Establishment of oxygen glucose deprivation reperfusion model of senescent SH-SY5Y cells

Obejective:To explore the establishment of an oxygen glucose deprivation/reperfusion model of senescent SH-SY5Y cells.Methods:SH-SY5Y cells were randomly divided into control(D-galactose 0 mmol/L group),D-galactose(25 mmol/L,50 mmol/L,100 mmol/L,200 mmol/L,400 mmol/L)groups,and treated with corresponding concentrations of D-galactose for 48 h.The changes of cell morphology,β-galactosidase,the cell morphology,β-galactosidase activity by microscopic observation,cell proliferation rate by EdU kit and cell survival rate by CCK-8 assay were used to determine the decaying concentration of D-galactose and to establish the senescence model.The senescent SH-SY5Y cells were randomly divided into control group(oxygen glucose deprivation without treatment group),oxygen glucose deprivation treatment(0.5 h,1 h,1.5 h,2 h)group,followed by re-glucose reoxygenation for 24 h,and CCK-8 assay for the survival rate of senescent SH-SY5Y cells.Results:There were no significant changes in cell morphology and β-gal activity in the 25 mmol/L and 50 mmol/L groups compared with the control group(P>0.05),cytosolic hypertrophy was seen in the cells of the 100 mmol/L group,chromatin fixation in the cells of the 200 mmol/L group,and massive vacuolization in the cells of the 400 mmol/L group;the positive rate ofβ-galactosidase staining in the cells of the(100-400 mmol/L)group was significantly higher compared with the control group(P<0.05),with little difference between the 100 mmol/L and 200 mmol/L groups(P>0.05);the cell proliferation ability of the(100-400 mmol/L)group was significantly decreased in a concentration-dependent manner(P<0.05);the cell survival rate was decreased in a concentration-dependent manner(P<0.05),with IC_(50) between 100 mmol/L and 200 mmol/L.The survival of senescent SH-SY5Y cells showed a time-dependent decrease in oxygen-glucose deprivation(P<0.05),with an IC_(50) close to 1 h.Conclusion:D-gal concentration of 100 mmoL/L and 48 h of cell action could establish a survival rate of about 50%of senescent SH-SY5Y cells,and oxygen glucose deprivation of senescent SH-SY5Y cells for 1 h and reperfusion for 24 h could establish an oxygen glucose deprivation/reperfusion model of senescent SH-SY5Y cells with a survival rate close to 50%.

Dehydrocostuslactone protects against oxygen-glucose deprivation/reperfusion-induced injury by inhibiting autophagy and apoptosis in PC12 cells

OBJECTIVE TO investigate the neural protection of dehydrocostus lactone(DHL)against neuronal injury induced by oxygen and glucose deprivation/reperfusion(OGD/R)in differentiated PC12 cells.METHODS We used a cellular model of 2 h of OGD and 24 h of reperfusion to mimic cerebral ischemia-reperfusion injury.Cell viability was used to reflect the degree of OGD/R-induced injury.Cells were treated with DHL during the reperfusion phase.Cell Counting Kit(CCK-8)and LDH assays were performed to determine the optimal dose of DHL and cell viability.Flow cytometry analysis and Monodansylcadaverine(MDC)staining were then conducted to detect apoptosis rate and autophagosome formation after OGD/R in PC12 cells.Immunofluorescence and Western blotting analyses were used to detect the expres⁃sion of proteins associated with autophagy and apoptosis.RESULTS OGD/R significantly decreased cell viability and increased apoptosis rate.The expression levels of autophagy-related proteins,namely,LC3 and Beclin-1,and apoptosisrelated proteins,namely,Bax and caspase-3 increased,but that of the anti-apoptosis Bcl-2 protein decreased.However,DHL attenuated OGD/R-induced neuronal injury through inhibition of apoptosis and autophagy properties by modulating au⁃tophagy-associated proteins(LC3 and Beclin-1)and apoptosis-modulating proteins(caspase-3 and Bcl-2/Bax).CONCLU⁃SION Our data provide an evidence for the neuroprotective effect of DHL against ischemic neuronal injury.Hence,DHL could be a promising candidate for treatment of ischemic stroke.

Neuroprotective effects of salvianolic acid B against oxygen-glucose deprivation/reperfusion damage in primary rat cortical neurons

Background Cerebral ischemia-reperfusion injury is the main reason for the loss of neurons in the ischemic cerebrovascular disease. Therefore, to deeply understand its pathogenesis and find a new target is the key issue to be solved. This research aimed to investigate the neuroprotective effects of salvianolic acid B （SalB） against oxygen-glucose deprivation/reperfusion （OGD/RP） damage in primary rat cortical neurons.Methods The primary cultures of neonatal Wister rats were randomly divided into the control group, the OGD/RP group and the SalB-treatment group （10 mg/L）. The cell model was established by depriving of oxygen and glucose for 3 hours and reperfusion for 3 hours and 24 hours, respectively. The neuron viability was determined by MTT assay. The level of cellular reactive oxygen species （ROS） was detected by fluorescent labeling method and spin trapping technique respectively. The activities of neuronal Mn-superoxide dismutase （Mn-SOD）, catalase （CAT） and glutathione peroxidase （GSH-PX） were assayed by chromatometry. The mitochondria membrane potential （△ψm） was quantitatively analyzed by flow cytometry. The release rate of cytochrome c was detected by Western blotting. The neuronal ultrastructure was observed by transmission electron microscopy. Statistical significance was evaluated by analysis of variance （ANOVA）followed by Student-Newman-Keuls test.Results OGD/RP increased the level of cellular ROS, but decreased the cell viability and the activities of Mn-SOD, CAT and GSH-PX; SalB treatment significantly reduced the level of ROS （P ＜0.05）; and enhanced the cell viability （P ＜0.05）and the activities of these antioxidases （P ＜0.05）. Additionally, OGD/RP induced the fluorescence value of △ψm to diminish and the release rate of cytochrome c to rise notably; SalB markedly elevated the level of △ψm （P ＜0.01） and depressed the release rate of cytochrome c （P ＜0.05）; it also ameliorated the neuronal morphological injury.Conclusion The neuroprotection of SalB may be attributed to the elimination of ROS and the inhibition of apoptosis.

Inhibition of the immunoproteasome LMP_(2) ameliorates ischemia/hypoxia-induced blood–brain barrier injury through the Wnt/β-catenin signalling pathway

Background:Disruption of the blood–brain barrier(BBB)after a stroke can lead to brain injury and neurological impairment.Previous work confirmed the involvement of the immunoproteasome subunit of low molecular mass peptide 2(LMP2)in the pathophysiology of ischemia stroke.However,the relationship between the immunoproteasome LMP2 and the BBB remains unclear.Methods:Adult male Sprague–Dawley rats were subjected to transient middle cerebral artery occlusion/reperfusion(MCAO/R).Three days before MCAO,the rats were treated with lentivirus-mediated LMP2 shRNA preparations by stereotactical injection into the ipsilateral hemispheric region.The rat brain microvascular endothelial cell(RBMVEC)line was exposed to oxygen–glucose deprivation/reperfusion(OGD/R)to mimic ischemic conditions in vitro.The RNA interference-mediated knockdown of LMP2 orβ-catenin was analysed in vivo and in vitro.Analysis of the quantity of extravasated Evans blue(EB)and cerebral fluorescent angiography were performed to evaluate the integrity of the BBB.Immunofluorescence and Western blotting were employed to detect the expression of target proteins.Cell migration was evaluated using a scratch migration assay.The results of immunofluorescence,Western blotting and cell migration were quantified using the software ImageJ(Version 1.53).Parametric data from different groups were compared using one-way ANOVA followed by the least significant difference(LSD)test.Results:Cerebral ischemia led to lower levels of structural components of the BBB such as tight junction proteins[occludin,claudin-1 and zonula occludens(ZO-1)]in the MCAO/R group compared with the sham group(P<0.001).However,inhibition of the immunoproteasome LMP2 restored the expression of these proteins,resulting in higher levels of occludin,claudin-1 and ZO-1 in the LMP2-shRNA group compared with the control-shRNA group(P<0.001).In addition,inhibition of the immunoproteasome LMP2 contributed to higher microvascular density and decreased BBB permeability[e.g.,the quantity of extravasated EB:LMP2-shRNA group(58.54±7.37)μg/g vs.control-shRNA group(103.74±4.32)μg/g,P<0.001],and promoted the upregulation of Wnt-3a andβ-catenin proteins in rats following MCAO/R.In vitro experiments,OGD/R induced marked upregulation of LMP2,proapoptotic protein Bax and cleaved caspase-3,and downregulation of occludin,claudin-1,ZO-1 and Bcl-2,as well as inhibition of the Wnt/β-catenin pathway Wnt-3a andβ-catenin proteins in RBMVECs,compared with the control group under normal culture conditions(P<0.001).However,silencing of LMP2 gene expression reversed these protein changes and promoted proliferation and migration of RBMVECs following OGD/R.Silencing ofβ-catenin by transfection of RBMVECs withβ-catenin-si RNA aggravated the downregulation of tight junction proteins,and reduced the proliferation and migration of RBMVECs following OGD/R,compared with the control-siRNA group(P<0.001).LMP2-si RNA andβ-catenin-si RNA co-transfection partly counteracted the beneficial effects of silencing LMP2-siRNA on the levels of tight junction proteins in RBMVECs exposed to OGD/R.Conclusions:This study suggests that inhibition of the immunoproteasome LMP2 ameliorates ischemia/hypoxia induced BBB injury,and that the molecular mechanism involves the immunoproteasome-regulated activation of the Wnt/β-catenin signalling pathway under ischemic conditions.

Protective Effect of Salvianolic Acid A on Brain Endothelial Cells after Treatment with Deprivation and Reperfusion of Oxygen-glucose

Objective Salvianolic acid A（SAA） has a significant protective effect on ischemia/reperfusion injury of brain. However, it is not clear for SAA to exert its cerebral protection by targeting at the microvascular endothelial cells of blood brain barrier（BBB）. Our previous study demonstrated that SAA could hardly pass through the BBB. This present study was therefore designed to investigate the protective effect of SAA on brain microvascular endothelial cells（BMECs） induced by deprivation and reperfusion with oxygen-glucose. Methods Rat BMECs were treated with oxygen glucose deprivation（OGD）, followed by reperfusion（OGD/R）. Cell viability was assessed by MTT and the content of reactive oxygen species（ROS） in cells after OGD/R in the absence or presence of SAA. GC-MS based metabolomic platform was applied to evaluate the regulation of SAA on the cellular metabolic perturbation induced by OGD/R. Results OGD/R significantly increased the production of intracellular reactive oxygen species（ROS）, and decreased the activity of cells. SAA significantly reduced ROS and improve the cell viability. Metabolomic study revealed distinct perturbation of metabolic pathways of energy metabolism in the BMEC induced by OGD/R, while SAA significantly regulated the perturbed metabolism involved in energy metabolism pathways, especially for intermediates in TCA cycle. Conclusion SAA shows protective effects on BMECs involved in central nervous system.

Stigmasterol protects human brain microvessel endothelial cells against ischemia-reperfusion injury through suppressing EPHA2 phosphorylation

Stigmasterol is a plant sterol with anti-apoptotic,anti-oxidative and anti-inflammatory effect through multiple mechanisms.In this study,we further assessed whether it exerts protective effect on human brain microvessel endothelial cells(HBMECs)against ischemia-reperfusion injury and explored the underlying mechanisms.HBMECs were used to establish an in vitro oxygen and glucose deprivation/reperfusion(OGD/R)model,while a middle cerebral artery occlusion(MCAO)model of rats were constructed.The interaction between stigmasterol and EPHA2 was detected by surface plasmon resonance(SPR)and cellular thermal shift assay(CETSA).The results showed that 10μmol·L−1 stigmasterol significantly protected cell viability,alleviated the loss of tight junction proteins and attenuated the blood-brain barrier(BBB)damage induced by OGD/R in the in vitro model.Subsequent molecular docking showed that stigmasterol might interact with EPHA2 at multiple sites,including T692,a critical gatekeep residue of this receptor.Exogenous ephrin-A1(an EPHA2 ligand)exacerbated OGD/R-induced EPHA2 phosphorylation at S897,facilitated ZO-1/claudin-5 loss,and promoted BBB leakage in vitro,which were significantly attenuated after stigmasterol treatment.The rat MCAO model confirmed these protective effects in vivo.In summary,these findings suggest that stigmasterol protects HBMECs against ischemia-reperfusion injury by maintaining cell viability,reducing the loss of tight junction proteins,and attenuating the BBB damage.These protective effects are at least meditated by its interaction with EPHA2 and inhibitory effect on EPHA2 phosphorylation.