide synthase in the rat model of Alzheimer's disease. BMC Neurosci. 2006, 7: 78-10.1186/1471-2202-7-78.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhu F, He G, Xu J: Berberine chloride can increase the activation of microglia by inhibiting the expression of peroxisome proliferator-activated receptor gamma in the rat model of Alzheimer's disease. Eur J Neurol. 2008, 15 (suppl 3): 46.Google Scholar
- Zhu F, Ma Y, Sun Y: Effect of berberine on expression of insulin degrading enzyme in rat models with AD. Chin J Neuromed. 2010, 9 (12): 1201-1203.Google Scholar
- Shi A, Huang L, Lu C, He F, Li X: Synthesis, biological evaluation and molecular modeling of novel triazole-containing berberine derivatives as acetylcholinesterase and beta-amyloid aggregation inhibitors. Bioorg Med Chem. 2011, 19 (7): 2298-305. 10.1016/j.bmc.2011.02.025.View ArticlePubMedGoogle Scholar
- Guang Y, Li Y, Tian Q, Liu R, Wang Q, Wang JZ, Wang X: Brberine Attenuates Calyculin A-Induced Cytotoxicity and Tau Hyperphosphorylation in HEK293 Cells. J Alzheimers Dis. 2011, 24 (3): 525-535.Google Scholar
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