Enhanced Healthspan in Caenorhabditis elegans Treated With Extracts From the Traditional Chinese Medicine Plants Cuscuta chinensis Lam. and Eucommia ulmoides Oliv

Abstract

To uncover potential anti-aging capacities of Traditional Chinese Medicine (TCM), the nematode Caenorhabditis elegans was used to investigate the effects of Eucommia ulmoides and Cuscuta chinensis extracts, selected by screening seven TCM extracts, on different healthspan parameters. Nematodes exposed to E. ulmoides and C. chinensis extracts, starting at the young adult stage, exhibited prolonged lifespan and increased survival after heat stress as well as upon exposure to the pathogenic bacterium Photorhabdus luminescens, whereby the survival benefits were monitored after stress initiation at different adult stages. However, only C. chinensis had the ability to enhance physical fitness: the swimming behavior and the pharyngeal pumping rate of C. elegans were improved at day 7 and especially at day 12 of adulthood. Finally, monitoring the red fluorescence of aged worms revealed that only C. chinensis extracts caused suppression of intestinal autofluorescence, a known marker of aging. The results underline the different modes of action of the tested plants extracts. E. ulmoides improved specifically the physiological fitness by increasing the survival probability of C. elegans after stress, while C. chinensis seems to be an overall healthspan enhancer, reflected in the suppressed autofluorescence, with beneficial effects on physical as well as physiological fitness. The C. chinensis effects may be hormetic: this is supported by increased gene expression of hsp-16.1 and by trend, also of hsp-12.6.

Keywords: C. elegans; Cuscuta chinensis; Eucommia ulmoides; aging; healthspan; traditional Chinese medicine.

FIGURE 1

Heat stress resistance of C. elegans in the presence of different TCM extracts. C. elegans was treated with seven different TCM plant preparations (30 μg/ml), and DMSO as control, starting at the L4 stage. Heat stress (37 °C for 3 h) was applied at different ages of adulthood (3^rd, 7^th and 12^th day), and the survival after stress was monitored in the different treatment groups (A). The values represent the mean survival ± SEM from three biological replicates. Significant differences were determined by a log-rank test and Bonferroni correction, with *p < 0.05; **p < 0.01; ***p < 0.001 and ****p < 0.0001. In addition, representative survival curves are shown for nematodes treated with E. ulmoides and C. chinensis extracts, which were heat-stressed on the 3^rd (B), 7^th (C) and 12^th (D) day of adulthood.

FIGURE 2

Survival of C . elegans during exposure to the pathogenic bacterium P. luminescens.C. elegans was treated with seven different TCM plant preparations (30 μg/ml), and DMSO as control, starting at the L4 stage (A) Pathogenic stress due to P. luminescens exposure was initiated on the 3^rd or 7^th day of adulthood, and the survival during stress was monitored in the different treatment groups. The values represent the mean survival ± SEM from three biological replicates. Significant differences were determined by log-rank test and Bonferroni correction with *p < 0.05; **p < 0.01; ***p < 0.001 or ****p < 0.0001. In addition, survival curves are shown for nematodes treated with E . ulmoides and C . chinensis extracts, which were exposed to the pathogen starting on the 3^rd (B) and 7^th (C) day of adulthood (D) The growth of P. luminescens and (E) E . coli OP50 was monitored during extract exposure and the colony-forming units per time point are shown.

FIGURE 3

Effects of C. chinensis and E. ulmoides extracts on the lifespan of wild type C. elegans. (A) Representative survival curves as well as (B) the mean lifespan ± SEM from three biological replicates are shown; 100 µM quercetin was used as a positive control. Significant differences were determined by a log-rank test and subsequent Bonferroni correction with *p < 0.05; **p < 0.01; ***p < 0.001 or ****p < 0.0001.

FIGURE 4

Influences of E . ulmoides and C . chinensis extracts on (A) pharynx pumping rate and (B) intake of the E. coli strain OP50-GFP. Synchronized L4 larvae were transferred to NGM plates in the absence (control) or presence (30 μg/ml) of the respective plant extract. On the 7^th and 12^th days of adulthood, the pharyngeal pumping rate of individuals (n = 50) was measured under a stereoscopic microscope for 60 s. The results represent mean ± SEM and significant changes to the control are considered *(p < 0.05), **(p < 0.01), ***(p < 0.001) or ****(p < 0.0001) according to one-way ANOVA and post-hoc Bonferroni test.

FIGURE 5

Red autofluorescence of wild-type C. elegans treated with 30 μg/ml plant extract. On the 7^th and 12^th days of adulthood, treated and untreated nematodes were photographed in bright field as well as under red fluorescent light. (A) Representative control and C. chinensis-treated nematode on the 12^th day of adulthood (B) The fluorescence intensity of individuals was determined by densitometric analysis using the CellProfiler software. The results are the average intensity for two independent experiments (n ≥ 25 per experiment). The intensity was represented as mean ± SEM and the significant changes to the control are considered *(p < 0.05), **(p < 0.01), ***(p < 0.001), or ****(p < 0.0001) according to one-way ANOVA and post-hoc Bonferroni test.

FIGURE 6

Swim performance of C. elegans after C. chinensis and E. ulmoides treatment. Wave initiation rate, body wave number, brush stroke and activity index were determined on the 7th and 12th day of adulthood. Error bars are the standard error of the mean (SEM) and one bar represents n ≥ 50 from two independent trials. Statistical significance was determined according to one-way ANOVA and post-hoc Bonferroni test as *(p < 0.05), **(p < 0.01), ***(p < 0.001), or ****(p < 0.0001).

FIGURE 7

Impact of E. ulmoides and C. chinensis on body size and reproduction of C. elegans. (A) Body size of C. elegans on the 7^th and 12^th day of adulthood after TCM treatment (n ≥ 25 per experiment). (B) The number of offspring per day and in total during exposure to TCM treatment (n = 27–30 nematodes per treatment). The results display the average size for three independent experiments and error bars represent the SEM. Statistically significant differences to the control are considered with *p < 0.05 according to one-way ANOVA and post-hoc Bonferroni test.

FIGURE 8

Effect of C. chinensis and E. ulmoides treatment on the gene expressions levels of heat-shock proteins at the 12^th day of adulthood. Gene expression was determined via qPCR, and expression values were calculated according to Pfaffl (2001). Gene expression values of the reference genes act-1 and cdc-42 were used to normalize the data. The graph shows the mean of three biological replicates. The error bars show the standard error of the mean (SEM) and statistical significance was determined according to one-way ANOVA and post-hoc Bonferroni test with *(p < 0.05).