Scorpion Venom Heat-Resistant Synthesized Peptide Increases Stress Resistance and Extends the Lifespan of Caenorhabditis elegans via the Insulin/IGF-1-Like Signal Pathway

Abstract

Improving healthy life expectancy by targeting aging-related pathological changes has been the spotlight of geroscience. Scorpions have been used in traditional medicine in Asia and Africa for a long time. We have isolated heat-resistant peptides from scorpion venom of Buthusmartensii Karsch (SVHRP) and found that SVHRP can attenuate microglia activation and protect Caenorhabditis elegans (C. elegans) against β-amyloid toxicity. Based on the amino acid sequence of these peptides, scorpion venom heat-resistant synthesized peptide (SVHRSP) was prepared using polypeptide synthesis technology. In the present study, we used C. elegans as a model organism to assess the longevity-related effects and underlying molecular mechanisms of SVHRSP in vivo. The results showed that SVHRSP could prolong the lifespan of worms and significantly improve the age-related physiological functions of worms. SVHRSP increases the survival rate of larvae under oxidative and heat stress and decreases the level of reactive oxygen species and fat accumulation in vivo. Using gene-specific mutation of C. elegans, we found that SVHRSP-mediated prolongation of life depends on Daf-2, Daf-16, Skn-1, and Hsf-1 genes. These results indicate that the antiaging mechanism of SVHRSP in nematodes might be mediated by the insulin/insulin-like growth factor-1 signaling pathway. Meanwhile, SVHRSP could also up-regulate the expression of stress-inducing genes Hsp-16.2, Sod-3, Gei-7, and Ctl-1 associated with aging. In general, our study may have important implications for SVHRSP to promote healthy aging and provide strategies for research and development of drugs to treat age-related diseases.

Keywords: Caenorhabditis elegans; SVHRSP; aging; insulin-IGF-1 like signal pathway; stress resistance.

FIGURE 1

Scorpion venom heat–resistant synthesized peptide (SVHRSP) extends the lifespan of C. elegans. (A) survival curve of C. elegans N₂ at 20°C treatment with 20-, 40-, 100-, 150-, or 200-μmol/L SVHRSP or with ddH₂O (control) starting from the L4 stage (day 0), the survival number of worms in each group was recorded until all worms died; 40-μMol/L SVHRSP appeared to extend the longest lifespans (***p < 0.001, log-rank test). (B) effects of SVHRSP on food intake in N₂ worms. Surplus E. coli OP50 was detected by OD 600. Data were analyzed by ordinary one-way analysis of variance (ANOVA) using Prism 9.0. Values are presented as mean ± SEM. *p < 0.05. (C) representative images of C. elegans showing the accumulation of autofluorescent lipofuscin treatment with SVHRSP or not. The scale bar was 200 μm. Total fluorescence per worm was analyzed using ImageJ software. Data were analyzed by ordinary one-way ANOVA using Prism 9.0. Values are presented as mean ± SEM; 20-μM SVHRSP: *p < 0.05; 40-μM SVHRSP: ***p < 0.001.

FIGURE 2

Scorpion venom heat–resistant synthesized peptide (SVHRSP) significantly increased the resistance of nematodes to stress. (A) mean survival time of wild-type worms treated with 20- and 40-μM SVHRSP at 30% H₂O₂. Log-rank verifies the calculated p-value. *p < 0.05 (B) the mean survival time of wild-type worms treated with 20- and 40-μM SVHRSP at 37°C. Log-rank verifies the calculated p-value. *p < 0.05 (C) effects of 20- and 40-μM SVHRSP on reactive oxygen species levels in wild-type worms. (D) the effect of SVHRSP on fat accumulation. SVHRSP significantly inhibited the accumulation of excess fat in N₂ nematodes. Values are presented as mean ± SEM. All of these measurements were made at least three times. Log-rank and one-way analysis of variance verifies the calculated p-value. ns means none significant; *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 3

Scorpion venom heat–resistant synthesized peptide (SVHRSP)-mediated longevity effects depended on the insulin/IGF-1-like signal (IIS) pathway. (A) effects of 20- and 40-μM SVHRSP untreated treatments on the lifespan of CF1038 nematodes. (B) effects of 20- and 40-μM SVHRSP treatments on the lifespan of CB1370 nematodes. (C) Fluorescence images of TJ356 nematodes treated with 20-μM and 40-μM SVHRSP, and untreated under heat stress. (D) Daf-16 mRNA expression levels of wild-type nematodes treated with 20- and 40-μM SVHRSP untreated. Values are presented as mean ± SEM. All of these measurements were made at least three times. Log-rank and one-way analysis of variance verifies the calculated p-value. ns means none significant; *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 4

Scorpion venom heat–resistant synthesized peptide (SVHRSP) regulates downstream gene expression of the IIS pathway. (A) effects of 20- and 40-μM SVHRSP untreated treatments on the lifespan of PS3551 nematodes. (B) -effects of 20- and 40-μM SVHRSP untreated treatments on the lifespan of EU1 nematodes. (C) (a) fluorescence images of TJ375 nematodes treated with 20- and 40-μM SVHRSP, and untreated under heat stress. (b) the GFP strength from the TJ375 strain was treated with 20- and 40-μM SVHRSP. (c) Hsp-16.2 mRNA levels in worms treated with 20- and 40-μM SVHRSP and controls. (D) (a) fluorescence images of CF1553 nematodes treated with 20- and 40-μM SVHRSP, and untreated. (b) the GFP intensity of CF1553(muIs84) strain after 20- and 40-μM SVHRSP treatment was determined. (c) Sod-3mRNA levels in worms treated with 20- and 40-μM SVHRSP and controls. (E) (a) Ctl-1 mRNA levels in worms treated with 20- and 40-μM SVHRSP and controls. (b) Gei-7 mRNA levels in worms treated with 20- and 40-μM SVHRSP and controls. Values are presented as mean ± SEM. Log-rank and one-way ANOVA. ns means none significant; *p < 0.05; **p < 0.01; ***p < 0.001. All of these measurements were made at least three times.