Critical Analysis of Anti-Aging Methods

In 1972, researchers fed mice a diet containing the antioxidant vitamin E. One year later, they found that lipofuscin levels were indeed lower, but the mice's mortality rate did not decrease, indicating that the oxygen free radical theory of aging is wrong.

Methylene blue (MB) is a strong antioxidant with a significant ability to cross cell membranes and is distributed in different subcellular compartments, such as lysosomes and mitochondria, but it did not extend the average lifespan of mice [1].

Cellular reprogramming includes "pluripotent reprogramming," "partial reprogramming," and "direct reprogramming." In pluripotent reprogramming, somatic cells are completely reverted to a pluripotent state; in partial reprogramming, these cells may be rejuvenated but do not lose their cellular identity; and in direct reprogramming (transdifferentiation), somatic cells are directly converted into another type of differentiated cell.

The iPS cells generated by pluripotent reprogramming, according to the “Telomere DNA and Ribosomal DNA co-regulation model for cell senescence,” have been found to have significantly increased telomere length and rDNA array length compared to senescent somatic cells, indicating that the cellular age is reversed to 0 years. However, iPS cells or adult cells differentiated from iPS cells can also be subject to immune rejection. For example, on May 13, 2011, Nature reported that American scientists transplanted mouse iPS cells or adult cells differentiated from iPS cells into mice from which the cells were derived. It was originally thought that there would be no rejection due to the identical genetic background, but the mice rapidly rejected the transplanted cells [1]. On August 11, 2022, a paper published in Nature Genetics confirmed that nearly three-quarters of iPS cell lines have severely damaged DNA. Moreover, allogeneic iPS cells with the same homozygous human leukocyte antigen (HLA) haplotype as the patient can still cause immune rejection [2], indicating that even with the same HLA, allogeneic stem cells will still be rejected. In 2024, Deng Hongkui and others published a paper in Cell on the transplantation of islets derived from chemically induced pluripotent stem cells (CiPSCs) for the treatment of type 1 diabetes. The use of immunosuppressive agents indicates that small molecule reprogramming can also be rejected [3]. The original text is as follows: Before CiPSC-islets transplantation, the patient was maintained on immunosuppressive therapy with tacrolimus (2-2.5 mg/day), mycophenolate mofetil (1 g/day) and methylprednisolone tablets (8 mg/day) due to liver transplantation. For CiPSC-islets transplantation, Basiliximab (20 mg) was used for induction therapy on day 0 and day 4. Etanercept was administrated intravenously on day 0 (50 mg) and subcutaneously on day 3, day 7 and day 10 (25 mg) to alleviate inflammatory reactions. For maintenance therapy, the previously described immunosuppressive maintenance regimen for liver transplantation was continued. Cefazolin Sodium was administered for infection prophylaxis during the perioperative period.

Partial reprogramming is also not viable because it has been found that the telomere length does not increase or even slightly decreases in partial reprogramming [4], and it is estimated that the length of the rDNA array does not increase either. After the cessation of Yamanaka factor expression, the epigenetic age quickly reverts to the previous state, and aging symptoms accumulate rapidly. Multiple cycles of treatment only extended the lifespan of progeroid mice by 30%, but failed to extend the lifespan of wild-type mice, indicating that partial reprogramming did not reverse the underlying mechanisms of aging. If in vivo reprogramming is excessive, it can cause mouse death or the formation of iPS cells and fatal teratomas derived from iPS cells. Partial reprogramming uses doxycycline, which is mentioned below to significantly extend the lifespan of Caenorhabditis elegans and mice. The earliest in vivo partial reprogramming experiments were conducted in 2016, and to date, they have not extended the lifespan of normally aging mice. In 2016, Cell reported that cyclic transient expression of the four Yamanaka factors—Oct4, Sox2, Klf4, and c-Myc (abbreviated as OSKM)—in mice significantly extended the lifespan of progeroid mice, but did not extend the lifespan when OSKM was induced in normally aging mice (12 months old) [5]. Altos Labs, founded with $3 billion in investment from the world's richest man Jeff Bezos and Yuri Milner, reported in September 2024 that partial reprogramming only increased the median lifespan of wild-type mice by 12%, which is not as effective as small molecule anti-aging drugs. It can be said that the path of partial reprogramming has been declared a failure [6].

Direct reprogramming has not been found to reverse the aging phenotype [7].

In 2011, a group of American scientists [1] administered drugs to elderly mice (not progeria model mice) and found that they could eliminate senescent cells from many tissues in the body, making the mice younger and stronger: their muscles developed again, their subcutaneous fat thickened, and their cataracts healed, but their lifespans did not increase. I believe that the reason why eliminating senescent cells does not extend lifespan is that eliminating senescent cells may activate adult stem cells to divide to fill the gaps, and cell division can lead to telomere shortening. There are also reports that excessive elimination of senescent cells in the lungs can lead to stem cell depletion and pulmonary fibrosis. A paper titled "Sexual dimorphic responses of C57BL/6 mice to Fisetin or Dasatinib and Quercetin cocktail oral treatment" in GeroScience found that female mice that took dasatinib and quercetin to eliminate senescent cells since youth had accelerated aging [2]; a research paper in Aging in February 2024 used dasatinib and quercetin to eliminate human senescent cells and observed a significant increase in epigenetic age and a significant shortening of telomere length [3]; ABT-263 treatment to eliminate senescent cells accelerated ovarian aging in elderly female mice [4].

Unity Biotechnology's stock price has recently plummeted and the company has been deserted. The reason is that Unity released Phase II clinical data showing that the precise removal of senescent cells in the joints through the drug UBX0101 did not show any statistically significant difference compared with placebo in treating knee osteoarthritis pain [5].

Replication within adipocytes can produce multinucleated cells, leading to replicative senescence. A Nature review article titled “Mechanisms and consequences of endothelial cell senescence” states that the elimination of cardiac senescent cells can lead to the expression of aging markers and the appearance of multinucleated cardiomyocytes, as well as cardiomyocyte hypertrophy [6], which are manifestations of cell replicative senescence. In other words, the elimination of cardiac senescent cells will accelerate the aging of the heart.

It is generally believed that senescent cells accelerate the aging of young cells. However, in 1958, Hayflick and Moorhead mixed male fibroblasts that had divided 40 times and normal female fibroblasts that had divided 10 times, and used cells cultured alone as a control. When the cells cultured alone stopped dividing, the mixed culture cells were examined and found that only female fibroblasts remained. This experiment shows that the cessation of cell division is determined by factors within the cells themselves [7]. In addition, mice that eliminated senescent cells failed to extend their maximum lifespan, indicating that senescent cells do not accelerate the aging of young cells. Moreover, mice that continuously eliminated senescent cells for a long time will age faster, indicating that senescent cells actually inhibit the division of young cells, play a throttling role, and allow young cells to slowly use up the constant number of divisions. The actual role of senescent cells is not to accelerate aging, but to delay aging. The fact that telomeres in children or young people shorten faster than those in middle-aged and elderly people also shows that senescent cells or inflammatory or aging body fluids caused by aging can actually delay aging.

Lysosomal autophagy can clear various waste proteins from cells, but it does not extend lifespan. In fact, increased autophagy in the nematode brain or intestine can shorten lifespan. This suggests that the cause of aging is not waste accumulation. Experiments in mice have found that ovarian aging is associated with increased autophagy and apoptosis in granulosa cells, and this can be reversed by estrogen receptor inhibitors [1].

Geranylgeranylacetone（GGA） has an inducing effect on the expression of heat shock proteins in mammalian tissues, ensuring that proteins can be folded correctly. However, tests by the most authoritative National Institute on Aging in the United States have shown that GGA cannot prolong the lifespan of mice [1], indicating that the loss of protein homeostasis is not the cause of aging, but a result of aging [2].

This may be because while activating telomerase, it also activates a mechanism that accelerates telomere shortening. This is because small molecule telomerase activation positively correlates with the expression of the telomere-binding protein TRF2, which inhibits telomere extension. Cancer cells have very high telomerase activity, yet most have very short telomeres. This is primarily due to the positive correlation between telomerase and TRF2 expression, suggesting that small molecule telomerase activators may shorten telomeres.

TA-65 failed to extend the lifespan of mice and even slightly shortened it [1]. In 2014, Nature reported that TA-65, the first anti-aging drug that activates telomerase, was accused of commercial fraud. The small molecule telomerase activator TAC also failed to extend the lifespan of mice. This may be because the telomeres did not significantly extend, which is a common feature of small molecules [2]. How can cancer cells achieve immortality when half have p53 mutations and the other half do not? One of the reasons is that TRF2 can bind to P53. Therefore, when cancer cells overexpress TRF2, it will reduce P53 levels, causing the cancer cells to divide continuously.

For example, rapamycin has a life-extending effect, but it has significant side effects. This is because many cells in the blood, oral cavity, and intestines need to be rapidly renewed. Inhibiting cell division can affect tissue renewal and cause significant side effects, such as immunosuppression. In 20-month-old mice, rapamycin was given to extend the median lifespan of male mice by only 9% and that of female mice by 14% [1].

Rapamycin, a commonly used immunosuppressant in organ transplants, can cause tumors. One study reported that lung transplants led to cancer cell transmission due to the use of immunosuppressants. Some people abroad reported rapidly enlarging facial melanocytic nevi and developing basal cell carcinoma after taking rapamycin.

On June 4, 2024, the American Society of Aging in Wisconsin published a report on a decade-long life-extending study of rapamycin in 60 marmosets, finding that the median lifespan increased by 15%. The study used a daily dose of 1 mg/kg, a relatively high dose used only in experimental animals, as the marmosets were living in a pathogen-free environment and are not prone to cancer. Humans, however, are exposed to various pathogens and are susceptible to cancer, so high doses are not recommended. Therefore, the life-extending effect in humans may be minimal, and may even shorten lifespan.

Klotho protein acts as a circulating hormone, binding to cell surface receptors and inhibiting the intracellular signaling of insulin and insulin-like growth factor 1 (IGF1). This lifespan extension is essentially achieved by reducing metabolic rate, and therefore, like rapamycin, it also inhibits cell replication and immune function. For example, some studies have shown that α-Klotho has a lifespan extension effect on male mice, but not on females [2]. This may be because females have a lower metabolic rate than males, and further reducing the metabolic rate is harmful. It has also been found that excessive Klotho increases the risk of cancer in cancer survivors [3].