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Research article
Peirong Lu
Department of Ophthalmology, the First Affiliated Hospital of Soochow University
Corresponding Author
ORCiD: https://orcid.org/0000-0002-1423-1737
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Purpose: To investigate the effect of high glucose concentrations on the expression of p66Shc and phosphorylation of p66Shc Ser36 in R28 cells, and to elucidate the role of p66Shc in high glucose-induced oxidative damage and apoptosis of R28 cells. Methods: R28 cells were cultured with various concentrations of D-glucose, or with 30 mM D-glucose or 30 mM mannitol only for the indicated timepoints. The expression of p66Shc and phosphorylation of p66Shc Ser36 were subsequently detected. Additionally, R28 cells were divided into five groups: i. control (CTL) group; ii. high glucose group; iii. high glucose overexpressed p66Shc group; iv. high glucose p66Shc knockout (KO) group; and v. high glucose S36A mutant group. P66Shc, cytochrome C, and reactive oxygen species (ROS) content in mitochondria, mitochondrial DNA damage, and apoptosis were also evaluated. Results: With the increase of glucose concentration, the levels of p66Shc mRNA and expression of p66Shc protein gradually increased. P66Shc mRNA and protein levels, and phosphorylation of p66Shc Ser36 increased with the prolongation of exposure to high concentrations of glucose. Notably, after stimulation with 30 mM mannitol, the levels of p66Shc mRNA, p66Shc protein, and p66Shc S36 phosphorylation did not change. With the increase of p66Shc level in the mitochondria of R28 cells, ROS content in mitochondria increased, mitochondrial DNA damage was exacerbated, cytochrome C content in mitochondria decreased, and apoptosis increased. Conclusion: High glucose concentrations increased the expression of p66Shc in R28 cells in a time- and concentration-dependent manner. P66Shc regulated both oxidative damage and apoptosis in R28 cells.
Keywords
Glucose; p66Shc; R28 cells; reactive oxygen species; apoptosis
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This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research article
Peirong Lu
Department of Ophthalmology, the First Affiliated Hospital of Soochow University
Corresponding Author
ORCiD: https://orcid.org/0000-0002-1423-1737
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
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Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
The most recent version of this article is available here.
No community comments so far
Version 1
Posted 22 Feb, 2019
No comments provided
Metrics
Comments: 0
PDF Downloads: ...
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Subject Areas
Internal Medicine Specialties
License:
This work is licensed under a CC BY 4.0 License. Read Full License
The most recent version of this article is available here.
Purpose: To investigate the effect of high glucose concentrations on the expression of p66Shc and phosphorylation of p66Shc Ser36 in R28 cells, and to elucidate the role of p66Shc in high glucose-induced oxidative damage and apoptosis of R28 cells. Methods: R28 cells were cultured with various concentrations of D-glucose, or with 30 mM D-glucose or 30 mM mannitol only for the indicated timepoints. The expression of p66Shc and phosphorylation of p66Shc Ser36 were subsequently detected. Additionally, R28 cells were divided into five groups: i. control (CTL) group; ii. high glucose group; iii. high glucose overexpressed p66Shc group; iv. high glucose p66Shc knockout (KO) group; and v. high glucose S36A mutant group. P66Shc, cytochrome C, and reactive oxygen species (ROS) content in mitochondria, mitochondrial DNA damage, and apoptosis were also evaluated. Results: With the increase of glucose concentration, the levels of p66Shc mRNA and expression of p66Shc protein gradually increased. P66Shc mRNA and protein levels, and phosphorylation of p66Shc Ser36 increased with the prolongation of exposure to high concentrations of glucose. Notably, after stimulation with 30 mM mannitol, the levels of p66Shc mRNA, p66Shc protein, and p66Shc S36 phosphorylation did not change. With the increase of p66Shc level in the mitochondria of R28 cells, ROS content in mitochondria increased, mitochondrial DNA damage was exacerbated, cytochrome C content in mitochondria decreased, and apoptosis increased. Conclusion: High glucose concentrations increased the expression of p66Shc in R28 cells in a time- and concentration-dependent manner. P66Shc regulated both oxidative damage and apoptosis in R28 cells.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10
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