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Effect of yellow rice wine on anti-aging ability in aged mice induced by D-galactose

2020-05-22RongLiuZukangFuFengjieZhangQingzhongMaoChunguangLuanXinglinHanJieXueDeliangWangSiQinFeikeHao

食品科学与人类健康(英文) 2020年2期

Rong Liu,Zukang Fu,Fengjie Zhang,Qingzhong Mao,Chunguang Luan,Xinglin Han,Jie Xue,Deliang Wang,Si Qin,Feike Hao,**

a College of Food Science and Technology,Hunan Agricultural University,Changsha,410128,China

b Kuaijishan Shaoxing Rice Wine CO.Ltd.,Shaoxing,312030,China

c China National Research Institute of Food and Fermentation Industries,Beijing,100015,China

ABSTRACT The present study is focused on investigation of the health protective effect of yellow rice wine (YRW)in mice and the potential underlying mechanism.Symptoms of aging were induced in mice by using Dgalactose(D-gal),and different doses of YRW(4,8,and 12 mL/kg BW)were orally administrated to D-galtreated mice for a period of six weeks.The in vivo data obtained in the present study preliminary indicated that YRW markedly increased the activity of antioxidant enzymes and reduced the concentration of MDA both in the brain and liver.Furthermore,a forced swim test showed that moderate intake of YRW(8 mL/kg BW)significantly decreased the duration of immobility,reduced the blood content of urea nitrogen and lactic acid,and increased hepatic glycogen storage.Besides,results from the Morris water maze test suggested that YRW significantly reversed cognitive impairment and also alleviated neuroapoptosis in the experimental mice by regulating the gene expressions of Bax/Bcl-2 and caspase-3.The above results indicated YRW has a potential anti-aging effect in mice and provided us certain molecular evidence for this action.

Keywords:Yellow rice wine Aging Antioxidant Antifatigue Cognitive impairment Apoptosis

1.Introduction

Aging is an inevitable and multifactorial process which features a progressive decline in antioxidant function,and therefore an increase in oxidative damage caused by reactive oxygen species(ROS)and other oxidant products[1,2].Accumulation of ROS negatively affects the functions of the liver,brain,heart,lungs,and other organs.Furthermore,oxidative stress aggravates pathological conditions such as liver lesions,cognitive impairment,and fatigue[3,4].Although cells and organism produce various antioxidants that counteract the potentially harmful effects of oxidizing agents,these systems cannot fully repair oxidative damage [5,6].Therefore,it is necessary to supply additional appropriate scavengers to protect the body from excess ROS.

The physical attenuation that accompanies aging is likely to cause fatigue easily.Fatigue is a state of feeling tired,weak,and lacking in energy,and is a strong predictor of functional limitations,disability,mortality,and other adverse outcomes in elderly people [7].Another indicator of aging in humans and in animal models is a deficit in spatial cognitive memory[8,9].This faculty is closely linked to the hippocampus,which is crucial for learning and environmental navigation in both human and rats[10].Moreover,structural and functional changes in the hippocampus are the earliest signs of age-related changes in brains[11].Therefore,neuronal dysfunction in the hippocampus due to aging may lead to a decline in spatial cognitive ability.

D-galactose (D-gal) is a reducing sugar that is naturally generated in the body.Although it can be completely metabolized at lower concentrations [12,13],it is converted to aldose,hydrogen peroxide,and galactose oxidase at higher concentrations.The presence of these species accelerates the production of superoxide anions and oxygen radicals,which impair the functions and abilities of macromolecules and cells [14].InjectingD-gal into rodent models induces changes such as oxidative stress,accumulation of metabolites,and impaired cognition,all of which are consistent with natural aging [15,16].Hence,D-gal has been widely used to establish the aging model[17,18].

Yellow rice wine(YRW,with 8%-15%alcohol)is one of the oldest alcoholic beverages in the world,with a history of more than 5000 years in China.Characterized by its unique flavor and high nutritional value,YRW has been a popular beverage for centuries.Traditional YRW is made from cereals through semi-solid fermentation,mediated by a Chinese qu starter culture [19,20],which enables YRW to retain more nutrients,including protein and carbohydrates.Many potential bioactive components are also generated during the fermentation process,including peptides,functional amino acids,oligosaccharides,phenolics,vitamins,and minerals.Some of these nutrients are comparable in content to those found in red wine [21].According to the pharmacopoeia of Chinese traditional medicine,YRW has been used as a medicinal inducer since ancient times,and is believed to aid in dredging blood vessels,protecting the stomach,and moisturizing the skin.Although YRW has long been used in Chinese traditional medicine due to its health-promoting effects,the functional mechanisms of YRW remain unclear.

The aim of the present study is to investigate the anti-aging effects of YRW in mice and to analyze the possible underlying mechanisms,which will improve understanding of the health effects of traditional Chinese YRW on humans.

2.Materials and methods

2.1.Chemicals and reagents

The YRW samples (base liquor,15% alcohol by volume) were obtained from International Joint Research Center of Quality and Safety of Alcoholic Beverages (Beijing,China).The samples were filtered and stored at 4°C for experimental use.

D-galactose was purchased from Biotopped Science & Technology Co.Ltd (Beijing,China).Vitamin C (VC) was obtained from Sinipharm Chemical Reagent Co.Ltd (Beijing,China).The superoxide dismutase (SOD),catalase (CAT),glutathione peroxidase (GSH-Px),malondialdehyde (MDA),and bicinchoninic acid(BAC)protein assay kits were all obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing,China).The Eastep®Total RNA Extraction Kit and the Reverse Transcription System were purchased from Promega (Madison,WI,USA).SYBR GREEN was purchased from Kapa biosystems(Wilmington,MA,USA).All other chemicals were at least of analytic grade.

2.2.Animals and experimental design

Twelve-week-old male Kuming mice((30±2)g)were obtained from Beijing Vital River Laboratory Animal Technology (Beijing,China).The present study was approved by the Ethics Committee of Joekai Biotechnology Co.Ltd(JK(2019)-W-002,Beijing,China).All animals were individually housed in a gnotobiotic isolator under standard laboratory conditions (temperature:(22 ± 1)°C,humidity:50%±10%,12 h light-dark cycle)and were allowed free access to food and water.All protocols were carried out in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals,enacted by the Ministry of Science and Technology of China.

After seven days of acclimation,a total of 120 mice were randomly divided into six experimental groups:control group(control);aging model group(model);VC group(VC);and groups with low,medium,and high doses of YRW.Half of the mice in each experimental group were subjected to a forced swimming test(FST);the other half were subjected to the Morris water maze(MWM)test.The model group,VC group and YRW groups received daily subcutaneous injections ofD-gal (300 mg/kg BW).An equal volume of physiological saline(PS)was injected into control mice.Mice in the three YRW groups received daily intragastric administrations of YRW at doses of 4,8,12 mL/kg(BW),respectively.The VC group received doses of 300 mg/kg(BW),and the control and model groups each received intragastric administrations of distilled water at doses of 8 mL/kg (BW).These treatments lasted for six weeks.Body weight was examined weekly and samples administration to supervise the general health of all groups.

2.3.Morris water maze test

The MWM test was used to evaluate spatial learning and memory [22,23].The MWM system includes:a circular steel pool(diameter of 140 cm,60 cm high),filled with clean tap water((25±2)°C)to a depth of about 35 cm;a video probe;and a computer monitor.The pool was divided into four equal quadrants,and a hidden platform was set in one of the quadrants about 1.5 cm below the water surface.

We followed the protocol according to Zhou [24],with some adjustments.The test included learning trials and a probe trial.In the place navigation test,each mouse underwent four daily training trials for six consecutive days.On the first day,each mouse was permitted to swim 60 s freely without the platform.The platform was set up from the second day on.The mice were allowed to remain on the platform for 15 s after locating it within 60 s.The time taken to reach the platform (swim latency) was recorded by ANY-maze software.Latencies over 60 s were recorded as 60 s and the mouse was placed on the platform for 20 s.On day 7,the probe trial was carried out without the platform.Time spent in the target quadrant and the number of times the mouse entered into the platform area (number of entries) were recorded for the duration of each 60 s trial.All mice were sacrificed after the probe trial.Samples of liver and brain were collected on ice and stored in liquid nitrogen for various biochemical assays.All surgeries were performed under sodium pentobarbital anesthesia.

2.4.Forced swim test

The(FST)was performed as described by Porsolt[25],with slight modifications.Each mouse was placed in a plastic pool (height:40 cm,diameter:20 cm),which was filled to a depth of about 10 cm depth with clean tap water((25±2)°C),for 6 min.The mouse was identified as immobile when it ceased struggling,floated in an upright position,and made only small movements to keep its head above water [16].The duration of immobility was recorded by ANY-maze software during the last 4 min of the 6 min testing period.

The immobility time for each mouse was measured following 6-week period of YRW administration.After the second FST test,the mice were forced to swim for 60 min and were then rested for 90 min.The mice were sacrificed,and blood and liver were preserved in sterile tubes using liquid nitrogen.Serum was prepared (3000 r/min for 10 min at 4°C)for analysis of hepatic glycogen(HG),blood lactic acid(BLA),and blood urea nitrogen(BUN).

2.5.Determination of biochemical parameter

The SOD,GSH-Px,and CAT activities,as well as the MDA content,were determined according to the manufacturer’s protocol.

Table1 The sequence of primers used for qRT-PCR assay sequences.

Table2 Effects of yellow rice wine on body weight in mice.

2.6.Quantitative reverse transcription polymerase chain reaction(qRT-PCR)analysis for apoptosis-related factor

We isolated RNA from the hippocampus with Eastep®Super Total RNA Extraction Kit.cDNA was obtained using a Reverse Transcription kit,and analyzed using the ABI7500 Real-Time fluorescence quantitative PCR system (Thermo,Waltham,MA,USA)using a fluorescent dye (SYBR Green PCR Master Mix).Mouse β-actinwas used as an internal reference and the relative gene expressions ofBax,Bcl-2andcaspase-3were measured according to the 2-ΔΔCtformula.The primers used are listed in Table1.

2.7.Statistical analysis

The data were all expressed as mean ± standard error of means(SEM)and analyzed by SPSS 18.0 software.Significant differences among groups were tested by one-way analysis of variance(ANOVA),withP<0.05 regarded as significant.

3.Results

3.1.General conditions of mice

Body mass reflects health status to a certain extent.It has been shown thatD-gal-treated mice exhibit symptoms such as inhibited weight gain,reduced activity,sluggishness,and shing hair[26,27].The body mass of animals was monitored before and after treatment.As shown in Table2 and Fig.1,weight gain in the model group was significantly reduced compared with the control group(P <0.05).However,administration of VC,an important antioxidant,reversed the weight loss (P <0.05).Administration of low,medium,and high doses of YRW alleviated weight gain inhibition caused byD-gal,with the high dose producing a significant effect(P <0.05).Both VC and YRW intake also reversed the symptoms of activity attenuation,reduced vigor and hair loss induced byD-gal(data not shown here).

Fig.1.The effect of YRW on weight gain in D-gal-treated mice.

3.2.YRW improved the antioxidant property of liver tissue in D-gal induced aging mice

Oxidative stress is closely related to the process of aging[20].In order to investigate the antioxidant effect of YRW onD-gal-induced aging in mice,we identified several important ROS-scavenging antioxidant enzymes in the brain and liver,including SOD,GSH-Px and CAT.As shown in Fig.2,the enzyme activities of SOD(Fig.2A),GSH-Px (Fig.2B),and CAT (Fig.2C) in the brain and liver were remarkably reduced in the model group compared with the control group(P <0.05 or 0.01).However,the antioxidant activities of these enzymes increased in direct correlation to YRW dosage,with significant effects at medium and high doses of YRW intake.Significant effects also occurred in the VC group.

The levels of MDA in brain and liver homogenates increased significantly inD-gal-treated mice compared with the control group(P <0.05,Fig.2D).Both VC and YRW treatment attenuated the MDA increase in both the brain and the liver,with the medium YRW dose producing a significant reduction.

3.3.YRW reduced the immobility time on D-gal induced aging mice in FST

Physical decline accompanies the aging process,rendering the body prone to fatigue.A common method for evaluating fatigue is the FST,followed by determination of associated biochemical indicators.An objective index to evaluate the degree of fatigue in FST is the time spent in immobility [28].As shown in Fig.3,mice treated withD-gal spent significantly more time immobile compared with the control group,suggesting a higher level of fatigue in this group(P<0.01).Administration of both VC and YRW(YRW high)resulted in increased activity time compared with the model group,as immobility time decreased significantly in FST(P<0.05).

3.4.YRW modified the level of BLA,BUN and HG in the D-gal induced aging mice after FST

Fatigue is closely related to the consumption of energy and the accumulation of metabolites [29].To further investigate the antifatigue effects of YRW onD-gal-treated mice,the blood lactic acid(BLA),blood urea nitrogen(BUN),and glycogen storage(HG)content in the liver of mice were assessed following the FST.As shown in Fig.4A and B,the levels of BLA and BUN in the model group were significantly higher than those in the control group (P <0.05 andP <0.001,respectively).In addition,the HG levels were significantly lower in the model group than in the control group(P <0.01,Fig.4C).Interestingly,YRW treatment(YRW medium and high)significantly decreased the levels of BUN and BLA(P <0.05,Fig.4A,B),and increased the HG level significantly compared with the model group(P <0.05,Fig.4C).Taken together,these results suggest that YRW can reduce fatigue inD-gal-treated mice in the FST.

Fig.2.Biochemical indicators in brain and liver.

Fig.3.The Effect of YRW on immobility in the FST.

3.5.YRW ameliorated the cognitive impairment and learning decline in D-gal-treated mice

Cognitive impairment along with learning and memory decline are symptoms of brain dysfunction that are closely related to aging.In order to test the spatial memory and learning ability of mice,the MWM test was performed.During the learning trial,mice in the model group spent more time in latency than those in the control group.However,administration of VC and YRW (YRW high)were both associated with significantly less time spent in latency(P <0.05,Fig.5A).There was no significant difference between the Vc group and the aging model group.In the probe trial,mice in the model group spent less time in the target quadrant and displayed fewer platform crossings compared with the control group(P <0.05,P <0.05,Fig.5B,C,respectively).Mice in the VC and YRW groups exhibited significantly more time in the target quadrant,with a higher number of platform crossings compared with the model group (P <0.05,Fig.5B,C).The typical traces,shown in Fig.5D,demonstrate that the mice in the YRW and VC treatment groups tended to select more direct routes to the platform,similar to the traces observed in the control group.These results suggest that YRW can reverse the spatial cognition and learning decline caused byD-gal.

Fig.4.Effects of YRW on the BLA,BUN,and HG.

Fig.5.Effects of YRW on the D-gal-induced cognitive,learning,and memory impairment in the MWM task.

3.6.Transcription of apoptosis-related genes in the hippocampus of D-gal-treated mice

After six weeks ofD-gal treatment,the cognitive and learning abilities of mice in the aging model group decreased significantly.Previous studies have implicated neuronal apoptosis in agingrelated cognitive decline and learning dysfunction[30].To further investigate the potential recovery mechanism of YRW on the cognitive impairment induced byD-gal,qRT-PCR was performed to detect the mRNA levels of apoptosis-related genes in the hippocampi.As shown in Fig.6,D-gal treatment caused a remarkable decrease in the gene expression ofBax/Bcl-2and increase in the gene expression ofcaspase-3in the model group compared with the control group (P <0.05).This suggested that neural cell apoptosis was induced in the hippocampus after continuous administration ofD-gal.However,treatment with YRW (YRW medium and YRW high) markedly improved the ratio ofBax/Bcl-2and the relative expression ofcaspase-3compared with the model group(P <0.05).These data suggest that YRW may help ameliorate the effects of neuronal apoptosis and recover some cognitive function inD-galtreated mice.

4.Discussion

Aging is a natural process that is both inevitable and complex.Accumulating research has shown that senescence,which is usually accompanied by physical attenuation,learning impairment,and cognitive decline,is associated with oxidative stress,neuroapoptosis,as well as other aging-related risks[31-33].In the present study,an aging model was established in mice through injection ofD-gal.Varying amounts of YRW were administered to different groups of mice in order to investigate the effects of YRW on aging.Some studies have indicated that certain organs are susceptible to attack by ROS in the aging process[34].Particularly affected are the liver,which plays a central role in metabolism,detoxification,and energy storage;and the brain,which is central to behavioral and consciousness regulation and is the body’s greatest consumer of oxygen.In the present study,six weeks ofD-gal treatment induced remarkable signs of aging,such as inhibited weight gain(Table2 and Fig.1),reduced activity,loss of fur,decreased enzymatic antioxidant activity,and increased MDA content in the model group compared with the control group(Fig.2).This indicated that the aging model was established successfully.However,these symptoms were improved in mice treated with VC and YRW,which indicates the antioxidant capability of YRWin vivoinD-gal-induced aging in mice.

Fig.6.Effects of YRW on expression levels of apoptosis-related proteins in hippocampus of D-gal-induced aging mice.

Due to the attenuation of physical strength,the aging body is vulnerable to fatigue during exercise.The FST is widely used to assess fatiguein vivo[35],with the degree of fatigue correlated with immobility time [28].Our results showed that the immobility time in the model group was significantly higher than that in the control group (Fig.3).Biochemical indicators are shown in Fig.4.One indicator of the degree of fatigue is BLA,which is the product of glycolysis under anaerobic conditions[36].Another biochemical indicator is BUN,which is formed in the liver as the final product of protein metabolism,and can indicate impaired glycolipid metabolism [37].Hepatic glycogen consumption also plays an important role in preventing fatigue by maintaining the blood glucose at a normal level [38].Results showed that BLA and BUN decreased in a dose-dependent manner in YRW-treated mice,and the levels of HG also improved compared with the model group.These results were consistent with the data from the FST;however,no significant changes occurred in the VC group.These observations indicate that moderate YRW administration can alleviate fatigue inD-gal-treated mice.The alcohol content of YRW is low,but it contains a large quantity of proteins,peptides,and amino acids,as well as a rich supply of functional oligosaccharides and polyphenols[39].All of these can provide continuous energy for life activities,and effectively protect the body from ROS damage.Based on this evidence,we hypothesize that the anti-fatigue effect of YRW inDgal-treated mice is associated with its ability to enhance HG storage and promote metabolite excretion during exercise.

In addition to fatigue,aging is accompanied by other symptoms such as cognitive impairment and learning decline.The MWM is a test designed to evaluate the hippocampal-dependent spatial learning and memory ability of the subjects[40].In this study,brain aging characteristics were evaluated using the MWM.The results show thatD-gal administration increased latency time,reduced time in the target quadrant,and reduced the number of entries into the platform area (Fig.5).Mice treated with YRW displayed significant improvement in cognitive function over mice in the model group (Fig.5).These results suggest that YRW can reverse the impairment of spatial memory and learning ability caused byD-gal.

It has been shown that cognitive dysfunction is correlated with neuronal apoptosis [41].To explore the mechanisms underlying the YRW-mediated recovery of cognitive abilities inD-gal-treated mice,we analyzed the hippocampi of the variously treated mice to identify activity in genes related to neuronal apoptosis.In the multi-step process of programmed cell death,activation and execution of apoptosis requires coordinated regulation of the Bcl-2 family of proteins and caspase signaling cascades.The Bcl-2 family plays a significant role in the regulation of cell apoptosis,in coordination with the pro-apoptotic protein BAX.Proteins from the Bcl-2 family can inhibit BAX in its in oligomer-forming role,which causes a change in the permeability of the mitochondrial membrane and the release of cyt c.Hence,BCL2 is regarded as an anti-apoptotic protein and BAX as a pro-apoptotic protein.Another important apoptotic protein iscaspase-3,which participates in the activation of the caspase cascade [42].In this study,we analyzed the relative gene expression ofcaspase-3and the ratio ofBax/Bcl-2(Fig.6),which is a key indicator of cell apoptosis.The results showed that the levels ofBaxandcaspase-3were up-regulated,while the gene expression ofBcl-2was down-regulated inD-galtreated mice.However,the medium-level dose of YRW alleviatedD-gal-induced neuronal apoptosis by decreasing theBax/Bcl-2ratio and suppressing the activation ofcaspase-3.Therefore,we conclude that the cognitive improvement seen inD-gal-treated mice upon YRW intake depends on both the regulation of the ratio ofBax/Bcl-2and the gene expression ofcaspase-3,which protects the neuronal cells from apoptosis.

Multiple studies have reported that YRW is rich in bioactive substances,such as polyphenols,peptides,functional oligosaccharides,and other by-products of fermentation.Hence,we speculate that the role of YRW in reducing fatigue mainly depends on the abundant bioactive components;however,few studies have focused on the discovery and mechanisms of specific substances.The identification of the main functional substances and their mechanisms remain to be discovered.

In summary,the results of this study show that continuous administration of YRW can reduce fatigue by increasing the storage of HG and preventing the accumulation of metabolites inD-galtreated mice.In addition,YRW can help reduce the effects of excessiveD-gal intake,including inhibited weight gain,oxidative damage,cognitive impairment,and increased neuronal apoptosis.Mice treated with YRW displayed enhanced cognitive performance,elevated antioxidant activity,and reduced neuronal apoptosis.Above all,these results demonstrate that YRW can promote positive health effects by reducing the symptoms of aging.

Funding

This work was partially supported by Natural Science Foundation of Hunan Province(2019)and Double First-Class Construction Project of Hunan Agricultural University(No.SYL201802025)to Si Qin.

Declaration of Competing Interest

We declare that we have no conflict of interests.

Acknowledgements

The authors appreciate technical support from Peking Union Medical College and declare no competing or financial interests.