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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 2, 2003 as doi:10.1096/fj.03-0376fje. |
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Department of Biological Sciences, the University of Southern Mississippi, Hattiesburg, Mississippi, USA;
Beaufour Ipsen, Paris, France; and
Institute for Behavioral Genetics, University of Colorado, Boulder, Colorado, USA
2Correspondence: Department of Biological Sciences, 2609 West 4th St., The University of Southern Mississippi, Hattiesburg, MS 39406-5018, USA. E-mail: yuan.luo{at}usm.edu
SPECIFIC AIMS
Small heat-shock protein hsp-16-2 is expressed only under conditions of stress. Ginkgo biloba extract EGb 761 has been demonstrated to have anti-oxidative and anti-stress effects. The original aim of this study was to delineate anti-stress mechanisms of EGb 761 in vivo. We used the hsp-16-2/GFP transgenic Caenorhabditis elegans strain to visualize the modulation of hsp-16-2 expression by EGb 761 in living animals in real time.
PRINCIPAL FINDINGS
1. Stress-induced expression of hsp-16-2/GFP was suppressed in the transgenic C. elegans fed with EGb 761
We previously showed that EGb 761 increases stress resistance in the model C. elegans. 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 hsp-16-2 expression. hsp-16-2/GFP gene expression was induced by the rise in temperature from 20°C to 35°C for 2 h observed as GFP fluorescence at the head of the transgenic worm, but not in wild-type controls. Induced expression of hsp-16-2 by heat shock was significantly suppressed by 33% in CL2070 worms fed with EGb 761 (n=total of 72 worms, P<0.05, Fig. 1
A). Exposure of transgenic worms to 40 µM juglone, a pro-oxidative stressor, for 24 h generated higher hsp-16-2 expression (Fig. 1B
, inset a) than that induced by heat shock (Fig. 1A
, inset a). The juglone-induced expression of hsp-16-2 was remarkably attenuated by 86% in worms pretreated with 100 µg/mL EGb 761 compared with untreated controls (n=total of 120 worms, P<0.001, Fig. 1B
).
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2. EGb 761 treatment increased survival rate in transgenic C. elegans under stress conditions
To provide evidence that EGb 761 attenuation of hsp-16-2 expression is beneficial, we conducted survival assays in CL2070 worms treated with or without EGb 761 prior to either a thermal or an oxidative stressor. Figure 2
A, B demonstrates that pretreatment of the CL2070 worms with 100 µg/mL EGb 761 increased their survival in response to the heat shock (n=189 total worms, Fig. 2A
) and to 160 µM juglone exposure (n=179 worms, Fig. 2B
). Thus, we interpret the suppression of hsp-16-2/GFP expression as an indication that EGb 761 decreases cellular stress resulting from exogenous treatments.
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3. Postjuglone treatment with EGb 761 suppressed the expression of hsp-16-2
To evaluate any poststress effects of EGb 761 against oxidative damage, the worms were treated with EGb 761 simultaneously with or 24 h after exposure to 40 µM juglone to induce hsp-16-2/GFP expression. EGb 761 suppressed hsp-16-2 expression induced by co-juglone treatment by 66% and by 50% when induced by postjuglone treatment. These results suggest that EGb 761 may function downstream of juglone-generated damage because it not only prevents stressor-induced hsp-16-2 gene expression, but also suppresses gene expression after the damage has been done. To further characterize the involvement of anti-oxidative properties of EGb 761 in the modulation of hsp-16-2 expression, worms were pretreated with known antioxidants L-ascorbic acid (100 µg/mL) or flavonoid fractions of EGb 761 (100 µg/mL), followed by exposure to 40 µM juglone. Juglone-induced hsp-16-2 expression was not significantly suppressed by either L-ascorbic acid or the flavonoid fractions, even with a higher concentration of flavonoids than present in the whole extract.
4. EGb 761 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. Profound induction of hsp-16-2 expression by juglone (Fig. 1B
) suggests that the sensing of oxidative stress triggers induction of hsp-16/GFP expression. To monitor ROS levels in the transgenic C. elegans strain CL2070, we performed a DCF-DA fluorescence assay. In the CL2070 C. elegans treated with EGb 761, basal ROS levels were significantly attenuated by 24%. By comparison, in CL2070 worms treated with L-ascorbic acid, H2O2-related ROS levels were attenuated by 31% and, in worms fed with the flavonoids of EGb 761, by 30%.
CONCLUSIONS AND SIGNIFICANCE
Our results demonstrate that expression of the hsp-16-2/GFP reporter gene induced by thermal and oxidative stressors was significantly suppressed by EGb 761 in the C. elegans strain CL2070 (Fig. 1)
. This effect of EGb 761 correlated with its ability to increase the organism’s resistance to thermal and oxidative stressors (Fig. 2)
. Thus, we interpreted the suppression of hsp-16-2/GFP reporter transgene expression to indicate that EGb 761 decreased cellular stress resulting from exogenous stressors. Furthermore, our results suggested that this effect of EGb 761 is beyond its known function as a scavenger for oxidative free radials. The GFP reporter of hsp-16-2 is an advantage in such studies, and a combination of specific gene regulation by EGb 761 with functional survival assays and oxidative free radical measurements in whole organism provide important insight into the functional interaction of cellular processes modulated by EGb 761. It provides novel evidence to support the hypothesis that EGb 761 augments the natural antioxidant system of C. elegans, thus increasing the organism’s resistance to stress.
Although the functional role of sHSPs in physiological conditions has yet to be clarified, the consequence of EGb 761 modulating hsp-16-2 expression is beneficial. If the function of hsp-16-2 is protective against stress, EGb 761 might protect against the deleterious process that leads to a decreased need for hsp-16-2 expression. Conceivably, strong sHSP expression could have deleterious longer term cellular consequences, to the point where EGb 761 attenuation of hsp-16-2 expression is directly beneficial. The results described here do not address the functional interaction between EGb 761 and hsp-16-2, but it is possible that EGb 761 functions downstream of an applied stressor. This is supported by our observation that EGb 761 was able to suppress the expression of hsp-16-2 after 24 h of juglone exposure. A better understanding of the mechanisms of EGb 761 is important in order to clarify the underlying stress response processes and to evaluate the effectiveness and complexity of this herbal medicine.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0376fje; doi: 10.1096/fj.03-0376fje 
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