延衰合剂对D-半乳糖衰老模型小鼠学习记忆及抗氧化能力的影响

栾增强，曹文富，彭 爽，黄和贤

重庆医科大学附属第一医院中西医结合科，重庆400016

Introduction

Aging is a momentous issue in life science research，and its mechanism is extremely complex.Traditional Chinese medicine has been widely applied in anti-aging field，and Chinese herbal formula has advantages in multi-levels and multi-targets effects.Yanshuai mixture (YSM)is composed of 12 Chinese materia medica indcluding Hongshen(Radix Ginseng Rubra)，Zhishouwu (Radix Polygoni Multiflori Preparata)，Huangjing (Rhizoma Polygonati)，Roucongrong(Herba Cistanches)，etc.YSM can invigorate spleen and kidney，replenish essence and marrow，nourish qi and blood，activate blood and dissipate binds，and so on.It has been shown that YSM can improve physical function，enhance immunity，promote illness recovery remarkably through long-term clinical application.In this study，D-galactose (D-gal)induced aging mice were established，and their learning and memory function and T-AOC，SOD，MDA leavels in serum and brain tissue were measured. The objective is to explore its anti-aging effect，provide experimental evidence for clinical application，and promote the development of new Chinese patent medicine.

Materials and Methods

Reagents

YSM is composed of 12 Chinese herbs indcluding Hongshen(Radix Ginseng Rubra)，Zhishouwu(Radix Polygoni Multiflori Preparata)，Huangjing(Rhizoma Polygonati)，Roucongrong(Herba Cistanches)，etc.YSM was decocted and concentrated to 2 g/mL(crude drug)，then stored at 4℃.Bushenyishou capsule (BSYS，Taiji Group Fuling Pharmaceutical Factory Co.，Ltd，batch number:09090095，preparation of crude drug concentration at 2 g/mL with normal saline)is a Chinese patent medicine can delay organism aging through tonifying the kidney and replenishing qi and its composition are Hongshen(Radix Ginseng Rubra)，Zhenzhu(Margarita)，Lingzhi(Ganoderma Lucidum)，Zhishouwu (RadixPolygoniMultiflori Preparata)，Gouqizi(FructusLycii)，Yinyanghuo (Herba Epimedii)，Danshen(Radix Salviae Miltiorrhizae)，Gancao(Radix Glycyrrhizae)，Huangjing(Rhizoma Polygonati).BSYS and the prepared slices of Chinese crude drugs were purchased from Tong Junge Dispensary.D-galactose(Amresco Inc.).The T-AOC kit，SOD kit，MDA kit and Coomassie brilliant blue protein (G250)assaykit werepurchasedfrom Nanjing Jiancheng Biological Engineering Institute.

Instruments

Morris Water Maze analysis system(Beijing Shuo Lin Yuan Technology Co.，Ltd.);electric water bath incubator(Zhongda Jintan Jiangsu Instrument Factory);electric tissue homogenizer(Miltenyi Biotec);desktop centrifuge(5810 R，Eppendorf);electronic balance (AUW120D，SHIMADZU);microplate reader(United States Bio-Rad).

Animal and Treatment

60 clean closed group Kun Ming mice at the age of 3 months，weighting 27±3 g，were provided by Animal Experimental Center of Chongqing Medical University.After adaptive feeding for 3 d，60 mice were randomly divided into 6 groups:normal control group，model group，BSYS group，YSM low dose(YSM-L)group，medium dose(YSM-M)group，and high dose(YSMH)group(n=10).The mice were injected with D-gal (120 mg/kg·d)subcutaneously on the back neck to induce aging model mice，while the normal control mice were injected with equal volume of normal saline.At the same time，the mice were administered intragastrically with low(9 g/kg)，medium(18 g/kg)and high (36 g/kg)dose of YSM once daily for 10 weeks.BSYS group mice were administered intragastrically with bushenyishou capsule solution(18 g/kg)，while the normal and model groups with same volume of normal saline.

Detection indicators

Aging signs of the mice were observed such as hair color，skin，vitality，food intake，body weight，etc.

Morris Water Maze test

By Morris water maze analysis system，the mice in each group were taken the place navigation test and spatial probe test.Morris water maze was a diameter of 120 cm and a high of 50 cm circular pool，and was divided intoⅠ，Ⅱ，Ⅲ，andⅣ four equal quadrants.It was filled with tepid water(25±1℃)that was made opaque by the addition of black ink.A white escape platform was located at 1 cm below the water surface near the center ofⅡquadrant.Outside the pool references were fixed such as ornaments，curtains，lights，etc.，and the experimenter remained the same location in each trial.The mice were released into the pool from four different mid-points of each quadrants and the time(i.e.escape latency)to find the escape platform within 60 s was recorded.If the mouse can not locate the escape platform within 60 s in any trial，then the experimenter placed the mouse on the platform，where it was remained for 15 s and recorded the latency as 60 s.Each mouse was trained 4 times a day(including four quadrants)continuously for 5 days between 10 am and 6 pm.In this experiment，the fifth day’s training results were regard as the test results on behalf of the learning and memory abilities of mice.Spatial probe test was taken after the completion of the place navigation test on the fifth day.After removing the platform，mouse was released into the water from the quadrant opposite the original plat-form quadrant(i.e.Ⅲ quadrant)，then the swimming time in the original quadrant within 60 s was recorded，and the Original quadrant swimming time percentage of total time was calculated.

Determination of T-AOC，SOD activity，MDA content in serum and brain tissue

After the behavior test，mice were anaesthenized with an intraperitoneal injection of 10%chloral hydrate.Serum was separated conventionally from the blood which collected by extirpating the eyeball under the condition of 4℃，centrifuged at 3000 r/min for 15 min，then preservated at–20℃.After mice were killed by cervical dislocation，brain were removed rapidly，rinsed in cold saline，weighed，cut into pieces.The brain pieces were made into 10%fresh brain tissue homogenate under low temperature with electric tissue homogenizer at 3500 r/min for 15 min.Then supernatant was collected and preservated at–20℃.T-AOC，SOD activity and MDA content were measured by kits.Protein content was assyed using Coomassie brilliant blue protein.The protocol were performed according to the manufacturer’s instructions strictly.

Statistical Methods

Viability was presented as mean with deviation(± s).Data analysis was performed using one-way ANOVA (the software SPSS18.0 version).Comparison of data from every two groups was done using student test，Significance was assumed at P＜0.05.

Results

Signs of aging

Given continuous injection of D-gal for 10 weeks，aging model group mice gradually showed obvious signs of aging，such as withered dull and caducous hair，poor skin elasticity，low spirit，fatigue and lethargy，decreased activity，decreased food intake，slowly weight gain，etc.Weight gain of mice is illustrated in Fig.1.

Fig.1 The weight growth curve of mice

Learning and memory function

The results of the place navigation test showed that the learning ability of D-gal induced aging mice was significantly decreased.Compared with the normal group，the escape latency of the other group mice was prolonged (P＜0.01).Compared with the aging model group，the escape latency of BSYS group and the low，middle and high dose YSM group was significantly shortened(P＜0.05 or P＜0.01).The spatial probe test results showed that spatial memory retention of the D-gal induced aging mice decreased significantly.The original quadrant swimming time of aging model mice was significantly shorter compared with the noaml control group(P＜0.05).The original quadrant swimming time of BSYS group and low，medium and high dose YSM groups was prolonged in comparison with aging model group(P＜0.05).Compared with BSYS group，the escape latency and original quadrant swimming time percentage of high dose YSM group were significant，whereas low and medium dose YSM group had no significant difference(P＞0.05).Detailed in Table 1.

Table 1 Effects of YSM on learning and memory in mice (±s，n=10)

Table 1 Effects of YSM on learning and memory in mice (±s，n=10)

Note:vs control group，*P＜0.05，＊＊P＜0.01;vs model group，ΔP＜0.05，ΔΔP＜0.01;vs BSYS group，#P＜0.05.The same as follows.

Group Escape latency (s) Original quadrant swimmingtime percentage(%) 26.32±2.48＊＊ 30.05±2.39＊＊BSYS 22.41±1.93Δ 35.65±4.77Δ YSM-L 23.02±2.07Δ 35.84±4.33Δ YSM-M 23.70±2.05Δ 37.53±2.03ΔΔ YSM-H 19.97±0.87ΔΔ# 41.21±3.09ΔΔ# 16.24±1.05 46.97±6.77 M N

Antioxidation indices in serum and brain tissue

The results showed that YSM can improve the T-AOC，SOD activity and decrease MDA content in serum and brain tissue supernatant significantly.Compared with the normal group mice，the T-AOC，SOD activities of aging group mice decreased，while MDA content increased significantly(P＜0.01);T-AOC，SOD activities in the serum and brain tissue of the BSYS group and high dose YSM mice significantly increased，while MDA levels decreased significantly compared with the aging model group(P＜0.01);Compared with BSYS group，SOD activity in serum and brain tissue of high dose YSM group increased，while MDA content in serum decreased significantly(P＜0.05).Detailed in Table 2，Table 3.

Table 2 Effects of YSM on T-AOC，SOD，MDA in serum of mice(±s，n=10)

Table 2 Effects of YSM on T-AOC，SOD，MDA in serum of mice(±s，n=10)

Group T-AOC (U/mL) SOD (U/mL) MDA (nmmol/mL) N 3.78±0.39 180.34±4.74 5.59±0.25 M 2.16±0.53＊＊ 133.61±3.58＊＊ 9.31±0.72＊＊BSYS 2.99±0.66Δ 163.80±5.13ΔΔ 6.69±0.39Δ YSM-L 2.31±0.60 150.78±4.29 7.09±0.56 YSM-M 2.41±0.70 158.77±3.14 6.54±0.80 YSM-H 3.35±0.82ΔΔ 173.05±2.79ΔΔ#5.84±0.95ΔΔ#

Table 3 Effects of YSM on T-AOC，SOD，and MDA in brain tissue of mice(±s，n=10)

Table 3 Effects of YSM on T-AOC，SOD，and MDA in brain tissue of mice(±s，n=10)

Group T-AOC (U/mgprot) SOD (U/mgprot) MDA (nmmol/mgprot) N 12.24±1.46 262.81±9.38 12.92±1.08 M 8.12±1.09＊＊ 184.48±4.47＊＊ 18.12±1.03* BSYS 10.49±1.45Δ 224.00±6.72 14.69±1.45Δ YSM-L 9.04±1.13 193.77±4.45 15.75±1.13 YSM-M 10.67±1.37Δ 222.38±5.79 15.31±1.37 YSM-H 11.70±1.14ΔΔ 246.00±8.35ΔΔ#14.38±1.14ΔΔ

Discussion

Aging is the function decline at the molecules，cells，organs，and even the whole body levels and is the accumulation of the complex physiological and pathological changes.In this study，the subacute aging model mice were induced by subcutaneous injection of D-gal.D-gal was reduced to galactitol under the catalysis of aldose reductase，but galactitol can not be further metabolised by the cells.Accumulation of galactitol affects the normal osmotic pressure in cells，leads to the cell swelling，dysfunction，metabolic disorders，then results in the occurrence of aging.The features of the model mice are almost the same with the natural aging.Thus aging model mice induced by D-gal can be used to study for behavior，anti-oxidation，immunology，anti-aging medicine，etc［1，2］.

The most prominent overall manifestations of aging are dysfunctions of the central nervous system such as poor learning and memory，forgetfulness，poor adaptability，poor spirit，fatiguability，etc［3］.Morris Water Maze test is an objective and repeatable method applied for assessing the learning and memory ability of mice［4］.The experimental results show that low，medium and high dose YSM could significantly shorten the escape latency and prolong the original platform quadrant swimming time compared with the aging model mice.It will be seen from this that YSM can improve learning and memory function of aging mice，and may play a role in delaying the process of brain aging.

Free radical theory of aging proposed in 1956 by Harman is generally acknowledged as one of the mechanisms of aging.The antioxidative capacity of the organism declines because of pathological or aging factors.The creating and removing of free radicals lose balance，then too much free radicals are created.Free radicals can damage macromolecules，causing molecule and cell level even tissue and organ level injury［5，6］.Although the weight of brain is not to 2% of body weight，but cerebral oxygen consumption accounts for 20%of body oxygen consumption.Brain tissue is rich in phospholipids and is vulnerable to oxygen free radicals attack［7］.Antioxidative defense system including enzymatic and non-enzymatic system.T-AOC could reflect the total antioxidative capacity of samples.SOD is a kind of key enzyme in scavenging free radicals in antioxidant enzyme defense system.Detection of SOD activity can reflect the ability of the organism to eliminate free radicals and protect cells from free radical damage.MDA is a lipid peroxide decomposition product under the action of oxygen free radicals，which can reflect the level of lipid peroxidation and the degree of cell damage indirectly［8］.Therefore，SOD and MDA are generally accepted as indicators of aging.Experimental results show that，high dose YSM can increase T-AOC，SOD levels and reduce MDA content in the serum and brain tissue significantly，which has good antioxidative capacity.

Traditional Chinese medicine believes that the basic mechanism of aging is complicated with deficency and excess.Deficency reffers to the deficency of zang-fu organ，in which the deficiency of kindey essence and qi is the main factor［9］.Brain is the house of the original spirit，the sea of marrow，and dominates mental awareness and memory.Kidneys take in charge of marrow producing and hold the will.So invigorating the kidney and brain can improve learning and memory function.YSM could reinforce the patient’s healthy qi and eliminating the invading pathogenic factors，regulate the body as a whole through both strengthening anti-pathogenic qi and eliminating pathogenic factors.The experimental results show that YSM can improve the general condition and learning memory function，increase TAOC，SOD levels and reduce MDA content in the serum and brain tissue.YSM is better than Bushenyishou capsule in improving learning memeory function and anti-oxoidation.In conculusion，YSM may delay brain and organism aging process through reducing free radical oxidative damage.But further research should be done to clarify the mechanism.

Acknowledgements:Thanks for the supportof Chongqing Key Laboratory of Ophthalmology.

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