Antioxidant and anti-aging effects of polysaccharide LDP-1 from wild Lactarius deliciosus on Caenorhabditis elegans

Background : Edible fungi (mushrooms) have attracted much more concerns due to their abundant nutritions and functional bioactive substances like polysaccharides. Objective : In this study, the anti-oxidation and anti-aging activities of polysaccharide fraction (LDP-1) from the wild Lactarius deliciosus fruiting bodies were systematically evaluated using Caenorhabditis elegans ( C. elegans ) as a model. Methods : Lifetime determination of C. elegans (survival status) was observed via microscope. Effects of LDP-1 on C. elegans induced by heat and oxidative stress were, respectively, performed in an artificial climate chamber and Juglone solution. Determination of lipofuscin levels in C. elegans was observed by laser confocal scanning microscopy. Determination of reactive oxygen species in C. elegans in vivo was conducted via fluorescence spectrophotometer reader. Results : The results revealed that LDP-1 could significantly extend the lifespan of C. elegans , and the lifetime of C. elegans treated with 1,000 μg/mL LDP-1 could be prolonged by 32.8% compared with the control. The survival rates of the experimental C. elegans under heat shock and oxidative stress conditions were clearly improved after treated with 1,000 μg/mL LDP-1 (40 and 19.8%, respectively), while under the same circum-stances all the C. elegans in the blank group died. Fluorescence microscopy analysis confirmed that LDP-1 could effectively reduce the accumulation of lipofuscin and reactive oxygen free radicals in C. elegans , where the respective maximum reduction reached 22.8 and 42.7% compared with the control. Conclusion : These results indicate that LDP-1 had favorable antioxidant and anti-aging effects, which could be explored as potential dietary additives to renovate oxidative damage and slow down aging process.

W ith the acceleration of the aging population, there is an increasing demand for health products. Delaying senility and preventing various diseases caused by aging are of great significance for prolonging the disease-free health period (1). As an irreversible, long, and gradual complex process (2), aging happens in different organs of the body naturally, which would impair the immune system, deteriorate the normal functions, and reduce the adaptability (3). More and more researches indicate that aging will greatly increase the incidence of some chronic diseases, such as hypertension, diabetes, atherosclerosis, and Alzheimer's disease (AD) (4,5). Many theories on aging (e.g. telomere theory and free radical theory) are put forward by far, and among them the free radical-oxidative stress is one of the widely accepted mechanisms (6). The reactive oxygen Popular scientific summary LDP-1 obtained from L. deliciosus had good antioxidant and anti-aging effects. Lipofuscin and ROS level of C. elegans was respectively reduced by 22.8 and 42.7%. LDP-1 significantly extended the average lifespan of C. elegans by 32.8%. Under heat stress, the life span of C. elegans treated with LDP-1 increased by 35%. species (ROS) like superoxide radicals and hydroxyl radicals are normal metabolites of organisms (7). However, excessive production of ROS will cause a certain degree of damages to the cell molecules like proteins, lipids, and nucleic acids that are manufactured by the body itself (8,9). The human body has an antioxidant defense system consisting of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) to eliminate harmful ROS (10). The oxidative stress stimulated by the imbalance elimination of ROS has become a major inducement of aging and disease occurrence (11). In order to maintain the homeostasis of the organism, scavenging free radicals is one of the effective ways to prevent diseases and delay aging. Recently, many synthetic chemical antioxidants have been explored for these purposes (8,12). However, they are limited due to potential liver toxicity and carcinogenesis (13). At present, researchers are focused on developing safe and potent antioxidants, especially original natural ones with low side effects to the human body (14,15).
Edible mushrooms are not only unique in flavor but also rich in bioactive ingredients containing a variety of natural functional compounds, such as polysaccharides (16), trace elements, and adenosine derivatives (17). It is reported that edible fungal polysaccharides have anti-tumor, anti-aging, immune regulation, and anti-inflammatory bioactivities (18,19). For instance, the fungal polysaccharides that are extracted from Agrocybe cylindracea (20), Tremella fuciformis (21), and Hericium erinaceus (22) have excellent antioxidant capacity and anti-aging activity. As a rare edible mushroom, the mycelia of Lactarius deliciosus are enriched with extracellular enzymes like lignin peroxidase and manganese peroxidase (23). Research has also indicated that polysaccharides from L. deliciosus Gray have favorable immune-regulating and antitumor activity in the in vitro and in vivo tests (24)(25)(26). In our previous work, a bioactive polysaccharide component named LDP-1 was obtained from the wild L. deliciosus fruiting bodies for the first time, and subsequent studies showed that it had a significant immunomodulatory effect on RAW 264.7 macrophages (27).
In this study, Caenorhabditis elegans (C. elegans) was chosen as the model to further explore the antioxidant and anti-aging activities of the polysaccharide LDP-1 (28,29). As known, C. elegans model has been widely researched on revealing the genetic and biochemical pathways associated with aging (30). It possesses many advantages like short life cycle, easy to cultivate, transparent body, and simple organs (31,32). Most importantly, C. elegans has many similarities with humans in terms of the aging mechanism (33,34). In addition, the longevity, reproductive ability, and behavioral response of C. elegans can be used as the vital physiological indicators to evaluate the effect of the bioactive polysaccharide (35). In this study, the antioxidant and anti-aging effects of LDP-1 on the lifespan, lipofuscin accumulation, and ROS levels of C. elegans were investigated, which are of great significance to developing potential reagents for preventing and delaying aging.

Materials
The polysaccharide of LDP-1 (Mw of 9.8 × 10 5 kDa) from the wild L. deliciosus fruiting bodies was obtained according to our previous report (27). LDP-1 with a purity level of 90.15% has well solubility in water and appears in yellow-brown color, which is an acidic polysaccharide fraction. C. elegans (N2, wild-type; belongs to Phylumnematoda, Secernentea, Rhabditida, Rhabditidae, and Caenorhabditis) and Escherichia coli OP50 strains were presented by Professor Ai-Fang Du from the College of Animal Sciences, Zhejiang University. 2, 7 dichlorodihydrofluorescein diacetate (H 2 DCFDA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Tryptone, yeast extract, NaCl, and MgSO 4 were bought from Sangon (Shanghai, China). Juglone and agar were purchased from Macklin (Shanghai, China). Other reagents were of analytical grades commercially produced in China.

Cultivation and synchronization of C. elegans
Culture and preservation of uracil-deficient E. coli Under aseptic conditions, uracil-deficient E. coli OP50 was added to the Luria-Bertani (LB) culture medium at a ratio of 1:100 (v/v) and cultured at 37°C under shaking at 180 rpm until reaching the logarithmic growth phase.
The E. coli OP50 was stored as follows: glycerin was added into the OP50 culture that reached the logarithmic growth phase with final concentration of 3% (v/v), put into eppendorf (EP) tubes, stored under -20°C freezing temperature (Siemens, KG29NS221C, Germany) overnight, and then transferred to -80°C condition for long-period preservation.

Cultivation of C. elegans
Under aseptic conditions, 100 μL of OP50 E. coli was pipetted into standard nematode growth medium (NGM) plate and spread evenly. The growth rate of uracil-deficient nematodes in NGM was much slower than that of E. coli, which can provide nematode food without overgrowth. A piece of agar containing adult eggs was cut and inoculated on the above NGM plate, and then placed into a 20°C biochemical incubator (XUTEMP TEMPTECH Co., Ltd., XT5107-IB150, Hangzhou, China). The obtained nematodes were subcultured every 4-5 days.

Synchronization of C. elegans
The nematodes cultured to the oviposition stage were washed with M9 buffer (KH 2 PO 4 3.0 g/L -1 , Na 2 HPO 4 6.0 g/L -1 , NaCl 5.0 g/L -1 , MgSO 4 1 mol/L -1 ). The supernatants were discarded, and the nematodes were collected within the buffer. Then the same amount of lysis solution was added and mixed thoroughly, and the eggs were obtained after centrifugation and rinsed twice with M9 buffer at last. The synchronized eggs were diluted with OP50 culture, transferred into NGM medium, and incubated at 20°C for further analysis.

Lifetime determination of C. elegans
The LDP-1 solution (5 mg/mL) was diluted with OP50 medium to obtain 125, 250, 500, and 1,000 μg/mL mixed cultures, and pipetted 100 μL onto the NGM plates, respectively. The LDP-1 interfered groups were prepared using the same method for the following experiments.
The synchronized L4 phase nematodes (36) were picked into the respective media of the blank group and the experimental group with thin platinum wire, and 40 nematodes were selected randomly for each group with three parallel contrasts. The experiments were performed in a constant incubator at 20°C, and the sample groups were taken out for record every other day. The survival status of the nematodes was observed via microscope, while the dead nematodes were picked out and recorded. Using a platinum wire the nematodes were gently touched, and if the head and body did not swing and bend, this indicated death. The survival rate (SR) % was calculated by using the following formula: where A 1 is the number of survival C. elegans, and A 0 is the total number of experimental C. elegans. Each measure was repeated at least eight times.
The following experiments were continued with the same procedures and criteria until all the nematodes in the experimental groups died.

Effects of LDP-1 on C. elegans induced by heat stress
The LDP-1 with concentrations of 250, 500, and 1,000 μg/mL was prepared as discussed in the previous section, and an amount of 100 μL was added to NGM media, respectively. Sixty nematodes that synchronized to L4 stage were picked into the blank group and the LDP-1-treated group media with three parallels. The experimental nematodes were transferred to fresh media every other day consecutively. The culture media were placed in a 35°C artificial climate chamber (Jiangnan Instrument Factory, RXZ-128A, Ningbo, China) under the same conditions. At predetermined time intervals, the nematodes were taken out for record, while the dead ones were picked out. The average growth time calculated by the growth time of each nematode was used as the average lifespan to characterize the influence of polysaccharides on the survival of nematodes.

Effects of LDP-1 on C. elegans induced by oxidative stress
The sterilized NGM medium was cooled to approximately 70°C, and the filtered Juglone solution was added to make a final concentration of 250 μg/mL. The synchronized nematodes were cultured to the L4 stage and transferred to different concentrations of LDP-1-treated groups and blank ones. After further cultivation at 20°C for 5 days, 60 nematodes were shifted to the media containing Juglone solution prepared above. Juglone can cause nematodes to die due to oxidative stress. The survived nematodes were recorded every 2 h until all nematodes died.

Determination of lipofuscin in C. elegans
C. elegans were cultured synchronously to the L4 stage and transferred to the LDP-1-treated group and blank group media at 20°C for continuing cultivation. The experimental media were replaced with fresh ones every other day. The lipofuscin levels of 15 nematodes that cultured for 5 and 10 days were observed by laser confocal scanning microscopy (LCSM) (FV1000, Olympus Corporation, Japan) with respective excitation and emission wavelengths at 365 and 420 nm. All photos were taken at 100× magnification, and the fluorescence intensity of each nematode was quantitatively analyzed using Image J software (Image-Pro Plus5.0, Media Cybernetics Inc., MD, USA).

Determination of ROS in C. elegans in vivo
The nematodes were cultured in the blank group and the LDP-1-treated group with the same procedures, and transferred to M9 buffer containing 1 mL of 1 mM H 2 DCFDA. After 3 h incubation at 20°C, the experimental nematodes were fixed on a 2% (w/v) agar plates. At the end of the specified treatment time, all of the C. elegans were collected and washed three times with M9 buffer. The nematodes were pipetted into 96-well microtiter plates containing H 2 DCFDA. The samples were recorded every 20 min using fluorescence spectrophotometer reader (Synergy Mx,BioTek, VT, USA) at excitation/emission wavelengths of 485 and 525 nm, respectively (11). The following formula was used to calculate the fluorescence ratio (FR, %) indicating the relative ROS level.
where F 0 is the fluorescence intensity of the blank group, and F 1 is the fluorescence intensity of the positive control group or the experimental group, with eight parallels in each group.

Statistical analysis
All measurements were performed at least in triplicate. The statistical significance (P < 0.05) between two samples was evaluated by one-way analysis of variance method, followed by the Turkey's test using OriginLab software (OriginPro 9.0.0, OriginLab Co., MA, USA). All data were expressed as mean ± standard deviation (SD).

Cultivation and observation of life cycle of C. elegans
The uracil-deficient E. coli OP50 grew slowly on NGM medium, which can be provided as food for nematode growth. Considering the reproducibility of the nematodes that are used, we investigated the growth character of OP50 to ensure the performed ones were in the logarithmic growth phase as inclusively as possible. As shown in Fig. 1a, OP50 grew up to the logarithmic growth phase at 37°C and 180 rpm after 3 h in LB medium. In view of the physiological-biochemical characteristics of the microbial growth, OP50 with absorbance value of 0.6 was used for all of the experiments.
The life cycle of the hermaphrodite individual C. elegans can be divided into worm eggs, larvae, and adults periods, while from the larvae to adults it showed four distinct phases, namely, L1, L2, L3, and L4 (Fig. 1b1-b4). When the individuals completed the gonad development, it produced the eggs during the L4 phase (Fig. 1b5). In a rich living environment, the average lifespan of nematodes lasted for 1 month, and it took only 3 days for the larvae developing to adults, and 4 days to reproducing next generation (37).
To evaluate whether the polysaccharide of LDP-1 had an effect on nematodes, the larvae were selected as the research model. At this stage, the larvae were in the critical period of development and more sensitive to interfering substance. Meanwhile, the adult nematodes could be used to evaluate the toxicity of the surroundings through indicators of lifespan, swallowing rate, motility, and spawning ability. Furthermore, under peroxidation conditions the nematodes will undergo certain degree of aging with extension of time, such as lipofuscin deposition, slow movement, and decreased feeding rate, which indicates that the nematodes were entering the aging process due to the imbalance of free radical homeostasis. These unique aging characteristics of C. elegans can display the potential influence of the target substance. In this study,

Survival and lifespan of C. elegans treated with LDP-1
The lifespan is one of the valuable indicators in evaluating the physiological characteristics of nematodes. It displays the growth status of nematodes and potential toxicity of the target substance. Herein, the polysaccharide of LDP-1 with concentrations of 125, 250, 500, and 1,000 μg/mL (w/v) were set as the parallel groups for treating the nematodes. The toxicity of the prepared LDP-1 solutions was investigated. As shown in Fig. 2, LDP-1 within concentrations of 125-1,000 μg/mL had no significant influence on the SRs of the treated nematodes (P > 0.05), indicating the polysaccharide of LDP-1 was nontoxic and safe to the wild-type nematodes. In subsequent experiments, three level concentrations of 250, 500, and 1,000 μg/mL were selected to further explore the effects of polysaccharide LDP-1 on the lifespan of wild nematodes.
As shown in Fig. 3a, all the nematodes in the blank group (Control) died after 20 days cultivation. In contrast, the lifespan of the experimental nematodes that were treated with LDP-1 was significantly longer than that of the blank group, and showed a concentration dependence. After 20 days, the SRs of the experimental nematodes treated with 250, 500, and 1,000 μg/mL LDP-1 solutions were 16, 17.6 and 21.2%, respectively. The average lifespan of the blank group and LDP-1-treated groups (250, 500, and 1,000 μg/mL) was, respectively, 11.7, 13.55, 15.157, and 15.538 days, with a maximum life extension of 32.8% compared with the blank one (Fig. 3b). Liu et al.'s research showed that the polysaccharide of CP2-c2-s2 from Cordyceps militaris could prolong the lifespan of nematodes by 16.58% with a concentration of 1,500 μg/mL treatment (32). The polysaccharide of LDP-1 showed favorable life-extending activity in nematodes.

Survival and lifetime of C. elegans under heat stress
The nematodes were transferred from the optimal surrounding (20°C) to a 'higher temperature' (above 30°C) environment. Under heat stress conditions, the protective effect of the active polysaccharide on the nematodes can be assessed through the SR. As can be seen from Fig. 4a, the LDP-1-treated nematodes had better resistance to heat stress than the normal cultured nematodes (Control). The nematodes in the blank group died after 6.5 days under heat stress treatment. In comparison, the SR of the 1,000 μg/mL LDP-1-treated group was still reached 40% in the same period. The average lifetime of the experimental nematodes that were treated with the active polysaccharide LDP-1 was significantly longer than that of the blank group (Fig. 4b).
The average lifetime of the nematodes treated with 250, 500, and 1,000 μg/mL LDP-1 was, respectively, increased by 14.5, 35, and 16.6% compared with the blank group. These results confirmed that the LDP-1 had good effects in anti-heating stress.
Regulation effect on C. elegans under oxidative stress Compared with the heat stress response, the oxidative stress to the nematodes induced by environment has a similar principle. That is to say, the outside oxidative stimulation combined with the nematode's autoimmune response system will initiate the resistance to the external environment, and thereby improve the tolerance so as to prolong their lifespan.
As shown in Fig. 5a, the nematodes in the blank group died after 14 h cultivation; comparatively, the experimental nematodes that were treated with different concentrations of LDP-1 showed good antioxidant capacity. After 16 h treatment, the SR of the nematodes (1,000 μg/mL LDP-1) was still up to 19.8%. Meanwhile, the average lifespan of the nematodes presented a rising trend from 6.8 to 10.8 h compared with the control (Fig. 5b). Comparatively, similar research was conducted on the Puer tea water extract by Fei et al. (38), which manifested that the concentration of 2,500 μg/mL had a significant antioxidant effect on nematodes. Thus, LDP-1 could improve the anti-oxidative stress ability of the nematodes effectively.

Determination of lipofuscin in C. elegans treated with LDP-1
Lipofuscin contained in C. elegans has spontaneous fluorescence characteristics, which can be observed under a fluorescence microscope. During the normal aging process, lipofuscin gradually accumulates and becomes increasingly strong in terms of fluorescence intensity, which is an important indicator for evaluating the aging process of the nematodes (39). As shown in Fig. 6a, compared with Fig. 4. Determination of C. elegans lifespan under a heat shock stress environment. (a). Survival rate of C. elegans and (b) average lifespan of C. elegans under heat stress conditions. Significant differences were compared from the Control at *P < 0.05 and **P < 0.01. the blank groups (Control), the fluorescence intensity of lipofuscin in the experimental nematodes became weaker clearly with the increasing concentrations in both 5 and 10 days later. Quantitative analysis shows that the lipofuscin accumulated in the experimental nematodes (1,000 μg/ mL) on the 5 th day was reduced by 20% compared to the blank groups. With the extension of the treatment time, the lipofuscin accumulation in the nematodes increased on the whole; however, it still presented a weaker image in the experimental groups. After 10 days treatment, the lipofuscin accumulated in the worms was reduced by 22.8% in the LDP-1 interfered groups compared with the blank group (Fig. 6b). These results indicate that LDP-1 could significantly delay the aging process of nematodes.

Analysis of ROS levels in C. elegans under oxidative stress
The free radical aging theory indicates that aging is caused by molecular damage due to the imbalance accumulation of oxygen free radicals (40). What is more is that the faster the individual's metabolic rate is, the more ROS oxidative stress conditions. Significant differences were compared from the Control at *P < 0.05 and **P < 0.01. it produces; under this circumstance, with the accumulation of oxidative damage it will speed up the aging process undoubtedly. Therefore, it is of great significance to assess the ROS levels in experimental nematodes.
As can be seen from Fig. 7, under the normal conditions, the ROS levels were tested in the blank group and LDP-1-treated groups after 5 days. It manifested that there were no significant differences in the ROS levels compared with the blank group (Control). However, when the experimental nematodes treated with 100 μg/mL Juglone solution for 3 h, the ROS levels of the LDP-1 interfered nematodes were significantly reduced than that of the blank group, with a maximum decrease by 42.7%. Wang et al. (41) showed that the maximum clearance rate of ROS via the bioactive peptides was greater than 20%. Comparatively, the clearance rate of LDP-1 was twice than that of it. It further confirmed that LDP-1 had favorable antioxidant activity.

Conclusions
In this article, the antioxidant and anti-aging activity of polysaccharide LDP-1 was mainly investigated with C. elegans as the model. The results showed that LDP-1 Fig. 6. Effects of the polysaccharide LDP-1 on the lipofuscin accumulation in C. elegans growth process. (a) Observation of the lipofuscin accumulation in C. elegans under fluorescent microscope; (b) Fluorescence intensity analysis of the produced lipofuscin. Significant differences were compared from the Control at **P < 0.01. had favorable life-extending characteristics. It proved that LDP-1 could increase the lifespan of nematodes under heat shock and oxidative stress environments. The fluorescence intensity observed from the lipofuscin accumulation of the LDP-1-treated groups became weaker, which confirmed that LDP-1 could clearly delay the aging process of nematodes and decrease the ROS levels. On the basis of our previous report, LDP-1 showed good antioxidant, anti-aging, and immunomodulatory effects. Thus, it could be further explored as potential additive agents applied in functional foods and health care products.