Anti‐ageing effects of red ginseng on female Drosophila melanogaster

Abstract Red ginseng (RG) was recently reported to extend the lifespan of Drosophila melanogaster. However, the mechanism underlying this effect has not yet been elucidated. The present study aimed to elucidate the molecular mechanisms of the RG‐mediated prolongation of the lifespan of female D melanogaster. In this study, protein changes in 36‐day‐old female D melanogaster were identified using isobaric tag for relative and absolute quantitation (iTRAQ), and levels of differentially expressed proteins were verified by quantitative real‐time PCR and Western blotting. Our studies have shown that RG concentrations of 12.5, 15 and 17.5 mg/mL significantly prolonged the lifespan. Eleven proteins were up‐regulated and 46 were down‐regulated between the RG and control groups; and Pebp1 expression was significantly down‐regulated. In addition, AKT and p‐AKT were down‐regulated, and ERK, p‐ERK and Raf1 were up‐regulated by RG. Therefore, RG significantly prolonged the lifespan of female D melanogaster by reducing the expression of Pebp1, up‐regulating ERK and inhibiting the AKT pathway. RG may be a potential drug for anti‐ageing treatment.


| Lifespan analysis of female Drosophila melanogaster
Wild-type D melanogaster was obtained from Jilin Agricultural University (Changchun, China). Single populations (200 flies each) of control-female D melanogaster were fed a basal food containing water. The RG group was fed the basal food supplemented with RG.

| Protein preparation
D melanogaster (36 days old, 20 flies each) was anaesthetized and collected. Samples were ground into fine powder in liquid nitrogen and then dissolved in SDT buffer (4% sodium dodecyl sulphate, 0.1 mol/L; dithiothreitol, 100 mmol/L; and Tris-HCl, pH 7.6).
The peptides were desalted on MILI-SPE Extraction disc cartridge (C18-SD), lyophilized and added to 40 µL of dissolution buffer.

| LC-MS/MS proteomic analysis
Each sample was injected for nano LC-MS/MS analysis coupled to an EASY nLC (Thermo Fisher Scientific). The sample was loaded into a Thermo Scientific Acclaim PepMap100 column (100 μm × 2 cm, nanoViper C18) using an automatic sampler and connected to an analytical column (Thermo Fisher Scientific EASY Column; 10 cm, ID75 μm, 3 μm, C18-A2) in buffer A (0.1% formic acid) and buffer B (84% acetonitrile and 0.1% formic acid) at a flow rate of 300 nL/min. LC-MS/MS analysis was performed using an Q Exactive mass spectrometer (Thermo Fisher Scientific).   Table 1).

| Western blotting
Female D melanogaster (36 days old, 20 flies each) of the control and RG groups was anaesthetized and collected. Pebp1, spartin, Ent2, CG9062, Tim17b and TSG101 differentially expressed proteins were selected for verification using Western blotting. The method referred to the lab's previous approach. 1 GAPDH (Proteintech) was used as a loading control.

| Statistical analyses
The comparison was made using Student's t-test. A P value of < .05 was considered statistically significant.

Gene
Primer sequences HOU et al.

| Lifespan analysis of female Drosophila melanogaster
Treatment with 10 mg/mL RG did not significantly extend the lifespan of female D melanogaster, while treatment with 12.5, 15 and 17.5 mg/mL RG significantly extended the lifespan of the flies (P = .0356, .0426 and .0015). However, 20 mg/mL RG reduced the lifespan of female D melanogaster ( Figure 1A).

| Identification of differentially expressed proteins
Using a threshold of >1.2 or <0.83 (P ≤ .05), 11 proteins were up-regulated and 46 proteins were down-regulated. Clustering heatmaps and volcano plot were used to identify significant changes in protein expression ( Figure 1B).

| Bioinformatics analysis of differentially expressed proteins
The differentially expressed proteins were subjected to gene ontology (GO)-based enrichment analysis using the Blast2GO program and Fisher's exact Test (P ≤ .05; Figure 1C).

F I G U R E 1 Effect of RG on the lifespan of Drosophila melanogaster.
A, Effects on the lifespan of RG-treated D melanogaster. B, The identified differentially expressed proteins by using clustering heatmaps and volcano plot. C, GO term annotation analysis of the differential proteins. D, Validation of differential proteins by qRT-PCR. E, Expression levels of Pebp1, spartin, Ent2, CG9062, Tim17b and TSG101 were confirmed using Western blotting. F, Expression levels of Raf1, ERK, p-ERK, AKT, p-AKT, mTOR and p-mTor were confirmed using Western blotting. G, Expression levels of EGFR were confirmed using Western blotting. Significance analysis was indicated by student's t-test (**P < .01, *P < .05). RG, red ginseng; Con, control; Pebp1, phosphatidyl ethanolamine-binding protein 1; AKT, protein kinase B; ERK, extracellular regulated protein kinases; mTOR, mechanistic target of rapamycin; EGFR, epidermal growth factor receptor

| Effect of Pebp1 on the ERK pathway and AKT/ mTOR pathway
Based on the experimental results of iTRAQ, qRT-PCR and Western blotting, Pebp1 was identified as an important protein among the identified differentially expressed proteins. Pebp1 is an inhibitor of Raf1, a kinase of the Raf/MEK (MAP/ERK kinase)/ERK signalling pathway, and mainly plays a role in cell proliferation and differentiation. 6,7 Moreover, Pebp1 reportedly regulates many other signalling pathways, such as AKT/mammalian target of rapamycin (mTOR). 8 We assessed the protein levels of Raf1, ERK, phosphorylated (p)-ERK, AKT, p-AKT, mTOR and p-mTOR by Western blotting.
Compared with the control group, RG treatment down-regulated the levels of AKT and p-AKT (P = .0412), and up-regulated the levels of ERK, p-ERK (P = .0204), and Raf1; in contrast, the expression levels of mTOR and p-mTOR remained unchanged ( Figure 1F).
icant variation of spartin. Spartin is present in organisms ranging from nematodes and flies to humans, 11 and it participate in endocytosis by interacting with epidermal growth factor receptor pathway substrate 15 (Eps15). 12 Knockout or overexpression of Spartin leads to a decrease in the degradation rate of epidermal growth factor receptor (EGFR), which affects the internalization of EGFR. EGFR degradation impairs endocytosis and synaptic growth, which may underlie the pathogenesis of Troyer syndrome. 13,14 Presently, Western blotting revealed the significantly lower level of spartin in the RG group compared to the level in the control group, with EGFR level remaining the same in the RG group ( Figure 1G). We suggest that EGFR is normally degraded or recycled without affecting downstream signalling pathways. Therefore, integrated iTRAQ, qRT-PCR and Western blotting analyses were used to comprehensively analyse the protein changes in 36-day-old (old-age) female D melanogaster. RG significantly prolonged the lifespan of the flies by reducing the expression of Pebp1, up-regulating ERK and inhibiting the AKT pathway. Therefore, RG may be a potential drug for anti-ageing treatment.

ACK N OWLED G EM ENTS
This work was supported by grants from the Jilin Science & Technology Development Plan (No.20180101261JC).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest.

F I G U R E 2
The possible mechanism of anti-ageing effects induced by RG in Drosophila melanogaster. RG, red ginseng; Con, control; Pebp1, phosphatidyl ethanolamine-binding protein 1; AKT, protein kinase B; ERK, extracellular regulated protein kinases; mTOR, mechanistic target of rapamycin; EGFR, epidermal growth factor receptor