Baicalein Exerts Beneficial Effects in d -Galactose-Induced Aging Rats Through Attenuation of Inflammation and Metabolic Dysfunction

Abstract

Baicalein is a flavonoid isolated from the roots of Scutellaria baicalensis Georgi. This study aimed to ascertain the effects and potential underlying mechanisms of baicalein in d-galactose (d-gal)-induced aging rat model by integration of behavior examination, biochemical detection, and ¹H nuclear magnetic resonance (NMR)-based metabolomic approach. Our findings suggest that baicalein significantly attenuated memory decline in d-gal-induced aging model, as manifested by increasing recognition index in novel object recognition test, shortening latency time, and increasing platform crossings in Morris water maze test. Baicalein significantly inhibited the releases of inflammatory mediators such as nitric oxide, interleukin-6, interleukin-1 beta, and tumor necrosis factor-α in d-gal-induced aging model. Metabolomic study revealed that 10 endogenous metabolites in cerebral cortex were considered as potential biomarkers of baicalein for its protective effect. Further metabolic pathway analysis showed that the metabolic alterations were associated with alanine, aspartate and glutamate metabolism, glycine, serine and threonine metabolism, inositol phosphate metabolism, and energy metabolism. These data indicate that baicalein improves learning and memory dysfunction in d-gal-induced aging rats. This might be achieved through attenuation of inflammation and metabolic dysfunction.

Introduction

Aging is characterized as a progressive loss of physiological integrity. Memory decline in normal aging is a conserved feature of growing older humans.¹ Episodic memory, including spatial memory, recollection, and interference, is thought to be particularly vulnerable to the effects of advancing age.² With the tremendous increase of older population, there is an urgent need to develop the agents that could ameliorate cognitive deficits in aging process.

Baicalein is a flavonoid extracted from the roots of Scutellaria baicalensis Georgi. Baicalein has extensive pharmacological properties, including antivirus,³ antibacteria,⁴ antitumor,⁵ and cardiovascular protection.⁶ Particularly, baicalein protects against several cerebral diseases such as traumatic brain injury,⁷ ischemic stroke,⁸ Parkinson's disease,^9

–12 and Alzheimer's disease.^13,14 Interestingly, we found that 0.04, 0.2, and 1 mg/mL baicalein extended the mean, median, and maximum life spans of Drosophila melanogaster,¹⁵ suggesting its potential antiaging roles. However, the antiaging effects of baicalein have not been deeply investigated.

Although the exact mechanism of aging remains a riddle, chronic inflammation plays important roles.¹⁶ It is appreciated that chronic low-grade inflammation, termed “inflammaging,” positively correlates with age.¹⁶ Accumulated data strongly suggest that continuous upregulation of proinflammatory mediators are induced during the aging process.^17
–19 Mitochondrial dysfunction, a hallmark of aging,²⁰ can accelerate aging in mammals. Mitochondria play a role in modulating the inflammatory response, and the production of proinflammatory mediators impairs mitochondrial function by increasing reactive oxygen species (ROS) production.

Metabolomics has significant potential in pharmaceutical research, including discovery of biomarkers for disease and the elucidation of the mechanism of action of drugs.²¹ Metabolomics can comprehensively and quantitatively determine the metabolites, whose levels can be regarded as the ultimate response of biological systems to genetic and environmental changes.²² With the utilization of a metabolomics approach, it has been discovered that the endogenous metabolites are correlated with aging.^23
–25

In the present work, the antiaging effects and the underlying mechanisms of baicalein were investigated in d-galactose (d-gal)-induced aging rats model. ¹H nuclear magnetic resonance (NMR)-based metabolomics was utilized to characterize the metabolic alterations in cerebral cortex. Our results support that baicalein has beneficial effects in d-gal-induced aging rats model.

Materials and Methods

Animals and drug administrations

Sixty male Sprague Dawley rats (180–220 g) of 8-week-old were commercially obtained from Beijing Vital River Laboratories (Beijing, China). The rats were adapted to the new environment for 1 week before experiments were carried out. All rats were accommodated in room temperature (20°C–25°C) and constant humidity (40%–50%) under a 12 hours light–dark cycle with food and water ad libitum. All recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health were stringently followed in all procedures of this study.

Sixty rats were randomly divided into five groups (n = 12 per group): control group, d-gal (Sigma-Aldrich, St. Louis, MO) administration group, and three d-gal administration plus baicalein (purity 98%, purchased from Jingzhu Bio-Technology Co., Ltd., Nanjing, China) groups [50 mg/(kg·d), 100 mg/(kg·d), 200 mg/(kg·d)]. d-gal [150 mg/(kg·d)] was injected subcutaneously daily into rats, and the rats in control group were injected subcutaneously 0.9% saline every day. The rats in d-gal administration plus baicalein groups were intragastricly administered baicalein, and the rats in control and d-gal groups were intragastricly administered distilled water. d-gal and baicalein were administered for 56 days before behavioral tests, and were continuously administered in the period of behavioral tests.

Behavioral tests

Novel object recognition test

The object recognition test was carried out as method presented.²⁶ The apparatus consisted of a square open box (55 cm long, 20 cm high, and 35 cm wide). The objects to be discriminated were in two different shapes: block and ball, and they could not be displayed by rats. This test included periods of adaptation, familiarity, and test. During the adaptation, each rat was placed in an empty box for 6 minutes. On the familiarity day, two identical objects (AA) were put in the test case and the rat was put into the box from the same position to the object (6 minutes). On the test day, one of the other set of objects replaced an object used in the familiarity day in the box (AB) and the rat was put into the box (6 minutes).

The time spent exploring the objects during the experimental sessions was defined as follows: sniffing or touching the object with nose. The recognition index was used to evaluate the ability of object recognition of rats. The “recognition index” was calculated by equation TB/(TA + TB), where TA is the time spent exploring the familiar object, and TB is the time spent exploring the novel object.

Morris water maze test

The spatial learning and memory ability was evaluated by the Morris water maze test, which included a 5-day place navigation training and a test day.²⁷ The maze consisted of a black circular water tank (120 cm in diameter, 38 cm in height), containing water (24°C ± 1°C) to a depth of 30 cm and a hidden platform submerged 1 cm below the water surface. The water cistern was divided into four quadrants, one of which contained a circular escape platform (12 cm diameter) placed at a fixed position. Place navigation trials were executed twice a day for 5 days before the test day, and there was no intertrial time interval between the two trials of the same day. For each trial, the rat was placed in the water facing the pool wall at one of four starting quadrant points and the starting quadrant was varied randomly over the trials.

For all training trials, the time that rats spent to reach the fixed platform (escape latency) was recorded to assess spatial learning capacity.²⁸ If a rat did not find the platform within 60 seconds, it was placed on the platform for 20 seconds at the end of the trial, and the escape latency was recorded as 60 seconds. On the test day, the platform was removed. The numbers of target crossings were recorded as well as the escape latency before reaching the target place. Data were collected by the video tracking equipment and processed by a computer equipped with an analysis management system.²⁸

Inflammatory mediators detection

Serum samples were harvested for detection of inflammatory mediators. The content of nitric oxide (NO) was measured by NO assay kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) according to the manufacturer's instructions.

The concentrations of interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and tumor necrosis factor-α (TNF-α) were determined using commercially available enzyme-linked immunosorbent assay (ELISA) kits obtained from Sangon Biotech (Shanghai, China). In brief, the samples were pipetted into the 96-well plates that contained rat-specific antibodies. After incubation and washing, the absorbance was determined by following the instructions specified in the kit.

Preparation of cerebral cortex extracts and acquisition of ¹H NMR spectral data

Metabolite extraction from the cerebral cortex of rats was achieved using methanol/chloroform/water.^29,30 After weighing, ice-cold methanol (4 mL/g) and double-distilled water (0.85 mL/g) were added to homogenize the cerebral cortex. The sample was vortexed, and then chloroform (2 mL/g) was added, and the sample was vortexed again. Subsequently, 2 mL/g chloroform and 2 mL/g water were added to the sample. The homogenate was left on ice for 15 minutes and centrifuged at 1000 g for 15 minutes at 4°C. Transfer the upper methanol/water phase (with polar metabolites) into separate glass vials. Remove the solvents from the samples under a stream of nitrogen.

The tissue extracts were reconstituted in 600 μL of NMR buffer (100 mM sodium phosphate buffer, pH 7.4, in 10% D₂O, containing 0.01% sodium 3-trimethlysilyl [2,2,3,3-d4] propionate [TSP] as an internal NMR chemical shift standard) and vortexed and then centrifuged at 12,000 g for 5 minutes. Then, 550 μL of the supernatant was transferred into a 5 mm NMR tube for NMR spectroscopy.

NMR analysis of cerebral cortex extracts

The ¹H NMR spectra of cerebral cortex extracts were acquired on a Bruker 600 MHz AVANCE III NMR spectrometer (Bruker) equipped with a Bruker 5 mm double resonance BBI probe at a temperature of 25°C. Spectra were recorded using the noesygppr1d pulse sequence.

The ¹H NMR spectra were processed using MestReNova software (version 10.0.1; Mestrelab Research, Santiago de Compostela, Spain). All the spectra were manually phased and baseline corrected. The spectra of cerebral cortex extracts were referenced to the chemical shift of TSP with a chemical shift at δ 0.00 ppm. The regions containing the resonance from residual water (δ 4.52–5.20) were excluded to avoid the influence of water. All spectra were divided and then the signal integral computed in 0.01 ppm across the region δ 0.09–8.90 for cerebral cortex extract samples. The remaining spectral segments for cerebral cortex extracts were normalized to a total sum of the spectral intensity.

Pathway analysis

MetaboAnalyst 3.0,³¹ a web-based analysis module, can perform metabolic pathway analysis by integrating two analysis approaches, including pathway enrichment analysis and pathway topology analysis. The main metabolic pathways regulated by baicalein were analyzed using this tool.

Statistical analysis

All data are expressed as a mean ± standard error of the mean. Repeated measures analysis of variance (ANOVA) followed by Fisher's least significant difference test was used for analysis of the data in acquisition phase of Morris water maze test. One-way ANOVA followed by Dunnett post hoc test was used for statistical analysis to determine significant differences of other behavior and biochemical changes among control, d-gal, and baicalein groups, whereas t test was used to study the mean difference of each metabolite between two groups. All preprocessed NMR data were imported into the software package SIMCA-P 13.0 (Umetrics) for multivariate data analysis. Results were considered statistically significant when p < 0.05.

Results

Body weight and food intake

Compared with the rats in control group, rats treated with d-gal daily for 8 consecutive weeks had no significant alteration on body weight. Compared with d-gal treated group, baicalein administration had no significant effect on body weight (Fig. 1A). Meanwhile, there was no significant difference in food intake among control, d-gal, and baicalein treatment groups (Fig. 1B). These results showed that 8 consecutive weeks administration of baicalein did not have significant effects on body weight and food consumption in rats induced by d-gal.

FIG. 1.

Effects of baicalein on body weight and food intake in d-gal-induced rats. (A) Body weight; (B) food intake. d-gal, d-galactose.

Effects of baicalein on the behavior of d-gal-treated rats

Novel object recognition test showed that recognition memory was significantly declined in the d-gal administration group (p < 0.05), however, in contrast to the d-gal group, it was significantly increased in rats treated with 100 or 200 mg/kg baicalein for 8 weeks (p < 0.001) (Fig. 2A). The present results suggest that memory acquisition was impaired following exposure to d-gal, and this decline could be attenuated after treatment of baicalein.

FIG. 2.

Effects of baicalein on the behaviors of d-gal-treated rats. (A) Novel object recognition behavior: recognition index of rats; (B) escape latencies during the 5-day place navigation training: the results of repeated measures ANOVA showed that the escape latencies were significantly higher in d-gal group in comparison with control group (p = 0.033), while there was no significant difference among d-gal and baicalein treatment groups; (C) latency time; (D) the number of platform crossings in 1 minute. All values given are the mean ± SEM. *p < 0.05, **p < 0.01 versus control group. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 versus d-gal group. ANOVA, analysis of variance; SEM, standard error of the mean.

The results of repeated measures ANOVA showed that the escape latency was significantly higher in d-gal group in comparison with control group (p = 0.033). Although there was no significant difference among d-gal and baicalein treatment groups, rats in the baicalein treatment groups exhibited shorter escape latencies compared with rats in the d-gal group on the 5th day (Fig. 2B).

On the test day, rats in d-gal administration group spent more time to find the target than those in the control group, while rats in the d-gal administration plus baicalein (100, 200 mg/kg) treatment groups spent less time to find the target than the d-gal administered rats (Fig. 2C). In addition, rats in the d-gal group crossed the location fewer times compared with rats in the control group. However, rats in d-gal administration plus baicalein (200 mg/kg) treatment groups displayed remarkable increases of the target crossing times compared with those in the d-gal-treated group (Fig. 2D). These results indicate that rats induced by d-gal had impairments in spatial learning and memory, while the treatment of baicalein could attenuate the age-related cognitive impairment.

Effects of baicalein on the levels of inflammatory mediators

The NO level was significantly increased in rats of the d-gal group, while it was significantly decreased in rats of the d-gal administration plus baicalein (100, 200 mg/kg) groups (Fig. 3A). IL-6, IL-1β, and TNF-α levels were significantly higher in the serum of rats in the d-gal group compared with those in the rats of the control group, demonstrating that baicalein significantly suppressed the d-gal-induced increases in IL-6 (Fig. 3B), IL-1β (Fig. 3C), and TNF-α (Fig. 3D).

FIG. 3.

Effects of baicalein on the levels of inflammatory mediators in the serum. (A) NO level; (B) IL-6 level; (C) IL-1β level; and (D) TNF-α level. All values given are the mean ± SEM (n = 4). *p < 0.05, **p < 0.01 versus control group. ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001versus d-gal group. IL-1β, interleukin-1 beta; IL-6, interleukin-6; NO, nitric oxide; TNF-α, tumor necrosis factor-α.

Metabolomic study of cerebral cortex of rat brain

The typical¹H NMR spectra of cerebral cortex obtained from the control rats are shown in Figure 4. The resonances in these spectra were assigned to individual metabolite by matching to the chemical shifts of compared standards, and those recorded in the literature,^30,32 and NMR databases such as the Human Metabolome Database (HMDB), Biological Magnetic, and Resonance Data Bank (BMRB). Identified endogenous metabolites in cerebral cortex of rats are listed in Table 1. Those detectable metabolites include numerous organic acids, amino acids, carbohydrates, tricarboxylic acid cycle intermediates, and nucleotide derivatives, together with some other metabolites.

FIG. 4.

Typical ¹H NMR spectrum of cerebral cortex in control rat. NMR, nuclear magnetic resonance.

Table 1.

¹ H Nuclear Magnetic Resonance Assignments of Major Metabolites from Rat Cerebral Cortex

No.	Metabolite	Chemical shift (ppm)
1	Isoleucine	0.95 (d), 1.01 (d)
2	Leucine	0.97 (t)
3	Valine	0.99 (d), 1.05 (d)
4	Lactate	1.33 (d), 4.12 (q)
5	Alanine	1.49 (d)
6	γ-Aminobutyric acid	1.91 (m), 2.30 (t), 3.01 (t)
7	N-acetylaspartate	2.03 (s), 2.50 (q), 2.68 (dd)
8	Glutamate	2.06 (m), 2.35 (m), 3.76 (m)
9	Glutamine	2.14 (m), 2.45 (m)
10	Pyruvate	2.37 (s)
11	Aspartate	2.67 (dd), 2.82 (dd), 3.91 (m)
12	Dimethylamine	2.72 (s)
13	Creatine	3.04 (s), 3.94 (s)
14	Choline	3.21 (s)
15	Phosphotyl choline	3.22 (s)
16	Taurine	3.27 (t), 3.43 (t)
17	Myo-inositol	3.29 (t), 3.55 (dd), 3.63 (t), 4.08 (t)
18	Glycine	3.57 (s)
19	Uracil	5.81 (d), 7.55 (d)
20	Adenosine	6.10 (d), 8.25 (s), 8.35 (s)
21	Hypoxanthine	8.21 (d)
22	Niacinamide	7.60 (dd), 8.72 (d), 8.94 (s)

Multivariate data analysis

To obtain the more detailed metabolic differences between the control, d-gal, and d-gal administration plus baicalein groups, all NMR data were subjected to multivariate data analysis. Significant differences between the control and d-gal groups were observed in partial least squares discriminant analysis (PLS-DA) score plot (Fig. 5A), indicating that the metabolic profiles were markedly altered following d-gal administration. The PLS-DA model was validated using the response of the permutation test through 200 permutations, in which all R² and Q² values were lower than original ones. The good PLS-DA model (R²X = 0.709, Q² = 0.891) indicates an excellent predictive power (Fig. 5B).

FIG. 5.

(A) PLS-DA score plot, significant differences between control and d-gal groups were observed in PLS-DA score plot, indicating that the metabolic profiles were markedly altered following d-gal administration; (B) PLS-DA validation plot of rat cerebral cortex between the control and d-gal group, the quite good PLS-DA model (R²X = 0.709, Q² = 0.891) indicated an excellent predictive power; (C) OPLS-DA score plot and (D) corresponding S-plot of rat cerebral cortex between the control and d-gal group, which were used to find the differential metabolites between control and d-gal groups. OPLS-DA, orthogonal projection to the latent structure with discriminant analysis; PLS-DA, partial least squares discriminant analysis.

Orthogonal projection to the latent structure with discriminant analysis (OPLS-DA) was further performed to find the differential metabolites between the control and d-gal groups (Fig. 5C). The corresponding S-plot (Fig. 5D) combined with variable importance in the projection (VIP) was used to find the differences of metabolites, and then independent t test was carried out to compare the relative peak areas of these metabolites. The abundance levels of 13 metabolites were significantly changed in the d-gal group, including higher levels of glutamate (Glu), pyruvate (Pyr), myo-inositol (m-Ins), and taurine (Tau) and lower levels of glutamine (Gln), N-acetylaspartat (NAA), aspartate (Asp), glycine (Gly), choline (Cho), creatine (Cr), lactate (Lac), hypoxanthine, and γ-Aminobutyric acid compared with the control group.

As shown in Figure 6, the principal component analysis (PCA) score plot showed good separations among the control, d-gal, and baicalein treatment groups. Baicalein treatment can attenuate the changes of Glu, Gln, NAA, Asp, Gly, Cho, Cr, Pyr, m-Ins, and Lac induced by d-gal at various degrees (Fig. 7).

FIG. 6.

PCA score plot derived from ¹H NMR spectra of cerebral cortex from rats in control group, d-gal group and d-gal administration plus baicalein groups. PCA, principal component analysis.

FIG. 7.

Comparison on relative peak areas of some potential differential metabolites in rat cerebral cortex. *p < 0.05, **p < 0.01, ***p < 0.001 versus control group. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 versus d-gal group.

Metabolic pathways regulated by baicalein

Metabolites with significant abundance changes between sample groups were subjected to MetaboAnalyst 3.0 as well as the KEGG database (www.genome.jp/kegg) for pathway analysis. The most relevant metabolic pathways regulated by baicalein include alanine, aspartate, and glutamate metabolism (Glu, Gln, NAA, Asp), glycine, serine, and threonine metabolism (Gly, Cho, Cr, Pyr), Pyruvate metabolism (Pyr, Lac), inositol phosphate metabolism, and glycolysis or gluconeogenesis (Fig. 8). Schematic diagram of perturbed metabolic pathways was constructed based on the relationship between the biomarkers and metabolic pathways (Fig. 9).

FIG. 8.

MetPA of metabolic pathway. The “metabolome view” presents all metabolic pathways arranged according to the scores based on enrichment analysis (y axis) and topology analysis (x axis).

FIG. 9.

Schematic diagram of the perturbed metabolic pathways regulated by baicalein for the beneficial effects in d-gal-induced rats. Square frame represents reduction, and ellipse represents increase in baicalein-treated rats compared with d-gal-treated rats.

Discussion

It was reported that caloric restriction or dietary restriction can prolong lifespan and improve the behavioral characteristics in later life of mice.^33,34 Our results demonstrate that the body weight and food consumption have not been significantly altered by baicalein in d-gal-induced aging rats, which was also supported by the report that 29 weeks administration of 400 mg/(kg·day) baicalein did not have significant effects on food intake and body weight of normal mouse.³⁵

d-gal is a reducing sugar and can be metabolized at the physiological level. However, at high levels, it can be converted into aldose and hydroperoxide, which have neurotoxic effect, resulting in the accumulation of ROS and oxidative stress.^36,37 It has also shown that d-gal could cause aging-related behavioral and neurochemical changes.^28,38 d-gal-induced aging models have been widely used in pharmacological studies of antiaging agents.^39,40 Literatures showed that subcutaneous injection each day with d-gal at dose range from 100 to 300 mg/kg for 6–8 weeks will establish an aging model.^28,38,41

In our research, we studied the effects of baicalein on the chronic d-gal injection-induced cognition deficits by behavioral tests. We found that d-gal could lead to the dysfunction of learning and memory in rats. However, both novel object recognition and Morris water maze test suggested that baicalein had a beneficial effect on the amelioration of brain functions during aging. The current results indicated that oral administration of baicalein attenuated the impairment of spatial learning and memory induced by d-gal. This result was in accordance with previous reports, which found that baicalein could rescue the memory deficits in a mouse model of Alzheimer's disease¹³ and in epilepsy-like tremor rats.⁴²

NO is a neurotransmitter and a marker of central nervous system inflammation. The higher concentrations of NO are neurotoxic, and it can induce defect in spatial memory,⁴³ inhibit mitochondrial respiration,⁴⁴ and diminish the ability of the cells to deal with oxidative stress.⁴⁵ Following d-gal subcutaneous injection for 8 weeks, inflammatory mediators NO and various inflammatory cytokines such as IL-6, IL-1β, and TNF-α were released in the aged rats.^28,46 In agreement with the previous reports, our results confirmed that d-gal significantly stimulated the releases of NO, IL-6, IL-1β, and TNF-α, which may be related to the injury of learning and memory ability. However, baicalein treatment could significantly diminish the productions of NO, IL-6, IL-1β, and TNF-α.

Glu, Gln, NAA, and Asp are involved in alanine, aspartate, and glutamate metabolism. Glu is the most abundant excitatory neurotransmitter in the human cortex, and its synthesis primarily occurs in astrocytes. Glu could potentially trigger excitotoxic cell injury and mitochondrial dysfunction. The high level of Glu is thought to be the primary contributor to cell damage.⁴⁵ Gln is synthesized from Glu and is vital in cerebral function. The level of Gln is correlated with cell density, and the reduced concentration might be related to cell loss with aging in cortical areas.⁴⁷ NAA is synthesized in the neuronal mitochondria, and is the marker of neuronal injury and cellular dysfunction.⁴⁸ NAA is related to neuronal integrity in many disorders, and it is involved in anti-inflammatory activity.⁴⁹

The decreases in Gln, NAA, and Asp concentrations were related to neuronal dysfunction and neuronal mitochondria deficit. Consistent with these changes, our results showed that the content of Glu was increased, and the levels of Gln, NAA, and Asp were decreased in d-gal-induced aging rats. These results indicate that alanine, aspartate, and glutamate metabolism, including glutamate–glutamine cycle, was impaired after d-gal injury, which might cause cell injury as well as mitochondrial dysfunction. However, baicalein has an apparent regulating function on alanine, aspartate, and glutamate metabolism pathway through decrease of Glu level and increases of Gln, NAA, and Asp levels, thus may protect against cell injury and mitochondrial dysfunction in d-gal-induced aging rats.

Gly, Cho, Cr, and Pyr participate in glycine, serine, and threonine metabolism. Gly could inhibit oxidative stress, neuroinflammation, and apoptotic neurodegeneration in postnatal rat brain.⁵⁰ Cho is an essential nutrient for all cells because it plays a role in the synthesis of the membrane phospholipid components of the cell membranes as well as in the synthesis of neurotransmitter acetylcholine.⁵¹ The decrease of Cho level suggests a decreased turnover of cell membrane that may be related to hypometabolism or dysfunction.⁵²

Cr is a naturally occurring nitrogenous organic acid, which participates in metabolic reactions within cells and plays an important role in maintaining cellular energy homeostasis. Research has found that Cr has potential neuroprotective effects in various models of neurological disease. Cr supplementation can alleviate cerebral energy depletion, exacerbated oxidative stress and mitochondrial dysfunction in neurodegenerative disorders.⁵³ Furthermore, Cr supplementation can improve cognitive performance in elderly people⁵³ and improve memory in vegetarians and omnivores.⁵⁴

In accordance to these reports, our data found that the levels of Gly, Cho, and Cr in the d-gal group were lower than those in the control group, indicating that impairment of the glycine, serine, and threonine metabolism may be involved in d-gal-induced aging. However, baicalein has an apparent regulating function on glycine, serine, and threonine metabolism pathway through elevation of Gly, Cho, and Cr levels. The content of m-Ins was increased during processes of aging,⁵⁵ which may indicate the upregulation of metabolic processes within glial cells.⁴⁷ In the current study, our results are consistent with previous findings that long-term treatment of d-gal leads to higher concentration of m-Ins. However, baicalein could significantly decrease the level of m-Ins in d-gal-induced aging rats.

Furthermore, the energy metabolism, including pyruvate metabolism and glycolysis or gluconeogenesis, also might be involved in the beneficial effects of baicalein in d-gal-induced aging model.

The above results suggest that baicalein exerts beneficial effects in d-gal-induced aging rats. However, the solubility of baicalein is poor, resulting in its low bioavailability. Recently, preparation of nanocrystal of baicalein could improve its bioavailability.⁵⁶ Moreover, Tsai et al. revealed that baicalein was able to penetrate the blood–brain barrier.⁵⁷ These results indicate that baicalein may be a promising agent for treatment of aging-related cognitive dysfunction. The potential roles should be further validated in senescence-accelerated prone mouse 8.

Conclusion

The behavior examination, biochemical detection, and ¹H NMR-based metabolomic approach were combined to investigate the effects and molecular mechanisms of baicalein on d-gal-induced aging rats. The behavior results indicated that baicalein could significantly ameliorate cognitive deficits induced by d-gal. Through biochemical detection, it was discovered that baicalein could significantly inhibit the releases of NO, IL-6, IL-1β, and TNF-α in serum, thus inhibit inflammaging. Metabolomic study showed that 10 differential endogenous metabolites in cerebral cortex were regulated by baicalein for its protective effect. The protective effect of baicalein was related to alanine, aspartate, and glutamate metabolism, glycine, serine, and threonine metabolism, pyruvate metabolism, inositol phosphate metabolism, and glycolysis or gluconeogenesis.

In short, baicalein improves learning and memory dysfunction induced by d-gal. These might be achieved through attenuation of inflammation and metabolic dysfunction.

Footnotes

Acknowledgments

This project was supported by the National Natural Science Foundation of China (81603319), and partly supported by Programs of Science and Technology and Higher Education of Shanxi Province (No. 2015118), Science and Technology Innovation Team of Shanxi Province (No. 201605D131045-18), and Key Laboratory of Effective Substances Research and Utilization in TCM of Shanxi province (201605D111004).

Author Disclosure Statement

No competing financial interests exist.

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Baicalein Exerts Beneficial Effects in d -Galactose-Induced Aging Rats Through Attenuation of Inflammation and Metabolic Dysfunction

Abstract

Introduction

Materials and Methods

Animals and drug administrations

Behavioral tests

Novel object recognition test

Morris water maze test

Inflammatory mediators detection

Preparation of cerebral cortex extracts and acquisition of 1H NMR spectral data

NMR analysis of cerebral cortex extracts

Pathway analysis

Statistical analysis

Results

Body weight and food intake

Effects of baicalein on the behavior of d-gal-treated rats

Effects of baicalein on the levels of inflammatory mediators

Metabolomic study of cerebral cortex of rat brain

Multivariate data analysis

Metabolic pathways regulated by baicalein

Discussion

Conclusion

Footnotes

Acknowledgments

Author Disclosure Statement

References

Preparation of cerebral cortex extracts and acquisition of ¹H NMR spectral data