When it comes to popular healthy foods today, cruciferous vegetables deserve a spot. Social media posts like "Broccoli Fights Cancer" have informed us that edible cruciferous plants mainly include broccoli, cabbage, kale, Chinese cabbage, rape, Chinese broccoli, shepherd’s purse, and various radishes. Beyond being labeled as "anti-cancer," these vegetables also offer health benefits such as boosting immunity, protecting cardiovascular health, and delaying aging.
Little did we know—they owe these benefits to an important signaling pathwa
1. Introduction to the Nrf2 Signaling Pathway
We have previously covered signaling pathways like AMPK, mTOR, FOXO, and Sirtuins, which play crucial roles in the body and are hot topics in anti-aging research. So, what is Nrf2? Simply put, Nrf2 (Nuclear Factor Erythroid 2-Related Factor 2) is a key transcription factor in the body that activates over 250 protective genes and mediates the body’s most important antioxidant pathway.
Readers familiar with advanced methods like gene therapy or artificial intelligence may not view antioxidants as a "powerful" anti-aging tool, but their role should not be underestimated. According to the free radical theory of aging, when the balance between free radical production and scavenging is disrupted, free radicals attack cell membranes, mitochondria, and DNA, disrupting normal cellular functions. Thus, excessive and uncontrolled free radicals are harmful to health. Moreover, Nrf2 has additional advantages:
- Unlike "one-for-one" antioxidants such as vitamin C, vitamin E, or coenzyme Q10, Nrf2 regulates an entire antioxidant signaling network. This means it controls the expression of a series of antioxidant proteins (e.g., SOD, CAT, GSH) to enhance cellular antioxidant capacity—an efficiency far beyond that of individual antioxidants.
- Nrf2 also participates in critical processes like detoxification, inflammation inhibition, metabolism regulation, and radiation protection. It converts various forms of cellular stress into adaptive cell-protective responses, helping cells survive under stress. For example, when cells need to resist exogenous toxic substances, Nrf2 can upregulate glutathione S-transferase to exert detoxification effects[1].
2. Molecular Regulation of Nrf2
For Nrf2 to exert its functions, it first needs to be activated.
Under normal physiological conditions, Nrf2 is anchored in the cytoplasm by its inhibitor, Keap1 (Kelch-like ECH-Associated Protein 1). As a substrate of the E3 ubiquitin ligase complex, Keap1 promotes the ubiquitination of Nrf2, leading to its rapid degradation by the proteasome—resulting in very low Nrf2 expression levels[2].
When cells are exposed to oxidative stress or other stimuli, multiple regulatory mechanisms can boost Nrf2 expression:
1. The Classic Keap1-Nrf2-ARE Mechanism
Reactive oxygen species (ROS) inhibit the activity of Keap1 and hydrolases, prompting Nrf2 to dissociate from Keap1 and quickly translocate into the nucleus. There, it forms a heterodimer with MAF proteins, binds to the ARE (Antioxidant Response Element) sequence in DNA, and activates the transcription of related genes[3].
Schematic Summary:
- Unstressed state: Keap1, Cul3 (a component of the E3 ligase complex), and ubiquitin (Ub) mediate Nrf2 ubiquitination and proteasomal degradation.
- Stressed state: Stressors inhibit Keap1-dependent Nrf2 ubiquitination; free Nrf2 enters the nucleus, binds to ARE via Nrf2-small MAF heterodimers, and activates gene transcription.
2. Autonomous Activation of Nrf2
Nrf2’s basic scaffold and leucine zipper regions contain a nuclear localization signal (NLS) and a nuclear export signal (NES), respectively. The Neh5 domain of Nrf2 has an oxidation-sensitive NES, suggesting that Nrf2 may spontaneously respond to external signals and translocate into the nucleus to perform transcriptional functions independently of Keap1[4].
3. Positive Regulation of Nrf2 by p21 and p62
- p21 (a protein that coordinates the cell cycle) competes with Keap1 for binding to Nrf2, inhibiting Keap1-dependent Nrf2 ubiquitination and increasing Nrf2 protein levels[5].
- p62 (a ubiquitin-binding protein) is an endogenous Nrf2 inducer that plays a key role in regulating Nrf2-dependent protein synthesis, autophagy-related protein degradation, and maintaining protein homeostasis[6].
4. Oncogenes Upregulate Nrf2 Gene Transcription
Oncogenes such as K-Ras, B-Raf, and Myc activate Nrf2 transcription via the Mek-Erk-Jun signaling pathway and enhance Nrf2 protein stability, thereby triggering Nrf2-mediated antioxidant programs[7].
Once Nrf2 expression increases, it binds to DNA response elements (e.g., AREs and Electrophile Response Elements, EPRES) to regulate gene expression and induce extensive biological effects.
3. Physiological Functions of Nrf2
Nrf2 is a multifunctional transcription factor—its activation triggers a wide range of physiological effects, including antioxidant defense, anti-inflammation, detoxification, DNA repair, metabolism regulation, and autophagy promotion. Below is a summary of Nrf2’s main target genes and their physiological functions (genes marked with "↓" are downregulated by Nrf2):
| Gene | Protein | Main Role | Physiological Function |
|---|---|---|---|
| Antioxidant & Redox Homeostasis | |||
| GCLC | Glutamate-cysteine ligase catalytic subunit | Rate-limiting enzyme for glutathione synthesis (catalytic subunit) | Maintains cellular homeostasis |
| GCLM | Glutamate-cysteine ligase modifier subunit | Rate-limiting enzyme for glutathione synthesis (modifier subunit) | Maintains cellular homeostasis |
| GPX2 | Glutathione peroxidase 2 | Intracellular antioxidant; detoxifies H₂O₂ | Maintains cellular homeostasis |
| PRDX1 | Peroxiredoxin 1 | Reduces peroxides; regulates intracellular H₂O₂ concentration | Maintains cellular homeostasis |
| SRXN1 | Sulfiredoxin 1 | Thioredoxin-based antioxidant system; reduces oxidized protein sulfhydryls | Maintains intracellular thiol balance; cellular homeostasis |
| TXN1 | Thioredoxin 1 | Participates in disulfide-dithiol exchange; reduces protein sulfenic acid | Maintains intracellular thiol balance; cellular homeostasis |
| Detoxification & Drug Metabolism | |||
| ABCB6 | MDR/TAP (Mitochondrial transporter) | ATP-dependent transport of heme and porphyrins; critical for blood metabolism | Phase III drug metabolism; bile transport/hepatic excretion |
| ABCC2 | MRP2 (Multidrug resistance-associated protein 2) | Excretion of certain anticancer drugs; multidrug resistance | Phase III drug metabolism |
| AKR1B10 | Aldo-keto reductase 1B10 | Converts retinal to retinol; reduces aromatic and aliphatic aldehydes | Phase I drug metabolism; retinal metabolism |
| AKR1C1 | Aldo-keto reductase 1C1 | Converts 4-hydroxy-2-nonenal to 1,2-dihydroxynonene; inactivates progesterone | Phase I drug metabolism |
| AKR1C3 | Aldo-keto reductase 1C3 | 17β-hydroxysteroid dehydrogenase type 5; prostaglandin F2α synthase | Phase I drug metabolism |
| CES1G | Carboxylesterase 1G | Catalyzes transesterification of exogenous substances; hydrolyzes long-chain fatty acids | Phase I drug metabolism; fatty acid oxidation/degradation |
| CES1H | Carboxylesterase 1H | Same as CES1G | Phase I drug metabolism; fatty acid oxidation/degradation |
| GSTA1 | Glutathione S-transferase A1 | Detoxifies electrophilic compounds; metabolizes bilirubin and anticancer drugs; activates glutathione peroxidase | Phase II drug metabolism; cellular protection |
| GSTM1 | Glutathione S-transferase M1 | Detoxifies electrophilic compounds | Phase II drug metabolism |
| NQO1 | NAD(P)H:quinone oxidoreductase 1 | Reduces quinones to hydroquinones; prevents one-electron reduction of free radical-forming quinones | Phase I drug metabolism |
| Heme Metabolism | |||
| FECH | Ferrochelatase | Catalyzes iron insertion into protoporphyrin IX during heme synthesis | Heme metabolism |
| HMOX1 | Heme oxygenase 1 | Degrades heme to biliverdin during heme catabolism | Heme metabolism |
| Lipid & Glucose Metabolism | |||
| AWAT1 | Acyl-CoA wax alcohol acyltransferase 1 | Catalyzes wax ester synthesis from long-chain alcohols and fatty acids (enriched in skin) | Lipid metabolism |
| FABP1↓ | Fatty acid-binding protein 1 | Intracellular transport of bile acids, long-chain fatty acids, and their CoA derivatives | Fatty acid uptake and intracellular transport |
| LIPH | Lipase H | Membrane-bound triglyceride lipase; hydrolyzes phosphatidic acid to 2-acyl lysophosphatidic acid | Platelet aggregation; smooth muscle contraction; cell proliferation/migration |
| PPARγ | Peroxisome proliferator-activated receptor γ | Transcription factor regulating lipid metabolism; key regulator of adipocyte differentiation and glucose homeostasis | Lipid mobilization; fatty acid β-oxidation; adipocyte differentiation; glucose metabolism |
| ACOT7 | Acyl-CoA thioesterase 7 | Hydrolyzes palmitoyl-CoA and other long-chain fatty acyl-CoAs into free fatty acids and CoA | Fatty acid oxidation/degradation |
| ACOX2 | Acyl-CoA oxidase 2 | Degrades branched-chain fatty acids and bile acid intermediates in peroxisomes | Fatty acid oxidation/degradation |
| ACLY↓ | ATP-citrate lyase | Catalyzes conversion of CoA and citrate to acetyl-CoA and oxaloacetate | Lipogenesis; cholesterol synthesis; gluconeogenesis |
| FASN↓ | Fatty acid synthase | Synthesizes long-chain fatty acids (e.g., palmitate) from malonyl-CoA and acetyl-CoA (requires NADPH) | Lipogenesis |
| SCD1↓ | Stearoyl-CoA desaturase 1 | Introduces double bonds into stearoyl-CoA to produce monounsaturated fatty acid (oleic acid) | Lipogenesis |
| SREBP1 | Sterol regulatory element-binding protein 1 | Transcription factor controlling LDL receptor expression; regulates genes involved in glucose/lipid synthesis | Lipogenesis; glucose metabolism |
| HMGCS1↓ | 3-Hydroxy-3-methylglutaryl-CoA synthase 1 | Catalyzes condensation of acetyl-CoA and acetoacetyl-CoA to form HMG-CoA (substrate for HMG-CoA reductase) | Cholesterol synthesis |
| FGF21↓ | Fibroblast growth factor 21 | Stimulates glucose uptake in adipocytes; regulates insulin sensitivity | Glucose uptake/clearance; insulin signaling |
| NADPH Generation & Pentose Synthesis | |||
| G6PD | Glucose-6-phosphate dehydrogenase | Generates NADPH in the pentose phosphate pathway; maintains glutathione redox state | NADPH generation |
| IDH1 | Isocitrate dehydrogenase 1 | Catalyzes oxidative decarboxylation of isocitrate to α-ketoglutarate (uses NADP⁺) | NADPH generation |
| TALDOL | Transaldolase 1 | Produces ribose-5-phosphate (required for nucleic acid synthesis) | Pentose phosphate pathway |
| TKT | Transketolase | Directs excess sugars from the pentose phosphate pathway to glycolysis (via glyceraldehyde-3-phosphate) | Pentose phosphate pathway; glycolysis |
| Other Functions | |||
| CD36 | Scavenger receptor/ Fatty acid translocase | Binds long-chain fatty acids; mediates fatty acid transport/regulation | Fatty acid transport; cell adhesion |
| P62/SQSTM1 | Selective autophagy adapter protein | Required for polyubiquitin body formation and autophagic degradation; acts as a scaffold protein | Autophagy; immunity |
| NOTCH1 | Notch1 (Transmembrane protein) | Contains EGF-like repeats; participates in signaling for development and tissue regeneration | Cell development signaling |
| AHR | Aryl hydrocarbon receptor | Binds planar aromatic compounds; upregulates transcription of xenobiotic metabolism-related genes (including CYP family members) | Xenobiotic metabolism regulation |
| Keap1 | Kelch-like ECH-associated protein 1 | E3 ubiquitin ligase substrate adapter; targets proteins for 26S proteasome degradation; known negative regulator of Nrf2 | Targeted ubiquitination; inhibition of Nrf2 activation; antioxidant response regulation |
Nrf2 activation also improves various chronic diseases, including metabolic disorders, respiratory diseases, gastrointestinal diseases, cardiovascular diseases, and neurodegenerative diseases. These seemingly unrelated diseases all share the Nrf2 "switch" that protects cells from internal and external damage.
Some may ask: Isn’t broccoli’s biggest selling point its anti-cancer effect? It must be noted that Nrf2’s role in cancer remains unclear—it is considered both a tumor suppressor and a tumor promoter. On one hand, Nrf2’s target genes are key mediators of DNA repair and xenobiotic metabolism, playing an important role in preventing cancer initiation and carcinogen accumulation. On the other hand, cancer cells can enhance Nrf2 activity to create a more favorable survival environment, increasing their viability—making Nrf2 an important target for anti-cancer drugs[8].
Note: Cancer cells are highly adaptable—they can exploit almost any health-promoting mechanism. As we have discussed in previous articles, however, there is no need to abandon beneficial practices out of fear.
4. Activation of Nrf2
As an invisible transcription factor, people are most concerned about how to activate it. As mentioned earlier, Nrf2 is usually in an "inactive" state. Only under low-level oxidative stress does it undergo the process of "Keap1 releasing its grip → Nrf2 entering the nucleus → Nrf2 binding to ARE → activating transcription of related genes" to exert effects at the genetic and protein levels.
What constitutes a "low-level stress environment"? Exercise and calorie restriction are well-known examples...
In addition to "controlling diet and increasing physical activity," many active substances in our daily diet can activate Nrf2, such as plant polyphenols, terpenes, organosulfur compounds, n-3 polyunsaturated fatty acids, and carotenoids. Among these, sulforaphane (SFN, an isothiocyanate)—found in broccoli and other cruciferous vegetables—exerts the most significant Nrf2 activation effect.
However, scientists point out that to obtain potential health benefits from broccoli alone, one would need to eat nearly 3 kg (6 pounds) of raw broccoli every day...
In terms of drugs, many new Nrf2-activating drugs have entered clinical trials for various indications:
- Sulforaphane and curcumin (for cancer prevention)[9, 10];
- Dimethyl fumarate (for treating multiple sclerosis)[11];
- Bardoxolone-methyl (for treating diabetic nephropathy)[12];
- Oltipraz (for delaying senescence of mesenchymal stem cells)[13].
These so-called "Nrf2 activators" should more accurately be called "Keap1 inhibitors," as they actually interact with cysteine residues in Keap1. The reactivity of cysteine thiols depends on the immediate environment of adjacent amino acid residues, which varies greatly within and between proteins—thus, different drug concentrations are expected to produce different biological effects.
Appendix: Nrf2 Activators
| Compound | Mechanism of Action | Indication | Clinical Phase | Identifier (NCT Number) |
|---|---|---|---|---|
| Bardoxolone-methyl (CDDO-Me) | Electrophilic modification of Keap1-Cys-151 | Chronic kidney disease, advanced solid tumors, lymphoid malignancies | 2/3 | NCT01351675, NCT03019185 |
| Familial hematuric nephritis, pulmonary hypertension | 3 | NCT03068130, NCT02657356 | ||
| Renal insufficiency, type 2 diabetes | 2 | NCT01053936 | ||
| Omilancor | Electrophilic modification of Keap1-Cys-151 | Mitochondrial myopathy | 2 | NCT02255422 |
| Ocular inflammation, cataract surgery | 2 | NCT02128113 | ||
| Melanoma | 1/2 | NCT02259231 | ||
| Breast cancer | 2 | NCT02142959 | ||
| Dimethyl fumarate | Electrophilic modification of Keap1-Cys-151 | Rheumatoid arthritis | 2 | NCT00810836 |
| Adult glioblastoma | 1 | NCT02337426 | ||
| Cutaneous T-cell lymphoma | - | NCT02546440 | ||
| Chronic lymphocytic leukemia, small lymphocytic lymphoma | - | NCT02784834 | ||
| Oltipraz | Electrophilic modification of Keap1-Cys-151 | Non-alcoholic steatohepatitis (NASH) | 3 | NCT02068339 |
| Lung cancer | 1 | NCT00006457 | ||
| ALKS-8700 | Electrophilic modification of Keap1-Cys-151 | Multiple sclerosis | 3 | NCT02634307 |
| Ursodeoxycholic acid | Electrophilic modification of Keap1-Cys-151 | Diarrhea | 4 | NCT02748616 |
| Cholelithiasis | 3 | NCT02721862 | ||
| Primary biliary cholangitis | 4 | NCT01510860, NCT00200343 | ||
| Chronic hepatitis C, type 2 diabetes | 2 | NCT02033876 | ||
| Sulforaphane | Electrophilic modification of Keap1-Cys-151 | Schizophrenia | 2/3 | NCT02880462 |
| Healthy volunteers | 1 | NCT01008826, NCT02023931 | ||
| Melanoma, prostate cancer, breast cancer, lung cancer | 1/2 | NCT01568996, NCT01228084, NCT00843167, NCT03232138 | ||
| Environmental carcinogenesis, aging, Helicobacter pylori infection | 2 | NCT01437501, NCT03126539, NCT03220542, NCT02801448 | ||
| Sulfonamide (SFX-01) | Electrophilic modification of Keap1-Cys-151 | Subarachnoid hemorrhage | 2 | NCT02614742 |
| Breast tumors, prostate cancer | 1/2 | NCT02970682, NCT02055716, NCT01948362 | ||
| Curcumin | Electrophilic modification of Keap1-Cys-151 | Type 2 diabetes, prediabetes, insulin resistance, cardiovascular risk | 4 | NCT01052025 |
| Chronic kidney disease (type 2 diabetes-related), tumors | 2/3 | NCT03262363, NCT02944578 | ||
| Crohn’s disease, chronic schizophrenia, mild cognitive impairment | 3/4 | NCT02255370, NCT02298985, NCT01811381 | ||
| Prostate cancer, major depressive disorder | 3/4 | NCT02064673, NCT01750359 | ||
| Resveratrol | Electrophilic modification of Keap1-Cys-151 | Non-alcoholic fatty liver disease (NAFLD) | 2/3 | NCT02030977 |
| Non-ischemic cardiomyopathy, endometriosis | 3/4 | NCT01914081, NCT02475564 | ||
| Chronic renal insufficiency, metabolic syndrome, Alzheimer’s disease, Huntington’s disease | 2/3 | NCT02433925, NCT02114892, NCT01504854, NCT02336633 | ||
| CXA-10 | Electrophilic modification of Keap1-Cys-151 | Acute kidney injury | 1 | NCT02248051 |
| Pulmonary arterial hypertension (PAH), focal segmental glomerulosclerosis (FSGS) | 2 | NCT03449524, NCT03422510 | ||
| Teglitazar | GSK-3 inhibition | Autism spectrum disorder, myotonic dystrophy type 1, Alzheimer’s disease | 2 | NCT02586935, NCT02858908, NCT01350362 |
| Nordihydroguaiaretic acid (NDGA) | GSK-3 inhibition | Prostate cancer, brain and central nervous system tumors | 1/2 | NCT00678015, NCT00404248 |
| Telomestatin (NDGA derivative) | GSK-3 inhibition | High-grade glioma, leukemia (acute myeloid leukemia, acute lymphoblastic leukemia) | 1 | NCT02575794, NCT00664677 |
| Refractory solid tumors, lymphoma | - | NCT00664586 |
References
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