| [Background]The patients of type 2 diabetes mellitus have increased rapidly in the world especially in the developing countries,which has been a threat to human's health as an epidemically disease.Since chronic diabetic complications can result in sight loss, uraemia and amputation,it has become difficult and important to prevent and treat diabetic complications.Accumulated evidences have revealed advanced glycation end products(AGEs) as an important mechanism of chronic diabetic complications.AGEs describes a heterogeneous group of proteins,lipids,and nucleic acids that are formed nonenzymatically.AGE formation is enhanced in the presence of hyperglycemia and oxidative stress.AGE bind to their cognate cell-surface receptor,RAGE,resulting in the activation of postreceptor signaling,generation of intracellular oxygen free radicals,and the activation of gene expression.Thus,AGE are not only markers,but act also as mediators of late diabetic complications.The main mechanisms of diabetic complications include the ignition of four metabolic pathways:advanced glycation end products(AGEs),hexosamine,protein kinase C(PKC) and polynol pathway. Brownlee have found impressant of generation of reactive oxygen species(ROS) could hinder the four metabolic pathways as above mechanisms of diabetic complications.His theory considers high glucose could induce oxidative stress which results in the ignition of the four metabolic pathways(as fig1). During the course of normal oxidative phosphorylation,however,between 0.4 and 4%of all oxygen consumed is converted into the free radical superoxide(·O2-) and is then either detoxified to H2O and O2 by glutathione peroxidase(in the mitochondria),or diffuses into the cytosol and is detoxified by catalase in peroxisomes.However,in the presence of reduced transition metals such as Cu or Fe, H2O2 can be converted to the highly reactive·OH radical.High glucose can result in electronicas increased which can result in the injury of DNA.After series of reactions the four metabolic pathways were ingited,which was a malignant circle.At the same time anti-oxygenics were comsumed.Oxidative stress was developed thus which can induce cells apoptosis.Accumulated evidences have proved hyperglycemia could result in the elevation of ROS output and induce oxidative stress since there were not enough anti-oxidative chemicals in diabetes.Oxidative stress can ignite the pathways sensitive to oxidative stress and result in some gene products.Thus chronic diabetic complications were developed.Though we have known some knowledgements about the mechnisms of chronic diabetic complications,there are limited methods directed against the mechanisms. Considerable researches have proved the HO system play an important role in anti-oxidative stress.But the role of the HO system in diabetes,inflammation,heart disease,hypertension,neurological disorders,transplantation,endotoxemia and other pathologies is still a burgeoning area of research.The myriad metabolic systems that have subsequently been shown to be responsive to the up-regulation of HO activity make it clear that enhancing HO activity by either pharmacological or genetic means offers potential for the development of new therapeutic modalities,which could moderate the course of various pathological processes in humans.Heme oxygenase is the rate-limiting enzyme in the catabolism of heme,a process that leads to formation of equimolar amounts of the bile pigment biliverdin,iron,and carbon monoxide(CO). Biliverdin formed in this reaction is rapidly converted to bilirubin.Both CO and bilirubin are bioactive molecules,and the iron generated by HO-1 and HO-2 is immediately sequestered by associated increases in ferritin.HO-2 is a constitutive enzyme,whereas HO-1 is inducible by heavy metals,cytokines,UV light,oxidative stress,inflammatory cytokines and many drugs.It is therefore possible that the induction of HO-1 may be an essential event for some types of acute reactions and for cellular protection after injury.Because the major source of CO in animals is the degradation of heme by HO,it is now becoming apparent that CO serves as an important cellular signal molecule.Nitric oxide(NO) is generated by nitric-oxide synthase(NOS),a heme containing enzyme.It has been proposed that certain NO effects can be duplicated by CO,specifically;the action of certain neurotransmitters and muscle relaxants can be regulated by both molecules.The biological actions of bilirubin may be especially relevant to the prevention of oxidant-mediated cell death. Bilirubin at a low concentration scavenges ROS in vitro,thereby reducing oxidant-mediated cellular damage and attenuating oxidant stress in vivo.Bilirubin inhibits NADPH oxidase and PKC activity.Both enzymes have been shown to mediate angiotensinⅡ-induced vascular injury.Recently,biliverdin and bilirubin have been shown to preserve endothelial cell integrity,prevent endothelial cell death and sloughing,enhance vascular reactivity,and prevent restenosis.Bilirubin is also implicated in reducing oxidative stress in experimental diabetes,in part,by increasing the bioavailability of NO needed for endothelial cell integrity.Plasma iron is bound to transferrin,which can transfer iron to the intracellular milieu of endothelial cells via cell surface receptor binding.The up-regulation of HO-1 increases ferritin synthesis to sequester the iron released from the HO-1 pathway.Hyperglycemia-mediated local formation of ROS is considered to be a major contributing factor to endothelial dysfunction which play a key role in developemnt of chronic diabetic complicaitons.This includes endothelial cell apoptosis,abnormalities in cell cycling due to lack of HO-1 induction and increased(?)formation.Some of these perturbations can be reversed by antioxidant agents or by increased levels of antioxidant enzymes.Increased levels of HO-1 through gene transfer in hyperglycemic rats resulted in a decrease of endothelial cell sloughing.Delivery of the human HO-1 gene to endothelial cells attenuated glucose-mediated oxidative stress,DNA damage,and cell death.HO-1 induction has been shown to provide vascular cytoprotection against oxidative stress.Thus,HO-1 plays a pivotal role in mitigating the detrimental effects of hyperglycemia.Increases in HO-1 expression in diabetic obese mice were paralleled by increases in serum adiponectin levels,insulin sensitivity,decreases in visceral and abdominal fat content,and decreased plasma TNFα,IL-6,and IL-1βlevels.Induction of HO-1 also resulted in decreased adipocyte superoxide production.Up-regulation of HO-1 by CoPP treatment caused a decrease in adipogenesis in bone marrow,both in vivo and in vitro in cultured mesenchymal stem cells and in increases in secretion of adiponectin in the culture media.Diabetic complications have been related to abnormalities in mitochondrial function as well as to increased endothelial cell death and detachment.Specific human HO-1 gene transfer to diabetic rats has also resulted in the restoration of mitochondrial carriers,including ADP/ATP and decarboxylate.The increase in HO-1 in diabetic rats is associated with increased pAKT.An increase in AKT phosphorylation is critical to cell survival in diabetes.Increases in AKT phosphorylation and Bcl-xL levels have been shown to prevent the loss ofβ-cells in diabetes.It is interesting to note that the alteration in mitochondrial function in vitro and in vivo correlates with the levels of activation of AKT and the Bcl-2 family of proteins.A decrease in Bcl-2 family members has been reported to contribute to apoptosis and the translocation of cytochrome c from the mitochondria to cytosol. Activation of AKT has been shown to augment ATP synthesis and promote the association of hexokinase with the voltage-dependent anion channel and,in so doing, promote voltage-dependent anion channel closure,thus blocking release of cytochrome c.Though the last decade has witnessed an explosion in the elucidation of the role that the HO system plays in human physiology,effects of HO-1 on oxidative stress in diabetes mellitus were not completely clarified yet.The use of pharmacological agents and genetic probes for manipulating HO has led to new insights into the prevention of chronic diabetic complications.Chapter 1Expressions of heme oxygen ase-1 in THP-1 cells incubated with high glucose and AGEs[Objectives]1.To investigate into the effects of glucose and advanced glycation end products (AGEs) on ROS outputs in THP-1 cells. 2.To investigate into the effects of high glucose combinded with AGEs on oxidative stress in THP-1 cells.3.To investigate into the effects of glucose and AGEs on expressions of HO-1 in THP-1 cells.4.To investigate if p38MAPK signal pathway were involved in oxidative stress in THP-1 cells induced by high glucose and AGEs.[Methods]1.Cell culture:THP-1 cells were cultured at 37℃,5%CO2 with RPMI-1640 containing 10%FCS,penicillin(100units/mL),and streptomycin(100μg/mL).2.Preparation of AGEs:Briefly,BSA and AGEs were prepared by incubation of 3.55 mg/mL BSA in the presence or absence of glucose and 0.5 mmol/L sodium azide in PBS(pH 7.4) at 37℃for 8 weeks.3.Determination of ROS outputs:Cells were resuspended in medium containing 10mmol/L of DCFH-DA(Molecular Probes),and incubated for 30min.And then flow cytometric analysis was used by FACScan(Becton-Dickinson,Mountain View,CA) at excitation 488 nm and emission 525nm to detect the mean fluorescence intensity(MFI) as ROS output.4.MDA assay:By TBA assay method.5.TNF-a ELISA assay:By enzyme linked immunosorbent assay(ELISA).6.RT-PCR for HO-1mRNA:Total RNA was isolated using TRIzol reagents (Invitrogen).The electrophoresis of the amplified products was operated with 1.5%agarose gel.7.Western blot analysis for HO-1 expression:Cytoplasmic extracts were separated from the nuclei.An equal amount of protein(30mg/well) for each sample was boiled for 5 min,loaded,and run for electrophoresis in a 4-20%Tris-Glycine gel. The gel was transferred electrophoretically to nitrocellulose membranes,and the blots were blocked with 5%milk in TBST.Membranes were then probed with a polyclonal rabbit antibody against HO-1 and with a horseradish peroxidaseconjugated anti-rabbit IgG.For quantification,bands in photo-graphs were scanned by a densitometer linked to a computer system. 8.p38MAPK signal pathway:There were 4 groups:15mmol/L-GLU group, SB+15mmol/L-GLU group,100μg/mL-AGEs group and SB+100μg/mL-AGEs group.SB group means the cells were stimulated by 10μmol/L SB203580 (inhibitor of p38MAPK signal pathway) before incubation with 15mmol/L GLU or 100μg/mL AGEs for 24h.9.Statistical analysis:Statistical analyses were carried out with the SPSS 13.0.All data are present as means and standard deviation((?)±s) of multiple measurements.Factorial analysis ANOVA was used,t test for independent specimen.Pearson coefficient correlation was used too.α=0.05.[Results]1.Concentration of AGEs:The fluorescence intensity of AGEs showed 7.40-fold (excitation 360 nm,emission 430nm) higher than that of BSA control.The concentration of AGEs was 101.29U/mg protein,while BSA was 13.68U/mg.2.ROS output(mean fluorescence intensity,MFI):GLU+AGEs group(107.21±8.94) has significantly higher ROS outputs than GLU group(65.88±1.61) and AGEs group(67.46±3.78)(P＜0.05),while that of AGEs group and GLU group were all significantly higher than control group(41.95±1.36)(P＜0.05).3.MDA(nmol/mL):GLU+AGEs group(3.26±0.32) has significantly higher MDA than GLU group(1.59±0.53) and AGEs group(1.56±0.97)(P<0.05),and that of GLU group was significantly higher than control group(P＜0.05).But there was no significantly difference between AGEs group and control group(0.65±0.23) (P＞0.05).4.TNFa(pg/mL):GLU+AGEs group(51.25±1.72) has significantly higher TNFa than GLU group(25.38±1.95) and AGEs group(24.43±2.65)(P＜0.05),and that of GLU group and AGEs group were significantly higher than that of control group(14.97±1.49)(P＜0.05).5.HO-1mRNA:Expression of HO-1mRNA of GLU+AGEs group(0.89±0.12) was significantly higher than that of GLU group(0.42±0.02) and AGEs group (0.48±0.03)(P＜0.05).And GLU group and AGEs group have significantly higher HO-1mRNA than control group(0.15±0.01)(P＜0.05).6.HO-1 protein:GLU+AGEs group(0.81±0.02) has significantly higher HO-1 protein than GLU group(0.39±0.01) and AGEs group(0.45±0.03)(P＜0.05).And GLU group and AGEs group have significantly higher level than control group (0.15±0.01)(P＜0.05).7.Correlation of ROS,MDA,TNF-a and HO-1mRNA and its protein:There was significantly positive correlation between the output of the expression of HO-1 mRNA and ROS,MDA(r=0.858,P=0.000) and TNFα(r=0.952,P=0.000).There were alos correlation between ROS output and HO-1mRNA(r=0.990,P=0.000), expression of HO-1 protein,MDA(r=0.873,P=0.000) and TNFα(r=0.964, P=0.000).And there were correlation between TNFαand ROS,MDA(r=0.851, P=0.000),HO-1mRNA and its protein.8.There were no significant difference in expressions of HO-1 mRNA between GLU group,SB+GLU group,AGEs group and SB+AGEs group.[Conclusions]1.ROS output of THP-1 cells has been exacerbated when incubated with high glucose or AGEs and presented as dose-,time-dependent increasing. Furthermore,AGEs combined with high glucose group has much more ROS output than high glucose or AGEs group.These suggest that there is a synergistic relationship between high glucose and AGEs in oxidative stress.AGEs combined with high glucose could induce more MDA and TNFαin THP-1 cells.2.The expression of HO-1mRNA and protein in THP-1 cells treated with high glucsoe combined AGEs has been increased significantly,which hint HO-1 could be an effective protective factor for oxidative stress induced by high glucose and AGEs in THP-1 cells.And some methods which can induce high expression of HO-1 may help to resist oxidative injury induced by high glucose and AGEs.3.p38MAPK signal pathway may not be involved in the transcription of HO-1 in THP-1 cells treated by high glucose and AGEs. Chapter 2Effects of ZnPP(inhibitor of HO-1) on oxidative stress in THP-1 cells induced by high glucose and AGEs[Objective]1.To investigate into the effects of ZnPP(inhibitor of HO-1) on oxidative stress induced by high glucose and AGEs in THP-1 cells.[Methods]1.Groups:There were 4 groups as control group,15mmol/LGLU+100μg/mL AGEs group,ZnPP group,ZnPP+15mmol/LGLU+100μg/mLAGEs group.The concentration of glucose in the cell culture solution of control group and ZnPP group were all 5mmol/L.2.Determination of ROS output,MDA,TNF-a,HO-1mRNA and proteins:Just the same as Chapter 1.3.Statistical analysis:Statistical analyses were carried out with the SPSS 13.0.All data are present as means and standard deviation((?)±s) of multiple measurements.Factorial analysis ANOVA was used,t test for independent specimen.α=0.05.[Result]1 ROS output(mean fluorescence intensity,MFI):ZnPP+GLU+AGEs gorup (128.81±5.53) has significantly higher ROS output than GLU+AGEs group (107.21±8.94) and ZnPP group(51.56±2.09)(P＜0.05).ROS output of GLU+AGEs gorup and ZnPP group were significantly higher than that of control group(41.95±1.36)(P＜0.05).2 MDA(nmol/mL):ZnPP+GLU+AGEs group(3.75±1.25) has significantly higher MDA than ZnPP group(1.61±0.10)(P＜0.05),but there were no significantly difference between ZnPP+GLU+AGEs group and GLU+AGEs group(3.26±0.32) (P＞0.05).MDA of GLU+AGEs group and ZnPP group were significantly higher than that of control group(0.65±0.23)(P＜0.05). 3 TNFa(pg/mL):ZnPP+GLU+AGEs group(141.10±12.84) has significantly higher TNFa than GLU+AGEs group(38.09±14.97) and ZnPP group(27.34±10.18) (P＜0.05).TNFa of GLU+AGEs group and ZnPP group were significantly higher than that of control gorup(18.55±7.26)(P＜0.05).4 HO-1mRNA:ZnPP+GLU+AGEs group(0.39±0.02) has significantly lower expression of HO-1mRNA than GLU+AGEs group(0.89±0.09)(P＜0.05),and significantly higher than ZnPP group(0.24±0.02)(P＜0.05).GLU+AGEs group and ZnPP group have higher HO-1mRNA than control group(0.15±0.01) (P＜0.05).5 HO-1 protein:ZnPP+GLU+AGEs group(0.38±0.00) has significantly lower HO-1 protein than GLU+AGEs group(0.81±0.02)(P＜0.05),and significantly higher than ZnPP group(0.23±0.03)(P＜0.05).GLU+AGEs group and ZnPP group have higher HO-1 protein than control group(0.15±0.01)(P＜0.05).[Conclusion]1.ZnPP itself could induced oxidative stress,injury and inflammation in THP-1 cells.ZnPP,as an inhibitor of HO-1,could aggravate oxidative stress and inflammation in THP-1 induced by high glucose and AGEs.Chapter 3Effects of CoPP(inducer of HO-1) on oxidative stress in THP-1 cells induced by high glucose and AGEs[Objectives]1.To investigate into the effects of CoPP(inducer of HO-1) on oxidative stress induced by high glucose and AGEs in THP-1.[Methods]1.Groups:There were 4 groups as control group,15mmol/LGLU+100μg/mLAGEs group,CoPP group,CoPP+15mmol/L-GLU+100μg/mL-AGEs group.The concentration of glucose in the cell culture solution of control group and CoPP group were all 5mmol/L. 2.Determination of ROS output,MDA,TNF-a,HO-1mRNA and proteins:Just the same as Chapter 1.3.Statistical analysis:Statistical analyses were carried out with the SPSS 13.0.All data are present as means and standard deviation((?)±s) of multiple measurements.Factorial analysis ANOVA was used,t test for independent specimen.α=0.05.[Results]1 ROS output(mean fluorescence intensity,MFI):GLU+AGEs has the main effect on ROS output(F=287.930,P=0.000),while CoPP has not the main effect (F=0.433,P=0.529),and there were interact between GLU+AGEs and CoPP (F=8.632,P=0.019),which means with the GLU+AGEs,CoPP could lower the ROS output.ROS output of GLU+AGEs group was significantly higher than control group(41.95±1.36)(P＜0.05),and there were no significantly difference between CoPP group and control group(P＞0.05).2 MDA(nmol/mL):CoPP+GLU+AGEs group(1.28±0.61) has significantly lower MDA than GLU+AGEs group(3.26±0.32)(P＜0.05),and there were no significantly difference between CoPP group(0.73±0.45) and control group (0.65±0.23)(P＞0.05).GLU+AGEs group has significantly higher MDA than control group(P＜0.05),and there were no difference between CoPP group and control group(P＞0.05).3 TNFa(pg/mL):There were no significantly difference between CoPP+GLU+ AGEs group(31.90±12.56),GLU+AGEs group(43.60±12.40) and CoPP group (16.60±7.99)(P＞0.05).GLU+AGEs group has higher TNFa than control group (14.97±1.49)(P＜0.05),while there were no difference between CoPP group and control group(P＞0.05).4 HO-1mRNA:There were no significantly difference between CoPP+GLU+ AGEs group(0.91±0.01) and GLU+AGEs group(0.89±0.12) and CoPP group (16.60±7.99)(P>0.05).CoPP+GLU+AGEs group has higher HO-1mRNA than CoPP group(0.24±0.01)(P＜0.05).CoPP group and GLU+AGEs group have higher level than control group(P＜0.05).5 HO-1 protein:CoPP+GLU+AGEs group(0.87±0.01) has higher HO-1 protein than GLU+AGEs group(0.81±0.02) and CoPP group(0.22±0.02)(P＜0.05). CoPP group and GLU+AGEs group has higher level than control group (0.15±0.01)(P＜0.05).[Conclusions]1.CoPP,an inducer of HO-1,could effectively decrease oxidative stress in THP-1 induced by high glucose and AGEs,while CoPP itself has not cause oxidative stress.Though CoPP has decreased the level of TNFa in THP-1 cells treated by high glucose and AGEs there was no significantly differece.Certainly CoPP significantly enhanced the expression of HO-1 protein but not HO-1mRNA.2.HO-1 as a stress protein could protect THP-1 cells against oxidative injury induced by high glucose and AGEs.Methods which could increase the expression of HO-1 would help to resist oxidative injury induced by high glucose and AGEs.Chapter 4Relationship between Transcription of HO-1 in Peripheral Blood Mononuclear Cells of Newly Diagnostic Type 2 Diabetic Patients with Chronic Complications and Oxidative Stress[Objectives]1.To investigate into the relationship between transcription of HO-1 in peripheral blood mononulclear cells(PBMC) of newly diagnostic type 2 diabetic patients with chronic complications and oxidative stress[Methods]1.Subjects were all newly diagnostic type 2 diabetes from our department.The diagnosis and classification criteria of type 2 diabetes mellitus was 1999 WHO criteria.Any acute metabolic disturbance and any drug intake were excluded. Diabetic chronic complications were determined by retinal photograph,feeling check and 24h urine albumin excretion.There were three groups:normal control group,non-complications group and complications group.2.Peripheral blood mononuclear cells(PBMC):All of subjects were taken blood samples after fasting for 8～14 hours.PBMC were seperated by ficoll-urografin density gradient centrifugation.Serum of samples were kept in-20℃for MDA analysis.3.ROS output of PBMC,serum MDA and expression of HO-1mRNA:Just the same as Chapter 1.4.Expression of HO-1 protein:Immunohistochemistry was used to detect the expression of HO-1 protein.Disposition of HO-1 in the kytoplasm was analyzed by fluorescence microscope.cytoplasm has green fluorescent which means positive which were calculated.5.Statistical analysis:Statistical analyses were carried out with the SPSS 13.0.All data are present as means and standard deviation((?)±s) of multiple measurements.The difference between two groups was analyzed by two-independent t test.Pearson Correlation was used to analyse the correlation among multiple variables.The received level of significance isα=0.05.[Results]1.Compared with non-complications group,complications group has higher fasting blood glucose(16.26±6.67 vs 10.61±3.61),HbAlc(12.54±2.10 vs 10.51±1.86), ROS output(419.60±216.31 vs 172.38±145.24),serum MDA(66.04±13.67 vs 0.77±0.23),HO-1mRNA(1.02±0.27 vs 0.77±0.23) and protein(85.47±10.89 vs 61.45±21.81)(P＜0.05).2.There was positive correlation between HO-1mRNA and protein(r=0.750, P=0.000),MDA(r=0.449,P=0.010),FPG(r=0.364,P=0.041) and 2hPG(r=0.477, P=0.016).Expression of HO-1 protein was correlated with ROS output(r=0.608, P=0.000),MDA(r=0.623,P=0.000).There were positive correlation between MDA and ROS output(r=0.788,P=0.000),HbAlc and 2hPG(r=0.412,P=0.041), 2hPG and FPG(r=0.811,P=0.000).[Conclusions]1.There are obvious oxidative stress in newly diagnostic type 2 diabetic patients with chronic complications and hyperglycemia may induce oxidative stress which then results in diabetic chronic complications.2.Correlation analysis hint that oxidative stress induced by diabetes mellitus could upregulate HO-1.Up-regulation of HO-1 could help to resist against oxidative injury induced by diabetes mellitus though it may not be enough to entirely protect cells.It would be a new pathway to prevent diabetic chronic complications by upregulating HO-1.
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