1School of Life Science and Technology, Tongji University, Shanghai 200092, China 2Shenqi Institute for Ethnomedicine, Tongji University, Shanghai 200092, China 3School of Medicine, Tongji University, Shanghai 200092, China 4Shanghai Engineering Research Center for Model Organisms, SRMOC/SMOC, Shanghai 201203, China 5Standard Testing Lab (Shanghai) Co., Ltd., Pudong, Shanghai 201203, China
Liuwei Dihuang (LWDH), a famous traditional Chinese medicine, is widely used in the clinical treatment of aging-related diseases in China. However, its pharmacological mechanisms are not clear. In the present study, we evaluated the lifespan extension effect of LWDH in C. elegans and mice and revealed its underlying mechanisms. The results showed that LWDH significantly extended the lifespan of C. elegans in a dose-dependent manner. LWDH also conferred protection to nematodes against oxidative stress and reduced their fat storage. Genetics analysis and microarray data showed that the longevity effect of LWDH was attributed to the regulation of the innate immune response, proteolysis, lipid metabolism, and the oxidation-reduction process and was dependent on daf-16. Among the six herbs in the formula, Radix Rehmanniae Preparata and Fructus Macrocarpii contributed most to the longevity effect of this medicine, while the other four components had a synergistic effect on the longevity effect of the prescription. The lack of any single herb reduced the efficacy of the complete formula. LWDH also extended the lifespan and reduced both the weight and oxidant stress status in aged mice. Taken together, these results suggested that LWDH might function in a multi-target manner to extend the lifespan in both C. elegans and aged mice, and the best effect was achieved with the complete formula.
Table 1 List of the primers used in the present study.
Mean life span ± SEM (days)
Number of animals
20.41 ± 1.010
23.74 ± 0.8559
26.97 ± 1.031
< 0.001 (***)
28.98 ± 1.321
< 0.001 (***)
29.84 ± 1.549
< 0.001 (***)
26.85 ± 2.496
< 0.001 (***)
7.368 ± 1.157
< 0.001 (***)
3.393 ± 0.2096
< 0.001 (***)
50 μM Mianserin
29.06 ± 0.9107
< 0.001 (***)
Table 2 LWDH extended the lifespan of C. elegans at different concentrations.
Figure 1. LWDH extended the lifespan of wild-type C. elegans at different concentrations
LWDH at different concentrations or 50 μM mianserin (positive control) were added into the medium on the first day of adulthood. The survival of worms was monitored every two days until all of the worms were dead.
Figure 2. LWDH improved the resistance of C. elegans against stress
Worms were pretreated with 1 mg/ml LWDH or not for five days. Subsequently, the worms were subjected to oxidative and heat stress. For oxidative stress, the worms were exposed to 50 mM paraquat (A) or 10 mM K2Cr2O7 (B), and for heat stress, the worms were transferred from 20 °C to 35 °C (C). The statistical significance between curves with different time points was calculated by two-way ANOVA. (D-F) LT50 was calculated and compared between worms pretreated with LWDH or not.
Figure 3. LWDH decreased the fat storage in C. elegans. Fat storage was measured with Nile Red after a five-day treatment in the absence (A) or presence (B) of LWDH. (C) Statistical comparisons between the two groups. (D) Relative expression levels of lipl-4 on day 5 after LWDH treatment.
Figure 4. The effects of LWDH in aging-related mutants
eat-2(ad465) (A), daf-2(e1370) (B), daf-16(mu86) (C) and wild-type N2 were treated with 1 mg/ml LWDH or not on the first day of adulthood to evaluate the effect of lifespan extension.
Representative images for the localization phenotype of DAF-16::GFP in transgenic TJ356 strain after a ten-day treatment in the absence (A) or presence (B) of LWDH, and the worms maintained at 35 °C for 2 h were regarded as the positive control (C). The GFP fluorescence intensity (D) and the number of DAF-16::GFP particles (E) were measured according to the images.
Figure 6. LWDH extended the lifespan of C. elegans in a multi-target manner
(A) The symbol of C and D in the group names represented worms treated with the absence (control) or presence (1 mg/ml) of LWDH, respectively. Days 10 and 22 of adulthood were represented as 10 and 22, respectively. DEGs were analyzed between groups, and numbers of up- or downregulated genes were provided. (B) GO analysis was performed with the downregulated genes in C22 vs. C10 and terms of biological process (BP) were shown. (C) The heat map showed the expression pattern of DEGs from C22 vs. C10 and D22 vs. C22. Red denoted higher expression level, and green denoted lower expression level. (D) Dynamic expression pattern of DEGs from C22 vs. C10 and D22 vs. C22, and genes were classified into 8 clusters. Numbers on top left corner indicated the cluster number. Numbers on bottom left corner indicated the number of DEGs. The colored clusters (1, 2 and 6) indicated significantly enriched ones by DEGs (P < 0.05). (E) GO analysis showed the BP terms of DEGs from cluster 2.
Figure 7. The effects of individual herbs and formulas lacking one herb in wild-type C. elegans
The complete formula, each individual herb (the first six graphs) or formulas consisting of five herbs (the rest ones, herb with (-) at the end represented its lacking in the formula) were added to the medium on the first day of adulthood. Black lines represented the control group, red lines represented the complete formula, and green lines represented the individual herbs or formulas used. The survival of the worms was monitored every two days until all of the worms were dead.
Figure 8. LWDH extended the lifespan of aged mice
(A) Mice that were 22-23 months old were treated with different doses of LWDH. Day 1 represented the first day of taking LWDH. n = 9, 7, and 10 in control, low-dose and high-dose group, respectively. (B) Body weight was measured three times a week and compared between the different groups or between the first and last week in the same group. (C, D) The 21-month-old mice were treated with the low dose of LWDH. Serum SOD and MDA were measured after a 12-week treatment. n = 8 in each group.
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