ABSTRACT
Objective:To evaluate the relationship between protein kinase RNA-like endoplasmic reticulum kinase (PERK) pathway-mediated endoplasmic reticulum stress and the reduction of cerebral ischemia-reperfusion (I/R) injury by dexmedetomidine in mice by the in vivo experiment and the cell experiment. Methods:In the in vivo experiment, 20 healthy clean-grade male mice, aged 6-8 weeks, weighing 20-30 g, were divided into 4 groups ( n=5 each) using a random number table method: sham operation group (group S), sham operation+ dexmedetomidine group (group SD), cerebral I/R group (group IR) and cerebral I/R+ dexmedetomidine group (group IRD). Cerebral I/R was established by two-vessel occlusion plus hypotension.Dexmedetomidine 25 μg/kg was intraperitoneally injected at 10 min of ischemia in group IRD and at the corresponding time point in group SD.Neurological function was assessed using modified neurological severity score at 1 h of reperfusion.The animals were then sacrificed and brain tissues were taken for determination of the expression of endoplasmic reticulum stress-related proteins such as immunoglobulin heavy chain-binding protein (BIP), eukaryotic translation initiation factor 2α (EIF-2α), phosphorylated EIF-2α (p-EIF-2α), PERK and phosphorylated PERK (p-PERK) (by Wester blot). In the cell experiment, a mouse hippocampal neuronal cell line was selected and divided into 4 groups ( n=12 each) using a random number table method: control group (group C), oxygen-glucose deprivation/restoration (OGD/R) group (group OGD/R), OGD/R+ dexmedetomidine group (group OGD/R+ D) and OGD/R+ ISRIB (PERK pathway inhibitor) group (group OGD/R+ ISRIB). Cells were exposed to 94%N 2-5%CO 2-1%O 2 and incubated in a low-glucose DMEM medium for 6 h followed by restoration to establish OGD/R model.At 30 min before OGD, dexmedetomidine (final concentration 5 mmol/L) was added in group OGD/R+ D, and ISRIB (final concentration 10 mmol/L) was added in group OGD/R+ ISRIB.After 12-h restoration was completed, the cell survival rate was detected by CCK-8 assay.At 24 of restoration, the expression of endoplasmic reticulum stress-related proteins was determined by Wester blot. Results:In the in vivo experiment, compared with group S, neurobehavioral score was significantly increased and the expression of BIP, p-EIF-2α and p-PERK in brain tissues was up-regulated in group IR ( P<0.05). Compared with group IR, neurobehavioral score was significantly decreased and the expression of BIP, p-EIF-2α and p-PERK in brain tissues was down-regulated in group IRD ( P<0.05). In the cell experiment, compared with group C, the expression of BIP, p-EIF-2α, PERK and p-PERK was significantly up-regulated, and the cell survival rate was decreased in group OGD/R ( P<0.05). Compared with group OGD/R, the expression of BIP, p-EIF-2α, PERK and p-PERK was significantly down-regulated, and the cell survival rate was increased in OGD/R+ D, OGD/R+ ISRIB groups ( P<0.05). Compared with group OGD/R+ ISRIB, the expression of PERK was significantly down-regulated ( P<0.05) and no significant change was found in the other parameters in group OGD/R+ D ( P>0.05). Conclusion:The mechanism by which dexmedetomidine reduces cerebral I/R injury may be related to inhibiting PERK pathway-mediated endoplasmic reticulum stress in mice.
ABSTRACT
Objective:To evaluate the relationship between silence information regulator 1 (SIRT1) and signal transducers and activators of transcription 3 (STAT3) acetylation during high glucose-induced cardiac microvascular endothelial cell injury.Methods:Cardiac microvascular endothelial cells of Sprague-Dawley rats were cultured.The cells at the logarithmic growth phase were selected and divided into 3 groups ( n=24 each) using a random number table method: control group (C group), high glucose group (HG group) and high glucose+ SIRT1 agonist SRT1720 group (HG+ SRT group). The cardiac microvascular endothelial cells were seeded in a 6- or 96-well cell culture plate at a density of 2×10 5 cells/ml.When the cell density reached 50%, the culture medium was then replaced with high-glucose (glucose 33 mmol/L) DMEM culture medium containing with 10% fetal bovine serum and 1% double antibody in HG and HG+ SRT groups.In group HG+ SRT, 20 μmol/L SRT1720 was added simultaneously, and the cells were cultured at 37 ℃ in an incubator with 5% CO 2 for 24 h. The cell viability was determined by CCK-8 assay, the activity of superoxide dismutase (SOD) was detected using a spectrophotometer, the levels of lactic dehydrogenase (LDH), interleukin-6 (IL-6) and tumor necrosis factor-β (TNF-β) in the supernatant were detected by enzyme-linked immunosorbent assay, and the expression of SIRT1, acetylated STAT3 (ac-STAT3) and phosphorylated STAT3 (p-STAT3) was determined by Western blot. Results:Compared with C group, the cell viability and SOD activity were significantly decreased, levels of LDH, IL-6 and TNF-β in the supernatant were increased, expression of SIRT1 was down-regulated, and expression of ac-STAT3 and p-STAT3 was up-regulated in group HG and group HG+ SRT ( P<0.05). Compared with group HG, the cell viability and SOD activity were significantly increased, levels of LDH, IL-6 and TNF-β in the supernatant were decreased, expression of SIRT1 was up-regulated, and expression of ac-STAT3 and p-STAT3 was down-regulated in group HG+ SRT ( P<0.05). Conclusion:SIRT1 can alleviate high glucose-induced cardiac microvascular endothelial cell injury by promoting STAT3 deacetylation.
ABSTRACT
Objective:To evaluate the role of protein O-linked beta-N-acetylglucosaminylation (O-GlcNAcylation) modification in oxidative stress injury in nerve cells of mice subjected to oxygen-glucose deprivation and restoration (OGD/R).Methods:The standard mouse hippocampal neuron cell line was inoculated on a culture plate or dish at a density of 5×10 4 cells/ml and divided into 4 groups ( n=20 each) using a random number table method: normal group (N group), O-(connection)N-acetylglucosamine hydrolase (OGA) inhibitor Thiamet G group (T group), OGD/R group (D/R group) and Thiamet G+ OGD/R complex sugar group (T-D/R group). The cells were exposed to a mixed gas of 94% N 2-5% CO 2-1% O 2 for 6 h in a low-glucose medium, then medium was replaced with a common medium for restoring oxygen and glucose, and the cells were cultured for 12 h. Thiamet G at a final concentration of 1 mmol/L was added to the culture medium at 4 h before OGD/R in T-D/R group, and the medium was replaced with a medium containing Thiamet G at a final concentration of 1 mmol/L at 4 h before extraction of cellular proteins.After oxygen and glucose restoration was completed, the accumulation of cellular ROS was measured using DCFH-DA staining, mitochondrial membrane potential was measured using Jc-1 staining, O-GlcNAc modification was determined by immunofluorescence, and the expression of nuclear factor E2-related factor 2 (Nrf2), c-Jun N-terminal kinase (JNK), phosphorylated JNK (p-JNK), and p53 tumor suppressor gene (p53) was detected using Western blot. Results:Compared with group N, the expression of O-GlcNAc in nerve cells was significantly up-regulated in group T, and the accumulation of ROS in nerve cells was significantly increased, JC-1 monomer was increased, JC-1 polymer was decreased, Nrf2 expression was down-regulated, and the expression of p-JNK and p53 was up-regulated in group D/R, and the expression of O-GlcNAc in nerve cells was up-regulated, the accumulation of ROS was increased, the polymerization of JC-1 monomer and JC-1 was increased, Nrf2 expression was down-regulated, and the expression of p-JNK and p53 was up-regulated in group T-D/R ( P<0.05). Compared with group D/R, the expression of O-GlcNAc in nerve cells was significantly up-regulated, the accumulation of ROS was decreased, JC-1 monomer was decreased, JC-1 polymer was increased, the expression of Nrf2 was up-regulated, and the expression of p-JNK and p53 was down-regulated in group T-D/R ( P<0.05). Conclusion:When mouse nerve cells are subjected to OGD/R, the protein O-GlcNAc modification as an endogenous protective mechanism is enhanced, which can reduce oxidative stress injury, and the mechanism may be related to regulating the Nrf2-mediated JNK pathway.