Extract Of Ginkgo Biloba Induces The Phase 2 Enzymes Through Keap1-Nrf2-ARE Signaling Pathway

Drugs and other xenobiotics normally experience two metabolizing phases in the body, which are called phase 1and phase 2 metabolism. During the phase 1 metabolism, drugs was catalyzed or detoxified mainly by P450s but some toxic substances were produced/activated in some cases, such as reactive oxygen species (ROS) and/or electrophilics which can functionally modify DNA or critical proteins resulting in diseases. In the phase 2 metabolism, these toxic substances can be detoxified by many enzymes such as glutathione-S-transferase (GST), quinone reductase(QR), epoxide hydrolase, sulfotranferase, acetyltransferase etc, which are called phase 2 enzymes or detoxication enzymes or phase 2 antioxidant enzymes. Induction of these enzymes is widely accepted as an efficient strategy for reducing the risk of a variety of diseases related to exposure to toxins, mutagens and carcinogens.The expression of phase 2 enzymes is mainly regulated by the Keap1-Nrf2-ARE signaling system. The antioxidant response element (ARE) is a cis-acting motif located in the 5'-flanking promoter region of nearly all phase 2 genes and mediates the transcriptional up-regulation of phase 2 genes caused by different classes of chemical compounds, including Michael reaction acceptors, diphenols, quinones, isothiocyanates, peroxides, mercaptans, trivalent arsenicals, heavy metals, and dithiolethiones. The inducer signal is transmitted to the ARE by two important proteins—Nrf2, a transcription factor homologous to Drosophila cap'n'collar proteins, and Keap1, a cytosolic protein homologous to the Drosophila actin-binding protein Kelch, which plays an active role in Nrf2 regulation, by directing it for proteosomal degradation. Inducers of Phase 2 genes are believed to interact with critical cysteines in the Keap1 causing a de-repression in Nrf2 activity, although the exact mechanism for this process is still unclear.Since the phase 2 enzymes exert important detoxification and other antioxidant functions in the body, inducers with lower side effects and higher efficacy have long being pursued by pharmacologists. Some synthesized chemical inducers have been employed but the side effects and potential damages in humans are unavoidable. Natural substances such as plants with relatively lower toxicity have been selected as the alternatives by some researchers and clinicians. Many traditional Chinese medicines such as Ginkgo, green tea etc. were proved to possess antioxidant function. But the mechanism underlying this effect remains unclear, which limits their prescription accordingly.AIMWe selected the extract of Ginkgo biloba (EGb) in this study to investigate its induction on the phase 2 enzymes and the related molecular mechanism. EGb is used widely to treat cerebrovascular and peripheral vascular insufficiency, neurosensory problems, and disturbances in vigilance, short-term memory and other cognitive functions that are associated with dementias, ageing and senility. In recent decades, EGb has been reported to possess antioxidant activity and to prevent oxidative damage in both cell lines and animals. But its effects on the phase 2 enzymes and the molecular mechanism underlying it have not been determined previously. METHODS1. Detection of the induction of GCLC and GST-P1 at the transcription level by EGb. Hepa1c1c7 cells were split to 2×106/well in 6-well plates and cultured for 24 hours. Cells were then washed in Hank's balanced salt solution (HBSS) and incubated in complete DMEM containing EGb at the different concentrations for 4 hours. Total RNA was isolated using TRIzol reagent and 1μg RNA was reverse transcribed using TaqMan reagents according to the manufacturer's instructions. GCLC, GST-P1 and GAPDH mRNA levels were quantified by real-time quantitative PCR.2. Detection of the induction of GCLC and GST-P1 at the protein level by EGb. Hepa1c1c7 and HepG2 cells were treated by EGb at different concertrations for 24 hours. Cells were then digested and total cytosolic proteins were extracted and the targeted proteins were detected by Western blot.3. Detection of induction of GCLC and GST-P1 at the level of enzyme activity by EGb. We further assessed the induction of GCLC and GST-P1 by EGb through measurement of changes in enzyme activity. GCLC is the rate-limiting enzyme for synthesis of GSH, and we therefore determined the cellular GSH levels in cells following EGb treatment. GST-P1 catalyses the detoxification of electrophiles by conjugation with reduced GSH, and we detected the GST activity.4. NQO1-ARE reporter assay. Hepa1c1c7 cells were seeded at 10,000 cells per well into a 96-well culture plate, in complete growth medium. The following day, the cells were transfected with either a wild type NQO1 ARE reporter, created by insertion of 1016 bp of 5'upstream sequence of the mouse NQO1 gene into a pGL3-Basic luciferase reporter vector to create-1016/nqo5′-luc, or a mutated NQO1 ARE reporter, in which the ARE sequence has been scrambled.5. Detection of nuclear translocation of Nrf2. Hepa1c1c7 and HepG2 cells were treated by EGb at different concertrations for 2 hours. Cells were then digested and total nuclear proteins were extracted and Nrf2 was detected by Western blot. 6. Detection of the inhibition of Keap1 by EGb. Hepa1c1c7 and HepG2 cells were treated by EGb at different concentrations for 24 hours. Cells were then digested and total cytosolic proteins were extracted and the targeted protein was detected by Western blot.RESULTS1. Two typical phase 2 enzymes, GCLC and GST-P1were induced by EGb at the levels of transcription, protein expression and enzyme activity.2. In the NQO1-ARE reporter assay, EGb activated the activity of the wild type but not the one with ARE mutated.3. Nrf2 was stimulated by EGb to translocate into nucleus.4. Cytosolic Keap1 was inhibited by EGb. CONCLUSIONS 1. EGb induces typical phase 2 enzymes (GCLC, GST-P1 and NQO1). 2. EGb induces phase 2 enzymes through ARE. 3. EGb dissociates the Nrf2-Keap1 complex, facilitating the release and nuclear translocation of Nrf2. Nrf2 can bind the ARE to enchance the transcription of phase 2 genes. 4. EGb inhibites the cytosolic Keap1, which also enables Nrf2 nuclear translocation. In summary, EGb was demonstrated to induce typical phase 2 enzymes (GCLC, GST-P1 and NQO1) through the Nrf2-Keap1-ARE passway.