XIONG Yun-yun, ZHOU Yan-ning, XIE Chang-cai, ZHOU Kai, YU Dao-rui

1. The First Affiliated Medical College of Hainan Medical University, Haikou, Hainan 571199

2. Shool of Basic and Life Scinece, Hainan Medical University, Haikou, Hainan 571199

Keywords:Hainan papaya extract D galactose Anti aging Cognitive impairment

ABSTRACT Objective: To explore the effects of Hainan papaya extract on learning and memory impairment and anti-aging in D-galactose-induced aging mice. Methods:A total of 72 Kunming mice with normal cognitive ability screened by water maze test were randomly divided into negative control group, model group, piracetam group, high, medium and low dose groups of Hainan papaya extract (400 mg/kg,200 mg/kg ,100 mg/kg), with 12 mice in each group. Hainan papaya extract and piracetam group were given the above drugs by gavage every day, The negative control and model groups were given the same amount of 0.9% NaCl solution in the same way. Mice in each group were weighed once a week; At the same time,except for the negative control group, mice in each group were intraperitoneally injected with 2% D-galactose every day, and the negative control group was intraperitoneally injected with normal saline for 7 weeks. After 7 weeks, We observed each group of mice’s capacity of learning and memory by Morris water maze behavioral test; Then, the content of superoxide dismutase (SOD), malondialdehyde (MDA),catalase (CAT), and nitric oxide synthase (NOS) were measured; On the other hand, we observed the hippocampus’ histopathological changes by hematoxylin-eosin staining, and measured the protein expression of nuclear factor-E2-related factor (Nrf2) in brain tissue of mice in each group by Western blot. Results:After the intervention of Hainan papaya extract on aging model mice, the high, medium and low dose groups could shorten the swimming time and swimming distance of mice to varying degrees, increase the activities of SOD, CAT and NOS in mouse brain tissue and reduce the content of MDA, The performance of high dose group was better than piracetam group (P＜0.01). At the same time, it can improve the histopathological changes of neurons in mouse hippocampus by reducing neuronal nuclear pyknosis,and increase the expression of Nrf2 protein in mouse brain in a dose-dependent manner. Conclusion: Hainan papaya extract is able to postpone various physical signs of subacute aging mice caused by D-galactose, and possesses definite anti-aging and antioxidant effects, which may be related to the regulation of Nrf2 signal pathway.

1. Introduction

With the improvement of social life and economic level, the aging population is increasing day by day, and many diseases caused by aging are the focus of modern social medicine. So far, one of the hot research problems in the contemporary scientific community is to explore new effective anti-aging drugs to improve the living standard of the elderly[1]. Aging is a degenerative change of the body with the general weakening of the functions of various organs of aging, which involves multiple factors, multiple organs and other complex processes. It has the characteristics of chronic irreversibility. The main reason for aging is the production of free radicals and the imbalance of oxygen free radical metabolism[2,3].As an important organ of the human body, the brain is also greatly affected by aging, which is manifested as decreased immunity,shortened life span, and decreased learning and memory ability[4].Although there are many reports on aging, the mechanism of antiaging is not clear, and there is also a lack of effective anti-aging drugs. Therefore, it is of great significance to explore the aging mechanism and develop effective anti-aging drugs, so as to improve the quality of life of the elderly[5,6,7].

D-galactose is often used to construct animal models of aging, and its effects have been recognized by researchers at home and abroad,and D-galactose has been widely used in anti-aging drug research[8].There is literature suggesting that[9] the extent of the D-galactoseinduced aging model is similar to that of normal mice aged 16 to 24 months, and these changes are consistent with natural aging. Carica papaya belongs to the Papaya family, also known as papaya[10]. It is mainly distributed in the tropics and subtropics [11]. At present,studies have shown that papaya has many biological activities,including bacteriostatic, antioxidant and anti-inflammatory,antibacterial, anti-cancer, analgesic, etc[12,13,14]. Recent studies have found that papaya has antioxidant properties, can effectively scavenge free radicals and other biological activities[15]. However,the research progress on whether Hainan papaya can delay aging is relatively backward, and the specific delay mechanism is not yet understood. Therefore, this experiment explores the anti-aging effect of Hainan papaya extract in a subacute aging mouse model, which provides experimental support for Hainan papaya in terms of antiaging.

2. Materials and methods

2.1 Main reagents, drugs and Instruments

Piracetam, batch number: 190705, was purchased from Guangdong South China Pharmaceutical Group Co., Ltd; D-galactose, batch number: G0625, was purchased from Sigma; Hainan papaya extract,was provided by the Biopharmacology Department of Hainan Medical College; 0.9% NaCl solution was configured into 400 mg/kg d, 200 mg/kg d and 100 mg/kg d. The doses of the three dose groups were increased by 2 times the amount of commercially available papaya powder. The doses of the three dose groups were increased by 2 times the amount taken by humans in commercially available papaya powder; [Superoxide Dismutase (SOD), Catalase(CAT), batch number: 20180118; Nitric Oxide Synthase (NOS) Lot No: 20180432, Malondialdehyde (MDA) batch number: 20180275]kit, both purchased from Nanjing Jiancheng Institute of Biological Engineering; anti-nuclear factor-E2-related fac- tor (Nrf2) kit,both purchased from Nanjing Jiancheng Institute of Biological Engineering; anti-nuclear factor erythroid-derived factor 2-related fac- tor (Nrf2) kit, both purchased from Nanjing Jiancheng Institute of Biological Engineering. (Nrf2), batch number: ab137550, was purchased from Abcam; Morris water maze device, was purchased from Chengdu Taimeng Technology Co. Biotinylated sheep antirabbit IgG (1:500), purchased from Sigma; Mini-PROTEAN Tetra vertical electrophoresis transfer system, purchased from Bio-Rad;Western Blot auto analyzer, purchased from proteinsimple.

2.2 Experimental animals

There were 72 experimental mice, weighing 18-25 g, Kunming clean grade, purchased by Changsha tianqin Biotechnology Co., Ltd.all mice in each group were fed in cages at room temperature and allowed to drink and eat freely. These experimental animals research is based on the standards of the ethics committee of Hainan Medical College, follows the "3R" experimental design principle, gives the animals as much as possible in a comfortable environment during the experimental process, standardizes the operation of experimental steps to alleviate the pain of the experimental animals, and after the experiment, the animals are euthanized, and finally entrusted to the school animal management center to dispose of all animal carcasses.Animal certificate number: SCXK (Xiang) 2014-001.

2.3 Preparation and administration of animal models

The mice were allowed to drink and eat freely at room temperature and fed for 1 week for adaptation, and then 72 mice with normal cognitive function were screened by water maze test [16] after first training as the formal experiment. Then they were randomly divided into positive control group, negative control group, model group and high, medium and low dose groups of Hainan papaya extract,12 mice per group. The mice in the high, medium and low dose groups (400mg/kg·d, 200mg/kg·d, 100mg/kg·d) and the positive control group (600mg/kg·d) were administered by gavage; the remaining two groups were given equal doses of 0.9% NaCl by gavage. This method was used for 7 weeks and the rats were weighed once a week and the dose was adjusted according to their body weight.

2.4 Morris water maze test

After the 7th week, the mice underwent two-day adaptive training.The training content was to place the mice in the water next to the end point, and the mice found the platform and climbed the steps twice. The test was started on the third day, and the data were used as memory results. The mice in each group were divided into two periods in the morning and afternoon. The large pool was divided into four symbolic limits and marked on average. The mice faced the pool wall and were put water from four different marked points. The mice in each group were divided into two periods in the morning and afternoon. The large pool was divided into four symbolic limits and marked on average. The mice faced the pool wall and put water from four different marked points. At the same time, their swimming distance and escape latency were recorded. The platform was found within 90 s. If the platform was not found within 90 s, the tester guided the mice to rest and withdraw from the pool after the platform, and 90 s was taken as their latency. Repeat the experiment at an interval of not less than 30 minutes. After 4 days of continuous experiment, remove the platform, and then let the mice face the pool wall at any water entry point, and record their swimming time[17].

2.5 Histomorphological changes of brain tissue of mice in HE

After mice were subjected to Morris water maze experiment, their brains were taken after cervical dislocation and executed, and the small amount of blood on the surface of brain tissue was washed with ice 0.9% NaCl aqueous solution and blotted dry with filter paper. The other half was fixed in 4% formaldehyde and stained with HE to observe the morphological changes of brain tissue. The remaining brain tissues were stored in the freezer at -20 ℃ for the determination of SOD, CAT, NOS and MDA in brain tissues.

2.6 Detection of SOD, NOS, MDA and CAT content in mouse brain tissue

4 ℃ 0.9% NaCl solution was added to the mouse brain tissue stored in advance, placed in a homogenate tube with a brain weight volume ratio of 1:9 into a slurry, put into a cryogenic centrifuge,adjusted to 4 ℃, centrifuged for 15 min (speed: 3000 r/min),after the centrifugation, take out the supernatant, according to the instructions for the next step, measure its SOD, NOS, MDA, CAT respective content.

2.7 Determination of Nrf2 Protein Expression in Hippocampal Tissues of Each Group of Mice

The other half of hippocampal tissues stored in the refrigerator at -80 ℃ was taken out and cut up, and it was mixed with RIPA lysis solution and PMSF protease inhibitor as required, ground and made into homogenate, lysed at low temperature for about 30 minutes, centrifuged for 40 min (speed: 12000 r/min) in the next step, and the supernatant was taken for protein quantification, and the calculated amount of supernatant was mixed with buffer, shaken well, and boiled at 100 ℃ Boil for 10 minutes. The protein samples were separated by electrophoresis with 40 μg of the upper sample,then the membrane was transferred and closed for 1 h. The primary antibody was incubated overnight in the refrigerator at 4 ℃, and the secondary antibody was incubated in ECL luminescent solution for 1 h at 37 ℃ the next day.

2.8 Statistical analysis

SPSS23.0 software package was used for statistical processing of the experimental data ;The representation of metrological data is±s; differences between groups were expressed by analysis of variance; two independent samples t- test was used for pairwise comparison.Finally, P＜0.05 indicated that the difference in experimental results was statistically significant.

3. Results

3.1 General observation

From the 15th day of administration, compared with the model group and the control group, the hair color of the mice was yellowish, glossless and accompanied by hair loss, and there were behaviors such as slow action, mental exhaustion and significantly smaller feeding. After 28 days, Hainan papaya extract high, medium and low dose groups appeared hair loss phenomenon, but after 42 days hair loss gradually reduced, and hair color, gloss and other aspects have greatly improved.

3.2 Effect of Hainan papaya extract on swimming time of morris water maze in aging model mice

By analysis of variance, it was concluded that there were differences among the groups as a whole. The latter two comparisons found that there was no significant difference in swimming time among the groups in the water maze experiment on the first day.From the third day, compared with the negative control group, the swimming time of the model group was significantly longer than that of the negative control group, [F3d= 0.015; F4d= 1.864; t3d=-12.38; t4d= -16.28; P3d＜0.05, P4d＜0.01]. Compared with the model group, the swimming time of each administration group showed a dose-dependent shortening trend with the increase of experimental time. On the 4th day, the swimming time of mice in each dose group of Hainan papaya extract was significantly lower than that of the model group [(Flow= 0.005, Fmedium=0.379, Fhigh=0.021; tLow=5.75,tmedium=9.84, thigh= 18.88, Plow＜0.05, Pmedium＜0.05, Phigh＜ 0.01)], and piracetam group compared with the model group, The swimming time of piracetam group was significantly lower than that of model group (F=0.036; t= 12.19; P＜0.01), but higher than that of high-dose group, suggesting that Hainan papaya extract is better than piracetamin improving memory ability in a certain range, as shown in Table 1

Table 1 Effects of hainan papaya extract on swimming time in water maze in aging mice(s,n=12, ±s)

Table 1 Effects of hainan papaya extract on swimming time in water maze in aging mice(s,n=12, ±s)

Note:Compared with the negative control group, #P＜0.05,##P＜0.01; compared with the model group, *P＜0.05,**P＜0.01.

Group Dose (mg/kg) swimming time Day 1 Day 2 Day 3 Day 4 Negative control group - 83.31±2.34 72.25±2.42 67.31±2.21 48.83±2.29 Model group - 92.88±3.19 89.23±3.38 79.81±2.29# 75.72±4.69##Piracetam formation 600 85.16±3.50 76.42±3.28 61.16±3.56* 39.78±3.07**Hainan papaya extract group 100 88.24±2.94 75.52±3.82 71.36±3.08 58.48±4.67*Hainan papaya extract group 200 86.52±3.23 74.83±3.89 59.46±4.35* 48.19±3.60*Hainan papaya extract group 400 81.67±2.86 59.29±3.49* 53.22±4.36* 35.71±4.78**

3.3 Effect of Hainan papaya extract on the swimming distance of morris water maze in aging model mice

After the ANOVA, the overall difference between the groups was obtained, and then by two-by-two comparison, it was found that: in the first day of the water maze experiment, there was no significant difference in the swimming distance between the groups. From the second day, the swimming distance of mice in the model group was significantly longer than that in the negative control group, and there were significant differences, [F2d=0.008; F3d=3.217; F4d=2.180;t2d=-4.21; t3d=-9.81; t4d=-8.58; P2d＜0.05; P3d＜0.01; P4d＜0.01 ].Compared with the model group, the swimming distance of each Hainanese papaya extract administration group showed a dosedependent decrease with the prolongation of the experimental time,and the shortened swimming distance of the mice in the high dose group was the most obvious in each experimental time, and the swimming distance of the mice in the middle and high dose groups of Hainanese papaya extract was significantly shorter than that of the mice in the model group on day 4, [ ( Fmedium=1.175, Fhigh=3.669 ) ;tmedium=6.11, thigh=7.78, Pmedium＜0.05, Phigh＜0.01) ] ; Piracetam groupwas also significantly shorter, but slightly higher than the high dose group (F=1.118; t=6.51; P＜0.01), see Table 2.

Table 2 Effects of hainan papaya extract on swimming distance of aging model mice in water maze test(m,n=12,±s)

Table 2 Effects of hainan papaya extract on swimming distance of aging model mice in water maze test(m,n=12,±s)

Note:Compared with the negative control group, #P＜0.05,##P＜0.01; compared with the model group, *P＜0.05,**P＜0.01.

Group Dose (mg/kg) Swimming Distance Day 1 Day 2 Day 3 Day 4 Negative control group - 19.53±2.85 14.71±3.02 7.80±1.48 6.56±1.43 Model group - 22.36±4.87 20.94±3.54# 18.81±3.22## 16.70±3.41##Piracetam formation 600 20.34±4.27 15.17±4.40 11.24±5.38* 7.67±7.15**Hainan papaya extract group 100 20.58±5.12 19.82±4.75 17.24±5.36 17.37±7.45 Hainan papaya extract group 200 19.65±3.90 16.62±6.60 13.24±4.98* 9.39±4.01*Hainan papaya extract group 400 18.58±4.46 12.86±5.15* 8.73±2.87** 7.11±1.91**

3.4 Changes of SOD, NOS, cat and MDA in mouse brain

There are differences between the groups as a whole. The comparison between the two groups shows that the contents of SOD,NOS and cat in the model group are significantly lower than those in the negative control group, while the content of MDA is significantly higher, indicating that the model is successfully prepared. Compared with the model group, the three dose groups of Hainan papaya extract reversed the contents of SOD, NOS, CAT and MDA in the aging model in a dose-dependent manner, and the effect increased with the increase of dose. Among them, the effect of the high-dose group was the strongest and statistically significant [FSOD=0.342;FNOS=1.411; FCAT=0.273; FMDA=0.120; tSOD=-24.08; tNOS=-17.17; tCAT=-22.37; tMDA=22.12; P＜0.01], which was similar to the positive drug piracetam group. It is better than piracetam group in increasing SOD content and reducing MDA level, suggesting that Hainan papaya may improve learning and memory ability by regulating these two activity levels, as shown in Table 3.

3.5 Effect of Hainan papaya extract on hippocampal morphology of aging model mice

In the result of HE staining: in Figure 1a (negative control group),normal nerve cells in CA1 area can be seen in hippocampal tissue,and the cells are arranged orderly. In Figure 1B (model group), the degeneration and necrosis of nerve cells in CA1 area are obvious,the volume of some cells becomes larger, the cytoplasm is deeplystained, the nucleus of neurons shrinks and axons disappear, the boundary of cell membrane nuclear membrane is not clear enough,and the number of cells decreases; In Figure 1C (piracetam group),the morphology of nerve cells in CA1 area showed an improvement trend. In Figure 1D (Hainan papaya extract low dose group),degeneration, necrosis and decrease in the number of nerve cells in CA1 area can be seen in the hippocampus, but it is lighter than that in the model group. In Figure 1e-f (medium and high dose groups of Hainan papaya extract), the degree of degeneration and necrosis of nerve cells in CA1 area of hippocampal tissue is light and the number is slightly reduced, and the boundary of cell membrane and nuclear membrane is much clearer. The high dose group is better,and the degree of improvement is dose-dependent. It is suggested that Hainan papaya extract may delay aging and improve memory by protecting hippocampal CA1 neurons. See Figure 1.

Table 3 Effects of papaya hainanensis extract on SOD, NOS, CAT and MDA contents in brain tissue of aging model mice(n=12,±s)

Table 3 Effects of papaya hainanensis extract on SOD, NOS, CAT and MDA contents in brain tissue of aging model mice(n=12,±s)

Note:Compared with the negative control group, #P＜0.05,##P＜0.01; compared with the model group, *P＜0.05,**P＜0.01.

Group Dose (mg/kg) SOD (U/mg) NOS (U/mg prot) CAT (U/mg) MDA (nmol/mg)Negative control group - 119.48±4.39 7.84±0.41 15.56±0.87 8.36±1.01 Model group - 84.67±2.78## 3.64±0.41## 8.53±0.64## 16.65±0.62##Piracetam formation 600 146.39±3.56** 6.78±0.54* 16.32±0.65** 10.32±0.66**Hainan papaya extract group 100 101.61±4.75* 6.12±0.95* 11.38±0.85* 15.81±0.96 Hainan papaya extract group 200 109.82±3.89* 7.37±0.43** 12.63±0.64* 13.72±0.83*Hainan papaya extract group 400 135.70±4.92** 7.48±0.58** 14.49±0.55** 9.97±0.72**

Figure1 Effects of hainan papaya extract on hippocampal morphology of d-galactose induced aging mice(HE×400)

3.6 Effect of Hainan Papaya Extract on Nrf2 Protein Expression in Brain Tissue of Aging Model Mice

The results of immunoprotein blotting showed that the expression of Nrf2 [(1.81±0.39) vs (0.92±0.23)] protein in the brain tissues of mice in the model group was significantly lower compared with that of mice in the negative control group, and the difference was statistically significant [(F=2.666, t=6.18, P＜0.05); compared with the model mice, the expression of Nrf2 [(2.69±0.25) vs. (0.92±0.23)]protein in the brain tissues of mice in the positive control piracetam group was significantly lower compared with that of mice in the model group. Nrf2 expression was significantly increased in the brain tissues of mice in the positive control piracetam group compared with the model mice [(2.69±0.25) vs. (0.92±0.23)] [(F=0.022, t=-16.43, P＜0.01)]; while Nrf2 protein expression in the brain tissues of mice in the low, medium and high dose groups of Hainan papaya extract showed a dose-dependent increase, with the most prominent in the high dose group, and the difference was The differences were statistically significant, [(1.72±0.33), (1.91±0.39), (2.51±0.33)vs. (0.92±0.23), (Flow=1.188, Fmedium=2.667, Fhigh=0.319; tlow=-6.29,tmedium=-6.88, thigh=-12.46, Plow＜0.05, Pmedium＜0.05, Phigh＜0.01)]. And the increase was the same as that in the positive drug piracetam group, see Figure 2.

Figure 2 Effects of papaya hainanensis extract on Nrf2 protein expression in brain tissue of aging model mice

4. Discussion

At present, the phenomenon of aging is gradually aggravating,and many problems caused by aging and later have attracted people's attention. Although aging is a normal physiological phenomenon, if it develops too fast, NS will bring pathological degenerative diseases such as AD and PD, causing serious economic losses to patients and their families [18]. If D-galactose is ingested for a long time, it will cause metabolic disorder, and then affect various organs and systems, reducing their normal function. Including memory loss, immune organ degeneration, obvious lack of physical strength and other symptoms, while the accumulation of free radicals and the increase of lipid peroxide and lipofuscin are invisible in the body, the lipid of cell membrane is damaged, and then the activity of type B monoamine oxidase in the body is increased. The above changes were similar to those in elderly mice.

The activity of superoxide dismutase in the body is the main factor in aging. With the increase of superoxide dismutase activity,the ability to scavenge free radicals will be strengthened. The degradation product MDA is produced by free radical, and the degree of cell damage increases with the increase of MDA content.Therefore, the content of the two directly affects the aging of the body and is an important indicator to measure aging. The results of this study showed that the three dose groups of Hainan papaya extract increased SOD activity and decreased MDA level in a certain trend, which indicated that it was helpful to enhance the body's ability to scavenge free radicals and greatly reduce the degree of tissue cell damage, so it may play a role in delaying aging[19,20]. NO is a small molecule substance with a variety of biological activities,which not only improves the learning and memory ability of the body, but also acts on the nervous system to mediate neurons to make amino acids have excitatory responses[21]. The results showed that the activity of NOS was increased in a dose-dependent manner in the extracts of Papaya hainanensis, which further indicated that Papaya hainanensis may delay aging by increasing the activity of NOS in vivo and decomposing more NO. Catalase can clear the body's hydrogen peroxide, block some free radical reaction, by inhibiting the production of reactive oxygen species and accumulation to protect the body organs caused by free radicals damage, thus has antiaging effect[22]. The results of this study showed that Hainan papaya extract of each dose group can greatly improve the activity of CAT in the brain tissue of aging mice, and with piracetam group.

The water maze test for neurocognition and therapeutic effect evaluation is a commonly used experimental method at present[23].The results of this study show that various doses of Hainan papaya extract significantly reduce the escape latency and swimming exploration distance, suggesting that Hainan papaya extract can improve the neurocognitive impairment of aging model mice.The histomorphology of mouse hippocampus further showed that the high, medium and low concentration groups of Hainan papaya extract improved the structure of nerve cells to varying degrees. Studies have shown that Nrf2 is an antioxidant factor,and its downstream antioxidant enzymes play an important role in antioxidant stress injury and indirectly delay aging[24,25]. The results of this study suggest that Hainan papaya can up regulate the expression level of Nrf2 protein, indicating that it can indirectly up regulate the content of its related antioxidant enzymes to increase the antioxidant capacity in vivo and delay aging.

Through the intervention of Hainan papaya extract on aging model mice induced by D-galactose, it is found that it has a certain effect on improving memory, and can improve neurocognitive function and pathological changes of hippocampal tissue to a certain extent. Its mechanism may be to effectively increase the contents of SOD, NOS and cat in brain tissue, reduce the level of MDA, and then increase the activity of Nrf2 protein to improve the learning and memory ability of mice, It provides a basis for clinical development of drugs. Although this experiment preliminarily revealed that Hainan papaya extract can delay the aging of mice to a certain extent, and preliminarily discussed some of its anti-aging mechanism, it is only for some indicators in animals, and it is not deep and comprehensive.In the future, it should be further improved through cell experiment and human experiment. In conclusion, Hainan papaya is worthy of further research and development.

Author contribution statement :

Yunyun Xiong: researched and organized literature, designed research proposal and thesis framework, drafted the paper;Yanning Zhou, Changcai Xie, Kai Zhou: participated in the study,implemented the research process, collected and organized data,statistical analysis; Daorui Yu: proposed the research topic, obtained technical or material support, mentoring support.