SVHRSP Alleviates Age-Related Cognitive Deficiency by Reducing Oxidative Stress and Neuroinflammation

Abstract

Background: Our previous studies have shown that scorpion venom heat-resistant synthesized peptide (SVHRSP) induces a significant extension in lifespan and improvements in age-related physiological functions in worms. However, the mechanism underlying the potential anti-aging effects of SVHRSP in mammals remains elusive.

Methods: Following SVHRSP treatment in senescence-accelerated mouse resistant 1 (SAMR1) or senescence-accelerated mouse prone 8 (SAMP8) mice, behavioral tests were conducted and brain tissues were collected for morphological analysis, electrophysiology experiments, flow cytometry, and protein or gene expression. The human neuroblastoma cell line (SH-SY5Y) was subjected to H₂O₂ treatment in cell experiments, aiming to establish a cytotoxic model that mimics cellular senescence. This model was utilized to investigate the regulatory mechanisms underlying oxidative stress and neuroinflammation associated with age-related cognitive impairment mediated by SVHRSP.

Results: SVHRSP significantly ameliorated age-related cognitive decline, enhanced long-term potentiation, restored synaptic loss, and upregulated the expression of synaptic proteins, therefore indicating an improvement in synaptic plasticity. Moreover, SVHRSP demonstrated a decline in senescent markers, including SA-β-gal enzyme activity, P16, P21, SIRT1, and cell cycle arrest. The underlying mechanisms involve an upregulation of antioxidant enzyme activity and a reduction in oxidative stress-induced damage. Furthermore, SVHRSP regulated the nucleoplasmic distribution of NRF2 through the SIRT1-P53 pathway. Further investigation indicated a reduction in the expression of proinflammatory factors in the brain after SVHRSP treatment. SVHRSP attenuated neuroinflammation by regulating the NF-κB nucleoplasmic distribution and inhibiting microglial and astrocytic activation through the SIRT1-NF-κB pathway. Additionally, SVHRSP significantly augmented Nissl body count while suppressing neuronal loss.

Conclusion: SVHRSP could remarkably improve cognitive deficiency by inhibiting oxidative stress and neuroinflammation, thus representing an effective strategy to improve brain health.

Keywords: SVHRSP; Sirt 1 pathway; cognitive deficiency; neuroinflammation; oxidative stress.

Figure 1

SVHRSP reduced memory impairment and improved synaptic functions. (A–H) The effects of SVHRSP on behavioral testing. (A) The escape latency of the passive avoidance experiment; (B) the number of mice that entered the dark room in the passive avoidance test; (C) a device structure-style diagram of the Y Maze; (D) the percentage of spontaneous alternate behavior in the Y-maze trial; (E) the discrimination index of each group of mice in the NOR test; (F) the identification index of each group of mice in the NOR test; (G) the escape latency to reach the platform in the MWM training phase; (H) the number of mice crossing the platform in the MWM probe trials; (I,J) The effects of SVHRSP on long-term potentiation including slope change and amplitude change; (K) The effects of SVHRSP on the synaptic ultrastructure in the hippocampus; The red arrows indicate the presence of synaptic vesicles and postsynaptic dense bodies. (L–N) the representative Western blot bands and the quantification of relative protein expression for SYN and PSD95. The bars represent the mean ± SD. * p < 0.05, ** p < 0.01, ^# p < 0.05, versus the indicated groups.

Figure 2

SVHRSP downregulated the expression of aging markers. (A,B) Representative SA-β-gal staining images and quantitative analysis in SAMP8 mice. (C) Representative WB bands. (D,E) Quantitative analysis of P21 and P16 in vivo. (F–H) Quantitative analysis of GSH-PX, SOD, and MDA enzyme activity in vivo. (I,J) Representative SA-β-gal staining images and quantitative analysis in H₂O₂-induced SH-SY5Y cells. (K) Representative WB bands. (L,M) Quantitative analysis of P21 and p1 in vitro. (N,O) Quantitative analysis of cell cycle arrest in the G0/G1 phase. The cell fraction in the G0/G1 phase, G2/M phase, and S phase was calculated. The bars represent the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, ^# p < 0.05, versus the indicated groups (n = 3 for each group).

Figure 3

SVHRSP can inhibit the SIRT1/P53 signaling pathway, leading to enhanced nuclear translocation of NRF-2 and subsequently promoting the expression of antioxidants in SAMP8 mice. (A) Representative WB bands. (B,C) Quantitative analysis of SIRT1 and P53 in vivo. (D) Representative WB bands. (E,F) Quantitative analysis of SIRT1 and P53 in vitro. (G–J) Representative WB bands and quantification analysis of nuclear NRF2 and cytoplasmic NRF2. (K,L) Immunofluorescence images and quantification analysis of NRF2 cells/total cells (scale bar = 10 μm). (M) Representative WB bands. (N,O) Quantitative analysis of SOD 1 and HO-1 in vivo. (P–R) mRNA quantitative analysis of SOD 1, Hmox-1, and NQO1 in vivo. (S–W) Representative WB bands and quantitative analysis of SOD 1, HO-1, and NQO 1 in vitro. The bars represent the mean ± SD. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 versus the indicated groups (n = 3 for each group).

Figure 4

SVHRSP exerts inhibitory effects on neuroinflammation in SAMP8 mice by modulating the MAPKs/NF-κB signaling pathway. (A–D) Representative Western blot bands and quantitative analysis of cytoplasmic NF-κB p65 and nuclear NF-κB p65. (E–H) Representative Western blot bands and quantification analysis of p-JNK, JNK, p-P38, and P38. (I–K) Quantitative analysis of the serum level of IL-1B, IL-6, and TNF-α measured by using ELISA. (L–N) Quantitative analysis of the mRNA level of IL-1B, IL-6, and TNF-α measured by using QRT-PCR. (O) Representative immunohistochemical images of Iba-1 (left) and GFAP (right). (P,Q) Representative image and quantitative analysis of Nissl staining. The bars represent the mean ± SD. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus the indicated groups. Scale bar = 50 μm (n = 3 for each group).

Figure 5

SVHRSP alleviates age-related cognitive deficiency by reducing oxidative stress and neuroinflammation. SVHRSP can enhance the expression of the SIRT1 protein, reduce P53 activity, promote the nuclear translocation of the NRF2 transcription factor, and induce the expression of antioxidant enzymes such as SOD1, NQO1, and HO-1 to counteract oxidative stress. Furthermore, SVHRSP inhibits the expression of senescence markers P16 and P21 proteins. Additionally, by suppressing NF-κB pathway activation, SVHRSP suppresses the release of inflammatory factors IL-1β, TNF-α, and IL-6. (The graphical abstract is depicted by Figdraw 2.0.)