The immune system is an important factor in the origin and mechanisms of different diseases, contributing significantly to biomedical research advancement (14). Conditions affecting the skin, gastrointestinal tract, respiratory system, joints, vital organs, and infectious illnesses are increasingly recognized as primarily influenced by immunological factors (15). A wide range of studies have shown that most of the major components of WS have immunomodulatory effects in mice (16) and humans (17). The immunomodulatory role of WS in various inflammatory complications such as inflammation (reducing type 2 cytokines, and inflammatory markers such as TNF-α and IgE) has been reported (18). The extract derived from WS root showcases robust hemopoietic, antioxidant, adaptogenic, and immune-stimulating properties (19). WS plays a role in immunomodulation by significantly improving the immune profile by altering innate and adaptive immune systems (20). In a randomized and controlled clinical trial, the immune-stimulatory effect of WS was evaluated and it was reported that WS enhanced CD4+/CD8+ and expression of CD3+/CD19+/CD45+, thus, increasing the antibody and antibody-forming cells (20). The extract from WS has demonstrated a notable ability to boost cell-mediated immunity in mice (21). Levels of interferon gamma (IFN-y), interleukin-2 (IL-2), and granulocyte macrophage colony-stimulating factor (GM-CSF) have been elevated in experimental mouse models when treated with WS; this indicates potential immune-enhancing and myelo-protective effects of root extracts of WS (22). Furthermore, it enhances the body’s immune system, specifically enhancing cell-mediated immunity, which is vital in defending against diseases (23).

WS amplifies the nitric oxide synthetase (NOS) activity in macrophages, consequently augmenting the microbial eradication capability of these immune cells. It has been elucidated that WS enhances the activity of nitric oxide via NOS induction (22). Indeed, WS boasts a wide array of beneficial effects on the body. Additionally, it promotes a balance in reproductive and sexual function contributing to an improved reproductive health (23). WS’s adaptogenic properties make the body more resilient to the harmful effects of stress (24). Furthermore, viscosalactone B and 27-O-glucoside (a derivative of WA) extracted from alcoholic leaf extract have demonstrated significant antiproliferative activity when treated with different cell lines viz. CNS cancer SF-268, breast cancer MCF-7, colon cancer HCT-116 and lung cancer NCI-H460 cell line (11).

Studies have shown antioxidant activities of WS concluding that it helps in prevention of glycation-induced pathogenesis as well as decreasing the levels of many oxidative stress markers indicating its potential therapeutic effects in managing healthy aging (25–27). WS’s potent antioxidant properties have an important part in protecting cells against free radicals (23). Hydroalcoholic root extracts of WS contains a diverse range of molecules namely saponins, particularly sitoinsides 7 and 8, withanolides and alkaloids which have anti-stress, anti-inflammatory and anti-oxidant properties, respectively, (28). Anti-inflammatory potential of withanolides and alkaloids is reported in in vitro study using RAW 264.7 cell line. Anti-stress properties of sitoindosides 7 and 8 were demonstrated using Wistar albino rats and albino mice (28). Also, studies conducted to investigate the effect of alkaloids in RAW 264.7 cells showed that the expression of iNOS and COX-2 proteins were upregulated (29). In addition, alkaloids inhibited LPS-induced inflammation, indicating a putative anti-inflammatory potential of alkaloids (29). Alkaloids from WS (withanamides A-I) are also reported to lower the rate of lipid peroxidation along with possessing anti-oxidant properties (30).

WS is known for its versatility in treating a range of conditions, such as immunomodulation, rejuvenation, enhancement of cognitive function, inflammation, enhancing concentration, etc. However, a synthetic review exploring its potential role in ameliorating aging and aging-related disorders is currently lacking. In this review, we summarize mechanistic pathways modulated by WS via coding and non-coding RNAs in aging relying on information gleaned from both in vitro and in vivo studies. The RNA biomarkers regulated by WS can potentially serve as promising targets to mitigate aging-related degenerative changes and, perhaps, to some extent, reverse biological aging. This may facilitate the development of various preventive and therapeutic strategies employing WS as a nutraceutical for healthy aging.

The primary causes of aging are related to various degradations, alterations and instabilities to our genomic framework and further cascading processes (39). Genomic instability and telomere shortening are major causes of DNA damage, while epigenetic alterations cause post-translational modification of histones and chromatin remodeling (40). Proteostasis irregulation can also have major impacts on aging due to refolding of proteins (41). Telomeres, protein structures which protect the terminal regions of the DNA, go through an attrition process as a part of cell cycle, while oxidative stress, inflammation and chronic stress are also known to increase the rate of attrition leading to degradation in the protective regions making DNA more susceptible to damage (42, 43). To add to it, genomic instability caused by both internal and external factors can alter the genetic code; this kind of DNA damage hinders the process of protein production used to damage repair and accelerates the process of aging (40, 44). Structural changes due to epigenetic alternations degrade the cellular function and are associated with a range of age-related diseases such as cancer, diabetes, osteoporosis, neurological diseases, and increased inflammation (45). Proteostasis, also known as protein homeostasis, helps maintain protein integrity to ensure proper functioning (46). Dysregulation due to various internal and external factors leads to refolding which can have serious health implications such as neurodegenerative disorders, impaired muscle function and low cardiac health (41).

Response of the cells to nutrients is considered as nutrient sensing (47). Mammalian target of Rapamycin (mTOR) is a crucial unit which incorporates nutrient sensing with different cellular processes that can lead to growth and proliferation of cells (48). In growing organisms, cell proliferation and cell growth are very crucial and, therefore mTOR is activated (49). mTOR is naturally decreased in aging (50). Over activating this pathway can cause increased aging and increase in cancer rate, heart conditions, metabolic disorders, bone loss, neurodegenerative conditions and sarcopenia (50). The mitochondrion serves as the cell’s energy hub, where most of the body’s energy is generated (51). Aging tends to decrease the efficiency of the mitochondria due to reduced biogenesis (51). Accumulation of reactive oxygen species and deficiencies (DNA polymerase γ) are the major causes of mitochondrial dysfunctions (52). Cellular senescence, or cell aging, is a process wherein the cell cycle is arrested and accompanied by characteristic phenotypic changes (53). The number of senescent cells tends to rise with age, leading to an increase in inflammatory markers that contribute to the aging process (1).

Adult stem cells are necessary for continuity of tissue homeostasis and regeneration (54). Consequently, the qualitative and quantitative decrease in functions of stem cells during the entirety of life are referred to as its exhaustion (1). Decline in stem cell regeneration is considered as a marker of aging since the proportion of stem cells and their pace of division gradually decreases over time (55). Stem cell exhaustion in adults can lead to cognitive decline, neurological degenerative diseases, delayed healing, lowered immune functions, and unfavorable heart conditions (55). Cells communicate with each other based on different chemical and electrical means such as endocrine, neuroendocrine and neuronal level. Thus, neurohormonal level is deregulated in aging as inflammatory reactions increase (56). One of the most prominent changes in cell signaling biomarkers is “inflammaging” indicating chronic inflammation development in elderly population (57).

One of the prevalent features related to aging is inflammation and, in the absence of any kind of open infection, this chronic inflammation is referred to as “inflammaging” (108). It poses a major risk factor in major health degradation and mortality in elderly populations (109). Human keratinocytes have a crucial role in inflammation by mediating tumor necrosis factor (TNF)-α and interleukins (IL) (110). Many skin diseases that are related to inflammation such as psoriasis, atopic dermatitis and allergic contact dermatitis are highly associated with keratinocytes and cytokines (111). Therefore, to study the anti-inflammatory effects of WS in modulation of these cytokines, Sikandan et al. (112) demonstrated the effect of Ashwagandha water extract (ASH-WEX) in human keratinocyte (HaCaT) cell line and 6-week-old male C57BL/6 J mice. RNA was isolated after the fifth day of treatment from mice skin and RT-qPCR was performed to check the transcriptional regulation of inflammatory cytokines in both HaCaT cells and C57BL/6 J mice. It was inferred that ASH-WEX inhibited TNF-α, and interleukins (IL-6, -8, -1β, and -12) while the expression of TGF-β1 was reduced in a dose-proportional manner (112). Also, mRNA expression studies performed in mice showed that ASH-WEX treatment inhibited TNF- α expression while it overexpressed TGF-β at mRNA level (112). Furthermore, a study was conducted (113) to demonstrate the beneficial effects of dry leaf extract of WS on anxiety and neuroinflammation which results from obesity. Using mRNA expression analysis by qRT-PCR, it was observed that WS led to inhibition of IKKα/β which further suppressed NF-κB signaling, and reduced the expression of iNOS, GFAP, IL-1β, TNF-α, PPARγ and MCP-1. Also, WS treatment led to upregulation of Bcl-xL and downregulation of Bad mRNA which further inhibited high fat diet-induced apoptosis in rats (113).

Signaling pathways including modulation of RNA by Withania somnifera in inflammation are summarized in Figure 5.

A key neurodegeneration hallmark includes accumulating advanced glycation endproducts (AGE) in the brain (114). Similarly, receptors for advanced glycation endproducts (RAGE) are overexpressed in brain (115). Gathering of amyloid-beta (Aβ) is considered as a major indicator of neurogenerative disease (116). Two crucial proteins, nuclear factor-κB (NF-κB) and (NLRP3), play significant roles in the neurodegenerative disorders development which include Alzheimer’s disease (AD) (117). Additionally, epigenetic modulation which includes histone deacetylase 2 (HDAC2) alteration is considered as a pivotal factor in Alzheimer’s disease (AD) pathogenesis (118). Hence, to investigate the therapeutic role of WS in modulation of these genes in AD, Venkata et al. (119) performed gene expression analysis using SH-SY5Y cells overexpressing amyloid precursor protein (SH-APP) cells. The results revealed that treatment with WA decreased NF-κB and IL-1β expression significantly, concluding that both of these genes have crucial roles in NF-κB-mediated neuroinflammation. Additionally, Withaferin A administration in SH-APP cells led to a downregulation of Aβ, an important mediator in the neuroinflammatory processes implicated in the pathogenesis of AD (119).

The memory process can be affected by a range of pathological factors, such as neurodegenerative disorders, stroke, tumors, head injuries, oxygen deficiency, cardiac surgery, nutritional deficiencies, neurological complexities such as depression and anxiety as well as side-effects of different medications and the natural process of aging (120). Scopolamine, a cholinergic antagonist, induces amnesia in humans as well as in rat models (121). These models have found extensive application in comprehending the molecular, biochemical, and behavioral alterations, serving as valuable tools for identifying potential therapeutic targets in the context of memory impairment (121). To evaluate the role of Withania somnifera in memory impairment, Konar et al. (122) assessed the therapeutic targets using alcoholic extracts of Withania leaves (i-Extract) in scopolamine-induced male Swiss albino mice of 12 weeks. From RT-PCR analysis, it was reported that scopolamine downregulated the mRNA expression of brain derived neurotropic factor (BDNF) and glial fibrillary acidic protein (GFAP) in mouse cerebrum in proportion to time and dose. Furthermore, it was also observed that i-Extract treatment attenuated the downregulation of scopolamine treatment in proportion to dose suggesting that WS serve as putative therapeutic agents to minimize the progression of neurodegenerative disorders (122).

Signaling pathways involving RNA in neurodegeneration and their modulation by W. somnifera are summarized in Figure 6.

The aging process is linked to alterations in endoplasmic reticulum (ER) chaperones and expression of folding enzymes (123). This leads to proteostasis disruption and elevation of misfolded proteins (123). Lowering the level of regulatory unfolded proteins results in ER stress which is linked to the unfolded protein response (124). Prolonged endoplasmic reticulum (ER) stress stimulates protein kinase-like ER-resident kinase (PERK), activates transcription factor-6 (ATF-6) and inositol requiring 1 (IRE1) apoptotic signaling (125). This leads to overexpression of C/EBP homologous protein (CHOP) expression, a proapoptotic-transcription factor (induced in ER stress), and is activated by p38 MAPK (126, 127). CHOP expression is usually regulated at mRNA level via PERK/eIF-2α/ATF6 pathway, ATF4 and IRE1/XBP1 pathway (128). Thus, to investigate the effect of Withaferin A in inducing apoptosis through ER stress, Choi et al. (129) conducted mRNA expression study of ER-stress specific X-box binding protein (XBP1) using human renal carcinoma cell line (Caki). Real-time PCR analysis showed that splicing of XBP1 mRNA was initiated following treatment with WA which in turn inactivates CHOP. Eukaryotic initiation factor-2α (EIF-2α) phosphorylation is activated by PERK which takes place during ER stress that leads to translational initiation and protein synthesis (130). These results suggest key signaling events implicated in ER-stress mediated apoptosis using WA which may facilitate chemopreventive and chemotherapeutic strategies based on Withaferin A (129).

“Adaptogens” are considered as plant-derived extracts or compounds which help organisms to adapt and survive during stress response (131). Activation of the gene that regulates stress response, which in turn postpones aging or ameliorates aging, is an effective way of tackling aging-associated diseases (132). Panossian et al. (133) studied adaptogens effect in stress and age-related diseases by using dunal root extract of WS in human T98G neuroglia cell line using mRNA expression profiling. It was reported that WS along with other adaptogens plays a major role in altering homeostasis. Also, it was observed that WS extracts downregulated PDE9A gene at transcriptional level. This indicated their potential to modulate transcriptional regulation in preventing aging-related and stress-induced disorders (133).

As people get older, the consistency or stability of circadian clock gene expression patterns is understood to potentially change or become less resilient (134). The suprachiasmatic nucleus (SCN) serves as the central master pacemaker, orchestrating the regulation of peripheral clocks found in all body tissues (135). In aging or in age-related complexities circadian timekeeping systems decrease (136). Circadian clocks operate through intricate transcriptional translational feedback loops (137). Two supplementary loops which include RORs, NFIL3, DBP and REV-ERBs, collaborate to produce discernible rhythms within a period of 24 h (138). Kukkemane et al. (139) evaluated WS influence on rhythmic alteration and levels on a daily basis with regard to age. They examined the expression of a hydroalcoholic leaf extract of WS using male Wistar rats. Through qRT-PCR analysis, they observed a significant chrono modulatory effect of WS. This effect was evident in the restoration of rhythms and pulses of clock genes namely Sirtuin1 and NRF2 daily. It was concluded that WS may offer therapeutic benefits in mitigating age-related disruptions in the circadian clock. This potential benefit is proposed to operate through two key pathways: SIRT1 modulation and antioxidant effects (139).

The regulation of signaling pathways in stress by Withania somnifera at RNA level are summarized in Figure 7.

Fibrosis is characterized by endpoint of pathological remodeling which triggers the progression of various chronic disorders and aging-associated organ damage (140). Hence, it is important to alleviate the symptoms or inhibit the downstream pathways to prevent fibrosis. To investigate the potential role of Withaferin A as a preventive constituent in fibrosis, a study was conducted by Gu et al. (141) using male 8-week-old C57/BL6 mice. Silent information regulator (SIR) protein family exhibits an important role in cell cycle regulation, mitochondrial homeostasis maintenance, caloric restriction and lipid metabolism to regulate glucose levels. Studies have elucidated that SIRT3 deficiency can aggravate liver injury while SIRT3 activation led to alleviate liver fibrosis (142). In this study, mRNA expression level of SIRT3 was assessed after treatment of WA in C57/BL6 mice. It was reported that WA led to decrease of liver fibrosis in a SIRT3-dependent manner (141).

Aged kidney is susceptible to kidney injury which is indicated by extended inflammation that can increase dysfunction of renal tissue (143). NF-κB signaling pathway is a mediator between inflammation and fibrosis in kidney (144). Several stimuli can trigger NF-κB activity in the kidney such as TNF (145) and angiotensin II (146), and both of these are linked to chronic kidney disease. The target genes of NF-κB are long but include CCL2 and CCL5 which are linked to renal fibrosis (147). Therefore, to assess the role of WS in regulating these inflammatory genes, Grunz-Borgmann et al. (148) studied the expression using hot water soluble extracts of WS in rat kidney NRK-52E cell line. Transcriptomic analysis was performed using real-time PCR by using different doses of WS. It was reported that WS pre-treatment gradually inhibited CCL2 and CCL5 expression NRK-52E cells. Also, pre-treatment with WS reduced the effect of LPS-induced NF-κB activity indicating anti-inflammatory effect of WS and alleviating renal fibrosis (148).

Hepatic toxicity has been proved to be aggravated during aging especially by the consumption of acetaminophen (APAP) (149). APAP is generally used as an analgesic and antipyretic drug (150). However, when used in large concentrations, it causes hepatotoxicity and necrosis in humans (151). Devkar et al. (152) studied the outcome of withanolide-rich fraction (WRF) in liver protection using male Swiss albino mice. Expression of TNF-α, IL-1β, iNOS and COX-II was assessed at the transcriptional level in APAP-treated mice. It was reported that WRF significantly downregulated TNF-α and IL-1β transcriptionally corelating with the dose level. However, mRNA expression of iNOS and COX-II was reduced at higher dose (200 mg/kg) of WRF. Reduced expression of COX-2, iNOS, IL-1β, TNF-α achieved by WRF intervention showed anti-inflammatory and antioxidant properties of WS leading to enhanced liver protection (152).

The modulation of RNA in signaling involved in organ fibrosis by Withania somnifera is summarized in Figure 8.

The decline in mass and function of skeletal muscle typically commences around the age of 40, with muscle mass potentially diminishing at a rate of 1–2% annually after reaching 50 years of age (153). This can lead to escalating complications in health-related outcomes which lead to functional restriction of the body (154). During aging, reduced muscle building capacity leads to loss of muscle mass (154). It is believed that the loss of muscle due to aging is influenced by apoptosis, however the mechanisms behind it remain unclear (153). The Bcl-2 protein family is implicated in apoptotic signaling pathway, consisting of Bax and Bcl-2 proteins, which regulate the process of mitochondria-mediated cell death (155). While Bcl-2 helps prevent cell apoptosis, Bax, a protein like Bcl-2, promotes cell death (156). To understand the effect of WS on the regulation of apoptotic proteins, Panda et al. (153) conducted transcriptomic analysis using hydroalcoholic extracts of WS in male Sprague Dawley rats. Their study revealed that expression of Bax was reduced after the treatment with WS, along with an increase in Bcl-2 expression. It was therefore inferred that WS may offer a therapeutic approach to address muscle weakness and deterioration (153).

Signaling pathway modulated by Withania somnifera in muscle impairment is depicted in Figure 9.

Telomerase shortening is considered as the main factor which escalates the rate of aging and promotes degeneration processes (157). Telomere is progressively shortened with each DNA replication, which leads to appearance of critically short telomeres (158). Low telomerase activity is targeted by both exogenous and endogenous factors which lead to DNA damage and low maintenance of cell function (159). Withanolide, an active constituent of WS indicated 29.7% extension in the lifespan of C.elegans via regulation of insulin/IGF-1 signaling pathway (160, 161).

Cellular senescence refers to cell-cycle suspension and secretion of inflammatory mediators which leads to aging and complications associated with aging (162). Obesity is an important comorbidity with increase in burden of senescent cell as well as neuropsychiatric disorders such as anxiety and depression (163). Withaferin A aids in alleviating several metabolic disorders. Therefore, to evaluate the protective effect of Withaferin A in conditions such as obesity, Abu Bakar et al. (164) performed a study using HFD-induced obese mice. They treated the mice with Withaferin A for 12 weeks. Pro-inflammatory cytokines were reduced following WA treatment. Additionally, using transcriptional analysis by real-time PCR, expression of all genes involved in inflammation such as TLR4, CCR2, NF-κB, COX2, TNF-α, IL-1β and CCL2/3 were downregulated following WA treatment. Also, WA downregulated the mRNA expression of main regulators of lipid metabolism (PPARα and γ, CD36, FAS and CPT1). These results suggested that WA treatment can help in regulating lipid and glucose metabolism in liver at the transcriptional level and provide a therapeutic response for WA in obesity (164).

Changes in vascular extracellular matrix in aging appear as one the major facets of dysregulated angiogenesis (165). In the process of angiogenesis, endothelial cells lead to the formation of new vasculature (166). Additionally, it is implicated in the progression of tumor cells and metastasis (167). The uniform release of VEGF, bFGF, PDGF and TGF lead to the activation of angiogenesis (168). Transcriptional as well as post-transcriptional mechanisms tightly regulate the expression level of VEGF mRNA (169). Recent research has unveiled the role of signaling pathways and genetic components governing this expression. The activation of the transcription factor Sp1 precedes VEGF promoter activity, showing that VEGF overexpression is regulated by Sp1 activation (170). This overexpression leads to angiogenesis and cancer progression (171). Sp1 has been evaluated as a crucial factor in tumor angiogenesis and aids in the pathogenesis of pancreatic adenocarcinoma (172). Santhekadur et al. (170) performed an experiment to explore the antiangiogenic effects elicited by WS, which resulted in a reduction in ascites fluid and VEGF expression, regulated by the Sp1 transcription factor, using Ehrlich ascites tumor (EAT) cells-bearing mice. Nuclear extracts were obtained from both untreated and withaferin A-treated EAT cells, and the binding activity of Sp1-DNA was studied using Electrophoretic Mobility Shift Assay (EMSA) with Sp1 oligonucleotides. The findings showed that Sp1 binding to VEGF gene promoter region was inhibited by withaferin A (170). Further, a study was carried out by Sajida and Prabhu (173) to analyze the activity of root extracts of WS using A549 human lung carcinoma cell line via inhibition mechanisms including antioxidant, autophagy and angiogenic inhibition. The expressions of VEGF, angiogenin and MMP-2 were assessed at the transcriptional level using qRT-PCR. It was reported that angiotensin and VEGF gene expression was significantly downregulated in WS extract-treated cells but there was a lower effect on MMP-2 expression (173).

The signaling pathways involving Withania somnifera in modulating RNAs in above-mentioned cellular events are illustrated in Figure 10. In addition, RNAs modulated by Withania somnifera are represented in Table 1 and summarized in Figure 11.