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. 2021 Jul 20;12(1):301-308.
doi: 10.1515/tnsci-2020-0178. eCollection 2021 Jan 1.

Tripchlorolide attenuates β-amyloid generation by inducing NEP activity in N2a/APP695 cells

Affiliations

Tripchlorolide attenuates β-amyloid generation by inducing NEP activity in N2a/APP695 cells

Yuqi Zeng et al. Transl Neurosci. .

Abstract

Background and purpose: Alzheimer's disease (AD) is a neurodegeneration disease. The previous work from our research group demonstrated the neuroprotective effects of tripchlorolide (T4) in AD animal models.

Materials and methods: Neprilysin (NEP) is known as an important physiological amyloid-β protein (Aβ) peptide-degrading enzyme in the brain due to its apparent rate-limiting function. In this study, we explored the effect of NEP on AD model N2a/APP695 cells. Western blots and enzyme-linked immunosorbent assays were performed to assess the expression of proteins, while quantitative real-time polymerase chain reaction assays were used to evaluate RNA levels. Cell vitality was detected by the MTT assay, and reactive oxygen species (ROS) levels were assessed using a ROS activity assay kit.

Results: We discovered that T4 was able to enhance the enzyme activity of NEP. T4 administration decreased the protein levels of the soluble amyloid precursor protein. In further experiments, we found that by using thiorphan the secretion of Aβ, oxidative stress, nitrosative stress, and inflammatory factors, which were suppressed by T4, were reversed. Due to its ability to attenuate Aβ generation and to protect neurons against the neurotoxicity of Aβ, T4 may be a potential therapy in the regulation of Aβ-related pathology in AD by affecting NEP activity.

Conclusion: Tripchlorolide attenuates Aβ generation by inducing NEP activity in N2a/APP695 cells.

Keywords: amyloid-β protein, N2a/APP695 cells; neprilysin; tripchlorolide.

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Conflict of interest statement

Conflict of interest: The authors state no conflict of interest.

Figures

Figure 1
Figure 1
T4 upregulated NEP mRNA and protein levels in N2a cells. N2a/WT and N2a/APP695 cells were treated with 5 nM or 10 nM T4 for 24 h. RT-PCR and western blots were used to determine NEP mRNA expression (a) and NEP protein expression (b). * P < 0.05, compared with untreated cells; # P < 0.05, compared with 5 nM T4-treated cells.
Figure 2
Figure 2
The NEP inhibitor, thiorphan, induced cell death in T4-treated N2a/APP695 cells. (a) The NEP specific inhibitor, thiorphan (500 nM), was used to suppress the NEP enzyme activity in N2a/WT or N2a/APP695 cells following T4 treatment. Western blots were used to detect NEP expression. * P < 0.05, compared with control cells; # P < 0.05, compared with thiorphan-treated cells. (b) NEP activity was detected following the manual protocol. * P < 0.05, compared with T4-untreated control cells; # P < 0.05, compared with T4-treated control cells. (c) MTT assay was performed to assess cell viability. * P < 0.05, compared with N2a/WT cells; # P < 0.05, compared with control group of N2a/APP695 cells.
Figure 3
Figure 3
NEP activation involved in T4 reduced Aβ secretion in N2a/APP695 cells. (a) The NEP specific inhibitor, thiorphan (500 nM), was used to suppress the NEP enzyme activity in N2a/WT or N2a/APP695 cells following T4 treatment. Western blotting was used to detect sAPPβ expression. (b) Aβ1-42 and (c) Aβ1-40 levels in the supernatant were also detected by ELISA. *P < 0.05, compared with untreated cells; # P < 0.05, compared with thiorphan-treated cells.
Figure 4
Figure 4
NEP activation involved in T4 reduced ROS and NO production in N2a/APP695 cells. Thiorphan (500 nM) was used to inhibit the NEP enzyme activity in N2a/APP695 cells following T4 treatment. ROS (a) and NO (b) levels in the supernatant were detected. * P < 0.05, compared with untreated cells; # P < 0.05, compared with thiorphan-treated cells.
Figure 5
Figure 5
NEP activation involved in T4 reduced neuroinflammation in N2a/APP695 cells. Thiorphan (500 nM) was used to suppress the NEP enzyme activity in N2a/APP695 cells following T4 treatment. Levels of inflammatory factors in the supernatant were accessed by ELISA: (a) level of IL-1β and (b) level of TNF-α. * P < 0.05, compared with untreated cells; # P < 0.05, compared with thiorphan-treated cells.

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