A cherry-picked DNA methylation study

This 2020 US/Sweden/Denmark human study measured twins during their old age:

“We evaluate individual differences in DNA methylation at individual CpG sites across the methylome across 10 years in two Scandinavian samples of same‐sex aging twins. We test two competing hypotheses about the longitudinal stability and change in DNA methylation:

  1. The contribution of genetic influences changes with age, reflecting diminishing influence across time; and
  2. Nonshared factors accumulate in importance, signaling an increasing diversity of response to environmental exposures.

Understanding epigenetic changes over time in the elderly may identify pathways of decline or plasticity (e.g., maintenance or even boosts in functioning) during the aging process and help with elucidating the biology of aging and survival.

Across time, stability in methylation is primarily due to genetic contributions, while novel experiences and exposures contribute to methylation differences. Elevated genetic contributions at age‐related methylation sites suggest that adaptions to aging and senescence may be differentially impacted by genetic background.”

https://onlinelibrary.wiley.com/doi/full/10.1111/acel.13197 “A decade of epigenetic change in aging twins: Genetic and environmental contributions to longitudinal DNA methylation”


Swedish subject measurements were taken at ages 62 and 72. Danish subject measurements were taken at ages 76 and 86.

One epigenetic clock that used 2019 technology was favored over three others, including Horvath’s 2013 original clock. For some reason this study didn’t use his 2018 skin-and-blood clock that had vastly improved technology such as an 18-fold increase in genomic coverage with Illumina 450k/850k bead arrays.

These researchers’ intentions became evident with:

“The 353 Horvath clock sites were selected as best predictors of chronological age using multiple tissues. The 71 Hannum clock sites best predicted age (adjusted for sex, BMI) based on methylation observed in whole blood while the 514 sites from the Zhang prediction model relied on methylation observed in blood and saliva samples (Zhang et al., 2019).

The current findings of moderately higher heritabilities in the Zhang and Hannum sites versus the other clock sites may be in part due to our use of blood tissue.”

The 18-fold increase improved accuracy in blood for the 2018 Horvath clock. Could these researchers ignore it and claim they did their due diligence in 2019 and 2020?


A larger issue was this study’s duality paradigm of either heritability or environment being solely responsible for observed changes. Consider what A blood plasma aging clock found at ages 60 and 78 peaks:

The above changes were due to life stage. Josh Mitteldorf did his usual excellent job of providing contexts for that study with New Aging Clock based on Proteins in the Blood, including:

“The implication is that a more accurate clock can be constructed if it incorporates different information at different life stages. None of the Horvath clocks have been derived based on different CpG sites at different ages, and this suggests an opportunity for a potential improvement in accuracy.”

Weren’t changes in subjects’ life stages relevant to their epigenetic changes? Why wouldn’t their life stages have been among the causes of observed effects?

Sulforaphane and RNAs

This 2020 Texas review subject was long non-coding RNAs:

“We review the emerging significance of long non-coding RNAs (lncRNA) as downstream targets and upstream regulators of the Nrf2 signaling pathway, a critical mediator of diverse cellular processes linked to increased cell survival.

It is believed that more than 3% of human genes are regulated by the Nrf2/Keap1 pathway. In addition to the classical cytoprotective and oxidative stress response genes transactivated by Nrf2, emerging evidence suggests a role for non-coding transcript regulation at the level of noncoding RNAs, [which] far outnumber protein-coding genes in the human genome.

One important distinction between miRNAs and lncRNAs is that the latter are often species-specific, meaning that a human lncRNA typically cannot be studied in the mouse or rat, and vice versa.

Sulforaphane (SFN) acts via multiple mechanisms to modulate gene expression, including the induction of Nrf2-dependent signaling. In addition to the established canonical targets of Nrf2, such as NQO1 and HMOX1, SFN altered the expression of multiple lncRNAs.

Given that SFN induces NMRAL2P [a lncRNA pseudogene] and several other lncRNAs in colon cancer cells, further studies are warranted on their respective roles as upstream regulators and/or downstream targets of Nrf2 signaling.

Pharmacological modulation of Nrf2 is considered a viable strategy against chronic conditions that are accompanied by oxidative stress and inflammation:

  • DMF [dimethyl fumurate] is the most successful Nrf2 activator, FDA-approved in 2013 for the treatment of relapsing remitting multiple sclerosis. However, DMF causes leukopenia and other side-effects.
  • Bardoxolone cleared Phase II clinical trials for the treatment of advanced chronic kidney disease and type 2 diabetes mellitus, but was halted in Phase III trials due to cardiovascular concerns.
  • SFN is relatively unstable at room temperature.

We used reported bioinformatics approaches to search for putative ARE [antioxidant response element] sequences among the entire set of 16,000+ annotated human lncRNAs. 13,285 promoter regions contained one or more potential binding sites for Nrf2.”

https://www.sciencedirect.com/science/article/pii/S0304383520303670 “Emerging crosstalk between long non-coding RNAs and Nrf2 signaling”


This study hyped lncRNAs in that only 7 have been validated as Nrf2 targets, and 8 validated as Nrf2 regulators. For regulators, “protein and/or miRNA interacting partners are yet to be fully corroborated” as well.

Also, there’s no need for a “SFN is relatively unstable at room temperature” problem. Just create sulforaphane right before consuming it.

Twice a day I microwave an average 65.5 grams of 3-day-old broccoli sprouts immersed in 100 ml water with a 1000W microwave on full power for 35 seconds to achieve 60°C. After microwaving I transfer broccoli sprouts to a strainer, and wait five minutes to allow further myrosinase hydrolization of glucoraphanin and other glucosinolates into sulforaphane and other healthy compounds.

Eat sauerkraut today!

This 2017 Spanish article reviewed health benefits of sauerkraut:

“During cabbage shredding and fermentation, a disruption of cabbage cells occurs, and GLS [glucosinolates] are hydrolyzed by myrosinase enzyme to a variety of GLS breakdown products. In particular, glucobrassicin is hydrolyzed into indol-3-carbinol (I3C) by myrosinase.

As the pH decreases during cabbage fermentation, I3C reacts nonenzymatically with ascorbic acid to yield ascorbigen (ABG). Studies have shown that ABG is the main GLS breakdown compound in sauerkraut, and it is present at levels between 3 and 18 μmol/100 g fw.

The antioxidant activity observed for sauerkraut in all studies was higher than that observed in raw cabbage.

It has been reported that doses between 53 and 150 μmol of ITCs [isothiocyanates] are enough to display anticarcinogenic effects. Taking into account that the content of ITCs in sauerkraut is in the range 22 μmol/100 g fw, it could be assumed that a weekly consumption of 200–250 g of sauerkraut would provide effective ITC doses to exert cancer chemopreventive effects.

Many studies reported that LAB [lactic acid bacteria] isolated from sauerkraut are potential probiotics.”

https://www.sciencedirect.com/science/article/pii/B9780128023099000248 “Sauerkraut: Production, Composition, and Health Benefits” (not freely available)


This introductory article presented interesting facts, but oversold sauerkraut. Dose and other conditional dependencies in order to achieve health and disease prevention benefits seemed to be beyond its scope.

A more considered view was offered in Fermented Food and Non-Communicable Chronic Diseases which referenced this article:

“Clinical data about the effects of sauerkraut on the human organism, health and disease are scarce. There is knowledge concerning particular compounds in sauerkraut and their impacts on diseases; however, a literature search revealed mostly cell line or rat experiments with very limited conclusions for humans.”


Earlier this month I started eating refrigerated sauerkraut twice a day with microwaved broccoli sprouts. I mix in three heaping teaspoons each time, and finish a 50 oz (1418 g) container in a week.

The mixture tastes better than just microwaved broccoli sprouts. It requires more chewing, which assists myrosinase hydrolization of broccoli sprout glucosinolates into sulforaphane and other healthy compounds.

Although sauerkraut isn’t a primary source, there may be beneficial amounts of probiotics etc. that increase what I get with broccoli sprouts and supplements.

I also started making my own sauerkraut using the commercial product’s juice as a starter. I add garlic but not salt. No results yet.

Take responsibility for your one precious life – Vitamin D3

Where to start among 6,489 studies and reviews published during the past five years, results from a PubMed search of “dihydroxyvitamin D3.” How about:

“Vitamin D plays a fundamental role in body calcium and phosphorous homeostasis, ensuring proper functioning of the skeletomuscular system. Pleiotropic activities include:

  • Anti-inflammatory and immunomodulatory properties (predominantly downregulation of adaptive and upregulation of innate immunity);
  • An important role in reproduction, pregnancy, placental functions and fetal and child development;
  • Important in neurodevelopment as well as in the functioning of the adult central and peripheral nervous system;
  • Regulation of global metabolic and endocrine homeostasis and the functions of different endocrine organs, as well as in the functioning of the cardiovascular system;
  • Inhibits malignant transformation, tumor progression and has anti-cancer properties on a variety of tumors;
  • Formation of the epidermal barrier and hair cycling; and
  • Ameliorating effects on skin cancer and on proliferative and inflammatory cutaneous diseases.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6342654/ “The serum vitamin D metabolome: What we know and what is still to discover”

Or maybe:

“A study in 6,275 American children and adolescents aged 1–21 years showed that 61% were 25-(OH)D3 insufficient and 9% deficient. In adults, up to 40% are 25-(OH)D3 insufficient and 6% deficient.

Once adequate vitamin D values are reached, to further preserve adequate vitamin D levels in adults, the IOM [Institute of Medicine] recommends a daily dose of 600 IU per day, while the Endocrine Society recommends a dose of 600–2000 IU per day (according to the amount of sunlight the individual is exposed to). There seems to be no additional health benefit in doses higher than 4000 IU/day.

Vitamin D supplementation was protective against acute respiratory tract infections in a 25-(OH)D3 deficient population, especially in those receiving daily or weekly supplementation. However, in children this protective effect could not be reproduced.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7281985/ “Vitamin D’s Effect on Immune Function”

Not to forget Advanced glycation end products alter steroidogenic gene expression by granulosa cells: an effect partially reversible by vitamin D:

“This study suggests that there is a relationship between AGEs (advanced glycation end products) and their receptors (RAGE and sRAGE) with vitamin D. Understanding the interaction between AGEs and vitamin D in ovarian physiology could lead to a more targeted therapy for the treatment of ovarian dysfunction.”

Or similarities to broccoli sprouts’ main effect of Nrf2 signaling pathway activation:

“1,25(OH)2D3 plays a role in delaying aging by upregulating Nrf2, inhibiting oxidative stress and DNA damage, inactivating p53‐p21 and p16‐Rb signaling pathways, and inhibiting cell senescence and SASP.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6516172/ “1,25‐Dihydroxyvitamin D exerts an antiaging role by activation of Nrf2‐antioxidant signaling and inactivation of p16/p53‐senescence signaling”


Why do we insist on giving ourselves non-communicable diseases?

I recently paid $22.53 after tax for a nearly two-year supply:

A better use of one’s money would be..?

My June 2020 serum 25-OH Vitamin D measurement was 76 on a scale of 0 to 100 from taking a total of 3,400 IU daily. It’s fat-soluble, so I take it along with 1 gram flax oil each time.

Take responsibility for your own one precious life.

Part 2 of Do broccoli sprouts treat migraines?

To follow up Do broccoli sprouts treat migraines? which used a PubMed “sulforaphane migraine” search, a PubMed “diindolylmethane” search came across a 2020 Czech human cell study Antimigraine Drug Avitriptan Is a Ligand and Agonist of Human Aryl Hydrocarbon Receptor that Induces CYP1A1 in Hepatic and Intestinal Cells that had this informative Introduction:

“The aryl hydrocarbon receptor (AhR) transcriptionally controls a wide array of genes. AhR is a critical player in human physiology (e.g., hematopoiesis) and also in many pathophysiological processes such as diabetes, carcinogenesis, inflammation, infection or cardiovascular diseases.

Suitable candidates for off-targeting AhR could be the antimigraine drugs of triptan class, which have an indole core in their structure. Indole-based compounds were demonstrated as ligands of AhR, including dietary indoles (e.g., indole-3-carbinol and diindolylmethane).”

Adding AhR to the search showed:

Changing the PubMed search to “icz migraine” pulled up a 2013 review Biomedical Importance of Indoles that described sumatriptan as an indole, and:

“Since DIM accumulates in the cell nucleus, it likely contributes to cell nuclear events that have been ascribed to I3C.”

Widening the search to “i3c ahr” added:

Changing the search to “i3c migraine” picked up a 2011 UK human study Effect of diindolylmethane supplementation on low-grade cervical cytological abnormalities: double-blind, randomised, controlled trial:

“In the study reported here, there was no statistically significant difference in serious adverse events between groups; in fact a higher proportion of women in the placebo group reported a serious adverse event. Although this study did not have sufficient power to study migraines, we did find a non-significant increase in reported headaches (18% on DIM, 12% on placebo, P=0.12).”

Returning to the original PubMed “sulforaphane migraine” search, Bioavailability of Sulforaphane Following Ingestion of Glucoraphanin-Rich Broccoli Sprout and Seed Extracts with Active Myrosinase: A Pilot Study of the Effects of Proton Pump Inhibitor Administration included one subject who took migraine medication. They weren’t a study outlier, however.


Although indole chemistry indicates a broccoli sprouts – migraine connection, I haven’t found relevant research. Maybe the known properties and actions of broccoli sprout compounds provide enough to affect causes of migraines?

Day 70 results from Changing to a youthful phenotype with broccoli sprouts

Here are my Day 70 measurements* to follow up Our model clinical trial for Changing to a youthful phenotype with broccoli sprouts, which had these findings:


Keep in mind that I’m not in the population represented by the clinical trial sample:

  1. My chronological age is above their inclusion range;
  2. My BMI is below their inclusion range; and
  3. I take supplements and meet other exclusion criteria.

I also didn’t take Day 0 measurements.

June 2019 BMI: 24.8

June 2020 BMI: 22.4

2020 IL-6: 1.0 pg / ml. See Part 2 of Rejuvenation therapy and sulforaphane for comparisons.

2020 C-reactive protein: < 1 mg / l.

2019 and 2020 No biological age measurements. Why aren’t epigenetic clocks standard and affordable?


I’ve made four lifestyle “interventions” since last summer:

  1. In July 2019 I started to reduce my consumption of advanced glycation end products after reading Dr. Vlassara’s AGE-Less Diet: How a Chemical in the Foods We Eat Promotes Disease, Obesity, and Aging and the Steps We Can Take to Stop It.
  2. In September I started non-prescription daily treatments of Vitamin D, zinc, and DHEA per clinical trial Reversal of aging and immunosenescent trends.
  3. Also in September, I started non-prescription intermittent quercetin treatments of Preliminary findings from a senolytics clinical trial.
  4. I started eating broccoli sprouts every day eleven weeks ago.

1. Broccoli sprouts oppose effects of advanced glycation end products (AGEs) provided examples of Items 1 and 4 interactions.

2. Two examples of Item 2 treatment interactions with Item 4 are in Reversal of aging and immunosenescent trends with sulforaphane:

  • “The effects of the combined treatment with BSE [broccoli sprout extract] and zinc were always greater than those of single treatments.”
  • “Vitamin D administration decreased tumor incidence and size, and the co-administration with SFN [sulforaphane] magnified the effects. The addition of SFN decreased the activity of histone deacetylase and increased autophagy.”

3. How broccoli sprout compounds may complement three supplements I take was in a 2020 review Central and Peripheral Metabolic Defects Contribute to the Pathogenesis of Alzheimer’s Disease: Targeting Mitochondria for Diagnosis and Prevention:

“The nutrients benefit mitochondria in four ways, by:

  • Ameliorating oxidative stress, for example, lipoic acid;
  • Activating phase II enzymes that improve antioxidant defenses, for example, sulforaphane;
  • Enhancing mitochondrial remodeling, for example, acetyl-l-carnitine; and
  • Protecting mitochondrial enzymes and/or stimulating mitochondrial enzyme activities, for example, enzyme cofactors, such as B vitamins and coenzyme Q10 .

In addition to using mitochondrial nutrients individually, the combined use of mitochondrial nutrients may provide a better strategy for mitochondrial protection.”

The review provided a boatload of mitochondrial multifactorial analyses for Alzheimer’s. But these analyses didn’t include effective mitochondrial treatments of ultimate aging causes. I didn’t see evidence of why, after fifteen years of treating mitochondrial effects with supplements, treating one more effect could account for my Week 9 vastly different experiences.


I nod to An environmental signaling paradigm of aging explanations. Its Section 10 reviewed IL-6, C-reactive protein, senescence, and NF-κB in terms of feedback loops, beginning with:

“It is clear that the increasing number of senescent cells depends on the post-adult developmental stage rather than chronological age. The coincidence that these processes result in particular forms of impairment in old age does not seem to be random as it is present in all mammals, and may be causative of many aspects of aging.”

A derived hypothesis: After sufficient strength and duration, broccoli sprout compounds changed my signaling environment, with appreciable effects beginning in Week 9.

I offered weak supporting evidence in Upgrade your brain’s switchboard with broccoli sprouts where a study’s insufficient one week duration of an insufficient daily 17.3 mg sulforaphane dosage still managed to change a blood antioxidant that may have changed four thalamus-brain-area metabolites. For duration and weight comparisons, I doubled my daily amount of broccoli seeds from one to two tablespoons just before Week 6 (Day 35), and from that point onward consumed a estimated 30 52 mg sulforaphane with microwaving 3-day-old broccoli sprouts every day.

Maybe a promised “In a submitted study, we will report that peripheral GSH levels may be correlated with cognitive functions” will provide stronger evidence? I’m not holding my breath for relevant studies because:

  • There wouldn’t be potential payoffs for companies to study any broccoli sprout compound connections with research areas such as aging, migraines, etc. Daily clinically-relevant broccoli sprout dosages can be grown for < $500 a year.
  • Sponsors would have to change paradigms, a very-low-probability event. They’d have to explain why enormous resources dedicated to current frameworks haven’t produced effective long-term treatments.

What long-term benefits could be expected if I continue eating broccoli sprouts every day?

The longest relevant clinical trial I’ve seen – referenced in Part 2 of Reversal of aging and immunosenescent trends with sulforaphane – was twelve weeks. Part 2 also provided epigenetic clock examples of changes measured after 9 months, which accelerated from there to the 12-month end-of-trial point.

Reviewing clinical trials of broccoli sprouts and their compounds pointed out:

“Biomarkers of effect need more time than biomarkers of exposure to be influenced by dietary treatment.”


A contrary argument: Perhaps people don’t require long durations to effectively change their signaling environments?

I apparently didn’t start eating an effective-for-me daily broccoli sprouts dosage until Day 35, when I changed from one to two tablespoons of broccoli seeds a day. If so, Weeks 6 through 8 may account for my substantial responses during Week 9.

  • Could eating broccoli sprouts every day for four weeks dramatically change a person’s signaling environment?
  • Do you have four weeks and $38 to find out? Two tablespoons of broccoli seeds = 38 21.4 g x 30 days = .642 kg or 1.42 lbs.

This is what twice-a-day one-tablespoon starting amounts of broccoli seeds look like through three days:


Maintaining the sprouting process hasn’t been a big effort compared with the benefits.

In the absence of determinative evidence, I’ll continue eating broccoli sprouts every day. Several areas of my annual physical have room for improvements. Extending my four lifestyle “interventions” a few more months may also provide hints toward inadequately researched connections.

* Results may not be extrapolatable to other people, to any specific condition, etc.

A review of sulforaphane and aging

This 2019 Mexican review stated:

“We describe some of the molecular and physical characteristics of SFN, its mechanisms of action, and the effects that SFN treatment induces in order to discuss its relevance as a ‘miraculous’ drug to prevent aging and neurodegeneration. SFN has been shown to modulate several cellular pathways in order to activate diverse protective responses, which might allow avoiding cancer and neurodegeneration as well as improving cellular lifespan and health span.

NF-κB is in charge of inflammatory response regulation. Under basal conditions, NF-κB is sequestrated into the cytosol by IκB, but when pro-inflammatory ligands bind to its receptors, the IKK protein family phosphorylates IκB to degrade it via proteasome, so NF-κB is able to translocate into the nucleus and transcript several inflammatory mediators. Sulforaphane is capable to inhibit IκB phosphorylation and NF-κB nuclear translocation.

SFN upregulated Nrf2 expression by reducing DNA demethylation levels of the Nrf2 promoter. In another model using the triple-transgenic mouse model of Alzheimer’s disease (3 × Tg-AD), the use of SFN regulates the expression of the Brain-derived neurotrophic factor (BDNF) via HDAC inhibition, thus increasing H3 and H4 acetylation on the BDNF promoter. Enhancing BDNF expression as an effect of SFN treatment increased the neuronal content of several synaptic molecules like MAP 2, synaptophysin, and PSD-95 in primary cortical neurons of 3 × Tg-AD.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6885086/ “Sulforaphane – role in aging and neurodegeneration”


I came across this review while searching PubMed for sulforaphane commonalities with presentation topics in Part 2 of Reversal of aging and immunosenescent trends with sulforaphane. The review outlined some aging aspects and presented relevant sulforaphane studies. Others such as eye and muscle decline weren’t addressed.

Since sulforaphane’s “a ‘miraculous’ drug” in the Abstract, I expected but didn’t see corresponding excitement in the review body. Just phrases like “it is known” and non-specific “more research is needed.”

Other papers published after this review were found by a PubMed “sulforaphane signal aging” search:


Part 2 of Reversal of aging and immunosenescent trends with sulforaphane

Reversal of aging and immunosenescent trends with sulforaphane covered only the first 13 minutes of a super informative presentation by the lead researcher of clinical trial Reversal of aging and immunosenescent trends.  Commonalities with sulforaphane research were found by PubMed searches of sulforaphane and each presentation topic, and used a 1/1/2015 publication date cutoff.

Continuing presentation topics from the 13:40 mark:

Cancer

Lymphocyte/monocyte ratio

CD38 monocytes

  • NQO1-induced activation of AMPK contributes to cancer cell death by oxygen-glucose deprivation

    “NQO1 plays a key role in the AMPK-induced cancer cell death in OGD through the CD38/cADPR/RyR/Ca2+/CaMKII signaling pathway. The expression of NQO1 is elevated by hypoxia/reoxygenation or inflammatory stresses through nuclear accumulation of the NQO1 transcription factor, Nrf2 (NFE2-related factor 2). Activation of the cytoprotective Nrf2 antioxidant pathway by sulforaphane protects immature neurons and astrocytes from death caused by exposure to combined hypoxia and glucose deprivation.”

Thymus – no recent sulforaphane studies

Renal function

  • Rapid and Sustainable Detoxication of Airborne Pollutants by Broccoli Sprout Beverage: Results of a Randomized Clinical Trial in China

    “Rapid and sustained, statistically significant increases in the levels of excretion of the glutathione-derived conjugates of benzene (61%), acrolein (23%), but not crotonaldehyde were found in those receiving broccoli sprout beverage compared with placebo. Excretion of the benzene-derived mercapturic acid was higher in participants who were GSTT1-positive compared to the null genotype, irrespective of study arm assignment. Measures of sulforaphane metabolites in urine indicated that bioavailability did not decline over the 12-week daily dosing period. Thus, intervention with broccoli sprouts enhances the detoxication of some airborne pollutants and may provide a frugal means to attenuate their associated long-term health risks.”

Hair rejuvenation

Epigenetic clocks – There are no sulforaphane studies that use epigenetic clocks, although broccoli compounds have epigenetic effects on aging, as reviewed in 2019:

  • Sulforaphane – role in aging and neurodegeneration

    “SFN has been shown to modulate several cellular pathways in order to activate diverse protective responses, which might allow avoiding cancer and neurodegeneration as well as improving cellular lifespan and health span.”


Both biomarker (Lymphocyte / monocyte ratio) and epigenetic clock (GrimAge) measurements done 6 months after the clinical trial ended suggested trial subjects’ aging phenotypes had been reset:

An environmental signaling paradigm of aging explained:

Apart from being slowed down or sped up, the body clock can also be reset. Organisms, organs, and their cells can be reset to different age-phenotypes depending on their environment.

This is not so much a principle as an application of principle that the environment determines age-phenotype.

There wouldn’t be a potential payoff for a company to study any broccoli compound / aging connections. People can achieve clinically relevant, daily doses of broccoli sprouts for < $500 a year.

What sponsor would be interested enough to put sulforaphane research on the clock?

Presentation topics are continued in Uses of the lymphocytes to monocytes ratio and A review of sulforaphane and aging.

Reevaluate findings in another paradigm

It’s challenging for people to change their framework when their paychecks or mental state or reputations depend on it not changing.

I’ll use The hypothalamus and aging as an example. The review was alright for partial fact-finding up through 2018. The review’s facts were limited, however, to what fit into the reviewers’ paradigm.

The 2015 An environmental signaling paradigm of aging provided examples of findings that weren’t considered in the review. It also presented a framework that better incorporated what was known at the time.


Here’s how they viewed the same 2013 study, Hypothalamic programming of systemic ageing involving IKK-β, NF-κB and GnRH (not freely available).

Paradigm: “The hypothalamus is hypothesized to be a primary regulator of the process of aging of the entire body.”

Study assessment:

“The age-associated inflammation increase is mediated by IκB kinase-β (IKK-β) and nuclear factor κB (NF-κB) in the microglia and, subsequently, nearby neurons through the microglia–neuron interaction in the mediobasal hypothalamus. Apparently, blocking the hypothalamic or brain IKK-β or NF-κB activation causes delayed aging phenotype and improved lifespan.

Aging correlates with a decline in the hypothalamic GnRH expression in mice and, mechanistically, activated IKK-β and NF-κB significantly down regulates the GnRH transcription. Notably, GnRH therapy through either hypothalamic third ventricularor subcutaneous injection leads to a significant recovery of neurogenesis in the hypothalamus and hippocampus and a noticeable improvement of age-related phenotype in the skin thickness, bone density, and muscle strength when applied in middle-aged mice.”

Paradigm: Environmental signaling model of aging

Study assessment:

“A link between inflammation and aging is the finding that inflammatory and stress responses activate NF-κB in the hypothalamus and induce a signaling pathway that reduces production of gonadotropin-releasing hormone (GnRH) by neurons. GnRH decline contributes to aging-related changes such as bone fragility, muscle weakness, skin atrophy, and reduced neurogenesis. Consistent with this, GnRH treatment prevents aging-impaired neurogenesis and decelerates aging in mice.

Zhang et al. report that there is an age-associated activation of NF-κB and IKK-β. Loss of sirtuins may contribute both to inflammation and other aspects of aging, but this explanation, also given by Zhang et al. merely moves the question to why there a loss of sirtuins.

The case is particularly interesting when we realize that the aging phenotype can only be maintained by the continuous activation of NF-κB – a product of which is the production of TNF-α. Reciprocally when TNF-α is secreted into the inter-cellular milieu, it causes the activation of NF-κB. In their study, Zhang et al. noted that the activation of NF-κB began in the microglia (the immune system component cells found in the brain), which secreted TNF-α, resulting in a positive feedback loop that eventually encompassed the entire central hypothalamus.

The net result of this is a diminution in the production of gonadotropin-releasing factor which accounted for a shorter lifespan because provision of GnRH eliminated that effect, while either preventing NF-κB activation (or that of the IKK-β upstream activator) or by providing gonadotropin-releasing factor directly into the brain, or peripherally, extended lifespan by about 20%.

In spite of the claim of Zhang et al. that the hypothalamus is the regulator of lifespan in mice, their experiments show that only some aspects of lifespan are controlled by the hypothalamus, as preventing NF-κB activation in this organ did not stop aging and death. Similar increased NF-κB activation with age has been seen in other tissues as well and said to account for dysfunction in aging adrenal glands. It was demonstrated that increased aging occurred as a result of lack of gonadotropin-releasing hormone and that increased lifespan resulted from its provision during aging.

In this manner:

  1. The aging of hypothalamic microglia leads to
  2. The aging of the hypothalamus, which leads to
  3. Aging elsewhere in the body.

So here we have a multi-level interaction:

  1. The activation of NF-κB leads to
  2. Cellular aging, leading to
  3. A diminished production of GnRH, which then
  4. Acts (through the cells with a receptor for it, or indirectly as a result of changes to GnRH-receptor-possessing cells) to decrease lifespan.

So the age state of hypothalamic cells, at least with respect to NF-κB activation, is communicated to other cells via the reduced output of GnRH.”


Not using the same frameworks, are they?

In 2015, the researcher told the world what could be done to dramatically change the entire research area. He and other researchers did so recently as curated in Part 3 of Rejuvenation therapy and sulforaphane which addressed hypothalamus rejuvenation.

Part 3 of Rejuvenation therapy and sulforaphane

Part 1 focused on the study’s clinical biomarkers. Part 2 highlighted its epigenetic clocks. Now we’ll look at rejuvenation of cognitive function.

Charts for this study’s most relevant human aging applications – measured by the new human-rat relative biological age clock – were in supplementary data due to combining the study’s untreated tissue samples into clock training data. Reanalyses showed:

“Using the final version of the epigenetic clocks, we find that the treatment effects become even more significant especially for the hypothalamus.”

Human-rat relative clock percentages of rejuvenation were:

  • “Blood 70.6%
  • Liver 79.4%
  • Heart 61.6%
  • Hypothalamus 20.9%”

The Discussion section addressed hypothalamus rejuvenation:

“Why does plasma fraction treatment not reduce brain epigenetic age by the same magnitude as it does the other organs? We can only begin to address this question after having first understood what epigenetic aging entails.

As it stands, our knowledge in this area remains limited, but it is nevertheless clear that:

  1. Epigenetic aging is distinct from the process of cellular senescence and telomere attrition,
  2. Several types of tissue stem cells are epigenetically younger than non-stem cells of the same tissue,
  3. A considerable number of age-related methylation sites, including some clock CpGs, are proximal to genes whose proteins are involved in the process of development,
  4. Epigenetic clocks are associated with developmental timing, and
  5. Relate to an epigenomic maintenance system.

Collectively, these features indicate that epigenetic aging is intimately associated with the process of development and homeostatic maintenance of the body post-maturity.

  • While most organs of the body turnover during the lifetime of the host, albeit at different rates, the brain appears at best to do this at a very much slower rate.
  • While most tissues harbor stem cells that are necessary for replenishment and turnover, stem cells in adult brain have only been detected in a defined and very limited area of the subventricular zone, olfactory bulb (in rats), hippocampus and hypothalamic proliferative region.

As such, if plasma fraction treatment’s rejuvenating effect is:

  • Mediated through the process of development and
  • Involves tissue stem cells

then its effect on the epigenetic age of the brain would appear to be modest, which indeed it does.

It is to be noted however, that improving brain function does not depend on neurogenesis as much as it does on synapse formation and factors such as NMDA receptors which decline in density with age.

Assessment of plasma fraction treatment on cognitive function (learning and memory). Rats were subjected to Barnes maze test – nine consecutive days of test where the time (in seconds) required by the rats to find the escape hole (latency) was recorded and plotted. The error bars depict 2 standard errors.

Within a month of plasma fraction treatment, the rats exhibited significantly reduced latency to escape, i.e., they learned and remembered better. After the second month, the treated rats began with a slightly reduced latency period compared to the untreated old rats, and once again, they learned much faster than the latter.

By the third month, it was clear that treated rats remembered the maze much better than the untreated ones even from the first day of test as their latency period was significantly reduced and by the end of the test period their latency was similar to that of the young rats. This feature was sustained and repeated in the fourth month.”

Not sure why there’s a 62-day gap between “Second month” and “Third month.” Maybe it had something to do with “First month” starting 10 days after the first treatment and “Third month” similarly starting 13 days after the second treatment?


Regarding cognitive function, a 2019 Italian paper Polyphenol Health Effects on Cardiovascular and Neurodegenerative Disorders: A Review and Meta-Analysis analyzed pathetic results of experiments with polyphenols other than broccoli sprout compounds:

“Current treatments to halt cognitive decline are limited to counteract symptoms and have a positive impact on cognition and behavior only in a transient manner, without affecting the underlying pathology.

Although some polyphenols might improve specific markers of cardiovascular risk and cognitive status, many inconsistent data are present in literature. Therefore, definitive recommendations for the use of these compounds in the prevention of cardiovascular disease and cognitive decline are currently not applicable.”


Many of us know older people who lived well past the time of good cognitive function. We see how they’re helpless and dependent. We see how others take advantage of them as they decline past the end of their healthspan.

We can make personal plans for that day, sure. But let’s also put some urgency into applying this study’s new human-rat relative biological age clock, and make:

“A step change in aging research. Although conservation of aging mechanism could be equally deduced from the existence of multiple individual clocks for other mammals (mouse, dog), the single formula of the human-rat clock that is equally applicable to both species effectively demonstrates this fact.”