Administration with Bushenkangshuai Tang alleviates UV irradiation- and oxidative stress-induced lifespan defects in nematode Caenorhabditis elegans

Abstract

During normal metabolism, oxidative byproducts will inevitably generate and damage molecules thereby impairing their biological functions, including the aging process. Bushenkangshuai Tang (补肾抗衰方, BT) is a traditional Chinese medicine widely used for clinically treating premature ovarian failure. In the present study, BT administration at high concentrations significantly increased lifespan, slowed aging-related decline, and delayed accumulation of aging-related cellular damage in wild-type Caenorhabditis elegans. BT administration could further largely alleviate the aging defects induced by UV and oxidative stresses, and BT administration at different concentrations could largely rescue the aging defects in mev-1 mutant animals. The protective effects of BT administration on aging process were at least partially dependent on the Ins/IGF-like signaling pathway. Moreover, BT administration at different concentrations obviously altered the expression patterns of antioxidant genes and suppressed the severe stress responses induced by UV and oxidative stresses, suggesting that BT-induced tolerance to UV or oxidative stress might result from reactive oxygen species scavenging. BT administration during development was not necessarily a requirement for UV and oxidative stress resistance, and the concentrations of administrated BT examined were not toxic for nematodes. Therefore, BT administration could effectively retrieve the aging defects induced by UV irradiation and oxidative stress in Caenorhabditis elegans.

Keywords

Bushenkangshuai Tang / UV irradiation / oxidative stress / tissue senescence / lifespan / Caenorhabditis elegans

Introduction

Klass first introduced Caenorhabditis (C.) elegans as “an excellent experimental system for the study of aging” for which he “identified some of the major biological and environmental factors influencing lifespan as a prelude to more detailed genetic and biochemical analyses” [1], and his prediction has been confirmed by the tremendous progress in biogerontology due to C. elegans studies carried out during the last three decades [2]. Among the genes influencing lifespan in this worm, those related to the Ins/IGF signaling pathway are among the best studied [2,3]. The forkhead transcriptional factor DAF-16, the master regulator of this pathway, can activate an enhanced life maintenance program in response to environmental and gonadal inputs or mutation, and DAF-16 up- and downregulates expression of many genes leading to metabolic alterations and increased stress and microbial stress [4-6]. During the last 20 years, the most important signaling pathways, together with their downstream targets that influence aging, have been identified and characterized in C. elegans.

For the aging control, Harman proposed that free radicals, inevitable byproducts of aerobic energy metabolism, are supposed to cause molecular damage, and accumulation of damaged macromolecules caused by reactive oxygen species (ROS) would lead to the decrease of cellular activity, and the death of organisms [7]. Based on this oxidative damage theory, species that produce plenty of free radicals should suffer more molecular damage which in turn will lead to a short life span, and the damage associated with ROS could be ameliorated by interventions that enhance resistance to oxidative stress [8,9]. There is increasing evidence to implicate the accumulation of macromolecular damage caused by ROS in the aetiology of a number of age-related disease states [10,11]. There is also a strong correlation between extended lifespan and resistance to oxidative stress, which is evident in a number of C. elegans mutant strains. Larsen and Vanfleteren were the first to indicate that mutation of age-1 results in the resistance to oxidative stress by upregulating superoxide dismutase (SOD) and catalase activity [12,13]. Further insight into the role of oxidative stress in aging has been gained by the analysis of mutants with altered mitochondrial function and lifespan. Mutation of mev-1 (succinate-Co-Q oxidoeductase in the mitochondrial electron transport chain) is isolated due to its hypersensitivity to methyl viologen (paraquat), and will cause the sensitive phenotype to oxidative stress and accelerate aging, suggesting that mev-1 governs the rate of ageing by modulating the cellular response to oxidative stress [14]. Moreover, it was shown that longevity mutants were also resistant to other stressors such as ultraviolet (UV) irradiation, elevated temperature and heavy metal exposure [15-18]. Therefore, it seems likely that stress resistance contributes to lifespan determination in C. elegans.

Based on the prediction of oxidative damage theory, aging could be ameliorated by interventions that enhance resistance to oxidative stress. Administration with antioxidant compounds can effectively reduce oxidative stress and thereby extend lifespan in C. elegans. Initial studies using vitamin E yielded encouraging results in relation to lifespan extension, although vitamin E treated populations also show slowed development and a reduction of fertility [19]. In addition, exogenously supplied coenzyme Q10 (CoQ10) can play a significant anti-aging function by lowering oxidative stress[20], and a dietary source of Co-Q is essential for growth of long-lived nematode clk-1 mutants [21]. The Ginkgo biloba extract EGb 761, a potential antioxidant, caused a modest extension of lifespan (8%), as well as increased resistance to oxidative and thermal stresses; however, a purified component, the flavonoid tamarixetin, extended lifespan by 25% [22]. Moreover, the oxidatively-challenged C. elegans strain mev-1’s short lifespan was significantly increased by the supplementation with trace manganese as a free radical scavenger [23]. More recently, a more spectacular lifespan increase (44%) was obtained by administration of the small, synthetic SOD/catalase mimetics (SCMs) of EUK-8 and EUK-134 [9].

Premature ovarian failure (POF) refers to the development of amenorrhoea due to cessation of ovarian function before the age of 40 years [24], and POF is a unique example of isolated organ senescence, with a population prevalence of approximately 1% [25]. In order to better the understanding of the molecular and physiological control of POF, premature gonadal senescence should be paid more attention to or considered in clinical treatment. Moreover, the important role of oxidative stress in regulating POF has been already suggested by some studies [26,27]. Bushenkangshuai Tang (补肾抗衰方, BT) is a traditional Chinese medicine which has been widely used for clinically treating POF. This medicine can alleviate the inhibitory action of excessive androgen on ovarian granulocyte and regulate the ovarian function by promoting follicular development, and increasing the levels of estrogen and progestogen. In the present study, we report the effects of administration of BT in effectively retrieving the aging defects induced by oxidative stress and UV irradiation in C. elegans. This analysis will be useful for our understanding the physiological control of POF and further modification of the compounds in this medicine for clinical POF treatment.

Materials and methods

BT preparation

BT consisted of 9 medicinal herbs: Cuscuta chinensis Lam. (Tusizi, 15 g), Polygonatum sibiricum Red. (Huangjing, 15 g), Rehmannia glutinosa Libosch. (Shudihuang, 15 g), Cistanche deserticola Y.C.Ma (Roucongrong, 10 g), Morinda officinalis How (Bajitian, 10 g), Angelica sinensis (Oliv.) Diels (Danggui, 10 g), Ligusticum chuanxiong Hort. (Chuanxiong, 6 g), Fluorite (Zishiying, 15 g), and Schisandra chinensis (Turcz.) Baill. (Wuweizi, 6 g). The herbs were boiled with water for 3 times, and the final concentration of this recipe was 0.33 g/mL for use in this project. All the chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Treatment media and culture conditions

The strains used in the current study were wild-type N2, and mutants of mev-1 (kn1), daf-16 (mu86), and daf-2 (e1370) originally obtained from the Caenorhabditis Genetics Center (Minneapolis, MN, USA). KC136 was the gift from Dr. King Chow’s lab. They were maintained on nematode growth medium (NGM) plates seeded with Escherichia coli OP50 at 20°C as described by Brenner [28]. Age synchronous populations of L2- or L4-larva stage nematodes were obtained by the collection as described [29]. The L2- or L4-larva stage animals were washed with double-distilled water twice, followed by washing with modified K medium once (50 mmol/L NaCl, 30 mmol/L KCl, 10 mmol/L NaOAc, pH 5.5) [30]. Exposures were performed in sterile culture plates (glass) for 2 h on L4-larva stage animals with 25%, 50%, 75%, and 100% of examined BT diluted with water. Approximately 100 animals were transferred in 100 μL to each exposure solution in the micropipette. All exposures were carried out in an incubator in the presence of food.

UV-irradiation experiments

Approximately 50 L2-stage larvae animals were irradiated on NGM plates without food at 20 J/m²/min as described [16, 31]. All UV-irradiation assays were performed at 20°C, and afterwards further maintained at 20°C. All assays were replicated more than three times.

Paraquat treatment

Approximately 50 L2-stage larvae animals were treated with 2 mmol/L paraquat solution for 2 h and survival at 20°C. All assays were replicated more than three times.

Lifespan assay

The lifespan assays were performed basically as described at 15°C, 20°C or 25°C and were initiated after BT administration [32,33]. In this test, the hermaphrodites were transferred daily for the first 4 days of adulthood. The worms were checked every 2-4 days and would be scored as dead when they did not move even after repeated taps with a pick. Approximately 50 animals were picked out and used for each lifespan trial. For life span, graphs were representative of at least three trials. The data were statistically analyzed using a 2-tailed 2 sample t-test (Minitab Ltd., Coventry, UK). Pharynx pumping rates were scored on adults at room temperature (24°C) under a Nikon stereomicroscope (Nikon, Melville, NY, USA) as described [34].

Photography of autofluorescence

The method was performed as described [3]. The images were collected for endogenous intestine fluorescence using a 525-nm bandpass filter and without automatic gain control in order to preserve the relative intensity of different animal’s fluorescence. Day 4, day 8 and day 14 adults were photographed on the same day to avoid the effects of light source variance on fluorescence intensity. Observations of the green fluorescent protein (GFP) were recorded and color images were taken for the documentation of results with Magnafire^® software (Olympus, Irving, TX, USA). Lipofuscin levels were measured using ImageJ Software (NIH Image) by determining average pixel intensity in each animal’s intestine. More than 50 animals were counted for the statistical analysis.

Analysis of transgenic strain

The method was performed as described [35]. To analyze the changes of hsp-16.2 expression patterns, the treated KC136 animals were allowed to settle for 10 min, and then pipetted onto an agar pad on a glass slide, mounted and observed for the fluorescent signals with a fluorescent microscope. Observations of the GFP were recorded and color images were taken for the documentation of results with Magnafire^® software (Olympus, Irving, TX, USA). The GFP levels were measured using ImageJ Software (NIH Image) by determining average pixel intensity in each animal’s intestine. More than 50 animals were counted for the statistical analysis.

Analysis of the expression of antioxidant genes to oxidative stress

The Poly (A)⁺ RNA of nematodes was prepared and analyzed as described [36,37]. Control and treated wild-type N2 animals were sampled for the semi-quantitative RT-PCR assay. The frozen animal pellets were harvested from 1 L of mixed stage liquid cultures. Purified Poly (A)⁺ RNA through two rounds of selection on oligo(dT)-cellulose was used. Gene-specific primers were designed for sod-1 (sod-1a, 5'- TCTCACTCAGGTCTCCAA -3'; and sod-1b, 5'-TTCTTCTGACCTTTGCGG -3'), for sod-2 (sod-2a, 5'-CGTTCGCTGTGTCTCAAA-3'; and sod-2b, 5'- TACAGTTACGATAAACCT-3'), for sod-3 (sod-3a, 5'- GAACCTGTAATCAGCCAT-3', and sod-3b, 5'- GTCTGATACAGGTACGAT-3'), for sod-4 (sod-4a, 5'-GTTCTGGCTCTCTCCGTT-3'; and sod-4b, 5'- TGACTACTGAACCCTGCG-3'), for ctl-1 (ctl-1a, 5'-TGGTTGGAAATAACACTC-3', and ctl-1b, 5'-TGTAAAGTCTGCTGACCT-3'), and for ctl-2 (ctl-2a, 5'-TTCTCTACAGTCGGTGGT-3'; and ctl-2b, 5'-CTCTCTTACACGGTCTTG-3'). act-1 was used to determine the equal loading for each sample, and primers specific for act-1 were used in control reactions (act-1a, 5'-CGAAGCTTACCGTCCCAATCTACGAAG-3'; act-1b, 5'-TGAGAATTCGAAGCACTTGCGGTGAAC-3'). Amplification of all DNA fragments for RT-PCR was performed for 30 cycles in a Perkin-Elmer 480 thermal cycler using a 55°C annealing temperature and a 1-min extension. Aliquots of individual PCR products were resolved by agarose gel electrophoresis and made visible with ethidium bromide under UV light. The gels were transferred onto a Nytran membrane with a Turboblotter (Schleicher and Schuell), followed by membrane baking at 80°C for 2 h and auto-crosslink in a UV Stratalinker. The amplified probe fragments (about 50 ng) were labelled with (α-³²P) dCTP using a Prime-it II Kit (Stratagene). Membranes were probed with labelled DNA fragments in 0.3 mol/L sodium phosphate buffer, pH 7.2, containing 7% (w/v) SDS, 1 mmol/L EDTA, pH 8.0, and 2% (w/v) BSA at 65°C for 16-24 h. Filters were washed at 65°C twice with 2×SSC and 0.1% (w/v) SDS for 30 min, once with 1×SSC and 0.1% (w/v) SDS for 20 min, and once with 0.5×SSC and 0.1% (w/v) SDS for 10 min. The membranes were exposed to a Kodak XAR film at -80°C for autoradiography.

Post-developmental treatment

Following paraquat treatment or UV irradiation at L2-larva stage, BT at different concentrations (25%, 50%, 75%, and 100%) was administered to experimental cultures after the nematodes had reached adulthood in the sterile culture plates (glass) for 2 h. The animals were scored for survival as described above.

Statistical analysis

All the data in this article were expressed as \overline{x}\pm s. One-way analysis of variance (ANOVA) followed by a Dunnett’s t-test was used to determine the significance of the differences between the groups. The probability levels of 0.05 and 0.01 were considered statistically significant.

Results

Administration with BT prolonged lifespan and slowed aging in C. elegans

We first examined the effects of administration with BT on the lifespan in adult wild-type N2 animals. As shown in Figure 1a, adult wild-type animals grown under our laboratory conditions had a mean lifespan of 10.4 days and average maximum lifespan of 15.8 days at 25°C. Administration with 25% of BT would not obviously affect the mean lifespan and maximum lifespan of treated wild-type animals, whereas administration with BT at concentrations of 50%, 75%, and 100% could significantly (P <0.01) prolong the mean lifespan of wild-type animals by 13%, 19%, and 26%, respectively, and increase the maximum lifespan of wild-type animals by 16%, 24%, and 32%, respectively.

Because lifespan in C. elegans can be affected by temperature [38], we also investigated the possible effects of BT administration on wild-type animals’ lifespan at 20°C and 15°C. Adult wild-type animals grown under our laboratory conditions had a mean lifespan of 14.7 days and average maximum lifespan of 27.2 days at 20°C, and a mean lifespan of 19.2 days and average maximum lifespan of 35.1 days at 15°C. Similarly, administration with 25% of BT would not obviously alter the mean lifespan and maximum lifespan of treated wild-type animals. However, administration with BT at concentrations of 50%, 75% and 100% all could significat (P < 0.01) prolong the mean lifespan and the maximum lifespan of wild-type animals compared to controls (Fig. 1b and c). Therefore, administration with BT at high concentrations could increase the lifespan in wild-type nematodes, and the effects were not temperature-sensitive.

To examine whether administration with BT could slow aging in C. elegans, we further analyzed the speed of pharyngeal pumping, which can predict the age-related changes of the physiological processes [34]. The pharyngeal pumping declined gradually in the wild-type animals with increasing age. In contrast to this, administration with BT at higher concentrations (50%, 75%, and 100%) was associated closely with higher pumping rates on adult days 8 and 10 (Fig. 1d). Therefore, administration with BT at high concentrations slowed aging, rather than simply improving survival at old age in C. elegans.

Administration with BT delayed the accumulation of aging-related cellular damage

Intestinal autofluorescence, caused by lysosomal deposits of lipofuscin, accumulates over time in the aging animal, and is a valuable marker for aging and cellular damage in aging cells [39]. To investigate whether the altered lifespan in BT administrated nematodes is due to accelerated or suppressed aging-related cellular damage, we further examined the lipofuscin levels in the intestines of control and BT administrated day 4, day 8 and day 14 adult animals. As shown in Fig. 2, at day 4 after L4, no obvious changes of intestinal lipofuscin were observed between control and BT administrated wild-type animals. At day 8 after L4, BT administration at lower concentration (25% and 50%) still did not alter the intestinal lipofuscin levels in wild-type animals, whereas administration with BT at concentrations of 75% and 100% could already significantly (P < 0.01) decreased the intestinal lipofuscin levels by 5% and 14%, respectively, in wild-type animals compared to controls. Moreover, at day 14 after L4, administration with BT at concentrations of 50%, 75%, and 100% could further significantly (P < 0.01) reduce the intestinal lipofuscin levels by 9%, 15% and 23%, respectively, in wild-type animals, although administration with 25% BT would not alter the intestinal lipofuscin level in treated wild-type animals compared to the controls. Therefore, administration with BT at high concentrations could delay the accumulation of aging-related cellular damage.

Administration with BT could alleviate the aging defects induced by UV irradiation and oxidative stress

One possible explanation for the beneficial effects of BT on the aging of wild-type nematodes is that it can increase cellular stress resistance. In addition, considering the fact that stress resistance may contribute to lifespan determination in C. elegans [2,14], and POF is a unique example of isolated organ senescence [24,25], we next examined the possible effects of BT administration on aging defects induced by UV irradiation and oxidative stress. UV irradiated adult wild-type animals grown under our laboratory conditions had a mean lifespan of 4.7 days and average maximum lifespan of 9.7 days at 20°C. As shown in Figure 3a, the mean lifespan in UV irradiated wild-type animals could be prolonged by 47%, 102%, 121%, and 140%, respectively, after administration with BT at concentrations of 25% to 100%, and the maximum lifespan in UV irradiated wild-type animals could be prolonged by 28%, 46%, 68%, and 80%, respectively, after administration with BT at concentrations of 25% to 100%. Moreover, the intestinal lipofuscin levels at day 14 after L4 could be significantly (P < 0.01) reduced by 40%, 66%, 72%, and 78%, respectively, after administration with BT at concentrations of 25% to 100% (Fig. 3c). Therefore, the resistance to UV irradiation was increased significantly by BT administration in wild-type nematodes.

Furthermore, it was found that BT administration could noticeably improve survival under oxidative stress. The resistance to oxidative stress was assayed by exposing animals to paraquat, an intracellular free-radical-generating compound. Paraquat treated adult wild-type animals grown under our laboratory conditions had a mean lifespan of 6.2 days and average maximum lifespan of 9.4 days at 20°C. As shown in Fig. 3b, the mean lifespan in paraquat treated wild-type animals could be prolonged by 18%, 36%, 57%, and 68%, respectively, after administration with BT at concentrations of 25% to 100%, and the maximum lifespan in paraquat treated wild-type animals could be prolonged by 23%, 45%, 68%, and 86%, respectively, after administration with BT at concentrations of 25% to 100%. In addition, the intestinal lipofuscin levels at day 14 after L4 could be significantly (P < 0.01) reduced by 51%, 61%, 79%, and 82%, respectively, after administration with BT at concentrations of 25% to 100% (Fig. 3d). Therefore, administration with BT could largely alleviate the aging defects induced by oxidative stress.

Administration with BT induced the changed stress response

The HSP-16 protein is encoded by a classical stress gene, and its expression can be induced by an array of environmental stresses including heavy metal exposure, heat and oxidative stress [35]. We reasoned that if the stress exposure was toxic, it would thus result in a stress response [35]. Again, we explored the stable transgenic line of hsp16-2-gfp to investigate the stress response induced by UV and oxidative stress. As shown in Fig. 4a and b, both UV irradiation and paraquat treatment could noticeably increase the percentage of the population expressing hsp16-2-gfp compared to wild-type. Moreover, the high percentages of the population expressing hsp16-2-gfp induced by UV irradiation and paraquat treatment could all be significantly (P < 0.01) decreased by BT administration at different concentrations compared to controls. Especially, administration with 100% BT could almost completely recover the defects of stress response formed in UV irradiated or paraquat treated wild-type animals. This observation further suggests that BT-induced tolerance to UV or oxidative stress may results from ROS scavenging.

Administration with BT altered the expression patterns of antioxidant genes

To confirm the alleviative role of BT administration in regulating stress-induced aging defects, we next examined the expression patterns of antioxidant genes BT and/or UV/paraquat treated wild-type animals. The investigated antioxidant genes were sod-1 (encoding a copper/zinc-superoxide dismutase, Cu/Zn-SOD), sod-2 (encoding a manganese-superoxide dismutase, Mn-SOD), sod-3 (encoding a MN-SOD), sod-4 (encoding an extracellular Cu/Zn-SOD), ctl-1 (encoding a cytosolic catalase), and ctl-2 (encoding a peroxisomal catalase). The expression of these antioxidant genes can reflect the response to oxidative damage in nematodes [40]. As shown in Fig. 5, there were considerable differences in the expression patterns of these 6 genes. The expression levels of sod-1, sod-2, sod-3, and sod-4 were decreased by 55%, 62%, 43%, and 31%, respectively, in UV irradiated wild-type animals, whereas the expression levels of ctl-1 and ctl-2 were increased by 89% and 200%, respectively, in UV irradiated wild-type animals compared to controls. Similarly, the expression levels of sod-1, sod-2, sod-3, and sod-4 were decreased by 49%, 58%, 35%, and 27%, respectively, in paraquat treated wild-type animals, whereas the expression levels of ctl-1 and ctl-2 were increased by 110% and 220%, respectively, in paraquat treated wild-type animals compared to controls. Furthermore, the decreased expression levels of sod-1, sod-2, sod-3, and sod-4 in UV irradiated or paraquat treated animals could be noticeably enhanced by administration with BT at different concentrations and the increased expression levels of ctl-1 and ctl-2 in UV irradiated or paraquat treated animals could also be markedly reduced by administration with BT at different concentrations from 25% to 100%. Nevertheless, administration with BT at different concentrations could not completely recover the altered expression patterns of examined 6 antioxidant genes formed after UV irradiation or paraquat treatment compared to those in normal wild-type animals. Therefore, administration with BT at different concentrations could largely rescue the deficits in expression patterns of antioxidant genes induced by UV and oxidative stresses in C. elegans.

Aging phenotypes in BT administrated mev-1 mutant

mev-1 is a short-lived strain with elevated oxidative stress, which provides a useful tool for testing compounds for antioxidant properties [14,23]. We therefore tested whether BT administration could confer oxidative stress resistance in mev-1 mutant. As shown in Fig. 6, administration with BT at concentrations of 25%, 50%, 75%, and 100% could prolong the mean lifespan of mev-1 mutant by 25%, 36%, 68%, and 83%, respectively, and prolong the maximum lifespan of mev-1 mutant by 18%, 31%, 50%, and 65%, respectively. Nevertheless, the defects of mean lifespan and maximum lifespan in mev-1 mutant could not be completely rescued by BT administration at different concentrations compared to those in wild-type animals (data not shown). Furthermore, our data demonstrated that the mean lifespan of BT administrated mev-1 mutant was significantly (P < 0.01) shorter than that of BT administrated wild-type animals. Therefore, administration with BT at different concentrations could largely recover the aging defects in mev-1 mutant animals. In addition, this observation further confirmed the alleviative role of BT administration in controlling oxidative stress-induced aging defects.

Aging phenotypes in BT administrated daf-2 and daf-16 mutants

Mutations in an Ins/IGF-like signaling pathway can lead to resistance to a variety of stresses including oxidative stress induced by paraquat [12,13]. To determine the possibility that the observed effects of BT administration were due to inhibition of this pathway, we further examined the lifespan in BT administrated daf-16 and daf-2 mutants. As shown in Fig. 7, administration with BT at concentrations of 25%, 50%, 75%, and 100% could prolong the mean lifespan of daf-16 mutant by 7%, 21%, 28%, and 42%, respectively, and prolong the maximum lifespan of daf-16 mutant by 7%, 15%, 27%, and 34%, respectively. Similarly, the defects of mean lifespan and maximum lifespan in daf-16 mutant could not be completely rescued by BT administration at different concentrations compared to those in wild-type animals (data not shown). More interestingly, administration with BT at concentrations of 25%, 50%, 75%, and 100% could also moderately prolong the mean lifespan of daf-2 mutant by 4%, 9%, 16%, and 24%, respectively, and prolong the maximum lifespan of daf-2 mutant by 4%, 8%, 13%, and 18%, respectively. Moreover, it was observed that the mean lifespan of BT administrated daf-16 mutant was significantly (P < 0.01) shorter than that of BT administrated wild-type animals, whereas the mean lifespan of BT administrated daf-2 mutant was significantly (P < 0.01) longer than that of BT administrated wild-type animals. Therefore, the protective effects of BT administration on aging process were at least partially dependent on the Ins/IGF-like signaling pathway in C. elegans.

Administration with BT after development conferred paraquat and UV irradiation resistance

We also considered the possibility that the BT affected development such that the resulting adult animals were resistant to UV and oxidative stresses. To exclude this possibility, we next investigated the effects of BT administration after development on lifespan of controls and UV irradiated or paraquat treated wild-type animals. As shown in Fig. 8, administration with BT at concentrations of 25%, 50%, 75% and 100% after development could markedly prolong the mean lifespan of wild-type animals by 9%, 14%, 19%, and 29%, respectively, and prolong the maximum lifespan of wild-type animals by 3%, 5%, 15%, and 23%, respectively. Similarly, administration with BT at concentrations of 25%, 50%, 75%, and 100% after development could obviously prolong the mean lifespan of UV irradiated wild-type animals by 26%, 87%, 115%, and 162%, respectively, and prolong the maximum lifespan of UV irradiated wild-type animals by 18%, 51%, 66%, and 88%, respectively. In addition, administration with BT at concentrations of 25%, 50%, 75% and 100% after development could noticeably prolong the mean lifespan of paraquat treated wild-type animals by 12%, 31%, 58%, and 100%, respectively, and prolong the maximum lifespan of paraquat treated wild-type animals by 18%, 40%, 66%, and 93%, respectively. Therefore, BT administration during development is not necessarily a requirement for UV and oxidative stress resistance.

BT was not toxic to the nematodes

Previous studies have suggested that non-lethal stresses can provide beneficial effects on stress resistance and longevity [8, 15]. To exclude the possibility that BT is mildly toxic and the observed increased stress resistance is due to an up-regulation of the cellular stress response, we then examined the cellular stress responses in BT administrated wild-type animals. As shown in Figure 4C, administration with BT at different concentrations would not alter the percentage of population with gfp expression in wild-type animals. In addition, it was observed that administration with BT at different concentrations throughout the development would not affect the brood size of treated wild-type animals, or reduce the brood size of treated mev-1, daf-2 and daf-16 mutant animals (data not shown). Therefore, BT administration was not toxic for examined nematodes.

Discussion

In the present study, we provide evidence to suggest that administration with BT, a clinical Chinese medicine used for POF treatment, can effectively alleviate the aging defects induced by UV and oxidative stresses in C. elegans. This observation is helpful for our understanding the pharmacology of this medicine in treating POF, and further modification of components in this medicine.

Administration with BT can slow the aging process

POF is a typical example for organ and tissue senescence involving a progressive decline in the number of oocyte-granulosa cell units to a critical threshold, and the phenotypic expression of POF is similar to that of age-appropriate menopause, although the underlying pathophysiological mechanisms are not entirely clarified [25, 41]. The pre-menopause is characterized by a significant reduction in the number of available oocyte-granulosa cell units, whereas at menopause there is nearly complete exhaustion of the oocyte pool [42]. Epidemiological data demonstrate that a higher oocyte mortality rate is associated with diminishing age at menopause [43]. Therefore, POF may represent an acceleration of the aging process in oocyte-granulosa cells, and slowing the aging process of ovary and oocyte is an important question for clinical treatment of POF.

In the current work, our data suggest that administration with BT at high concentrations could effectively prolong the lifespan of wild-type nematodes. In addition, administration with BT at high concentrations slowed the aging process, rather than simply improving survival at old age in C. elegans, since administration with BT at higher concentrations was associated closely with higher pumping rates at adult days 8 and 10. Especially, based on our assay on intestinal lipofuscin levels in BT treated wild-type animals, administration with BT at high concentrations could further delay the accumulation of aging-related cellular damage. Therefore, BT administration may alleviate the damage from POF by at least partially slowing the ovarian aging process and delaying the accumulation of aging-related cellular damage in ovarian tissue and oocytes.

Administration with BT may activate oxidative stress resistance

Anasti indicated that chemotherapy/irradiation is associated with exaggerated attrition of granulose cell-oocyte units by directing destroying dividing cells as well as by a secondary hit resulting from alteration of DNA, and it was found that usually the younger the patient, the lesser is the likelihood of complete cessation of gonad function [44]. Smoking has also been shown to accelerate the age related menopause by approximate 2 years [45]. Moreover, Nrf2 can protect against the ovarian toxicity of 4-vinylcyclohexene diepoxide (VCD) by controlling antioxidant response and ROS defense, and Nrf2-/- female mice exposed to VCD exhibited an age-dependent decline in reproduction leading to secondary infertility after 30 weeks of age, which suggest that exposure to oxidative or environmental stress may play an important role in the POF commonly associated with infertility and premature aging in women [26].

In the present study, the decreased expression levels of sod-1, sod-2, sod-3 and sod-4 in oxidative or UV stress treated animals could be noticeably enhanced by BT administration, and the increased expression levels of ctl-1 and ctl-2 in oxidative or UV stress treated animals could also be markedly reduced by BT administration at different concentrations from 25% to 100%, indicating that administration with BT at different concentrations could largely rescue deficits in expression patterns of antioxidant genes induced by UV and oxidative stresses in C. elegans. Furthermore, the high percentages of the population expressing hsp16-2-gfp induced by oxidative or UV stress could all be significantly decreased by BT administration at different concentrations, suggesting that BT-induced tolerance to UV or oxidative stress may result from ROS scavenging. These observations suggest that BT administration may be able to suppress the diversely adverse effects induced by oxidative stress in clinical treatment, and BT administration has an antioxidant property to a great degree. In addition, the beneficial effects from BT administration may also cause the resistance to other stresses, such as UV irradiation.

Administration with BT effectively alleviated the lifespan defects induced by oxidative stress

Although genetic influence remain the primary determinants of natural menopause, the environmental stress do impact on gonadal senescence. Oxidative stress theory, the most widely accepted theory explaining the molecular basis of aging, indicates that during normal metabolism, oxidative byproducts are inevitably generated and damage molecules thereby impairing their biological functions [2]. The stress-induced acceleration of reproductive senescence has been proposed as a theoretical model for POF [25]. In the present study, the mean lifespan and maximum lifespan in paraquat treated wild-type animals could be noticeably lengthened after administration with BT at concentrations of 25% to 100%. The intestinal lipofuscin levels in paraquat treated wild-type animals at day 14 after L4 could also be significantly reduced after administration with BT at concentrations of 25% to 100%. Especially, administration of BT at different concentrations could largely rescue the aging defects in mev-1 mutant animals. Therefore, BT administration can largely alleviate the aging defects induced by oxidative stress. The aging resistance to UV irradiation was also increased significantly by BT administration in wild-type nematodes. Our data support the idea that oxidative stress is a major cause of aging, since BT administration can obtain a kind of antioxidant property as discussed above. These observations further suggest that BT administration will be beneficial for alleviating the ovarian aging damage induced by oxidative stress, as well as pathophysiology. Nevertheless, we still do not know if simple upregulation of antioxidants or overexpression of antioxidant enzymes may extend nematode lifespan.

In addition, we noted that administration with BT could more significantly prolong the lifespans in oxidative or UV stress exposed animals than those in wild-type animals (data not shown). It was previously reported that the long-lived Ins/IGF mutants are expected to generate more ROS than wild-type, which seems to run counter to the oxidative stress theory of aging [2]. However, this conflict would be resolved if these mutants exhibit a strongly increased ROS degrading response. We propose that the increased ROS degradation activity might be activated in BT administrated animals subjecting to oxidative stress.

Genetic control activated by administration with BT in oxidative stress-induced lifespan

In C. elegans, the loss of function of the daf-16 gene decreases life span and stress resistance and ATP levels, and the long life of daf-2 and age-1 mutants is due to their greater resistance to oxidative stress [40]. In addition, the heterozygous Igf1r+/– mice lived on average 26% longer than their wild-type littermates, and displayed greater resistance to oxidative stress, suggesting that IGF-1 receptor also regulates lifespan and resistance to oxidative stress in mice [46]. Moreover, alterations in the neuro-endocrine pacemaker have been demonstrated in aging women suggesting its central contribution to the occurrence of natural age related menopause, and the alterations include an exaggerated response of the adrenals to stress with a rise in the basal cortisol levels [25, 47]. Here we provide evidence to demonstrate that the protective effects of BT administration on aging process are at least partially dependent on the Ins/IGF-like signaling pathway in C. elegans. The mean lifespans of BT administrated daf-16 mutant were markedly shorter than those of BT administrated wild-type animals, whereas the mean lifespans of BT administrated daf-2 mutant were obviously longer than those of BT administrated wild-type animals. These data suggest the possible involvement of Ins/IGF-like signaling pathways in the control of oxidative stress-induced ovarian aging in POF patients.

Moreover, because some evidence have been raised that the increased superoxide detoxification and low oxidative damage are sometimes not crucial for the longevity of the mutant nematodes [48], we suppose that, besides the mev-1 and insulin signaling identified, some other unknown signals may also be involved in the regulation of BT administration-mediated aging control.

Furthermore, Foxo3a, a member of FOXO subfamily of forkhead transcription factors in mice, also functions at the earliest stages of follicular growth as a suppressor of follicular activation, since Foxo3a-/- female mice exhibit a distinctive ovarian phenotype of global follicular activation leading to oocyte death, early depletion of functional ovarian follicles, and secondary infertility [49]. Therefore, insulin-like signaling may simultaneously regulate the ovarian aging and reproductive processes in POF patients.

Suggestions for the clinical use of BT administration in treating POF

It was shown that the concentrations of administrated BT examined in this project were not toxic for nematodes, as revealed by the assay on the brood size and aging process. Nevertheless, we suggest that the doses of BT used for specific POF populations or organisms should be advised with maximum limitation values. Similar question was also met for SCMs that high concentrations of SCMs appear to have toxic effects in housefly and C. elegans [50,51], although a number of other groups have investigated the effects of these compounds on aging and disease models with predominantly encouraging results [8]. Our data further demonstrated that BT administration during development is not necessarily a requirement for UV and oxidative stress resistance. Administration with BT at different concentrations after development could markedly lengthen the mean lifespan and maximum lifespan of wild-type animals, and UV irradiated or paraquat treated animals. This observation provides an important cue for developmental stage selection of clinical BT administration, and explains well the effectiveness of this medicine while administrating at different developmental stages in women.

In addition, since exogenous estrogen therapy may not have a uniformly beneficial effect on POF and there are many ways in which a woman might develop POF, it is not yet possible to predict the specific impact of this disease in clinical treatment [25, 52]. Our data suggests that the combination use of this medicine with specific antioxidant should be considered in clinical treatment to a great degree. In women with POF and uterine resistance to hormonal replacement therapy, combined treatment with 800 mg of pentoxifylline and 1000 IU of tocopherol daily for at least 9 months reduced fibroatrophic uterine lesions and improves the uterine response to hormonal replacement therapy, thus, allowing ongoing pregnancy [53]. Nevertheless, the added antioxidant(s) should still be tested for the suitable used amount, because our previous study suggested that high concentrations (400 μg/mL) of tocopherol exposure will result in adverse effects on memory behaviors [31].

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