Cellular senescence (CS) is initially defined as an irreversible growth arrest of normal somatic cells, and has been proposed to contribute to tissue and organismal aging itself, and to be an intrinsic safeguard against tumor progression. SCs have been shown to have diverse phenotypes, including cellular flattening and hypertrophy, senescence-associated β-galactosidase activity (SAβG), senescence-associated secretory phenotype (SASP), resistance to apoptotic cell death, alterations in nuclear structure and senescence-associated heterochromatic foci (SAHF), mitochondrial expansion, and signaling events, including upregulation of cell cycle inhibitors and -pro-survival effectors . SCs accumulated in various tissues in mice with age, and comprised 5–40% of the total cells, depending on the tissue types.  Although their number are relatively small in tissues, SCs can cause extensive dysfunction of the microenvironment and damage to surrounding cells and tissues, due to their pro-inflammatory senescence-associated secretory phenotype (SASP). Accumulating evidence suggests that CS plays an important role in diverse biological processes, such as embryonic development, diabetes, host immunity, wound healing, tissue renewal, as well as fibrosis, cardiovascular diseases, and cancer.

Since aging is one of major risk factors of human diseases, a variety of aging interventions have been developed to extend health span and to prevent or treat ARDs in in vitro and in vivo experimental models. Caloric restriction (CR) is the only intervention shown to increase health span as well as to decrease the risk of ARDs in nonhuman primates . Recently, clinical trials of CR in non-obese humans revealed that a 15% lower calorie intake for 2 years delayed metabolism accompanied by reduced oxidative damage, suggesting that CR could also slow down the aging process in humans. Although CR can enhance healthy aging, the inconvenience of most subjects to maintain CR for a longtime limits its application. Therefore, caloric restriction mimetics, and calorie restriction diets or fasting-mimicking diets have been proposed as alternatives. Elucidation of the mechanisms by which aging is regulated also suggested a variety of compounds and medicines, including sirtuin activators , AMP dependent protein kinase (AMPK) activators, mammalian target of rapamycin (mTOR) inhibitors , autophagy activators , that might be applicable for use in aging intervention. In addition, the use of geroprotectors, compounds and medicines that slow down aging, and thus lengthen the lifespan of model organisms has also been proposed . In present, a curated database of geroprotectors is available, and includes 259 compounds in 13 animal models from yeast to human, obtained from 2,408 literature (http://geroprotectors.org/). An old story tells the rejuvenation effects of young blood. Heterochronic parabiosis, in which an aged mouse and a young one were joined surgically, revealed that some factors in young blood, such as growth differentiation factor 11 with controversial reports and oxytocin enhanced tissue regeneration, and led to improvement of aging phenotypes . Similarly, transfusion of young serum also retarded age-related impairments in cognitive function and synaptic plasticity in aged mice .

Although CS is one of hallmarks of aging, and accumulation of SCs with age has been suggested to be associated with aging and ARDs , direct evidence of a causal relationship between CS and aging or ARDs has only recently been validated in rodent models. Furthermore, senotherapeutics, have been implicated as novel strategies for aging intervention in applications designed to extend healthy aging and to prevent or treat ARDs.

DIRECT LINKAGE OF CS TO AGING AND ARDs

Baker et al., reported the first direct evidence of a direct causal relationship of CS to ARDs in 2011 . For the clearance of senescent cells in mice, a transgenic strategy, was employed, using INK-ATTAC derived from p16Ink4a, a well-known marker of CS, in which senescent cells were selectively eliminated by apoptotic cell death upon administration of AP20187. The INK-ATTAC transgenic mice were bred onto a BubR1H/H-progeroid mouse background to obtain BubR1H/H;INK-ATTAC mice. The authors demonstrated that the animals treated with AP20187 from early (weaning time) or late (5 months) in life, had reduced numbers of p16Ink4a-positive senescent cells, and progression of p16Ink4a-mediated age-related phenotypes in adipose tissue and muscle was delayed . Five years later, Baker et al., reported more concrete evidence of the direct linkage of CS to aging itself and ARDs . This time, they demonstrated effects of the clearance of p16Ink4a-positive senescent cells in both male and female INK-ATTAC transgenic mice of two distinct genetic backgrounds (C57BL/6 and mixed). AP20187 treatment from 12 months to 18 months increased the median lifespan of both C57BL/6 and mixed background mice by 24%, and prolonged the heath span in C57BL/6 mice by 18%, and by 25% in mixed background mice. In addition, they demonstrated that AP20187 attenuated age-related functional and structural deterioration of multiple organs, without any detrimental side effects to adipose tissue, kidney, or heart . Genetic ablation of senescent cells, using the INK-ATTAC transgenic mice further revealed that clearance of p16Ink4a-positive senescent cells improved age-related lipodystrophy, hepatic steatosis , age-related cardiac function and bone loss , and tau-mediated cerebral pathologies . Studies on another p16Ink4a-based transgenic mouse, named the p16Ink4a-trimodality reporter (p16-3MR) mouse, in which p16Ink4a-positive senescent cells were cleared by treatment with ganciclovir, revealed that removal of senescent macrophages attenuated atherosclerotic plaque formation in LDLR−/− background mice , and osteoarthritis. In addition to genetic clearance of senescent cells, transplanting senescent ear fibroblasts into the knee region induced osteoarthritis in mice . Transplanting relatively small numbers of senescent cells into young mice was reportedly sufficient to cause persistent physical dysfunction, as well as to spread cellular senescence to host tissues, which led to reduced survival (28). Therefore, these proof-of-principle experiments revealed the direct linkage of CS to aging and ARDs, and suggest that therapeutic interventions to remove senescent cells or block their effects might represent a novel strategy to lengthen health span and prevent or treat ARDs in human.

SENOLYTICS

Since elimination of SCs using genetic approaches mitigated aging and ARDs, pharmacological intervention targeting SCs, named as senotherapeutics, has been proposed. Senotherapeutics are classified as senolytics, which selectively kill SCs; senomorphics which modulate SCs by blocking SASP; and senoinflammation, the immune system-mediated clearance of SCs .

Senotherapeutics targeting SCs. CS induced by diverse factors is involved in many biological processes, embryonic development, tissue homeostasis, and tissue dysfunction, thus contributing to age-related pathologies and lifespan. Therefore, senotherapeutics targeting SCs is an emerging strategy of aging intervention for extension of health span and prevention and treatment of ARDs. Senotherapeutics is comprised of 3 classes: senolytics which kill SCs selectively; senomorphics which modulates or even reverses the phenotypes of SCs to those of young cells by interfering with triggers of CS, targeting SCs directly, or blocking SASP: and mediators of the immune-system clearance of SCs.

In 2015, Zhu et al., reported the first senolytics, dasatinib, a protein tyrosine kinase inhibitor, and quercetin, a plant flavonoid . Based on transcriptomic analysis, they discovered that SCs increased the expression of pro-survival networks, which led to resistance to apoptosis. The authors screened 46 candidate drugs for their ability to eliminate SCs in vitro and found that dasatinib was effective against senescent human preadipocytes, and that quercetin was effective against senescent human endothelial cells and mouse bone marrow-derived mesenchymal stem cells (BM-MSCs). Finally, they showed that combination of dasatinib and quercetin reduced SC burden in chronologically aged, radiation-exposed, and Ercc1−/Δ-progeroid mice, which resulted in extension of health span and reduction of age-related pathologies. Their seminal demonstration on the feasibility of selectively ablating senescent cells by pharmacological intervention and the efficacy of senolytics for alleviating symptoms of frailty and extending health span in mice, prompted development of other senolytics.

Until now, seven classes of senolytics have been reported, including kinase inhibitors, a Bcl-2 family inhibitors, natural compounds, a p53 binding inhibitor, heat shock protein 90 (HSP90) inhibitors, UBX0101, and a histone deacetylase (HDAC) inhibitor . The combined administration of dasatinib and quercetin improved the pathologies of diverse ARDs, including cardiac aging, atherosclerosis. osteoporosis, pulmonary fibrosis, hepatic steatosis, and Alzheimer’s disease  by clearing SCs in the tissues. Recently, the combination of dasatinib and quercetin was reported to enhance physical function and to lengthen health span and lifespan in old mice.