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Objective To investigate the effect of intermittent high glucose on oxygen-glucose deprivation/refurnish (OGD/R) neuronal survival.Methods The primary cultured hippocampal neurons of mice were sub-cultured when the cell fusion reached about 80％.Cells in logarithmic growth phase were placed in a hypoxic incubator (37 ℃,5％ CO2,95％ N2) to simulate cell hypoxia.The culture medium was replaced by glucose-free Hank equilibrium salt solution (HBSS) to simulate cell hypoglycemia.The normal glucose and oxygen control group was set up.Cell morphology was observed under inverted phase contrast microscope after 6 hours of hypoxia and hypoglycemia treatment,and cell viability was detected by CCK-8 cell proliferation assay kit,and then grouping experiment was carried out.The cells were randomly divided into four groups.The cells were cultured in different concentration glucose medium under normal oxygen,5％ CO2 and 37 ℃ for 72 hours to prepare OGD/R model of cell ischemia/reperfusion.The low-glucose control group was cultured in medium containing 5.5 mmoFL glucose.The constant high-glucose group was cultured in medium containing 33.0 mmol/L glucose.The intermittent high-glucose group was cultured in medium containing 33.0 mmol/L glucose for 3 hours then in medium containing 5.5 mmol/L glucose for 2 hours alternately for 3 times during the day,and overnight in medium containing 33.0 mmol/L glucose at night.The hyperosmotic control group was made up of 5.5 mmol/L glucose medium and mannitol.The osmotic pressure was the same as that of the constant high-glucose group,and the effective glucose concentration was the same as that of the normal glucose and oxygen group,so as to eliminate the effect of osmotic pressure changes caused by the high-glucose medium on the results.Cell morphology was observed under inverted phase contrast microscope after 72 hours of cell culture in each group.Cell viability was measured by CCK-8 kit,and apoptotic rate was measured by flow cytometry.Results The inverted phase contrast microscope showed that the cells in the normal glucose and oxygen control group were plump and refractive,and had obvious nucleus,clear processes and high cell activity.After 6 hours of hypoxia and hypoglycemia treatment,the cells were shrunk,refractive index was poor,the nucleus was unclear,the processes were not clear,and the cell activity was significantly lower than that of normal glucose and oxygen control group (A value:0.34±0.06 vs.1.09±0.06,P ＜ 0.01),which indicated that the model of oxygen-glucose deprivation (OGD) was successfully prepared.After 72 hours of culture with different concentrations of glucose,the cells in the low-glucose control group were shrunk,the cell membrane was incomplete,the nucleus was unclear,and number of necrotic cells were more.In the constant high-glucose group,the refractive index of cells was poor,a large number of cells floated,and the nucleus was not obvious.In the intermittent high-glucose group,the cell morphology was normal,the refractive rate of cells was decreased slightly,and the necrotic cells were less.In the hypertonic control group,the cell status was close to that in the constant high-glucose group.Compared with the low-glucose control group or constant high-glucose group,the cell viability in the intermittent high-glucose group was significantly increased (A value:2.04±0.15 vs.0.64±0.18,1.16±0.16,both P ＜ 0.01),the apoptotic rate was significantly decreased [(59.60 ± 2.55)％ vs.(78.15 ± 15.77)％,(95.60± 0.14)％,both P ＜ 0.05].There was no significant difference in cell activity or apoptotic rate between the hypertonic control group and the constant high-glucose group [cell activity (A value):1.07 ± 0.07 vs.1.16 ± 0.16,apoptotic rate:(87.80 ± 4.53)％ vs.(95.60 ± 0.14)％,both P ＞ 0.05].Conclusion Intermittent high glucose within a certain range had protective effect on OGD/R neuronal survival.
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Objective: To investigate the effect of berberine on the apoptosis of human coronary artery endothelial cells (HCAECs) induced by intermittent high glucose in vitro. Methods HCAECs were isolated, identified, cultured, and divided into five groups: Group A was the normal glucose group (5.5 mmol/L glucose, NG); Group B was the persistent high glucose group (25 mmol/L glucose, PHG); Group C was the intermittent high glucose group (5.5 mmol/L and 25 mmol/L glucose fluctuated every 24 h, IHG); Group D was in a volatile hyperosmotic environment (5.5 mmol/L and 25 mmol/L mannitol alternating every 24 h to maintain the same osmotic pressure as IHG, OC); Group E was the fluctuation of hyperglycemia + berberine intervention group (5.5 mmol/L and 25 mmol/L glucose + 50 μmol/L berberine fluctuated every 24 h, IHG + BBR). The cells of each group were changed every 24 h, and the culture was stopped after 7 d, the cell viability and apoptosis were detected in each group. The expression of the apoptosis related protein Caspase-3 was determined by qRT-PCR and Western blotting. Results Compared with the persistent high glucose group, the apoptosis of HCAECs in the intermittent high glucose group was more significant (P < 0.05), but the apoptosis of HCAECs in the fluctuation of hyperglycemia + berberine intervention group was more significantly reduced than that in the intermittent high glucose group (P < 0.05). Berberine significantly reduced the expression of Caspase-3 induced by intermittent high glucose (P < 0.05) Conclusion Intermittent high glucose decreased the activity of HCAECs more easily than the persistent high glucose,and promoted the apoptosis of HCAECs. But berberine significantly reduced the apoptosis of HCAECs under intermittent high glucose, which had a significant protective effect on HCAECs.
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Aim To investigate the effect of salvianolic acid B (Sal B)on c-Jun N-terminal kinase (JNK)ac-tivation and apoptosis of INS-1 cells induced by inter-mittent high glucose.Methods INS-1 cells were pre-incubated with Sal B for 24 h,followed by exposure to intermittent high glucose (IHG,11.1 mmol·L-1 12 h,33. 3 mmol·L-1 12 h)for 72 h.Cell viability was assessed by MTT assay and cell apoptosis was evalua-ted by flow cytometry.Glucose induced insulin secre-tion capacity and intracellular reactive oxygen species (ROS)contents were measured by enzyme linked im-munosorbent assay (ELISA)and a fluorescent probe DCFH-DA,respectively.Levels of JNK activation and PDX-1 protein expression were determined by Western blot analysis.Results Sal B significantly alleviated IHG-induced cell injury and apoptosis,with glucose induced insulin secretion capacity improved evidently (P<0.05 or P<0.01).Preincubation with Sal B no-tably decreased intracellular ROS and JNK activation in INS-1 cells,while the level of PDX-1 protein was in-creased markedly (P<0.05 or P<0.01 ).Conclu-sion Sal B is capable of ameliorating IHG-induced cell injury and apoptosis in INS-1 cells,which might be derived from suppression of JNK activation and up-regulation of PDX-1 protein expression.
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The mechanism of damage in monocytes with the oxidation inhibitor α-lipoic acid (ALA) was observed under intermittent high glucose condition.The results showed that fluctuating high blood glucose levels led to more damage than that under constant high blood glucose level in monocyte,and antioxidant α-ALA could ameliorate the oxidative stress.The high level of nuclear factor-κB and monocyte chemotactic protein-1 are probably among the factors that cause vascular damage by deposit of monocytes.
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Objective To investigate the role of local aldosterone system in oncofetal fibronectin ( oncofetal FN) mRNA expression and reactive oxygen species (ROS) production in human mesangial cells (HMCs) exposed to short-term intermittent high glucose and the effect of Fenofibrate.Methods The HMCs were divided into 8 groups:normal glucose(NG) ;osmotic fluctuation(OF) ;mean glucose load (MGL) ;stable high glucose (SHG),short-term intermittent high glucose (IHG) ; intermittent high glucose plus eplerenone (IHGE) ; intermittent high glucose plus fenofibrate(IHGF) ; and normal glucose plus fenofibrate (NGF) groups.The mRNA expression levels of Aldosterone synthase ( CYP11 B2 ),11 β-hydroxysteroid dehydrogenase type 2 ( 11βHSD2 ) and oncofetal FN were determined by RT-PCR.The expression of CYP11B2 protein was determined by western-blot.Aldosterone level in cell culture supernatant was detected by radioimmunoassay.The expression and translocation of mineralocorticoid receptor (MR)protein were assayed with confocal laser scanning microscopy. ROS levels were determined by Fluorescence microscopy and fluorescence microplate reader.Results ( 1 ) MGL,SHG,and IHG groups showed a 2.41,3.63,and 4.45 times increase in CYP11B2 mRNA expression,and a 1.83,2.15,and 2.78 times increase in CYP11B2 protein expression,respectively,compared with NG group (P ＜ 0.05 ).The aldosterone levels of HMCs culture supernatant in MGL,SHG,and IHG groups were also increased,being 1.49,2.04,and 2.54 times of that in NG group ( P＜0.05 ),and the degree of elevation in IHG group was more marked than that in SHG group( P＜0.05 ).MR was activated and translocated from cytosol to nucleus in MGL,SHG,and IHG groups.Quantitative analysis showed the ratioes of cytosol/nucleus fluorescence intensity in MGL,SHG,and IHG groups were 15％,38％,and 53％ decreased as compared with that in NG group,and the decrease was more marked in IHG group ( P＜0.05 ).(2) Oncofetal FN mRNA expression and ROS levels in MGL,SHG,and IHG groups were increased,being 1.54,2.31,3.65 and 1.26,1.91,2.48 times of those in NG group,respectively ( P＜0.05 ),and this increase was more marked in group IHG ( P＜O.05 ).Compared with IHG group,oncofetal FN mRNA expression and ROS levels in group IHGE were significantly decreased by 54％ and 53％,and in group IHGF by 45％ and 39％. ( 3 ) CYP11B2 mRNA,protein,and aldosterone levels in IHGF group were decreased by 74％,59％,and 50％,and the activation of MR in group IHGF was inhibited when the ratio of cytosol/nuclear fluorescence intensity was increased 1.88 fold as compared with that in group IHG ( P＜0.05 ).Conclusions Increased expressions of oncofetal FN and ROS by HMCs induced by short-term intermittent high glucose were nore marked than those induced by stable high glucose.The mechanism was associated with activation of local aldosterone system.Fenofibrate may inhibit the activation of local aldosterone system and alleviate the injury to HMCs induced by intermittent high glucose.
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Objective To study the effects and mechanisms of resveratrol (Resv) on the expression of adiponectin receptors ( AdipoR1 and AdipoR2 ) in rat mesangial cells (MCs) cultured in intermittent high glucose medium.Methods The MCs cultured in normal glucose ( 5.6 mmol/L) medium were transfected by the plasmid vector of the FoxO1 short hairpin RNAs ( FoxO1 shRNA ),and then cultured in intermittent high glucose medium (5.6mmol/L or 30 mmol/L,alternately every 8 hours ) and resveratrol ( Resv 20 μmol/L).The MCs cultured in normal glucose (5.6 mmol/L) medium served as normal glucose control group (NG),and the MCs exposed to iutermittent high glucose medium were divided into five groups:intermittent high glucose group (IHG),IHG+Resv group,IHG+FoxO1 shRNA group,IHG+ Resv + FoxO1 shRNA group,IHG + Negative control group ( shNC ),each group was cultured for 72 h.The level of reactive oxygen species (ROS) was assessed by Fluorescence microplate reader.The mRNA levels of Sirt1,Foxo1,AdipoR1,and AdipoR2 were assessed by RT-RCR.The protein levels of FoxO1,phosphorylation FoxO1 ( p-FoxO1 ),AdipoR1,and AdipoR2 were assessed by Western blotting.Results ( 1 )Compared with NG,intermittent high glucose significantly decreased the mRNA expression of Sirt1,but markedly increased the level of p-FoxO1.Furthermore,the mRNA and protein expression levels of AdipoR1 were obviously decreased while the level of ROS was enhanced in IHG ( all P＜0.05 ).(2) Compared with IHG,the mRNA and protein expression levels of FoxO1 and AdipoR1 were inhibited,but the level of ROS was enhanced in group IHG+FoxO1 shRNA ( all P＜0.05 ).( 3 ) Compared with IHG,after treating with Resv,the mRNA expression level of Sirtl was increased,whereas the level of p-FoxO1 was decreased.Moreover,the mRNA and protein levels of AdipoR1 was increased while the level of ROS was lowered in group IHG+Resv (all P＜0.05 ).(4) Compared with group IHG+Resv,the mRNA and protein levels of FoxO1 and AdipoR1 were inhibited,but the level of ROS was increased in group IHG+Resv+FoxO1 shRNA (all P＜O.05).(5) In addition,the mRNA and protein expression level of AdipoR2 showed no significant difference among these groups ( P＞0.05 ).Conclusion ( 1 ) Resveratrol significantly increases the expression of AdipoR1 in MCs induced by intermittent high glucose.( 2 ) FoxO1 plays an important role in regulating the expression of AdipoR1 by resveratrol.
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Objective To investigate the effect of intermittent high glucose on proliferation, apoptosis, and cell cycle progression of INS-1 cells, and the possible intracellular pathways activated by intermittent high glucose. Methods Cell viability was evaluated by cell counting kit, the cell cycle was determined by flow cytometry,Annexin-V/PI double-labeled cell apoptosis detection kit was used to monitor cell apoptosis. Cell cycle related protein Skp2 and p27 expressions were detected by Western blot. Results ( 1 ) Both intermittent and constant high glucose significantly inhibited the growth of INS-1 cells, and the former effect was more significant. ( 2 ) Intermittent and constant high glucose levels significantly increased apoptosis in INS-1 cells, and the former effect was more significant. (3) Intermittent and constant high glucose levels significantly inhibited the cell process, the G0/G1 cell cycle arrest also was induced by intermittent high glucose, resulting in lowered proportion of the G2/M phase and S phase of INS-1 cells. (4) Intermittent and constant high glucose significantly decreased the level of protein Skp2 and increased the level of cell cycle related protein p27. Conclusion Intermittent high glucose levels affect INS-1 cell growth and proliferation, as well as induce cell apoptosis, probably by decreasing the level of protein Skp2 and increasing the level of p27 in the cells, resulting in arrest of progression through the G1 phase to the S phase of INS1 cells, and thus impairment of cell proliferation.
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Objective To explore the effect of glucose fluctuation on resistin. Methods The phorbol-12-myristate-13-acetate(PMA)-activated and differentiated U937 cells were exposed to experimental condition for 3 days, three groups of cells were formed, each one receiving the following fresh medium every 6 hours, respectively: (1) continuous 11.1 mmol/L glucose concentration medium (Con group), (2)continuous 22.2 mmol/L glucose concentration medium (CHG group), (3) alternating 11.1 mmol/L glucose concentration and 22. 2 mmol/L glucose concentration medium every 6 hours (IHG group). The supernatants of cell mediam at the last 6 hours were collected to test resistin concentration. Besides, 92 subjects were selected and classified into three groups according to the results of oral glucose tolerance test:normal glucose tolerance group ( NGT group, n =30), impaired glucose tolerance patients (IGT group, n =31) and newly diagnosed type 2 diabetes patients (T2DM group, n =31). Blood glucose and serum resistin levels were measured at 0 h and 1 h during oral glucose tolerance test ( OGTT) to compare the glucose fluctuation (△Glu1-0) and the change of serum resistin level (△lnRes1-0) among the three groups. Results Resistin concentration in the Con , CHG and IHG group was (73.62 ± 5.07)ng/L, (97.78 ±7.00)ng/L and(212.49 ± 28. 81 )ng/L respectively and in IHG group it was higher as compared with the other two groups (P<0.05). △Glu1-0 in NGT, IGT and T2DM group was(2.31 ±2.30)mmol/L,(5.70 ±2.08)mmol/L and (8.41 ±2.63)mmol/L respectively; △Glu1-0 increased gradually in all the three groups (P<0.05). Serum resistin level from 0 h to 1 h in the NGT group was 6.41 (1.52-15.76) μg/L to 6. 96( 1.52-22. 70) μg/L, in the IGT group 5.47( 1.49-24. 09)μg/L to 9. 12( 1.27-21.94)μg/L and in the T2DM group 5.77( 1.11-30.10) μg/L to 9. 27(1.02-48.15)μg/L In the IGT and T2DM group serum resistin level increased from 0 h to 1 h (P<0.05), but no difference was observed in the NGT group (P>0. 05).△lnRes1-0 in these 3 groups was (0.05 ± 0.05) μg/L, (0.25 ± 0.04) μg/L and (0.37 ± 0.03 )μg/L respectively and the change in the T2DM group was significant as compared with that in the NGT group,△lnRes1-0 was positively correlated with △Glu1-0 (r = 0.23, P = 0.02). Conclusion Glucose fluctuation induced monocyte/macrophage to secrete resistin, greater the glucose fluctuation, greater the change of amplitude of serum resistin.
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Aim To investigate the effect of pioglitazone on the AdipoR1 and AdipoR2 expression of HU-VECs induced by intermittent high glucose.Methods After exposed to intermittent high glucose for 5 d,HUVECs were incubated with pioglitazone(10,1 or 0.1 ?mol?L-1) for 24 hours.The AdipoR1 and AdipoR2 mRNA expression levels in HUVECs were also determined by real-time PCR.Results After exposed to intermittent high glucose for 5 days,HUVECs were treated with pioglitazone(10,1 or 0.1 ?mol?L-1) for 24 h.10,1?mol?L-1 of pioglitazone treatment enhanced AdipoR1 mRNA level(both P