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. 2018 Jul 18;24:4982-4991.
doi: 10.12659/MSM.909056.

Collapsin Response Mediator Protein-2 Ameliorates Sevoflurane-Mediated Neurocyte Injury by Targeting PI3K-mTOR-S6K Pathway

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Free PMC article

Collapsin Response Mediator Protein-2 Ameliorates Sevoflurane-Mediated Neurocyte Injury by Targeting PI3K-mTOR-S6K Pathway

Jiaxuan He et al. Med Sci Monit. .
Free PMC article

Abstract

BACKGROUND Collapsin response mediator protein-2 (CRMP-2) is the first member of the CRMP family that has been identified in primary neuronal cells; it was originally found and identified in the regulation of microtubule dimerization into microtubules. MATERIAL AND METHODS In the present study, we aimed to investigate the roles and mechanisms of CRMP-2 in sevoflurane-induced neurocyte injury. Cell viability, proliferation, and apoptosis were measured by Cell Counting Kit-8 (CCK-8) assay and flow cytometry. Colorimetry was performed to measure the activity of caspase-3. Western blot and quantitative real-time reverse transcription assays were used to evaluate the related mRNAs and proteins expression. RESULTS We found that CRMP-2 reversed the inhibitory effect of sevoflurane on the viability of nerve cells. Moreover, CRMP-2 accelerated the proliferation and suppressed the apoptosis of sevoflurane-induced nerve cells. CRMP-2 modulated the expression levels of apoptosis-associated protein in sevoflurane-induced nerve cells. Furthermore, it was demonstrated that CRMP-2 impacted the PI3K-mTOR-S6K pathway. CONCLUSIONS CRMP2 ameliorated sevoflurane-mediated neurocyte injury by targeting the PI3K-mTOR-S6K pathway. Thus, CRMP2 might be an effective target for sevoflurane-induced neurocyte injury therapies.

Conflict of interest statement

Conflict of interest

None.

Figures

Figure 1
Figure 1
Identification of the rat neurocytes. Primary cultured rat neurocytes for 2 days (A) and 6 days (B) were observed by inverted fluorescence microscope. Magnification 200×; n=3.
Figure 2
Figure 2
Sevoflurane (SEV) restrained the cell viability of nerve cells, and collapsin response mediator protein-2 (CRMP-2) was overexpressed in nerve cells transfected with CRMP-2. (A) CCK-8 assay was carried out to measure the cell viability of nerve cells treated with different concentration of SEV mixed gas (0.5%, 1.0%, 2.0%, 3.0%, 4.0%, and 5.0%). qRT-PCR (B) and western blot (C) assays were performed on the expression level of CRMP-2 in nerve cells, nerve cells transfected with empty vector, nerve cells transfected with CRMP-2, nerve cells treated with 3% SEV mixed gas, nerve cells transfected with empty vector and then treated with SEV, and nerve cells transfected with CRMP-2 and then treated with SEV. * P<0.05 and ** P<0.01 versus NC; ## P<0.01 versus NC+SEV; n=3.
Figure 3
Figure 3
Collapsin response mediator protein-2 (CRMP-2) enhanced the cell viability of nerve cells suffered from sevoflurane (SEV). CCK-8 assay was carried out to measure the cell viability of nerve cells, nerve cells transfected with empty vector, nerve cells transfected with CRMP-2, nerve cells treated with 3% SEV mixed gas, nerve cells transfected with empty vector and then treated with SEV, and nerve cells transfected with CRMP-2 and then treated with SEV. * P < 0.05 versus NC; # P < 0.01 versus NC+SEV; n=3.
Figure 4
Figure 4
The cell proliferation of sevoflurane (SEV)-induced nerve cells was promoted by transfecting with collapsin response mediator protein-2 (CRMP-2). Flow cytometry was performed on the cell proliferation of nerve cells, nerve cells transfected with empty vector, nerve cells transfected with CRMP-2, nerve cells treated with 3% SEV mixed gas, nerve cells transfected with empty vector and then treated with SEV, and nerve cells transfected with CRMP-2 and then treated with SEV. * P<0.05 versus NC; # P<0.05 versus NC+SEV; n=3.
Figure 5
Figure 5
PAP suppressed the apoptosis of nerve cells suffered from sevoflurane (SEV). Flow cytometry was carried out to detect the apoptosis ability of nerve cells, nerve cells transfected with empty vector, nerve cells transfected with collapsin response mediator protein-2 (CRMP-2), nerve cells treated with 3% SEV mixed gas, nerve cells transfected with empty vector and then treated with SEV, and nerve cells transfected with CRMP-2 and then treated with SEV. * P<0.05 and ** P<0.01 versus NC; # P<0.05 versus NC+SEV; n=3.
Figure 6
Figure 6
Collapsin response mediator protein-2 (CRMP-2) impacted the expression levels of apoptosis-associated proteins. Nerve cells were transfected with empty vector, transfected with CRMP-2, treated with 3% sevoflurane (SEV) mixed gas, transfected with empty vector and then treated with SEV, and transfected with CRMP-2 and then treated with SEV. (A) Colorimetry was performed on the activity of caspase-3 in nerve cells. qRT-PCR (B) and western blot (C) assays were performed on the expression levels of actived-caspase-3, Bax, and Bcl-2 in nerve cells. * P<0.05 and ** P<0.01 versus NC; + P<0.05 and ## P<0.01 versus NC+SEV; n=3.
Figure 7
Figure 7
Collapsin response mediator protein-2 (CRMP-2) modulated the PI3K-mTOR-S6K pathway. Western blot assay was performed to measure the expression levels of synapsin-I, p-mTOR, mTOR, p-S6K, S6K, p-S6, and S6 in nerve cells, nerve cells transfected with empty vector, nerve cells transfected with CRMP-2, nerve cells treated with 3% sevoflurane (SEV) mixed gas, nerve cells transfected with empty vector and then treated with SEV, and nerve cells transfected with CRMP-2 and then treated with SEV. * P<0.05, ** P<0.01 and *** P<0.001 versus NC; # P<0.05 and ## P<0.01 versus NC+SEV; n=3.

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