fj.03-0376fje.full.pdf

1、 The FASEB Journal express article10.1096/fj.03-0376fje. Published online October 2, 2003. Expression of the small heat-shock protein Hsp-16-2 in Caenorhabditis elegans is suppressed by Ginkgo biloba extract EGb 761 Amy Strayer,* Zhixin Wu,* Yves Christen, Christopher D. Link, and Yuan Luo* *Departm

2、ent of Biological Sciences, The University of Southern Mississippi, Hattiesburg, MS; Beaufour Ipsen, Paris, France; Institute for Behavioral Genetics, University of Colorado, Boulder, CO Corresponding author: Yuan Luo, Department of Biological Sciences, 2609 West 4th Street, The University of Southe

3、rn Mississippi, Hattiesburg, MS 39406-5018. E-mail: ABSTRACT EGb 761, a standardized extract of Ginkgo biloba leaves, has been shown to have antioxidative properties. We have previously demonstrated that EGb 761 increases stress resistance and mean life span in the model organism Cae

4、norhabditis elegans. In this study, the molecular mechanism of EGb 761 on alleviating effects of oxidative stress is further investigated using transgenic C. elegans expressing a jellyfish green fluorescent protein (GFP)-tagged inducible small heat-shock protein gene (hsp-16-2). The expression of hs

5、p-16-2 induced by the pro-oxidant juglone and by heat shock was significantly suppressed by 86% and 33%, respectively, in the transgenic nematode fed with EGb 761. These effects of EGb 761 correlate with its ability to increase mean survival rate of the nematode in response to acute oxidative and th

6、ermal stresses, as well as to attenuate the basal levels of hydrogen peroxide in the organism. Thus, we interpret the suppression of hsp-16-2/GFP expression as an indication that EGb 761 decreases cellular stress resulting from exogenous treatments, therefore leading to a decreased transcriptional i

7、nduction of the reporter transgene. These results support the hypothesis that EGb 761 augments the natural antistress system of C. elegans, thus increasing stress resistance and life span. Key words: GFP reporter ROS oxidative stress ll organisms have developed the ability to respond to environmenta

8、l threats by the synthesis of highly conserved stress response proteins. A universal response to elevated temperature and other forms of stress is the induction of heat-shock proteins (HSPs). These include HSP-100, HSP-90, HSP-70, HSP-60, HSP-40, HSP-30, and the small HSPs (sHSPs) (1). The sHSPs bel

9、ong to a family of low molecular weight polypeptides (1243 kD) that have been highly conserved from yeast through humans and show sequence as well as functional similarities to the lens B crystallins (2). Under physiological conditions, sHSPs are among the most highly inducible HSPs during thermal s

10、tress or oxidative stress. Their expression correlates specifically with the presence of stressors (3) that induce protein damage (4). In the nematode C. elegans, 16 sHSPs have been identified. The major sHSPs of the 16 kD species (hsp-16-1, hsp-16-2, hsp-16-41, and hsp-16-48) are only expressed und

11、er stress conditions (5). A mutagenesis study of the hsp-16-2 promoter has demonstrated that HSF, and possibly other A transcription factors, control hsp-16-2 induction in response to heat shock (6). To follow sHSP regulation in vivo, we constructed transgenic reporter strains (e.g., CL2070) in whic

12、h the transcription of a jellyfish green fluorescent protein (GFP) is driven by the promoter of the hsp- 16-2 gene (7). Expression of the GFP in the CL2070 reporter strain parallels endogenous expression of hsp-16-2 protein, and thus the response of this strain to various stressors can be quantitati

13、vely assayed in living animals. The standard Ginkgo biloba leaf extract EGb 761 is a popular dietary supplement taken by the general population to enhance mental focus and by the elderly to delay the onset of age-related loss of cognitive function. During the past decade, in vivo and in vitro experi

14、ments in mammalian systems and clinical studies in humans demonstrated that EGb 761 exhibits a range of biochemical and pharmacological effects that include cognition enhancement and stress alleviation (8). In human studies, available data have confirmed the clinical efficacy of EGb 761 in primary d

15、egenerative dementia of Alzheimers type (911). Some data support the view that the extract enhances learning and longevity in rats (12) and has neuromodulatory and neuroprotective properties in several species (1316). However, the evidence of an effect on memory in healthy humans is still inconclusi

16、ve (17); some studies found an effect (18), and others did not (19). Currently there are no data about longevity in humans. We have previously reported that EGb 761 protects cultured neuronal cells from stress-induced cell death (20, 21), increases stress resistance and mean life span in model C. el

17、egans (22), and serves as a stress buffer in experimental mice (23). Because sHSPs are a family of molecular chaperones that are expressed only under stress conditions, we asked whether EGb 761 affects the expression of these proteins in C. elegans. Therefore, we used the CL2070 transgenic GFP repor

18、ter strain to visualize sHSP expression in living animals in real time. Here, we demonstrate that the stressor-induced expression of hsp-16-2 is suppressed in C. elegans fed with EGb 761, suggesting a modulatory role of the extract in the function of a stress-response gene. Furthermore, with the com

19、bination of the survival assay and the assay for levels of oxidative free radicals in C. elegans, our results indicate that the effect of EGb761 on the modulation of hsp- 16-2 expression is beyond its known function as a scavenger for oxidative free radicals. MATERIALS AND METHODS Gingko biloba The

20、standardized leaf extract EGb 761, consisting of two major active constituents (8), flavonols (24%) and terpene lactones (6%), was provided by Schwabe Pharmaceuticals (Karlsruhe, Germany). The isoflavone constituent was a gift from Beaufour IPSEN (Paris, France). The terpene lactones, including GA,

21、GB, GC, GJ, and BB, were obtained from Dr. Ikhlas Khan of the National Center for Natural Products Research (University, MS) (24). Stock solutions of EGb 761 (1000) were made in 100% ethanol. The final concentration of ethanol did not exceed 0.01% in the food (Escherichia coli strain OP50). Juglone,

22、 5-hydroxy-1,4-napthoquinone, and L- ascorbate, were obtained from Sigma Pharmaceuticals (St. Louis, MO). Caenorhabditis elegans strains, maintenance, and treatment The wild-type N2 strain was obtained from the Caenorhabditis Genetics Center, University of Minnesota (Minneapolis, MN). The transgenic

23、 strain hsp-16-2/GFP (CL2070) was generated and characterized by Dr. C. Link as described previously (7). CL2070 contains a jellyfish GFP reporter transgene that is under the control of the promoter for the sHSP gene hsp-16-2. The HSP hsp-16-2 is expressed by either heat shock (35C for 2 h) or by ex

24、posure to juglone (40 M for 24 h), a quinone known to induce superoxide radicals in C. elegans. All nematodes were cultivated on nematode growth medium (NGM) agar on 60 mm Petri plates and maintained at 20C in a temperature-controlled incubator. E. coli (OP50) was the food source and was added to th

25、e surface of NGM plates at 100 l. To obtain age-synchronized nematodes, we left the self- fertilizing hermaphrodites to lay eggs (for 48 h) on their third day of life and then removed them to set the eggs in synchrony. For the pretreatment protocol, the nematodes were fed EGb 761 on the day after ha

26、tching (except life span assay) for 48 h, and the stressors were applied on their third day of life when they passed into the adult stage. Stock solutions of juglone were made as 1 mg/1 ml in 100% ethanol. Juglone was mixed freshly with NGM to reach the desired final concentration and dried to admin

27、ister the stressor exogenously through the skin. Fluorescence microscopy and quantitation of hsp-16-2 expression Using the CL2070 strain, we used thermal or oxidative stress to induce hsp-16-2 gene expression. For heat shock, the hsp-16-2/GFP worms, synchronized and maintained as stated above, were

28、exposed to 35C for 24 h. Worms were then allowed to recover in their normal environment at 20C for 12 h before pictures were taken. The oxidative stress involves exposing the worms to 40 M juglone for 24 h. After both inductions, the expression of hsp-16-2 was measured by directly observing the fluo

29、rescence of the reporter GFP. Epifluorescence images were acquired at the same exposure parameter using the 40 objective of a microscope (BX 60, Olympus, Tokyo, Japan) equipped with a digital camera (Micropublisher 5.0, QIMAGING, Burnaby, BC, Canada). For quantifying a population of GFP reporter ani

30、mals, each 40 image was analyzed using Image-ProPlus 4.51 software (MediaCybernetics, Silver Spring, MD). Survival assays Thermotolerance was measured using a heat stressor. Worms (3 dishes of 30 worms each) were treated with 100 g/ml EGb 761 for 48 h and were then exposed to 35C for 24 h. The numbe

31、r of survivors was counted every hour until all were dead. Oxidative stress was induced by an acute, lethal concentration of juglone at 160 M. Worms were first treated with 100 g/ml EGb 761 on the day after hatching and then were transferred to fresh dishes after 2 days of treatment. Juglone was add

32、ed to liquefied NGM at 65C and then pipetted to 35 mm Petri plates. After the plates had solidified, OP50 was added at 70 l and the plates were dried in a fume hood. Worms were transferred within an hour of preparing the dishes and were counted every 30 min for survival until all were dead. They wer

33、e scored dead if they did not respond to a touch stimulus. Analysis of oxidative free radicals Intracellular hydrogen peroxide (H2O2)-related reactive oxygen species (ROS) were measured in C. elegans using 2,7-dichlorofluorescein diacetate (DCF-DA; Molecular Probes). Nonfluorescent DCF-DA is a cell-

34、permeable dye that is readily converted to 2,7-dichlorofluoroscein (DCF) by interacting predominantly with hydrogen peroxide (25). Age-synchronized C. elegans treated with or without EGb 761 on the day after hatching for 72 h were collected into 100 l PBST (PBS containing 0.1% Tween 20) with 30 worm

35、s from each group and 3 groups per treatment. The worms were then subjected to timed homogenization (Pellet Pestle Motor, MG Scientific) and sonication (Branson Sonifier 250, VWR Scientific) to break up the outer cuticle. Samples were vortexed, transferred into 96-well plates, and incubated with 50

36、M DCF-DA in PBS at 37C in a fluorescent microplate reader (Bio-Tek Instruments, Winookski, VT) for quantification of fluorescence at excitation 485 nm and emission 640 nm. Samples were read kinetically every 10 min for 2.5 h. Statistical analyses Statistical comparison between treatments was done wi

37、th unpaired Students t test using Origin 6.0 software (Microcal Software, Northampton, MA). Standard error of the mean was used in the figures. Differences of P0.05 were defined as statistically significant. RESULTS Stress-induced expression of hsp-16-2/GFP is suppressed in the transgenic C. elegans

38、 fed with EGb 761 We have previously shown that EGb 761 increases stress resistance in the model C. elegans (22). To determine whether this is due to EGb 761 regulating a specific stress-response gene, we used the transgenic C. elegans (CL2070) expressing GFP as a reporter transgene for inducible hs

39、p-16- 2 expression. Figure 1A shows the phenotype of wild-type (a) and the transgenic strain CL2070 (b), in which the hsp-16-2/GFP gene expression was induced by a rise in temperature from 20C to 35C for 2 h. Upon induction by thermal stress, the GFP fluorescence is visible at the head of the worm,

40、including the pharynx and the anterior nerve ring in the transgenic worm (b) but not in the wild-type controls (a). The expression of hsp-16-2 induced by heat shock was significantly suppressed by 33% in CL2070 worms fed with EGb761 (control, GFP mean pixel density 75 vs. EGb-treated, GFP mean pixel

41、 density 50, n=72 worms, P0.05, Fig. 1B). Figure 1B insets: representative hsp-16- 2/GFP expression induced by heat shock in CL2070 worms untreated (a) or treated with 100 g/ml EGb 761 (b). Exposure of the transgenic worms to 40 M juglone, an oxidative stressor, for 24 h generated higher hsp-16-2 ex

42、pression (Fig. 1C, inset a) than that induced by heat shock (Fig. 1B, inset a). Shorter exposure time to juglone also induced hsp-16-2 expression but to a lesser degree: an 11% and 30% induction were observed in worms exposed to juglone for 6 h and 12 h, respectively (24 h exposure set as 100%, data

43、 not shown). Most importantly, the juglone-induced expression of hsp-16-2 was remarkably attenuated by 86% in worms pretreated with 100 g/ml EGb 761, compared with untreated controls (EGb761, GFP mean pixel density 180 vs. control GFP mean pixel density 25, n=120 worms, P0.001, Fig. 1C). Similar res

44、ults were obtained in worms exposed to a higher concentration of juglone (60 M, data not shown). It is possible that the attenuation of hsp-16-2/GFP expression by EGb 761 could be due to the drugs interference with reporter GFP expression per se. To exclude this possibility, we used a chromosomally

45、integrated transgenic line (CL1234) that constitutively expresses GFP from the synaptobrevin snb-1 pan-neuronal promoter to serve as a control. The CL1234 worms were treated with the same concentration of EGb 761 for the same time as CL2070 worms, and GFP fluorescence was measured subsequently by fl

46、uorescence microscopy. Figure 1D insets show the representative GFP expression in control (a) and EGb 761-treated (b) CL1234 worms. For statistical analysis of GFP expression affected by EGb 761 treatment, only the anterior neurons within the head region of the worms were outlined and analyzed. From

47、 three independent experiments of 30 worms each, the fluorescence density was not significantly affected by the treatment of the CL1234 worms with EGb 761 (Fig. 1D graph), strongly suggesting that the attenuation of hsp-16-2/GFP by EGb 761 is not an artifact. EGb 761 treatment increased survival rat

48、e in transgenic C. elegans under stress conditions To provide further evidence that the attenuation of hsp-16-2 expression by EGb 761 treatment of the animals is a beneficial effect, we conducted survival assays in CL2070 worms treated with or without EGb 761 before exposure to either a thermal or a

49、n oxidative stressor. Figure 2A demonstrates that pretreatment of the CL2070 worms with 100 g/ml EGb 761 increased their survival in response to the heat shock (percent survival at 15 h, control 15.0% 10.1 vs. EGb 48.6% 0.9, n=189 worms). Figure 2B shows that pretreatment of the CL2070 worms with 10

50、0 g/ml EGb 761 increased their survival rate in response to exposure to the pro-oxidant juglone (160 M) (mean survival: control 4.6 h vs. EGb 5.6 h, n=179 worms). This result is consistent with our previous observation in the wild-type C. elegans treated with EGb 761 (22) and further indicates that

51、the attenuation of hsp-16-2/GFP expression by EGb 761 does not cause abnormal physiological changes, which may affect GFP reporter gene expression. Postjuglone treatment with EGb 761 suppressed the expression of hsp-16-2 EGb 761 has been known to have dual antioxidative actions, with its flavonoid c

52、onstituent able to scavenge oxidative frees radicals and its ginkgolide constituent able to prevent the formation of free radicals (8). To test any poststress effects of EGb 761 against oxidative damage, we treated the worms with EGb 761 either simultaneously with or 24 h after exposure to 40 M jugl

53、one to induce hsp-16-2/GFP expression. Figure 3A shows that EGb 761 suppressed hsp-16-2 expression induced by concomitant juglone treatment by 66% (control, GFP mean pixel density 240 vs. EGb 761, GFP mean pixel density 80, n=2, P0.05, total of 30 worms), and hsp-16-2 expression induced by postjuglo

54、ne treatment by 50% (control, GFP mean pixel density 240 vs. EGb 761, GFP mean pixel density 120, n=2, P0.05, total of 35 worms). These results suggest that EGb 761 may function downstream of juglone-generated damage, because not only does it prevent stressor-induced hsp-16-2 gene expression, but it

55、 also suppresses gene expression after the damage has been done. If the suppression of juglone-induced hsp-16-2 expression by EGb 761 is due to its antioxidative actions, then other antioxidants should exhibit the same effect. To further characterize the involvement of antioxidative properties of EG

56、b 761 in the modulation of hsp-16-2 expression, we pretreated the worms with the known antioxidants L-ascorbic acid (vit C, 100 g/ml) or the flavonoid fractions of EGb 761 (FLV, 100 g/ml) before exposure to 40 M juglone. Figure 3B demonstrates that juglone-induced hsp-16-2 expression was not signifi

57、cantly suppressed by either L-ascorbic acid or the flavonoid fractions, even with the higher concentration of flavonoids than present in the whole extract (note that the attenuation of hsp-16-2 expression by vitamin C is close to being significant but still less than that seen with EGB 761). EGb 761

58、 attenuated intracellular levels of hydrogen peroxide in C. elegans Oxidative free radicals have been postulated as a cause of aging and of some degenerative diseases (26, 27). Profound induction of hsp-16-2 expression by juglone (Fig. 1C) suggests that the sensing of oxidative stress triggers the i

59、nduction of sHSP expression. Recently, we were able to measure H2O2-related ROS levels in C. elegans, and we observed an age-dependent increase in the levels of H2O2 and an increased level of H2O2 in a transgenic C. elegans model expressing the A peptide (28). To monitor the ROS levels in the transgenic C. elegans CL2070 treated with EGb 761, we performed a DCF-DA fluorescence assay. Although we were unable to detect an increase in ROS induced by juglone in these worms, we consistently observed (Fig. 4) that in the CL2070 C.