Study on the Mechanism of Total Caffeoylquinic Acid Regulating PI3K/Akt/FoxO3a Pathway and Delaying Aging in Scutellaria baicalensis
Aging is a gradual physiological decline related to age, leading to loss of survival ability and increased vulnerability. Aging is influenced by environmental and genetic factors, and has twelve major characteristics: genomic instability, epigenetic changes, telomere depletion, nutritional perception disorders, protein homeostasis imbalances, mitochondrial dysfunction, cellular aging, abnormal intercellular communication, stem cell depletion, macroautophagy inactivation, chronic inflammation, and gut microbiota dysbiosis. Among these features, ‘nutritional dysregulation’ is the first to be confirmed at the molecular level, which affects lifespan by regulating downstream signaling pathways through the binding of insulin and insulin-like growth factor-1 (IGF-1) to insulin receptors. Insulin/IGF-1 binds to insulin receptors to activate phosphatidylinositol 3-kinase (PI3K), which catalyzes the production of 3,4,5-triphosphate phosphatidylinositol (PIP3) from 4,5-diphosphate phosphatidylinositol (PIP2). Subsequently, protein kinase B (Akt/protein kinase B, Akt/PKB) is recruited into the plasma membrane, and threonine in Akt is expressed through PIP3 dependent kinase-1 and -2 (PDK-1 and PDK-2), respectively. Phosphorylation occurs at positions 308 (Thr308) and 473 (Ser473) of serine, forming phosphorylated Ak (t phosphop Akt, p-Akt) and activating Akt. Forkhead box class O3a (FOXO3a) is one of the four members of the forkhead box transcription factor O subfamily. Activated Akt phosphorylates the threonine 32 site (Thr32) and serine 253 site (Ser253) of FoxO3a, forming phosphorylated FOXO3 (a phosphop-FOXO3a, p-FOXO3a), which promotes the binding of FoxO3a to 14-3-3 protein, thereby preventing FoxO3a from entering the nucleus and losing transcriptional activity. FoxO3a is a key transcription factor regulating aging, which may play a role in promoting longevity by promoting autophagy, enhancing oxidative resistance, and maintaining stem cell pluripotency. In terms of oxidative resistance, FoxO3a transcriptional activation enhances the expression of genes such as superoxide dismutase (SOD), glutathione peroxidase (GSH Px), and catalase (CAT), clears reactive oxygen species (ROS), and reduces the level of lipid peroxidation product malondialdehyde (MDA), thereby delaying body aging and prolonging lifespan. Studies in invertebrate and vertebrate models have shown that reducing insulin/IGF-1 levels and mutating insulin receptors, PI3K, and Akt can promote FoxO3a nuclear translocation, enhance its transcriptional activity, and delay aging.
Erigeron breviscapus (Vant.) Hand- Mazz.)， Also known as Dengzhan Hua, first recorded in “Dian Nan Ben Cao”, it has the effects of promoting blood circulation, unblocking meridians, relieving pain, dispelling wind and dispelling cold. It is mainly used for treating stroke hemiplegia, chest pain, rheumatism pain, headache, and toothache. Flavonoids and caffeoylquinic acid (CQA) are the main active components of Scutellaria baicalensis. Previous studies using network pharmacology and molecular docking have found that flavonoids and CQA compounds are potential active ingredients in the anti-aging properties of Scutellaria baicalensis, which may exert anti-aging effects by regulating four key targets: p53, AKT, HDAC1, and SIRT1. In molecular docking, caffeoyl compounds including single CQA components 4-CQA, 5-CQA, chlorogenic acid, and dicaffeoylquinic acid (DCQA) components 4,5-DCQA, 3,4-DCQA, 3,5-DCQA, 1,3-DCQA, and 1,5-DCQA have higher docking scores with AKT than their inhibitory ligands. The lifespan experiment found that these components all have the effect of prolonging the lifespan of nematodes. Among them, it has been confirmed through mutant strains and transgenic nematodes that 3,5-DCQA inhibits the activity of PI3K and AKT homologs AGE-1 and AKT-1 in nematodes, promotes the entry of FoxO3a homolog DAF-16 into the nucleus, and thus exerts a longevity promoting effect. The above findings suggest that CQA components in Scutellaria baicalensis may delay aging by inhibiting AKT activity and regulating the PI3K/Akt/FoxO3a pathway. The aim of this study is to observe the anti-aging effect of total caffeoylquinic acid from Erigeron breviscapus (EBCQA) on the basis of previous research, and to explore the anti-aging mechanism of EBCQA based on the PI3K/Akt/FoxO3a pathway.

Aging is a natural process of life, and although it is inevitable, it can be delayed through nutritional regulation, genetic manipulation, drug intervention, and other means. More importantly, these anti-aging interventions not only prolong lifespan but also significantly improve the overall health of the body. In previous studies, we found that EBCQA has potential anti-aging effects and may be related to regulating AKT activity. In this study, we confirmed that EBCQA has the effects of prolonging the lifespan of nematodes, enhancing oxidative and heat resistance, and improving motility. Through mutant strains of nematodes, we confirmed that the anti-aging effect of EBCQA is related to regulating the PI3K/Akt/FoxO3a pathway, enhancing antioxidant enzyme activity, and reducing MDA levels; In D-galactose-induced aging rats, EBCQA enhances learning and memory abilities, thymus and spleen coefficients, inhibits PI3K and Akt activation, as well as FoxO3a phosphorylation, enhances antioxidant enzyme activity, and reduces MDA levels.
CQA class components are an important class of phenolic active ingredients in Scutellaria baicalensis, and are also present in many foods (such as purple sweet potatoes, green coffee) and medicinal herbs. CQA compounds have neuroprotective, cardiovascular protective, antioxidant, anti-inflammatory, anti diabetes and other effects. In terms of anti-aging, green coffee extract and honeysuckle extract rich in CQA components extended the lifespan of nematodes, but it is still unclear whether their anti-aging mechanisms involve the PI3K/Akt/FoxO3a pathway; The anti-aging effects of CQA components in mammals are rarely reported. Consistent with previous reports that CQA components have the ability to prolong nematode lifespan, we found that EBCQA prolongs nematode lifespan with a maximum elongation rate of 17.7%, and enhances oxidative resistance, heat resistance, and motility. The D-galactose-induced aging rat model has been proven to effectively simulate natural aging, and is therefore widely used to study aging mechanisms and screen drugs that delay aging. Reduced learning and memory abilities are common physiological phenomena associated with aging, and some may develop into neurodegenerative diseases such as Alzheimer’s disease. As age increases or diseases occur, the body’s immune capacity usually gradually decreases. The thymus and spleen are important immune organs, and their organ indices can to some extent reflect the size of the body’s immune capacity. Therefore, learning and memory ability, as well as thymus/spleen organ index, are commonly used indicators for evaluating aging and anti-aging. D-galactose-induced aging rats showed decreased learning and memory abilities, as well as decreased thymus and spleen coefficients. EBCQA administration effectively improved learning and memory abilities, and increased thymus and spleen coefficients. These results indicate that EBCQA has a anti-aging effect in both invertebrate nematodes and vertebrate D-galactose-induced aging rats.
The PI3K/Akt/FoxO3a pathway is an evolutionarily conserved signaling pathway regulated by insulin, IGF-1, and other factors. In aging regulation, PI3K and Akt play a pro aging role, while FOXO3a plays an anti-aging role. In nematodes, AGE-1, AKT-1, and DAF-16 are homologs of mammalian PI3K, Akt, and FoxO3a, respectively. Nematodes with AGE-1 mutations have an average lifespan extension of 40% to 65%, those with AKT-1 mutations also have significantly longer lifespans, and DAF-16 mutations significantly shorten lifespans. In mammals, overexpression of AKT-1 induces cellular aging. Mice with AKT-1 mutations (Akt1+/-) have an 8% increase in male lifespan and a 15% increase in female lifespan. Research on long-lived populations has also found that genetic polymorphisms/mutations that lead to loss of function of IGF-1 receptors, insulin receptors, or their downstream factors (such as PI3K, AKT) are associated with prolonged human lifespan. The study of long-lived populations among Japanese Americans, Han Chinese, Californians, New Englanders, Ashkenazi Jews, Danes, Germans and Italians shows that the genetic polymorphisms of rs2764264, rs2802292, etc. of FOXO3a are related to longevity and healthy aging. In the regulation of the PI3K/Akt/FoxO3a pathway, Akt is activated upon phosphorylation of Thr308 and Ser473, while FoxO3a is inhibited upon phosphorylation of Thr32 and Ser253. In this study, EBCQA extended the lifespan of wild-type nematode N2 and promoted DAF-16 nuclear translocation, but had no effect on the lifespan of AGE-1, AKT-1, and DAF-16 mutants, indicating that the role of EBCQA in extending lifespan depends on the regulation of the PI3K/Akt/FoxO3a pathway. Rats treated with D-galactose showed upregulation of PI3K, Akt, p-Akt, and p-FOXO3a expression, while FOXO3a expression was downregulated. This is consistent with previous reports of changes in D-galactose-induced aging rats and naturally aging small/large rats. These changes in protein expression may lead to decreased learning and memory abilities, as well as decreased thymus and spleen coefficients, thereby inducing aging. After administration of EBCQA, these changes were inhibited, which may have an anti-aging effect.
In 1956, British scholar Harmna first proposed the theory of free radical aging, believing that the cellular dysfunction caused by free radical attacks on biomolecules is one of the fundamental causes of aging in the body. In this study, EBCQA increased the activities of SOD, GSH Px, and CAT in nematodes treated with paraquat and rats treated with D-galactose, while reducing MDA levels. The increase in the activity of these antioxidant enzymes may lead to a reduction in ROS and oxidative damage, thereby helping to delay aging. One of the downstream mechanisms by which the PI3K/Akt/FoxO3a pathway affects lifespan is the regulation of oxidative resistance. In nematodes, some long-lived mutant strains, such as daf-2 (e1368) and age-1 (hx546), exhibit enhanced activities of antioxidant enzymes SOD, GSH Px, and CAT, which depend on DAF-16. After DAF-16 mutation, the antioxidant enzyme activity of these mutant strains is almost similar to that of wild-type nematode N2. In mammals, FoxO3a regulates the expression of antioxidant enzymes such as SOD, GSH Px, CAT, and inhibits the activity of FoxO3a, leading to the accumulation of ROS in cells, accelerating atherosclerosis, and increasing the risk of cancer. EBCQA promotes the nuclear entry of nematode DAF-16 and inhibits the phosphorylation of rat FoxO3a, indicating that EBCQA may play an anti-aging role by enhancing FoxO3a transcriptional activity and antioxidant enzyme activity.
In summary, EBCQA can effectively prolong the lifespan of nematodes and antagonize D-galactose-induced aging in rats. Its mechanism of action may be by inhibiting PI3K and Akt activation, reducing FOXO3a phosphorylation, promoting FOXO3a nuclear and transcriptional activity, enhancing antioxidant enzyme activity, inhibiting oxidative stress, and thus delaying aging.