Protective effects of green tea polyphenol against reactive oxygen species-induced oxidative stress in cultured rat calvarial osteoblast

The injurious effects of reactive oxygen species on osteoblasts and the potential protective role played by green tea polyphenols (GtPP) were investigated using primarily cultured rat calvarial osteoblasts. Oxidative stress was induced in cultured osteoblasts, either by adding 100 mmol/L H2O2 or by the action of 40 U/L xanthine oxidase (XO) in the presence of xanthine (250 micromol/L). After incubation, the cellular viability, function and morphology were evaluated. Both treatments produced a significant reduction in osteoblast viability, as assessed by a two-colored fluorescence staining method combined with flow cytometric analysis and MTT assay. A significant reduction in the alkaline phosphatase activity was observed after H2O2 addition, whereas XO did not have the same effect. On the microscopic observations, the morphological changes and intracellular ultrastructural damages were remarkably induced by both treatments. The H2O2-induced alterations were prevented by pre-incubating the osteoblasts with 200 microg/ml GtPP for 1 h. When the oxidative stress was induced by XO, the cellular viability and morphology was also maintained at the same polyphenol concentration. These results demonstrate that GtPP can act as a biological antioxidant in a cell culture experimental model and protect cells from oxidative stress-induced toxicity.     

Targets for hydrogen-peroxide-induced damage to suspension and biofilm cells of Streptococcus mutans

Hydrogen peroxide (H2O2) is considered a major endogenous source of oxidative stress to oral bacteria and also is widely used in oral care products. Our study objectives were to identify specific targets for H2O2-induced damage to cells of Streptococcus mutans in suspensions and monospecies biofilms and to differentiate bacteriostatic and bactericidal actions of the peroxide. Streptococcus mutans was grown in suspension cultures and fed-batch biofilms for assessing relative sensitivities of viability, glycolysis, and protein synthesis to H2O2 damage. Biofilm cells were found to have essentially the same peroxide sensitivity as cells in suspensions. H2O2 at low concentrations of about 16.3 mmol/L was highly inhibitory for glycolysis and mainly bacteriostatic. The most sensitive target detected for glycolytic inhibition was glyceraldehyde-3-phosphate dehydrogenase with IC50 (50% inhibitory concentration) values of ca. 2.2 mmol/L for suspension cells and 2.3 mmol/L for biofilms with 15 min treatments. The phosphoenolpyruvate:glucose phosphotransferase pathway was less sensitive with an IC50 of ca. 10 mmol/L. Aldolase was not inhibited at bacteriostatic concentrations of the peroxide. For suspensions and biofilms, acidification somewhat diminished peroxide sensitivity, while increased temperature enhanced sensitivity. At concentrations above about 30 mmol/L, H2O2 became mainly bactericidal but not mutagenic for S. mutans. A major target for bactericidal damage was protein synthesis, thus rendering cells incapable of repairing or replacing oxidatively damaged proteins.     

Antioxidants N-acetyl-L-cysteine and manganese(III)tetrakis (4-benzoic acid)porphyrin do not prevent beta-cell dysfunction in rat islets cultured in high glucose for 1 wk

We previously showed that the stimulation of heme oxygenase-1 expression by high glucose and hydrogen peroxide (H(2)O(2)) in cultured rat islets is prevented by antioxidants and suggested that this effect of high glucose results from an oxidative stress. However, the role of oxidative stress in high-glucose-induced beta-cell dysfunction is unclear. We therefore compared the preventative effects of N-acetyl-l-cysteine (NAC), a free radical scavenger, and manganese(III)tetrakis (4-benzoic acid)porphyrin (MnTBAP), a superoxide dismutase/catalase mimetic agent, on the alteration of stimulus-secretion coupling induced in rat islets by overnight exposure to hydrogen peroxide (H(2)O(2)-treated islets) or 1-wk culture in 30 vs. 10 mmol/l glucose (High-glucose vs. Control islets). The features of beta-cell dysfunction differed between the two groups: reduced glucose-induced insulin secretion without changes in glucose sensitivity in H(2)O(2)-treated islets; increased sensitivity to glucose with parallel reductions in insulin content and maximal rate of glucose-induced insulin secretion in High-glucose islets. The latter alterations were accompanied by a decrease in preproinsulin without changes in pancreatic and duodenal homeobox gene 1 mRNA levels. The functional alterations induced by H(2)O(2) were significantly prevented by addition of NAC or MnTBAP in the culture medium. In contrast, neither NAC nor MnTBAP affected the functional alterations induced by high glucose. These results suggest that beta-cell dysfunction induced by 1-wk culture in high glucose does not result from an increase in oxidative stress.     

Intermittent high glucose enhances apoptosis in human umbilical vein endothelial cells in culture

To explore the effect of fluctuating glucose on endothelial cells, human umbilical vein endothelial cells were incubated for 14 days in media containing different glucose concentrations: 5 mmol/l, 20 mmol/l, or a daily alternating 5 or 20 mmol/l glucose. Apoptosis was studied by different methods: viability assay, cell cycle analysis, DNA fragmentation, and morphological analysis. Furthermore, the levels of Bcl-2 and Bax, well known proteins involved in apoptosis, were evaluated. Stable high glucose induced apoptosis in human umbilical vein endothelial cells, a phenomenon accompanied by a significant decrease of Bcl-2 and a simultaneous increase of Bax expression. However, apoptosis was enhanced in human umbilical vein endothelial cells exposed to intermittent, rather than constant, high glucose concentration. In this condition, Bcl-2 was not detectable, whereas Bax expression was significantly enhanced. These findings suggest that variability in glycemic control could be more deleterious to endothelial cells than a constant high concentration of glucose.     

Glucose as a lipolytic agent: studies on isolated rat adipocytes

In order to elucidate the direct effect of glucose on lipolysis in isolated rat adipocytes, cells were incubated in a buffer with different concentrations of this sugar: 2, 8 or 16 mmol/l. The increase in glucose concentration from 2 mmol/l to 8 or 16 mmol/l enhanced basal lipolysis by 30% and 47%, respectively. Epinephrine-induced lipolysis (1 micromol/l) was also increased by 31% and 32%, when glucose concentration was increased from 2 mmol/l to 8 or 16 mmol/l, respectively. The rise in lipolysis caused by glucose was restricted by H-89 (an inhibitor of protein kinase A, 30 micromol/l), but insulin (1 nmol/l) had no inhibitory action. The augmentation of lipolysis by glucose did not require its metabolism (as demonstrated using 2-deoxyglucose) and was due to the action of this sugar on the final steps of the lipolytic cascade, particularly on protein kinase A. However, short-term exposure of adipocytes to higher glucose concentrations did not restrict the inhibitory action of insulin on lipolysis induced by epinephrine.     

Gliclazide protects pancreatic beta-cells from damage by hydrogen peroxide

Oxidative stress is induced under diabetic conditions and possibly causes various forms of tissue damage in patients with diabetes. Recently, it has become aware that susceptibility of pancreatic beta-cells to oxidative stress contributes to the progressive deterioration of beta-cell function in type 2 diabetes. A hypoglycemic sulfonylurea, gliclazide, is known to be a general free radical scavenger and its beneficial effects on diabetic complications have been documented. In the present study, we investigated whether gliclazide could protect pancreatic beta-cells from oxidative damage. One hundred and fifty microM hydrogen peroxide reduced viability of mouse MIN6 beta-cells to 29.3%. Addition of 2 microM gliclazide protected MIN6 cells from the cell death induced by H(2)O(2) to 55.9%. Glibenclamide, another widely used sulfonylurea, had no significant effects even at 10 microM. Nuclear chromatin staining analysis revealed that the preserved viability by gliclazide was due to inhibition of apoptosis. Hydrogen peroxide-induced expression of an anti-oxidative gene heme oxygenase-1 and stress genes A20 and p21(CIP1/WAF1), whose induction was suppressed by gliclazide. These results suggest that gliclazide reduces oxidative stress of beta-cells by H(2)O(2) probably due to its radical scavenging activity. Gliclazide may be effective in preventing beta-cells from the toxic action of reactive oxygen species in diabetes.     

过氧化氢预处理对H9c2心肌细胞氧化应激损伤的保护作用

目的：活性氧介导的氧化损伤是缺血再灌注损伤的重要机制,本研究通过观察H2O2预处理对氧化损伤的H9c2心肌细胞存活率和细胞凋亡的影响,探讨其保护H9c2心肌细胞的作用机制。方法：体外培养H9c2心肌细胞,取对数生长期细胞用于实验研究。建立H2O2预处理抵抗高浓度H：O：诱导的细胞氧化损伤模型,实验分组如下：（1）正常对照组（CTL）;（2）损伤组（INJURY）;（3）预处理组十损伤组（PC）。应用CCK8法检测细胞存活率;试剂盒检测胞内MDA水平和T．sOD活性;Hoechst33258染色观察凋亡形态;Annexin-V／PI双染与流式细胞术检测细胞凋亡率。结果：25vLmol／L的H202预处理90rain能明显地保护H9c2心肌细胞抵抗400μmol／LH2O2诱导的氧化损伤,提高细胞存活率,下调MDA水平,上调SOD活性,抑制细胞凋亡,降低细胞凋亡率。结论：低浓度H2O2预处理能减轻H9c2心肌细胞的氧化损伤,抑制氧化损伤诱导的心肌细胞凋亡,具有很好的抗氧化损伤和抗心肌细胞凋亡的保护作用,其作用机制可能与细胞SOD活性上调有关。H2O2预处理为临床治疗心肌缺血／再灌注损伤提供了一项新策略。     

Relationship among oxidative stress, growth cycle, and sporulation in Bacillus subtilis.

The sensitivity of Bacillus subtilis to hydrogen peroxide (oxidative stress) was found to vary with the position of the culture in the growth cycle. The most dramatic change occurred at the stationary phase, when the cells became totally resistant to 10 mM H2O2, in contrast to the loss of 3 to 4 log units of viability when treated at the early log phase. Two of the eight proteins induced by a protective concentration of H2O2 (50 muM) were also induced (in the absence of oxidative stress) on entry into the late log phase of growth. The response of five isogenic spo0 mutants (spo0B, spo0E, spo0F, spo0H, and spo0J) to oxidative stress was identical to that of the wild-type parental strain. In an isogenic spo0A strain, mid-log-phase cells were 100-fold less sensitive to 10 mM H2O2 than was the wild type. Pretreatment with 50 microM H2O2 induced little further protection, suggesting that the response is constitutive in this strain. By comparison of proteins induced by 50 microM H2O2 in the wild-type, spo0A, spo0H, and spo0J strains, four proteins were identified that may be essential for protection against lethal concentrations of H2O2. The presence of multiple copies of the spo0H gene in a spo0A background converted the stress phenotype of the spo0A mutant to that of the wild type but left the sporulation phenotype unaltered.     

5′-磷酸腺苷对小鼠脾细胞氧化应激损伤的保护作用

目的研究5′-磷酸腺苷（5′-AMP）体外抗氧化和对体外氧化损伤脾细胞的损伤修复能力。方法用化学比色法测定5′-AMP体外清除二苯代苦味酰基自由基（DPPH自由基）的能力;建立过氧化氢（H2O2）氧化损伤体外培养小鼠脾细胞模型,用MTT法检测5′-AMP修复受损伤脾细胞的作用,并分析其对细胞抗氧化体系及抗氧化能力的影响。结果5′-AMP具有剂量依赖性的体外抗氧化和清除活性氧能力,添加0.5mmol/L、1mmol/L、5mmol/L和10mmol/L5′-AMP均能显著修复H2O2诱导的脾细胞氧化损伤（P〈0.05）,总抗氧化能力和抗氧化酶类活力（P〈0.01）,5′-AMP添加量大于1mmol/L时,可显著降低丙二醛（MDA）含量（P〈0.01）。其细胞培养液的氧自由基（ROS）水平逐渐降低,5′-AMP添加量为10mmol/L时,ROS水平接近对照组水平。结论5′-磷酸腺苷能显著修复氧化损伤,具有显著的抗氧化作用。     

Heme Oxygenase-1 Protects Retinal Endothelial Cells against High Glucose- and Oxidative/Nitrosative Stress-Induced Toxicity

Diabetic retinopathy is a leading cause of visual loss and blindness, characterized by microvascular dysfunction. Hyperglycemia is considered the major pathogenic factor for the development of diabetic retinopathy and is associated with increased oxidative/nitrosative stress in the retina. Since heme oxygenase-1 (HO-1) is an enzyme with antioxidant and protective properties, we investigated the potential protective role of HO-1 in retinal endothelial cells exposed to high glucose and oxidative/nitrosative stress conditions. Retinal endothelial cells were exposed to elevated glucose, nitric oxide (NO) and hydrogen peroxide (H(2)O(2)). Cell viability and apoptosis were assessed by MTT assay, Hoechst staining, TUNEL assay and Annexin V labeling. The production of reactive oxygen species (ROS) was detected by the oxidation of 2',7'-dichlorodihydrofluorescein diacetate. The content of HO-1 was assessed by immunobloting and immunofluorescence. HO activity was determined by bilirubin production. Long-term exposure (7 days) of retinal endothelial cells to elevated glucose decreased cell viability and had no effect on HO-1 content. However, a short-time exposure (24 h) to elevated glucose did not alter cell viability, but increased both the levels of intracellular ROS and HO-1 content. Moreover, the inhibition of HO with SnPPIX unmasked the toxic effect of high glucose and revealed the protection conferred by HO-1. Oxidative/nitrosative stress conditions increased cell death and HO-1 protein levels. These effects of elevated glucose and HO inhibition on cell death were confirmed in primary endothelial cells (HUVECs). When cells were exposed to oxidative/nitrosative stress conditions there was also an increase in retinal endothelial cell death and HO-1 content. The inhibition of HO enhanced ROS production and the toxic effect induced by exposure to H(2)O(2) and NOC-18 (NO donor). Overexpression of HO-1 prevented the toxic effect induced by H(2)O(2) and NOC-18. In conclusion, HO-1 exerts a protective effect in retinal endothelial cells exposed to hyperglycemic and oxidative/nitrosative stress conditions.     

Aldose reductase is implicated in high glucose-induced oxidative stress in mouse embryonic neural stem cells

Oxidative stress caused by hyperglycemia is one of the key factors responsible for maternal diabetes-induced congenital malformations, including neural tube defects in embryos. However, mechanisms by which maternal diabetes induces oxidative stress during neurulation are not clear. The present study was aimed to investigate whether high glucose induces oxidative stress in neural stem cells (NSCs), which compose the neural tube during development. We also investigated the mechanism by which high glucose disturbs the growth and survival of NSCs in vitro . NSCs were exposed to physiological d -glucose concentration (PG, 5 mmol/L), PG with l -glucose (25 mmol/L), or high d -glucose concentration (HG, 30 or 45 mmol/l). HG induced reactive oxygen species production and mRNA expression of aldose reductase (AR), which catalyzes the glucose reduction through polyol pathway, in NSCs. Expression of glucose transporter 1 (Glut1) mRNA and protein which regulates glucose uptake in NSCs was increased at early stage (24 h) and became down-regulated at late stage (72 h) of exposure to HG. Inhibition of AR by fidarestat, an AR inhibitor, decreased the oxidative stress, restored the cell viability and proliferation, and reduced apoptotic cell death in NSCs exposed to HG. Moreover, inhibition of AR attenuated the down-regulation of Glut1 expression in NSCs exposed to HG for 72 h. These results suggest that the activation of polyol pathway plays a role in the induction of oxidative stress which alters Glut1 expression and cell cycle in NSCs exposed to HG, thereby resulting in abnormal patterning of the neural tube in embryos of diabetic pregnancy.     

Scoparia dulcis, a traditional antidiabetic plant, protects against streptozotocin induced oxidative stress and apoptosis in vitro and in vivo

Oxidative stress is implicated in the pathogenesis of diabetic complications. The experiments were performed on normal and experimental male Wistar rats treated with Scoparia dulcis plant extract (SPEt). The effect of SPEt was tested on streptozotocin (STZ) treated Rat insulinoma cell lines (RINm5F cells) and isolated islets in vitro. Administration of an aqueous extract of Scoparia dulcis by intragastric intubation (po) at a dose of 200 mg/kg body weight significantly decreased the blood glucose and lipid peroxidative marker thiobarbituric acid reactive substances (TBARS) with significant increase in the activities of plasma insulin, pancreatic superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH) in streptozotocin diabetic rats at the end of 15 days treatment. Streptozotocin at a dose of 10 mug/mL evoked 6-fold stimulation of insulin secretion from isolated islets indicating its insulin secretagogue activity. The extract markedly reduced the STZ-induced lipidperoxidation in RINm5F cells. Further, SPEt protected STZ-mediated cytotoxicity and nitric oxide (NO) production in RINm5F cells. Treatment of RINm5F cells with 5 mM STZ and 10 mug of SPEt completely abrogated apoptosis induced by STZ, suggesting the involvement of oxidative stress. Flow cytometric assessment on the level of intracellular peroxides using fluorescent probe 2'7'-dichlorofluorescein diacetate (DCF-DA) confirmed that STZ (46%) induced an intracellular oxidative stress in RINm5F cells, which was suppressed by SPEt (21%). In addition, SPEt also reduced (33%) the STZ-induced apoptosis (72%) in RINm5F cells indicating the mode of protection of SPEt on RIN m5Fcells, islets, and pancreatic beta-cell mass (histopathological observations). Present study thus confirms antihyperglycemic effect of SPEt and also demonstrated the consistently strong antioxidant properties of Scoparia dulcis used in the traditional medicine.     

In vitro cytoprotective activity of cyanidin 3-glucoside extracts from Haematocarpus validus pomace on streptozotocin induced oxidative damage in pancreatic β-cells

Cyanidin-3-glucoside (C3Ghv) compounds were purified and isolated from the anthocyanins extract of Haematocarpus validus. C3Ghv were studied for antioxidant and cytoprotective properties on pancreatic β-cells of rat insulinoma cells (RINm5F) against the oxidative stress induced by streptozotocin (STZ). The exposure of RINm5F cells to C3Ghv at concentration of 100 and 200 μg/mL for 24 h reduced 10% and 23% cell viability, respectively, as compared to control cells. The pre-treatment of RINm5F cells with C3Ghv (50 µg/mL) increased the cell viability by 29% as compared to control, on being treated with STZ (10 mM) for 24 h. The pre-treatment of RINm5F cells with C3Ghv (50 µg/mL) for 24 h followed by exposure to STZ (10 mM) for 1 h decreased the generation of reactive oxygen species (ROS) by 57%, generation of nitric oxide by 22.8%, generation of malondialdehyde (MDA) by 32%, the production of p-ERK ½ by 83%, p-JNK by 82.6%, p-MEK by 57%, and p-p38 MAPK by 64%. The C3Ghv treatment also decreased the ratio of apoptotic proteins Bax to Bcl-2 by 61%, and improved the M2 phase of cell cycle by 75% as compared to STZ treated cells. The overall results suggest that C3Ghv protects pancreatic β-cells against oxidative stress-induced apoptosis, thereby implicating the significant role of C3Ghv as an antidiabetic agent.     

Dose-dependent effect of hydrogen peroxide on calcium mobilization in mouse pancreatic acinar cells.

We have employed confocal laser scanning microscopy to investigate how intracellular free calcium concentration ([Ca2+]i) is influenced by hydrogen peroxide (H2O2) in collagenase-dispersed mouse pancreatic acinar cells. In the absence of extracellular calcium, treatment of cells with increasing concentrations of H2O2 resulted in an increase in [Ca2+]i, indicating the release of calcium from intracellular stores. Micromolar concentrations of H2O2 induced an oscillatory pattern, whereas 1 mmol H2O2/L caused a slow and sustained increase in [Ca2+]i. H2O2 abolished the typical calcium release stimulated by thapsigargin or by the physiological agonist cholecystokinin octapeptide (CCK-8). Depletion of either agonist-sensitive or mitochondrial calcium pools was unable to prevent calcium release induced by 1 mmol H2O2/L, but depletion of both stores abolished it. Additionally, lower H2O2 concentrations were able to release calcium only after depletion of mitochondrial calcium stores. Treatment with either the phospholipase C inhibitor U-73122 or the inhibitor of the inositol 1,4,5-trisphosphate (IP3) receptor xestospongin C did not modify calcium release from the agonist-sensitive pool induced by 100 micromol H2O2/L, suggesting the involvement of a mechanism independent of IP3 generation. In addition, H2O2 reduced amylase release stimulated by CCK-8. Finally, either the H2O2-induced calcium mobilization or the inhibitory effect of H2O2 on CCK-8-induced amylase secretion was abolished by dithiothreitol, a sulphydryl reducing agent. We conclude that H2O2 at micromolar concentrations induces calcium release from agonist-sensitive stores, and at millimolar concentrations H2O2 can also evoke calcium release from the mitochondria. The action of H2O2 is mediated by oxidation of sulphydryl groups of calcium ATPases independently of IP3 generation.     

Elevated cAMP level attenuates 2-deoxy-d-ribose-induced oxidative damage in pancreatic beta-cells

Glucose toxicity to pancreatic beta-cells is defined as irreversible beta-cell damage, including apoptosis, caused by chronic exposure to high glucose levels in type 2 diabetes. Oxidative stress is an important mechanism for glucose toxicity to pancreatic beta-cells. Reducing sugars produce reactive oxygen species through autoxidation and protein glycosylation. 2-Deoxy-d-ribose (dRib) is a reducing sugar with high reactivity. We investigated whether cAMP-stimulating agents could protect beta-cells from dRib-induced oxidative damage. HIT-T15 cells were cultured with various concentrations of dRib for 24 h. We measured cell survival, intracellular cAMP and H2O2 levels, and apoptosis. dRib decreased cell survival in a dose- and time-dependent manner and markedly increased intracellular H2O2 levels and apoptosis. N-Acetyl-l-cysteine decreased dRib-induced rises in intracellular H2O2 and apoptosis to control levels. Forskolin, IBMX, and dbcAMP markedly elevated intracellular cAMP levels and significantly attenuated dRib-induced cytotoxicity and apoptosis, but had no influence on the dRib-induced rise in intracellular H2O2 levels. These results demonstrate that dRib produced oxidative stress and apoptosis in pancreatic beta-cells and that elevated intracellular cAMP levels reduced dRib-induced damage, independent of reactive oxygen species metabolism.     

热量限制对SH-SY5Y细胞氧化损伤的影响

目的观察热量限制培养条件下,SH-SY5Y细胞抗氧化应激损伤的能力。方法建立过氧化氢诱导的SH-SY5Y细胞损伤模型。体外培养SH-SY5Y细胞,分为对照组、损伤组（50、100、250、500、1 000μmol/L H2O2）、低糖组（2 g/L）、低糖＋损伤组,进行细胞形态观察、测定各组细胞的噻唑蓝（MTT）代谢率、乳酸脱氢酶（LDH）漏出率。结果与对照组比较,（50、100、250、500、1 000）μmol/L H2O2损伤1 h后MTT代谢率测定细胞活力,50μmol/L组与对照组比较差异无统计学意义（P〉0.05）;其他组与对照组比较,随着H2O2浓度的增加,细胞活力呈递减趋势,差异具有显著性（P〈0.01）;选定250μmol/L H2O2组为损伤应激源。用低糖预处理细胞24 h,给与250μmol/L H2O2损伤1 h后测定MTT代谢率显示,与对照组比较,损伤组活力明显下降,低糖组活力上升（P〈0.01）;与损伤组比较,低糖＋损伤组活力明显上升（p〈0.01）;继续培养至7 h发现,与对照组比较,低糖组活力上升（P〈0.01）;与损伤组比较,低糖＋损伤组活力明显上升（P〈0.01）。进一步检测LDH漏出率显示,损伤1 h后结果显示,与对照组比较,损伤组漏出率明显增加（P〈0.05）,低糖组漏出率稍有减少（P〉0.05）;与损伤组比较,低糖＋损伤组漏出率明显减少（P〈0.01）;继续培养7h显示,低糖7h组与低糖1 h组比较,漏出稍有增多（P〉0.05）,低糖＋损伤组7 h组与低糖＋损伤组1 h比较漏出率稍有增加（P〈0.05）;细胞形态学观察显示,未加损伤之前,低糖组的细胞形态,与对照组比较无明显改变。加入损伤药物1h后的细胞形态与对照组比较无明显改变。加入损伤药物7 h后的细胞形态,低糖组和对照组细胞突起伸展良好细长,损伤组可见细胞数目明显减少,死细胞多,突起回缩,细胞明显变圆,贴壁性不好,透光性差。结论热量限制能提高神经细胞的抗氧化应激能力,增加细胞生存率,降低死亡率。     

In vitro cytoprotective activity of cyanidin 3-glucoside extracts from Haematocarpus validus pomace on streptozotocin induced oxidative damage in pancreatic β-cells

Cyanidin-3-glucoside (C3Ghv) compounds were purified and isolated from the anthocyanins extract of Haematocarpus validus. C3Ghv were studied for antioxidant and cytoprotective properties on pancreatic β-cells of rat insulinoma cells (RINm5F) against the oxidative stress induced by streptozotocin (STZ). The exposure of RINm5F cells to C3Ghv at concentration of 100 and 200 μg/mL for 24 h reduced 10% and 23% cell viability, respectively, as compared to control cells. The pre-treatment of RINm5F cells with C3Ghv (50 µg/mL) increased the cell viability by 29% as compared to control, on being treated with STZ (10 mM) for 24 h. The pre-treatment of RINm5F cells with C3Ghv (50 µg/mL) for 24 h followed by exposure to STZ (10 mM) for 1 h decreased the generation of reactive oxygen species (ROS) by 57%, generation of nitric oxide by 22.8%, generation of malondialdehyde (MDA) by 32%, the production of p-ERK ½ by 83%, p-JNK by 82.6%, p-MEK by 57%, and p-p38 MAPK by 64%. The C3Ghv treatment also decreased the ratio of apoptotic proteins Bax to Bcl-2 by 61%, and improved the M2 phase of cell cycle by 75% as compared to STZ treated cells. The overall results suggest that C3Ghv protects pancreatic β-cells against oxidative stress-induced apoptosis, thereby implicating the significant role of C3Ghv as an antidiabetic agent.     

Citrus flavanone metabolites significantly modulate global proteomic profile in pancreatic β-cells under high-glucose-induced metabolic stress

Hesperidin and narirutin are the major citrus flavanones. Several studies have associated these compounds with pancreatic β-cell survival through their capacity to reduce oxidative stress, inflammation, and inhibit apoptosis. However, the molecular mechanisms of action of flavanones in pancreatic β-cells under high-glycemic stress is still largely unknown. Therefore, this study aimed to decipher molecular mechanisms of flavanone metabolites in pancreatic β-cells treated with high glucose concentration using untargeted shotgun proteomics. We identified 569 proteins differentially expressed in cells exposed to hesperetin 7-glucuronide (H7G) and 265 in cells exposed to 3-(4′-hydroxyphenyl) propanoic acid (PA). Comparison of global proteomic profiles suggest that these metabolites could counteract changes in protein expression induced by high glucose stress. The bioinformatic analyses suggested that H7G and PA modulated the expression of proteins involved in cell adhesion, cell signaling, metabolism, inflammation, and protein processing in endoplasmic reticulum (ER) pathways. Taken together, this study suggests that H7G and PA can modulate the expression of proteins that may prevent dysfunction of pancreatic β-cells under stress induced by high glucose.     

IGFs, basic FGF, and glucose modulate proliferation and apoptosis induced by IFNgamma but not by IL-1beta in rat INS-1E beta-cells.

We investigated the effects of glucose and beta-cell growth factors (IGF-I, IGF-II, bFGF) on growth and apoptosis in the presence and absence of apoptosis inducing cytokines (IFNgamma, Il-1beta, TNFalpha). Rat INS-1E beta-cell viability was measured by WST-1 viability assay and cell counting, apoptosis by FACS analysis of annexin-V-FITC and fluorescein-dUTP (TUNEL-staining)-positive cells. Glucose alone maintained INS-1E beta-cell viability at high physiological concentrations (6.2-12.5 mmol/l), addition of IGF-II alone or in combination with bFGF further increased these glucose effects. The cytokines IFNg and IL-1beta, but not TNFalpha strongly induced INS-1E beta-cell apoptosis. Interestingly, glucose alone induced apoptosis at extremely low or very high concentrations. In combination with IFNg, low glucose (1.6 mmol/l) increased apoptosis by 25.6% (1SD 5.0%) and high glucose (50 mmol/l) by 22.8% (1SD 2.8%) compared to 12.5 mmol/l glucose. In contrast, glucose failed to modulate IL-1beta-induced apoptosis. Most importantly, IGF-II and bFGF inhibited apoptosis induced by IFNg, but not by IL-1beta. Therefore, IGF signaling, supported by bFGF and optimal glucose levels, maintains beta-cell viability in vitro. Cytokines IFNg and IL-1beta differentially interfere with intracellular signaling cascades stimulated by IGFs and bFGF or glucose, respectively.