Figure 1.
Icariin and its bioactive form icariside II extend lifespan in C. elegans.
A. Chemical structures of icariin and its derivatives. The common structure is 8-prenylkaempferol. R1 and R2 are substituted by rhamnose (rha) or glucose. Removal of R1 (Rha) results in icariside I, while removal of R2 results in icariside II. Removal of both R1 and R2 results in icaritin. B. Survival curves of N2 hermaphrodites treated with DMSO control or icariin (15, 45 and 75 µM) from day 1 adulthood to death at 25°C. Maximum increase in lifespan was observed at 45 uM. C. Survival curves of N2 treated with DMSO control or icariside II (10, 20 and 40 µM). Maximum increase in lifespan was observed at 20 µM. D. Optimum dosage-response analysis of icariin (45 µM) and icariside II (20 µM). Icariside II treatment induces the similar extension with relatively lower dosage compared to icariin. E. HPLC profiles of icariin and icariside II in N2 treated with 45 µM icariin for 4 days initiated from day 1 in adulthood. HPLC detects high level of icariside II in vivo. Small figures (insert) are HPLC profiles of standard samples of icariin and icariside II. All the data above came from 1 representative experiment. For lifespan assay, the treatments were started at day 1 in adulthood continuing to death. Statistical detail and repetitions of the experiments were summarized in Table S1.
Figure 2.
Icariside II promotes stress resistance and slows age related decline in movement in C. elegans.
A. Icariside II pre-exposure elevated the N2 adults' survival significantly under thermostress. Animals were pre-treated with the DMSO control (1%) or 20 µM icariside II starting from day 1 adulthood for four days. Shown is the combination of two replicates. Mean survival time, DMSO control, 9.7 hrs; icariside II, 11.3 hrs. Animals tested: 87 (DMSO control); 95 (icariside II). P = 0.0151 (Log-rank (Mantel-Cox) Test). B. Pre-treatment with icariside II increased oxidative stress resistance significantly. Day 1 wild type adults were treated with DMSO control or 20 µM icariside II for 4 days and then soaked in different concentrations of H2O2 (10, 15, 20 mM) for 2 hrs. Survival percentages were scored after a 16 hours recovery window in regular NGM. Shown are average survival percentages in 3 experiments with 30–50 animals/experiment. Total number of animals tested: 102 (DMSO Control), 113 (icariside II 20 µM); error bars indicate SEM among three independent experiments; t-test, *P<0.05. C. Treatment with icariside II extends swimming healthspan in advanced age; Average swimming bends per 30 seconds in 15 animals were scored in two independent trials; error bars indicate SEM among individual animals scored; t-test, **P<0.01. D. Icariside II treatment slows the age-related deterioration of C. elegans body wall muscle. Age-related deterioration of C. elegans body wall muscle is indicated by GFP fluorescence decline of a pmyo-3NLS::GFP fusion in the strain PD4251. Representative images of whole-animals treated with DMSO control and icariside II are presented at the ages indicated (25°C). E. Quantitation of the number of fluorescent nuclei in PD4251 treated with DMSO or icariside II. 15 animals of each strain were scored in two independent trials. Error bars indicate SEM among individual animals scored; t-test, ***P<0.001.
Figure 3.
Icariside II ameliorates protein aggregation and protetoxicity-mediated paralysis phenotype.
A. Representative images of the whole-animal of Q35-YFP (Q35) transgenic strain AM140 treated with DMSO or icariside II from forth-stage larvae at 20°C are shown. AM140 shows an increasing aggregation phenotype age-dependently. Icariside II treatment slows the aggregation process obviously. B. Quantitation of the number of the fluorescent aggregation in AM140 treated with DMSO or icariside II. 15 animals of each strain were scored in two independent trials. Error bars indicate SEM among individual animals scored; t-test, ***P<0.001. C. Age associated paralysis caused by Q35 expression is significantly reduced in icariside II treated animals relative to DMSO control animals. Animal were treated under the conditions from forth-stage larvae until paralysis. Shown is the representative of two replicates. n = 38 (control); 37 (icariside II) animals, P = 0.0151 (Log-rank (Mantel-Cox) Test). D. The paralysis phenotype associated with muscle Aβ1–42 expression is suppressed by 20 µM icariside II treatment from hatching and E. L3 in the transgenic strain CL4176. Shown is the non-average paralysis percentages in 3 independent experiments with 30–50 animals/experiment in indicated time points after temperature upshift to 25°C; error bars indicates SEM among the non-paralysis percentages of three independent experiments; total number of animals tested: 112 (DMSO Control), 133 (icariside II 20 uM); t-test, *P<0.05, **P<0.01.
Figure 4.
Increased lifespan in C. elegans by Icariside II treatment is dependent on the insulin/IGF-1 signaling.
A. Survival curves of N2, daf-2(e1370), and daf-16(mu86) hermaphrodites treated with DMSO control or 20 µM icariside II are shown. Icariside II treatment does not increase the life span in daf-2(e1370) and daf-16(mu86) mutant but extends the life span in N2. This is the representative of 3 independent experiments with similar results. Detailed parameters are presented in Table S2. B. Icariside II increases the mRNA expression of FOXO/DAF-16 targets hsp12.3 and sod3 significantly. Shown is the representative of three independent experiments with similar results. Error bars indicate SEM among three replicates in qRT-PCR; t-test, ***P<0.001. C. Representative images of the whole-animal of SOD-3::GFP transgenic strain CF1553 treated with DMSO or 20 uM icariside II from day 1 in adulthood to day 4 are shown. Icariside II increased the GFP fluorescence significantly compared to DMSO control. D. Western blot analysis shows an increase of GFP induced by 20 uM Icariside II treatment in SOD-3::GFP transgenic strain CF1553. Picture shown is the representative of three independent experiments with similar results. Levels shown are the average relative density level in 3 experiments; error bars indicate SEM among the density level of three experiments; t-test, **P<0.01. E. Effect of icariside II is HSF-1 dependent. Survival curves of hsf-1 (sy441) treated with DMSO control and 20 uM icariside II are shown. Icariside II did not increase the lifespan in hsf-1 (sy441) mutant.
Figure 5.
Icariside II may not function as DR mimetic.
Survival curves of A. eat-2 (ad1113), B. rsks-1 (ok1255) hermaphrodites treated with DMSO control or 20 uM Icariside II is shown. Icariside II treatment caused similar lifespan extension in these mutants as in N2 animals. This is the representative of 2 independent experiments. Detailed parameters are presented in Table S3.