Orientin Prolongs the Longevity of Caenorhabditis elegans and Postpones the Development of Neurodegenerative Diseases via Nutrition Sensing and Cellular Protective Pathways

Abstract

Age is the major risk factor for most of the deadliest diseases. Developing small molecule drugs with antiaging effects could improve the health of aged people and retard the onset and progress of aging-associated disorders. Bioactive secondary metabolites from medicinal plants are the main source for development of medication. Orientin is a water-soluble flavonoid monomer compound widely found in many medicinal plants. Orientin inhibits fat production, antioxidation, and anti-inflammatory activities. In this study, we explored whether orientin could affect the aging of C. elegans. We found that orientin improved heat, oxidative, and pathogenic stress resistances through activating stress responses, including HSF-1-mediated heat shock response, SKN-1-mediated xenobiotic and oxidation response, mitochondria unfolded responses, endoplasmic unfolded protein response, and increased autophagy activity. Orientin also could activate key regulators of the nutrient sensing pathway, including AMPK and insulin downstream transcription factor FOXO/DAF-16 to further improve the cellular health status. The above effects of orientin reduced the accumulation of toxic proteins (α-synuclein, β-amyloid, and poly-Q) and delayed the onset of neurodegenerative disorders in AD, PD, and HD models of C. elegans and finally increased the longevity and health span of C. elegans. Our results suggest that orientin has promising antiaging effects and could be a potential natural source for developing novel therapeutic drugs for aging and its related diseases.

Figure 1

Orientin increases the longevity of C. elegans and slows the aging-related phenotypes. (a) The chemical structure of orientin. (b) The survival curves of the wild-type (N2) worms cultured at 20°C on NGM plates containing 0, 25, 50, 100, and 200 μM of orientin, respectively. (c) Dose-response analysis of the effect of orientin on the longevity in C. elegans. The assays were independently performed at least three times. (d) Aging-related movements of N2 worms treated with or without 100 μM of orientin. The mean body bending is in Table S2. (e) The intestinal autofluorescence of lipofuscin was analyzed on the 10th day of adulthood. The results of the mean lipofuscin accumulated are summarized in Table S3. Life span was analyzed by using the SPSS package and Kaplan-Meier, and p values were calculated by using the log-rank test. These results are represented as mean ± standard error of the mean (SEM), p < 0.05 was considered statistically significant, and detailed life span values are presented in Table S1 (supplementary information).

Figure 2

Orientin improves the resistance of heat stress. (a) The survival curves of N2 worms at 35°C. The detailed results are shown in Table S4. (b) QPCR analyses of the targeted genes of hsf-1 (hsp-12.6, hsp-16.1, hsp16.2, hsp-6, and hsp-60) and itself in the wild-type N2 worms exposed to 100 μM of orientin. The detailed results are in Table S9. (c) The survival curves of PS3551 hsf-1(sy441) I at 35°C. The detailed results are shown in Table S4. (d) The survival curves of PS3551 treated with or without 100 μM of orientin at 20°C. The statistical details of the mutant with error bars representing SEM are presented in Table S4.

Figure 3

Orientin enhances the antioxidant capacity of C. elegans. (a) The survival curves of N2 nematodes exposed to paraquat (20 mM) and orientin (100 μM); then, the death of individuals was counted every day and is summarized in Table S4. (b) Quantitation of intracellular level of ROS in N2 worms. The positive control is 2 mM of PQ (paraquat), and the negative control is 1 mM of NAC (N-acetyl-cysteine). (c) The accumulation of SOD-3::GFP in CF1553 treated with or without orientin for 7 days. (d) The representative pictures of SKN-1::GFP in LD1 transgenic worms treated with or without 100 μM of orientin for 7 days. These pictures were photographed by using a fluorescence microscope (Leica DFC 7000T) and examined by using the software ImageJ. (e) The survival curves of EU1 skn-1(zu67) IV treated with or without medicine (100 μM of orientin) at 20°C. Statistical details and repeats of these assays are presented in Tables S4 and S5.

Figure 4

Orientin improves the resistance of pathogenic stress and increases the expression of heat shock proteins. (a) The survival curves of N2 worms fed with P. aeruginosa (PA14). (b) The mRNA expression levels of immune-related genes F55G11.4 and irg-1. (c) The mRNA expression levels of genes pgp-8, ire-1, xbp-1, ubl-5, pek-1, atf-6, atfs-1, aak-2, and sir-2.1 in the nutrition sensing signal pathway. (d) The pictures of green fluorescence in the transgenic strain SJ4005 expressing HSP-4 were captured by using a fluorescence microscope (Leica DFC 7000T) and analyzed by using the software ImageJ. (e) The image and quantitation of the protein HSP-6 in SJ4100 worms. The detailed results are presented in Tables S4, S5, and S9. Each of these assays was repeated independently at least three times (mean ± SD; Student's t-test; n ≥ 20; ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001).

Figure 5

Orientin enhances autophagy activity. (a) The fluorescence intensity of SQST-1::GFP was analyzed at day 3 adulthood. The results with error bars representing SD are presented in Table S5. (b) The mRNA expression levels of genes bec-1 and lgg-1 expressed in autophagy. The columns represent the mean value of three independent experiments with error bars representing SD in Table S9. (c) The life span analysis of atg-18 mutant treated with or without 100 μM of orientin. The statistical details of the mutant with error bars representing SEM are presented in Table S7. (d) The survival curves of JIN1375 hlh-30(tm1978) IV treated with or without orientin (100 μM) at 20°C. The statistical details of the mutant with error bars representing SEM are summarized in Table S4. Each of these experiments was repeated independently at least three times.

Figure 6

Orientin delays the progression of neurodegenerative diseases in models of C. elegans. (a) The aggregation of α-synuclein in NL5901 treated with 100 μM of orientin was captured with a fluorescence microscope (Leica DFC 7000 T) and analyzed by using the software ImageJ. The results are presented in Table S5. (b) Aging-related movements of NL5901 on the 5th and 10th days. The mean body bending could be found in Table S6. (c) T5he fluorescence intensity of the head dopamine neurons in worms BZ555 rescued by orientin and NAE after 6-OHDA induction. After the induction of 6-OHDA, the fluorescence intensity was significantly reduced. 30 worms per condition were analyzed in each independent test, and the statistical details and the results of three repeated experiments are presented in Table S5. (d) The paralysis counts of the transgenic strain CL4176 at 25°C. Error bars represent SEM. The mean life span of CL4176 is summarized in Table S7. (e) The poly-Q protein aggregation of AM141 with or without orientin (100 μM); the detailed results are summarized in Table S5. These experiments were each performed at least three times (mean ± SD; Student's t-test; n ≥ 30; ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001).

Figure 7

Orientin depends on FOXO/DAF-16 to prolong the longevity of C. elegans. (a–e) The survival curves of daf-2, age-1, akt-1, akt-2, and daf-16, mutants in the absence or presence of orientin (100 μM). These results are represented as mean ± standard error of the mean (SEM). The results were considered statistically significant when p < 0.05. Statistical details of the longevity of the mutants and repeats of these experiments are presented in Table S7. (f) Effect of orientin on the nuclear localization of DAF-16. The representative fluorescence photomicrograph of transgenic TJ356 worms with cytosolic, intermediary, and nuclear staining. (g) The mRNA expression levels of genes downstream of daf-16 (ctl-1, ctl-3, sod-3, and dod-3) and itself in N2 worms exposed to orientin (100 μM) versus control worms. The columns are shown in Table S9.

Figure 8

The effect of orientin on the energy metabolism and fat metabolism of C. elegans. (a) The survival curves of eat-2 mutants in the NGM plates with or without orientin. The results are shown in Table S7. (b) The ADP : ATP ratio of N2 nematodes cultured with or without orientin at 20°C for 3 days was determined by HPLC. The figures are exhibited in Table S8. (c–e) The survival curves of aak-2, sir-2.1, and daf-15 mutants untreated or treated with orientin (100 μM) at 20°C. The statistical details of these mutants are presented in Table S7. (f) QPCR analysis of fat-related genes (fat-1, fat-3, fat-6, acs-2, and lipl-4) in the N2 worms treated with orientin (100 μM) versus control worms. The columns are shown in Table S9. (g) The relative Oil Red O intensity of wild-type N2 worms treated or untreated with orientin for 3 days was calculated by using ImageJ. (h) The relative Nile red fluorescence intensity of N2 worms cultured with or without orientin for 3 days was analyzed by using ImageJ. These experiments were each performed at least three times (mean ± SD; Student's t-test; n ≥ 30; ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001).

Figure 9

The effect of orientin on the mitochondrial and reproductive signaling pathway of C. elegans. (a–e) The survival curves of clk-1, isp-1, mev-1, glp-1, and daf-12 mutants in the absence (0 μM) or presence (100 μM) of orientin. Orientin could not further extend the life span compared with the control group (p > 0.05). The statistical details of these mutants with error bars representing SEM are summarized in Table S7.