Anti-aging effects of glycyrrhizin via maintaining Klotho levels

Abstract

Klotho, an anti-aging protein, controls the insulin-like growth factor-1 (IGF-1) and suppresses inflammation implicated in senescence. We previously reported that glycyrrhizin (GL), a medicinal ingredient of licorice root, significantly attenuates inflammatory responses by inhibiting high-mobility group box 1 (HMGB1). In this study, we investigated whether GL is effective in suppressing the decline in Klotho levels and aging-induced frailty. After oral administration of GL three times a week for 12 months, the following effects were observed: reductions in the increases in blood fats, steatoses, obesity, and kidney aging; maintenance of activity level and rotarod test performance. Furthermore, we found beneficial effects of GL on the blood levels of functional proteins; upregulation of anti-aging factors, IGF-1 and Klotho; decrease in cystatin-C, an indicator of declining kidney function; and decreases in IL-6 and TNF-α, inflammatory cytokines. GL also seemed to increase superoxide dismutase 2, an antioxidant enzyme, in the liver and kidneys. As GL can inhibit HMGB1, it may attenuate this inflammatory amplification loop, reducing the systemic senescence-associated secretory phenotype and thus mitigating multiple aging phenotypes by maintaining Klotho levels.

Introduction

Aging is associated with a progressive physiological decline, organ dysfunction, and systemic frailty. The current hypothesis is that aging-induced telomere shortening leads to chronic inflammation through the activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, which in turn reduces Klotho expression and exacerbates oxidative stress and frailty.^(1,2) Telomere shortening is a primary driver of cellular senescence. We previously reported that DNA damage caused by oxidative stress and inflammation accelerates telomere shortening and promotes aging.^(3,4) During aging, chromatin fragments, including telomeric DNA, accumulate and activate the cGAS-STING pathway. This initiates a proinflammatory cascade through cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-6, leading to mitochondrial dysfunction, further reactive oxygen species (ROS) production, and ultimately decreased Klotho expression.⁽⁵⁾

These inflammatory signals contribute to the establishment of a senescence-associated secretory phenotype (SASP), which is a defining feature of aging cells.^(6–8) High-mobility group box 1 (HMGB1) is a chromatin-associated protein that is passively or actively released from senescent cells, and acts in a damage-associated molecular pattern (DAMP). It amplifies SASP by binding to the receptor for advanced glycation end products (RAGE) and toll-like receptor (TLR) 4, resulting in the promotion of nuclear factor-kappa B (NF-κB) activation and secretion of cytokines, such as IL-6 and TNF-α.^(9,10)

Klotho is an anti-aging protein primarily expressed in the kidneys and brain. Klotho-deficient mice exhibit phenotypes resembling human aging, including a shortened lifespan, vascular calcification, cognitive decline, and sarcopenia.⁽¹¹⁾ A systematic review showed a relationship between Klotho activity and physical performance in middle-aged and elderly individuals.⁽¹²⁾ Klotho overexpression extends lifespan and confers resistance to oxidative stress. Klotho suppresses the insulin-like growth factor-1 (IGF-1) and NF-κB signaling pathways linked to aging, and enhances antioxidant defenses via the nuclear factor erythroid 2-related factor 2 (Nrf2) and forkhead box protein O (FOXO) transcription factors.⁽¹¹⁾ The activation of antioxidant enzymes by Klotho contributes to protection against age-related diseases, such as physical frailty and renal disease.⁽¹¹⁾ Maintaining the level of Klotho is an attractive target for therapeutic interventions against age-related diseases.⁽¹³⁾

We previously reported that glycyrrhizin (GL), a medicinal ingredient of licorice root, significantly attenuates the inflammatory response by inhibiting HMGB1–NF-κB signaling.⁽¹⁴⁾ As GL can inhibit HMGB1, it may attenuate this inflammatory amplification loop to reduce the systemic SASP and thus mitigate multiple aging-related symptoms. Age-related diseases include cancer, dementia, heart disease, and kidney disease. As GL has a hepatoprotective effect, much research has been conducted on liver cancer. However, there have been few reports on its anti-aging properties, including improvements in Alzheimer’s disease,⁽¹⁵⁾ inhibition of vascular endothelial cell aging,⁽¹⁶⁾ and improvements in kidney function in diabetic mice.⁽¹⁷⁾ However, there are no reports of improvements to motor function decline due to aging. Therefore, we investigated the therapeutic effects of GL on aging-related symptoms, including motor function decline, lipid accumulation, and inflammation, and discussed the underlying mechanisms.

Materials and Methods

Ethics

This study was performed in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the Suzuka University of Medical Science (approval number: 89).

Animals

Specific pathogen-free male ICR mice (7 and 9 weeks old) were purchased from SLC (Hamamatsu, Japan). Seven-week-old mice were used for the treatment experiments, and nine-week-old mice were used as young controls at the time of sacrifice. The mice were individually housed in cages at 23 ± 1°C in an air-conditioned room with controlled humidity (60 ± 10%) and a 12-h light/dark cycle. The mice were acclimatized for one week to the environment before the experiments.

Experimental procedure

Eight-week-old mice were quarantined for the first week and randomly divided by body weight into two groups: control (n = 5) and GL (n = 5). The dosage used in this study (15 ‍mg/kg body weight) was set to be sufficiently low from the toxicity threshold⁽¹⁸⁾ and within the safety margin used when evaluating the efficacy of the drug.^(19,20) Phosphate-buffered saline was used to dissolve GL because pH control was necessary. GL (15 ‍mg/kg body weight, glycyrrhizic acid monoammonium; COKEY Co., Ltd., Tokyo, Japan) dissolved in phosphate-buffered saline was orally administered to the GL group using a feeding needle three times per week for 12 months. Water was administered to the control group instead of GL solution. After 12 months, behavioral tests were performed and biological samples were collected. For comparison, 10-week-old mice were used in this study. Behavioral tests, sample collection, and autopsy findings were confirmed in at least two participants. All animals were euthanized by blood collection from the heart into heparinized tubes under deep anesthesia using pentobarbital, and all precautions were taken to minimize suffering. Liver, kidney, and blood samples were collected.

Behavioral tests

Exploratory behavior was examined using an open-field test (LOCOMO LS-5; MELQUEST, Toyama, Japan). Each mouse was placed in the center of an acrylic cage surrounded by infrared sensors. The amount of movement was measured and quantified by using these infrared sensors. Data were collected for 15 ‍min after each mouse was placed into the apparatus, a new environment for them. The grip strength tests were performed using a digital force meter (GPM-100B; MELQUEST). The mouse grasped the horizontal bar attached to the gauge with its front paws, and the experimenter slowly pulled its tail back and measured the tension when the mouse removed its front paws from the horizontal bar. Forelimb grip strength was measured horizontally in three repeated tests, and the maximum of the three measurements was used for the analysis. Rotarod tests were performed on a rotarod treadmill for rats and mice (MK-670; Muromachi Kikai Co., Ltd., Tokyo, Japan). The rotation speed was set to 10 ‍rpm, and the mouse was placed in the roller lane of the rotarod (90-mm diameter). The time taken for the mouse to fall was recorded as fall latency. Measurements were obtained after twice of the pre-test training. The cut-off value was 360 ‍s.

Biochemical tests

Blood was analyzed after the separation of plasma using commercially available kits for triglyceride and cholesterol (FUJIFILM Wako Chemicals, Osaka, Japan), IGF-1 (R&D Systems, Minneapolis, MN), Klotho (CUSABIO TECHNOLOGY LLC, Houston, TX), cystatin-C (BioVendor-Laboratorni medicina s.r.o., Brno, Czech Republic), IL-6 (BioLegend, San Diego, CA), and TNF-α (Proteintech, Rosemont, IL).

Histopathological study

Liver and kidney tissues were cut, fixed in 10% buffered formalin, and embedded in paraffin. Histopathological changes were assessed in paraffin sections stained with hematoxylin and eosin (HE) or periodic acid-methenamine-silver (PAM) following standard methods. Stained tissue sections were histopathologically analyzed by two investigators.

Immunohistochemical study

Paraffin sections were heated for 10 ‍min at 95°C in 5% urea for antigen retrieval. The sections were treated with 0.1% avidin to block endogenous biotin, then with 3% hydrogen peroxide to block endogenous peroxidase, then with skim milk, and then reacted with the primary antibody. Anti-superoxide dismutase (SOD) 2/MnSOD antibody [EPR2560Y] (ab68155; Abcam K.K., Tokyo, Japan) 1:100 was used as the primary antibody. DAB staining was performed using the VECTASTAIN (R) Elite ABC kit (Funakoshi Co., Ltd. Tokyo, Japan), in accordance with the manufacturer’s instructions. Staining was visualized with a Peroxidase Stain DAB Kit (Brown Stain) (Nacalai Tesque, Inc., Kyoto, Japan) and counterstained with hematoxylin.

Statistical analysis

Data are presented as means with SD. Comparisons of data between groups were performed using Student’s t test. Significance was set at p<0.05.

Results

Effects of GL on obesity and fatty liver

All 12-month-old mice appeared healthy, except for being obese. The changes in body weight at 12 months are shown in Fig. 1A, and the final body weights are shown in Fig. 1C. The increase in body weight was significantly suppressed in GL-treated mice compared with that in control mice. In the livers of 12-month-old mice, fat accumulation was observed as a white-spotted pattern, but it was decreased by the oral administration of GL (Fig. 1D). The 12-month-old mice showed accumulation of lipid droplets (shown as white circles) in liver cells compared with the 10-week-old mice, and this accumulation was suppressed by the oral administration of GL (Fig. 2A). At 12 months, four of the five control mice exhibited steatosis (Table 1). None of the GL-treated mice exhibited steatosis. These results suggest that GL improves obesity and excessive visceral fat accumulation.

Fig. 1.

Effects of GL on body weight and organ changes in 12-month-old mice. (A) Changes in body weights of control and GL-treated mice (n = 5). A statistically significant difference (p value <0.01) was observed between Control and GL from the 7th month onward. (B) Representative image of mice taken from behind at the end-point. (C) Body weights of 10-week-old mice (10 w, n = 4), 12-month-old mice in control group (12 m, n = 5), and 12-month-old mice in GL group (12 m + GL, n = 5) at the end-point. (D) Representative image of livers. Lower panels are enlarged images of the yellow squares in the upper panel. (E) Representative image of kidneys. Graphs represent the average score (bar: SD). **p<0.01.

Fig. 2.

Effects of GL on fatty liver tissues and kidney tissues. (A) HE staining of liver tissues. The 12-month-old mice showed lipid droplet accumulation (shown as white circles) in liver cells compared with the 10-week-old mice, which was suppressed by GL. (B) PAM staining of kidney tissues. The lower panel is an enlarged image of the yellow square in each upper panel. *, enlargement of the tubular lumen (in upper middle panel). Green arrowhead, PAM-positive particles. Blue arrowhead, basement membrane strongly stained by PAM. (C) Immunostaining with SOD2. The three images on the left are the livers, and the three on the right are the kidneys. Representative images are shown.

Table 1.

Number of steatoses in mice

Group	Steatoses mice/all mice
10 weeks old	0/4
12 months old	4/5
12 months old + GL	0/5

Effects of GL on kidneys

The kidneys of all the mice were grossly normal (Fig. 1E). Kidney weight per unit body weight of 12-month-old mice was tended to be lower than that of 12-month-old mice treated with GL (data not shown). This finding suggests that GL improves aging-induced kidney degeneration. Figure 2B shows the kidney tissues stained with PAM. The glomeruli tended to be larger in 12-month-old mice than in 12-month-old GL-treated mice. In the 12-month-old mice, tubular lumen enlargement (*) was frequently observed. The glomerular basement membrane and mesangial cells were stained black. The 10-week-old mice exhibited little glomerular basement membrane staining. The 12-month-old mice exhibited strongly positive staining in the glomerular basement membrane, compared with the 12-month-old + GL-treated mice (blue arrowhead). The 12-month-old mice exhibited PAM-positive particle accumulation, such as AGE in kidney cells (green arrowhead). This accumulation was suppressed by GL. To summarize, the aging of the kidneys of GL-treated mice was slower than that of control mice at 12 months old.

Antioxidant enzyme SOD2

In the 10-week-old mice, positive staining of SOD2 was observed in the cytoplasm of numerous hepatocytes near blood vessels and of numerous renal tubular cells (Fig. 2C). The 12-month-old mice showed decreased positive staining cells compared with the 10-week-old mice. The decline seemed to be suppressed by GL administration, suggesting that GL maintains the antioxidant capacity.

Behavioral tests improvements

The activity level test evaluates the cognitive ability and curiosity of mice by measuring their exploratory behavior in a novel environment. Neuromuscular function can be assessed in mice by measuring their maximal grip strength. The rotarod test was used to assess coordination, balance, and motor learning. The 12-month-old mice showed decreased activity levels, grip strength, and rotarod test results compared with the 10-week-old mice (Fig. 3). These declines were suppressed by GL administration, suggesting that GL slows the decline in motor and cognitive functions associated with aging.

Fig. 3.

Effects of GL on behavioral tests of 12-month-old mice. The 12-month-old mice showed decreased activity levels (A), grip strength (B), and rotarod test results (C) compared with the 10-week-old mice, and these decreases were suppressed by GL. Graphs represent average scores (bar: SD). *p<0.05, **p<0.01.

Blood test improvements

The 12-month-old mice showed increased blood triglyceride and cholesterol volumes compared with the 10-week-old mice, and these increases were suppressed by GL administration (Fig. 4A and B). This suggests that GL suppressed the increase in blood fat content.

Fig. 4.

Effects of GL on blood components of 12-month-old mice. (A) Triglyceride, (B) cholesterol, (C) IGF-1, (D) Klotho, (E) cystatin-C, (F) IL-6, and (G) TNF-α in blood. Graphs represent average scores (bar: SD). *p<0.05, **p<0.01.

The 12-month-old mice showed decreased levels of IGF-1 (Fig. 4C) and Klotho (Fig. 4D), which have anti-aging properties, in the blood compared with the 10-week-old mice. These effects were significantly suppressed by GL administration.

The 12-month-old mice showed increased cystatin C levels in the blood, indicating poor renal function compared with the 10-week-old mice (Fig. 4E). This increase was significantly suppressed by GL administration. Figure 4F and G show the levels of the inflammatory cytokines IL-6 and TNF-α in blood. The 12-month-old mice showed increased IL-6 and TNF-α compared with the 10-week-old mice. GL administration significantly suppressed this increase. These results suggest that GL suppresses aging by improving renal function and inflammation.

Discussion

In this study, GL moderated frailty associated with aging. Twelve-month-old mice exhibited increased body and organ weights compared with 10-week-old controls, but these increases were suppressed by GL administration. Similarly, age-related decreases in IGF-1 and Klotho levels were prevented by GL. Hepatic fat accumulation, renal aging, and motor function decline were also ameliorated, suggesting a broad protective role of GL. The mechanism by which GL improves these age-related symptoms is shown in Fig. 5.

Fig. 5.

Possible anti-aging mechanism of GL.

GL prevented the age-associated decline in Klotho. Reduced Klotho levels are correlated with diminished IGF-1 signaling, contributing to muscle weakness and frailty. As the growth hormone (GH)/IGF-1 axis is critical for maintaining muscle and bone integrity, preserving Klotho levels aids in sustaining many metabolic functions, including the proper development and growth of bones and skeletal muscles.⁽²¹⁾ This idea reasonably explains the observed improvements in physical condition and muscle preservation in GL-treated mice. Song et al. recently reported that STING signaling in inflammation is a novel target for the treatment of musculoskeletal diseases.^(22,23) Our data suggest that GL indirectly modulates Klotho-related IGF signaling by inhibiting HMGB1 and inflammation.

Our histological analysis showed that GL suppressed aging-mediated kidney damage, such as glomerular enlargement and AGE accumulation. Age-related kidney damage and dysfunction reduce energy-intensive processes such as tubular reabsorption and filtration, contributing to systemic aging and frailty.⁽²⁴⁾ This study also demonstrates that the 12-month-old mice increased the blood level of IL-6 and TNF-α, compared with the 10-week-old mice and that the increase was significantly suppressed by GL administration. It has recently been reported that inflammatory cytokines, such as TNF-α, downregulate renal Klotho expression via NF-κB, exacerbating kidney aging.⁽²⁵⁾ It is considered that GL can contribute to maintaining Klotho levels by reducing inflammatory signals via HMGB1 inhibition. This study also demonstrated that 12-month-old mice exhibited increased cystatin C levels in the blood, indicating poor renal function, compared with 10-week-old mice, and that the increase was significantly suppressed by GL administration. Oxidative stress and Klotho deficiency in the kidneys form a vicious cycle that accelerates senescence. Klotho disrupts this cycle by enhancing the expression of antioxidant enzymes, such as SOD2. These enzymes neutralize ROS, which explains the renal protective effects observed in the GL-treated animals.

GL improves lipid deposition in rats with high-fat-diet-induced obesity.⁽²⁶⁾ This study showed that 12-month-old mice exhibited accumulation of lipid droplets in liver cells compared with 10-week-old mice, and this accumulation was suppressed by GL. Decreased Klotho is associated with obesity and metabolic syndrome.⁽²⁷⁾ The ability of GL to preserve Klotho likely contributes to improved metabolic health and decreased fat accumulation in the liver tissues of aged mice. The findings of this study demonstrate that GL administration ameliorates multiple age-related pathologies, including organ hypertrophy, frailty, oxidative stress, inflammation, renal dysfunction, and lipid accumulation. This is the first study to demonstrate that GL has strong potential as a therapeutic candidate for age-related diseases.

The protective effects of GL may be mediated by the suppression of the HMGB1-SASP axis, inhibition of the cGAS-STING inflammatory loop, and preservation of klotho expression. GL may reduce inflammation by suppressing the decline in Klotho levels by not only inhibiting HMGB1, but also activating peroxisome proliferator-activated receptor (PPAR) γ and AMP-activated protein kinase (AMPK). Klotho suppresses the activation of the proinflammatory NF-κB pathway.⁽²⁸⁾ NF-κB is also involved in inflammation that contributes to aging. GL blocks inflammation by inhibiting HMGB1 and maintaining Klotho levels. Klotho prevents the nuclear translocation of NF-κB, but it may also inhibit the TNF-α-induced activation of NF-κB.

The mechanism by which GL increased Klotho can be considered as follows. GL is an agonist to PPAR-γ and can induce PPAR-γ upregulation.⁽²⁹⁾ PPAR-γ itself functions as a key regulator of glucose and lipid metabolism and may play an important anti-inflammatory role.⁽³⁰⁾ It has been reported that Klotho is upregulated by PPAR-γ.⁽³⁰⁾ The main function of PPARs during inflammatory responses is the promotion of NF-κB inactivation.⁽³¹⁾ As a result, PPARγ indirectly prevents the reduction of Klotho through the inactivation of NF-κB.

AMPK may control the aging process via an integrated signaling network.⁽³²⁾ GL activates AMPK,⁽³³⁾ which is involved in the suppression of obesity through PPARγ expression. AMPK increases the expression of the nuclear receptor PPARγ, thereby helping to prevent metabolic syndrome.⁽³⁴⁾ AMPK is a potent inducer of Klotho. Both Klotho and AMPK have beneficial effects on similar organs. GL, an AMPK activator, enhances Klotho expression.⁽³⁵⁾

Through these mechanisms, GL suppresses the decline in Klotho levels and protects against age-related symptoms. Reducing the dosage may weaken the effect, but since humans can take the medication for longer periods than mice, a stronger effect may be expected.

Author Contributions

SO conceived the hypotheses, designed and performed the experiments, interpreted the results, and wrote the manuscript. KH performed the experiments and participated in critical discussions. NY and SK participated in critical discussions.

Abbreviations

AGE

advanced glycation end products

AMPK

AMP-activated protein kinase

DAMP

damage-associated molecular pattern

FOXO

forkhead box protein O

GAS

guanosine-adenosine synthase

GH

growth hormone

GL

glycyrrhizin

HE

hematoxylin and eosin

HMGB1

high-mobility group box 1

HO

heme oxygenase

IGF

insulin-like growth factor

IL

interleukin

NF-κB

nuclear factor-kappa B

Nrf2

nuclear factor erythroid 2-related factor 2

PAM

periodic acid-methenamine-silver

PPAR

peroxisome proliferator-activated receptor

RAGE

receptor for AGE

ROS

reactive oxygen species

SASP

senescence-associated secretory phenotype

SOD

superoxide dismutase

STING

stimulator of interferon genes

TLR

toll-like receptor

TNF

tumor necrosis factor

Conflict of Interest

SO, KH, and SK have no conflicts of interest. NY is an employee of Cokey Co., Ltd. Cokey Co., Ltd. manufactures glycyrrhizin for medicinal drugs or cosmetics.