責任著者 Corresponding author. キーワード: catechins , diabetes , Goto-Kakizaki rat , glucose tolerance , oxidative stress. ジャーナル フリー. PDFをダウンロード K メタデータをダウンロード RIS形式 EndNote、Reference Manager、ProCite、RefWorksとの互換性あり.

引用文献 関連文献 0. 電子付録 0. 前の記事 次の記事. Timeline of in vivo experiments. Rabbits were randomized into six groups. Group 1 Control : control rabbits given saline intragastrically after injured carotid artery operation.

Group 2 DM : diabetic rabbits given saline intragastrically after injured carotid artery operation. Group 4 DM-sham : injured carotid artery of diabetic rabbits washed and then incubated 10 min by saline after injured operation.

Group5 DM-EPC : autologous L-EPCs transplanted into the area of injured carotid artery of diabetic rabbits after injured carotid artery operation. Group 6 DM-EGCG-EPC : EGCG pre-treated autologous L-EPCs transplanted into the area of injured carotid artery of diabetic rabbits after injured carotid artery operation.

The diabetic rabbits were then given normal diet for 4—6 weeks with fasting blood glucose level checked once per week. Rabbit carotid injury was performed by using a 3. Figure 1 shows the timeline of the in vivo experiment. After that, a total of 1 × 10 6 cells were resuspended in μl of 4°C saline.

Immediately after the balloon withdrawal, cells were injected into the carotid lumen, and were kept for 10 min before the incision was closed. The pictures were taken and the area of denudation was identified using the NIH Image J software by an investigator blinded to the group assignment.

In CM-Dil labeled L-EPC auto-transfusion groups, the homing of transplanted EPCs to the site of injured vascular was analyzed using fluorescent microscopy Olympus, Tokyo, Japan. Then, cells were randomized into four groups: control, mannitol, high glucose, and high glucose group with EGCG 40 μM treatment.

For the high glucose group, cells were co-incubated with 30 mM glucose, whereas cells in the osmotic pressure control mannitol group were co-incubated with 30 mM mannitol. To detect the exact proliferation rates of L-EPCs an EDU 5-ethynyldeoxyuridine incorporation assay was executed with the Cell-Light TM EdU in vitro Imaging Kit Ribobio, Guangzhou, China according to the manufacturer instructions.

After the EDU staining, cell nuclei were stained with Hoechst and observed with an inverted fluorescent microscope Olympus, Tokyo, Japan. For each group, 6 random fields were photographed. The proliferation rate refers to the number of EDU stained cells divided by the number of Hoechst stained cells.

Late endothelial progenitor cells migration assay was performed with a modified Boyden chamber assay as described previously Qiu et al. Cells were examined under a fluorescent microscope Olympus, Tokyo, Japan at a × total magnification and 6 random fields were photographed for each sample.

The average number of migrated cells in the 6 random fields was computed and used as the migration number for the group. Total RNA of the cultured L-EPCs was extracted with standard TRIZOL Invitrogen, Carlsbad, CA, United States method.

β-actin was used as endogenous controls for mRNAs expression. Primers of Akt, eNOS, and β-actin were synthetized by Invitrogen Carlsbad, CA, United States.

Cells in a 6-well plate were scraped in a RIPA lysis buffer Beyotime, Shanghai, China supplemented with 1 mM PMSF. After incubations in an enhanced chemiluminescence reagent Amersham, Haemek, Israel , images were captured and analyzed on the LAS image reader system Fujifilm, Tokyo, Japan.

Data were presented as means ± SEM. Analyses were conducted with SPSS A total of about 3 × 10 6 —1 × 10 7 of PBMCs was isolated from each 20 ml blood sample. Most of the cells were double-positive for Dil-acLDL uptake and FITC—lectin binding Figure 2B. There were FIGURE 2. Identification of L-EPCs.

A The panel shows the sequential changes of L-EPCs. All the pictures were taken under × magnification.

B The L-EPCs shows both Dil-uptaken red and lectin binding green ability × magnification. C Flow-cytometry reveals the L-EPCs expressed abundant CD34 and KDR molecule. Numbers are presented as Mean ± SEM. Percentage of positive cells for all experiment is determined by comparison with corresponding negative control labeling.

There was no statistical difference on rabbit weight and age among all the three groups data not shown. Diabetic model was rendered successfully by using an alloxan method. The average fasting blood glucose level was 5. Compared with the control group, diabetic rabbits showed poor reendothelialization ratios after carotid injury FIGURE 3.

Reendothelialization of injured carotid arteries is promoted by EGCG. A Reendothelialization of injured carotid arteries is promoted by intragastrically-given of EGCG. The blue area, which was rendered by Evans Blue, represent the area of carotid artery where lack of endothelium.

The control group shows the best reendothelialization, whereas diabetes rabbits show very poor reendothelialization. After intragastrically given EGCG for 7 days, the reendothelialization rates risen. The number of rabbits in each group was 6.

DM: diabetic rabbits were given with saline intragastrically after injured carotid artery operation. B EGCG pre-treated autologous L-EPCs transfusion improved diabetic rabbits carotid reendothelialization. DM-sham: injured carotid artery of diabetic rabbits were washed and then incubated 10 min by saline after injured operation.

DM-EPC: autologous L-EPCs were transplanted into the area of injured carotid artery of diabetic rabbits after injured carotid artery operation. DM-EGCG-EPC: EGCG pre-treated autologous L-EPCs were transplanted into the area of injured carotid artery of diabetic rabbits after injured carotid artery operation.

C Representative figure of autologous L-EPC tracking in vivo under fluorescent microscope. Picture shows the extent to which L-EPCs are involved in reendothelialization 7 days after carotid artery injury. CM-DiI-labeled L-EPCs red are attached to endothelium FITC-lectin-stained, green.

Nuclei of L-EPC were stained with DAPI blue. To identify whether EGCG benefits carotid reendothelialization through improving the functions of L-EPCs, we transfused autologous L-EPCs to the carotid injured rabbits. Compared with diabetic sham-operated group, diabetic autologous L-EPCs transplantation locally to the injured carotid artery showed an accelerated trend of the reendothelialization process, but statistical difference was not found After EGCG pre-treatment for 72 h, autologous L-EPCs transfusion strongly improved the reendothelialization of diabetic rabbits Moreover, autologous L-EPCs transfusion group EGCG pre-treated displayed clearly more CM-Dil labeled L-EPCs attached to the endothelium, compared with the non-pre-treated ECGG group Figure 3C.

Late endothelial progenitor cells viability was determined by a CCK-8 test. We first examined the toxicity of EGCG to L-EPCs. Total confluent L-EPCs cultured in well plate were added different concentrations of EGCG and their absorbance at nm was examined.

As shown in Figure 4A , up to 40 μM concentration of EGCG showed no poisonousness to L-EPCs. The typical IC50 of EGCG is μM. High glucose treatment for 72 h directly impaired the proliferation of L-EPCs, while this impairment was greatly blocked in a time-and-dose dependent manner by EGCG.

EGCG treatment of 40 μM for 72 h could most effectively recover the proliferation of L-EPCs impaired by high glucose Figures 4B,C. FIGURE 4. EGCG improves L-EPCs proliferation and migration under high glucose condition.

A CCK-8 test reveals that up to 40 μM EGCG shows no poisonousness to L-EPCs, the typical IC50 of the chemical is μM. B,C L-EPCs incubated in high glucose environment 30 mM were co-incubated under different concentrations of EGCG 1—40 μM for different time 24—72 h.

Histogram reveals that high glucose treatment for 72 h directly impairs the proliferation of L-EPCs, this impairment could be block by EGCG in a time and dose dependent manner. D EDU incorporation assay × magnification. The magnification is × The mobility of L-EPCs was examined by a Boyden chamber assay.

The mRNA level of both Akt and eNOS remained unmodified by 72 h high glucose treatment. Also the EGCG treatment did not affect the Akt and eNOS mRNA level Figure 5A.

Compared with the control group, high glucose blocked the phosphorylation of Akt and eNOS in diabetic rabbits by half as shown in the western blot test, and the EGCG treatment restored the level of p-Akt and p-eNOS completely Figures 5B,C. FIGURE 5. Current issue.

Effects of epigallocatechin gallate on total antioxidant capacity, biomarkers of systemic low-grade inflammation and metabolic risk factors in patients with type 2 diabetes mellitus: the role of FTO-rs polymorphism. Seyedahmad Hosseini 1. Meysam Alipour 1. Mehrnoosh Zakerkish 2. Bahman Cheraghian 3.

Pegah Ghandil 4. Nutrition and Metabolic Diseases Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. Department of Endocrinology and Metabolism, Health Research Institute, Diabetes Research Center, Jundishapur University of Medical Sciences, Ahvaz, Iran.

Department of Statistics and Epidemiology, Faculty of Public Health, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.

Department of Medical Genetics, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. Meysam Alipour.

body mass index. Metabolic Syndrome. Introduction: Type 2 diabetes mellitus T2DM is known as one of the most common metabolic diseases and FTO polymorphism has been implicated in the susceptibility to this disease. Epigallocatechingallate EGCG has shown favorable effects on risk factors related to T2DM.

The present study aimed to investigate the effects of EGCG on total antioxidant capacity, biomarkers of systemic low-grade inflammation and metabolic risk factors in patients with T2DM considering the role of FTO polymorphism.

Material and methods: In this double-blind randomized clinical trial, 60 patients with T2DM 20—60 years were randomly allocated to three groups. Group 1 received mg of EGCG TT genotype.

We genotyped FTO rs and measured body mass index BMI , blood pressure, profile lipid, interleukin-6, high sensitivity C-reactive protein and total antioxidant capacity, before and after the intervention, at 2 months. Results: In carriers of A allele, EGCG intervention caused a significant decrease in BMI, diastolic blood pressure DBP , mean arterial pressure and serum cholesterol level compared with placebo p 0.

Conclusions: These findings suggest that carriers of the risk alleles A of FTO-rs have a better response to EGCG in improving BMI and DBP in patients with T2DM.

Introduction Type 2 diabetes mellitus T2DM is known as one of the most common metabolic diseases. Material and methods Participants Sixty-six patients with T2DM, screened from an endocrine clinic, were recruited. Ethical approval The study was approved by the Ethics Committee of Ahvaz Jundishapur University of Medical Sciences ID: IR.

Study design and randomization This study was a randomized double-blind placebo-controlled trial conducted in Ahvaz, Iran, from August to March Figure 1 Flowchart of the study. Anthropometric and blood pressure measurement Weight accuracy of 0. Biochemical measurements The blood samples 5 ml were collected during the study 2 months in the hour fasting condition.

Genotyping A 5 ml blood sample was collected in EDTA containing tubes and stored at —80°C for extraction of DNA. Statistical analysis All analyses were performed with SPSS Results Of the volunteers, were eligible and underwent genotyping, anthropometric and biochemical assessments.

Table I Baseline characteristics of study patients. a Post-baseline changes within groups. Table II Effects of epigallocatechingallate EGCG on anthropometric indexes and blood pressure, regarding FTO -rs Table III Between-group analysis of epigallocatechingallate EGCG effects, regarding FTO -rs a Changes between the three groups.

c Significant changes in EGCG group TT compared to placebo. Discussion In the current study, we investigated the modulation of metabolic and inflammatory responses to EGCG supplementation by polymorphism of FTO -rs in patients with T2DM.

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Emerging therapies for raising high-density lipo-protein cholesterol HDL-C and augmenting HDL particle functionality Best Pract Res Clin Endocrinol Metab.

Łabuz-Roszak B , Machowska-Majchrzak A , Skrzypek M , et al. Antiplatelet and anticoagulant therapy in elderly people with type 2 diabetes mellitus in Poland based on the PolSenior Study Arch Med Sci.

Rezazadeh K , Rahmati-Yamchi M , Mohammadnejad L , Ebrahimi-Mameghani M , Delazar A. Effects of artichoke leaf extract supplementation on metabolic parameters in women with metabolic syndrome: influence of TCF7L2-rs and FTO-rs polymorphisms Phytother Res.

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Khella MS , Hamdy NM , Amin AI , El-Mesallamy HO. The FTO gene polymorphism is associated with metabolic syndrome risk in Egyptian females: a case-control study BMC Med Genet. Xi B , Takeuchi F , Meirhaeghe A , et al. Alipour M , Malihi R , Hosseini SA , et al.

The effects of catechins on related risk factors with type 2 diabetes: a review Progress in Nutrition. Kim HS , Montana V , Jang HJ , Parpura V , Kim JA. Epigallocatechin gallate EGCG stimulates autophagy in vascular endothelial cells: a potential role for reducing lipid accumulation J Biol Chem.

Suzuki T , Pervin M , Goto S , Isemura M , Nakamura Y. Beneficial effects of tea and the green tea catechin epigallocatechingallate on obesity Molecules. Liu HW , Chan YC , Wang MF , Wei CC , Chang SJ.

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For Group5 DM-EPC and Group 6 DM-EGCG-EPC , autologous L-EPCs and EGCG pre-treated autologous L-EPCs were transplanted into the area of injured carotid artery of diabetic rabbits after procedure.

FIGURE 1. Timeline of in vivo experiments. Rabbits were randomized into six groups. Group 1 Control : control rabbits given saline intragastrically after injured carotid artery operation.

Group 2 DM : diabetic rabbits given saline intragastrically after injured carotid artery operation. Group 4 DM-sham : injured carotid artery of diabetic rabbits washed and then incubated 10 min by saline after injured operation.

Group5 DM-EPC : autologous L-EPCs transplanted into the area of injured carotid artery of diabetic rabbits after injured carotid artery operation.

Group 6 DM-EGCG-EPC : EGCG pre-treated autologous L-EPCs transplanted into the area of injured carotid artery of diabetic rabbits after injured carotid artery operation. The diabetic rabbits were then given normal diet for 4—6 weeks with fasting blood glucose level checked once per week.

Rabbit carotid injury was performed by using a 3. Figure 1 shows the timeline of the in vivo experiment. After that, a total of 1 × 10 6 cells were resuspended in μl of 4°C saline.

Immediately after the balloon withdrawal, cells were injected into the carotid lumen, and were kept for 10 min before the incision was closed. The pictures were taken and the area of denudation was identified using the NIH Image J software by an investigator blinded to the group assignment.

In CM-Dil labeled L-EPC auto-transfusion groups, the homing of transplanted EPCs to the site of injured vascular was analyzed using fluorescent microscopy Olympus, Tokyo, Japan.

Then, cells were randomized into four groups: control, mannitol, high glucose, and high glucose group with EGCG 40 μM treatment. For the high glucose group, cells were co-incubated with 30 mM glucose, whereas cells in the osmotic pressure control mannitol group were co-incubated with 30 mM mannitol.

To detect the exact proliferation rates of L-EPCs an EDU 5-ethynyldeoxyuridine incorporation assay was executed with the Cell-Light TM EdU in vitro Imaging Kit Ribobio, Guangzhou, China according to the manufacturer instructions.

After the EDU staining, cell nuclei were stained with Hoechst and observed with an inverted fluorescent microscope Olympus, Tokyo, Japan. For each group, 6 random fields were photographed. The proliferation rate refers to the number of EDU stained cells divided by the number of Hoechst stained cells.

Late endothelial progenitor cells migration assay was performed with a modified Boyden chamber assay as described previously Qiu et al.

Cells were examined under a fluorescent microscope Olympus, Tokyo, Japan at a × total magnification and 6 random fields were photographed for each sample.

The average number of migrated cells in the 6 random fields was computed and used as the migration number for the group. Total RNA of the cultured L-EPCs was extracted with standard TRIZOL Invitrogen, Carlsbad, CA, United States method. β-actin was used as endogenous controls for mRNAs expression.

Primers of Akt, eNOS, and β-actin were synthetized by Invitrogen Carlsbad, CA, United States. Cells in a 6-well plate were scraped in a RIPA lysis buffer Beyotime, Shanghai, China supplemented with 1 mM PMSF.

After incubations in an enhanced chemiluminescence reagent Amersham, Haemek, Israel , images were captured and analyzed on the LAS image reader system Fujifilm, Tokyo, Japan. Data were presented as means ± SEM. Analyses were conducted with SPSS A total of about 3 × 10 6 —1 × 10 7 of PBMCs was isolated from each 20 ml blood sample.

Most of the cells were double-positive for Dil-acLDL uptake and FITC—lectin binding Figure 2B. There were FIGURE 2. Identification of L-EPCs.

A The panel shows the sequential changes of L-EPCs. All the pictures were taken under × magnification. B The L-EPCs shows both Dil-uptaken red and lectin binding green ability × magnification.

C Flow-cytometry reveals the L-EPCs expressed abundant CD34 and KDR molecule. Numbers are presented as Mean ± SEM. Percentage of positive cells for all experiment is determined by comparison with corresponding negative control labeling.

There was no statistical difference on rabbit weight and age among all the three groups data not shown. Diabetic model was rendered successfully by using an alloxan method.

The average fasting blood glucose level was 5. Compared with the control group, diabetic rabbits showed poor reendothelialization ratios after carotid injury FIGURE 3. Reendothelialization of injured carotid arteries is promoted by EGCG.

A Reendothelialization of injured carotid arteries is promoted by intragastrically-given of EGCG. The blue area, which was rendered by Evans Blue, represent the area of carotid artery where lack of endothelium.

The control group shows the best reendothelialization, whereas diabetes rabbits show very poor reendothelialization. After intragastrically given EGCG for 7 days, the reendothelialization rates risen. The number of rabbits in each group was 6.

DM: diabetic rabbits were given with saline intragastrically after injured carotid artery operation. B EGCG pre-treated autologous L-EPCs transfusion improved diabetic rabbits carotid reendothelialization.

DM-sham: injured carotid artery of diabetic rabbits were washed and then incubated 10 min by saline after injured operation. DM-EPC: autologous L-EPCs were transplanted into the area of injured carotid artery of diabetic rabbits after injured carotid artery operation.

DM-EGCG-EPC: EGCG pre-treated autologous L-EPCs were transplanted into the area of injured carotid artery of diabetic rabbits after injured carotid artery operation. C Representative figure of autologous L-EPC tracking in vivo under fluorescent microscope.

Picture shows the extent to which L-EPCs are involved in reendothelialization 7 days after carotid artery injury. CM-DiI-labeled L-EPCs red are attached to endothelium FITC-lectin-stained, green. Nuclei of L-EPC were stained with DAPI blue. To identify whether EGCG benefits carotid reendothelialization through improving the functions of L-EPCs, we transfused autologous L-EPCs to the carotid injured rabbits.

Compared with diabetic sham-operated group, diabetic autologous L-EPCs transplantation locally to the injured carotid artery showed an accelerated trend of the reendothelialization process, but statistical difference was not found After EGCG pre-treatment for 72 h, autologous L-EPCs transfusion strongly improved the reendothelialization of diabetic rabbits Moreover, autologous L-EPCs transfusion group EGCG pre-treated displayed clearly more CM-Dil labeled L-EPCs attached to the endothelium, compared with the non-pre-treated ECGG group Figure 3C.

Late endothelial progenitor cells viability was determined by a CCK-8 test. We first examined the toxicity of EGCG to L-EPCs. Total confluent L-EPCs cultured in well plate were added different concentrations of EGCG and their absorbance at nm was examined. As shown in Figure 4A , up to 40 μM concentration of EGCG showed no poisonousness to L-EPCs.

The typical IC50 of EGCG is μM. High glucose treatment for 72 h directly impaired the proliferation of L-EPCs, while this impairment was greatly blocked in a time-and-dose dependent manner by EGCG.

EGCG treatment of 40 μM for 72 h could most effectively recover the proliferation of L-EPCs impaired by high glucose Figures 4B,C.

FIGURE 4. EGCG improves L-EPCs proliferation and migration under high glucose condition. A CCK-8 test reveals that up to 40 μM EGCG shows no poisonousness to L-EPCs, the typical IC50 of the chemical is μM. B,C L-EPCs incubated in high glucose environment 30 mM were co-incubated under different concentrations of EGCG 1—40 μM for different time 24—72 h.

Histogram reveals that high glucose treatment for 72 h directly impairs the proliferation of L-EPCs, this impairment could be block by EGCG in a time and dose dependent manner.

D EDU incorporation assay × magnification. The magnification is × The mobility of L-EPCs was examined by a Boyden chamber assay. The mRNA level of both Akt and eNOS remained unmodified by 72 h high glucose treatment. Also the EGCG treatment did not affect the Akt and eNOS mRNA level Figure 5A.

Statistical analysis was performed using GraphPad Prism © software version 6. Myocardial alterations were assessed by measurements of heart to body weight ratio as well as HR and QTc. Serum CK-MB and troponin-I levels were also estimated as markers of myocardial damage.

EGCG group showed no alteration in these parameters compared to normal group. STZ increased heart to body weight ratio and QTc whereas it decreased HR. Moreover, this group showed elevation of serum CK-MB and troponin-I levels.

On the other hand, administration of EGCG to diabetic mice markedly deteriorated these parameters compared to diabetic group confirming that EGCG preparation potentiated diabetes-induced myocardial damage Table 1. Moreover, EGCG administration to normal mice showed no histopathological alteration compared to the normal control mice.

However, diabetic mice showed moderate congestion of myocardial blood vessels whereas EGCG administration to diabetic mice caused severe congestion of myocardial blood vessels and moderate intermuscular edema verifying that EGCG preparation potentiated diabetes-induced myocardial damage Fig.

A Heart of a normal mouse showing normal cardiomyocytes. B Heart of a mouse from EGCG group showing normal cardiomyocytes. C Heart of a diabetic mouse showing moderate congestion of myocardial blood vessels. D Heart of a diabetic mouse receiving EGCG showing severe congestion of myocardial blood vessels.

E Heart of a diabetic mouse receiving EGCG showing moderate intermuscular edema. In normal mice, EGCG significantly decreased NADPH oxidase, TBARS and nitrotyrosine contents with increases in TAC, GSH, GPx, Nrf2, HO-1, and HSP 90 contents indicating restrained ROS generation. On the other hand, diabetic mice receiving EGCG have shown increased NADPH oxidase, TBARS and nitrotyrosine associated by decreases in TAC, GSH, GPx, GR, Nrf2, HO-1 and HSP 90 cardiac contents.

These effects were more profound than those in STZ animals, signifying the worsening of the oxidative stress status with a shifted balance towards a pro-oxidative milieu by STZ and EGCG administration Figs 2 and 3.

Effect of EGCG on STZ-induced changes in A NADPH oxidase, B TBARS, C TAC, D GSH, E GPx and F GR. Effect of EGCG on STZ-induced changes in A Nrf2, B HO-1 and C HSP EGCG administered to normal mice did not affect NF-κB and TNF-α as well as iNOS protein expression compared to the normal control group.

Induction of diabetes increased the myocardial NF-κB and TNF-α contents as well as iNOS protein expression, while administration of EGCG preparation to diabetic animals showed marked increment in the previously mentioned markers indicating a severe inflammatory status Fig.

Effect of EGCG on STZ-induced changes in A iNOS protein expressionas well as b nitrotyrosine, C NF-κB and B TNF-α contents.

Normal mice treated with EGCG preparation showed no alteration from the normal control animals regarding active caspase-3 expression, whereas diabetic mice receiving EGCG preparation showed significant elevation of active caspase-3 expression compared to diabetic control mice.

This signifies EGCG prominent apoptotic potential in the presence of diabetes Fig. Effect of EGCG on STZ-induced changes in immunohistochemical expression of active caspase-3 expression In an earlier study, we documented the involvement of inflammation, oxidative stress and apoptosis in EGCG-induced nephrotoxicity in the presence of diabetes Mouse maybe a better predictor of EGCG toxicity than rat due to higher bioavailability in both human and mouse compared to rat 25 , Moreover, i.

p route of administration offers a higher bioavailability compared to oral route poor bioavailability and extensive biotransformation which allows better investigation of the potential biological activity and toxicity of EGCG as estimated using previous studies 27 , Moreover, higher EGCG bioavailability has been linked to higher unconjugated EGCG level which is linked to its related toxicity Myocardial hemodynamic and biochemical alterations were clearly observable in the EGCG treated and non treated diabetic groups, which were lower with diabetes alone.

Moreover, the microscopic appearances, featuring severe myocardial blood vessels congestion and moderate intermuscular edema in diabetic mice receiving EGCG outweighed the effect of diabetes alone.

The increase in heart to body weight ratio in EGCG treated and non treated diabetic mice indicated hypertrophy of myocardial tissues. Hypertrophy may contribute to impairment of electromotive forces 30 as demonstrated by decrease in HR and increase in QTc in the present study.

HR variability could be also related to metabolic alterations due to hyperglycemia and insulinopenia or downregulation of cardiac adrenergic receptors Short-term diabetes mellitus induced by STZ has previously demonstrated to modify autonomic control of HR and cause bradycardia which was reversible by insulin administration confirming that these cardiovascular changes were related to metabolic perturbations and not to the direct toxic effect of diabetogenic agent; STZ Serum CK-MB is a useful early diagnostic index for myocardial injury and infarction, while troponin-I is a specific and more sensitive cardiac injury biomarker that predicts the risk of both cardiac cell death and subsequent infarction Therefore, probable reason for the rise in serum levels of CK-MB and troponin-I by EGCG could be cell death, namely apoptosis as will be elaborated later that may impose diabetic mice to the possible risk for myocardial injury.

Noteworthy, CK-MB and troponin-I elevations are linked to increased cell permeability caused by inflammation and free radical damage 34 , Such effects can be clarified by the core findings of the present work as EGCG preparation, in diabetic mice, activated NFκ-B with the association of TNF-α and NADPH oxidase elevation as well as iNOS overexpression that surpassed diabetic animals.

Of note, NADPH oxidase is a major source of ROS 36 which cause oxidative damage, justified in the present study by the marked increase in oxidative stress markers TBARS and nitrotyrosine in addition to marked depletion of endogenous anti-oxidants namely, GSH, GPx, GRx and TAC.

The aforementioned events support EGCG deleterious effects in diabetic kidney 19 and reinforce its reported pro-oxidant effect which was previously linked to its nephro- and hepatotoxicity 16 , The elevation of inflammatory cytokines enhances the expression of iNOS contributing to the production of the highly reactive oxidant peroxynitrite in the presence of oxidative stress and resulting in reduced nitric oxide NO level and endothelial dysfunction Of note, the pro-inflammatory cytokine TNF-α 35 and free radicals 39 were documented to increase the transcriptional activity of NFκ-B, thus contributing to an endless feed forward cycle that propagates and potentiates both inflammation and oxidative stress leading hence to excessive CK-MB and troponin-I leakage as documented herein in mice receiving EGCG preparation and STZ.

Apart from the involvement of NF-κB in oxidative and inflammatory damage, the current more conspicuous reduced level of HO-1 in EGCG-treated diabetic animals plays also a role in the catechin-mediated myocardial toxicity. This enzyme catalyzes the degradation of heme which has both pro-oxidant and pro-inflammatory properties; hence in the vicinity of a sharp decrease of HO-1, free radicals and inflammation that lead to cellular injury are augmented The decrease in HO-1 might be clarified by the present inhibition of Nrf2 in EGCG-treated diabetic mice that was again more prominent than diabetic mice.

These effects go in line with our previous report of its nephrotoxic effect in the presence of diabetes Moreover, it has been earlier reported that high doses of EGCG reduced the expression of HO-1 20 , The transcription factor Nrf2 is an important machinery that upregulates the HO-1 gene.

This enzyme initiates antioxidant and anti-inflammatory processes, hence Nrf2 down-regulation triggers and progresses various diabetic complications 42 , Moreover,it was reported that STZ-induced diabetes in Nrf2 knockout mice rapidly progressed to severe myocardial lesions associated by robust inflammatory response, oxidative stress, and apoptosis These data indicate that Nrf2 and its downstream target HO-1 serve as defense factors against cardiac diabetic complications; hence their excessive reduction by EGCG preparation in diabetic animals seen in this study can explain, in part, the polyphenol cardiotoxic effect.

Besides the upregulation of HO-1, Nrf2 initiates the transcription of a number of anti-oxidative genes to reduce the pathological oxidative stress in the heart In response to various forms of stress, cells activate a highly conserved heat shock response in which a set of HSPs are induced to participate in cellular repair and protective mechanisms 45 , Several studies noticed that diabetes is associated with significant reduction of HSPs levels 47 , 48 , Moreover, in diabetic patients, cells are more vulnerable to damage if HSPs levels are decreased which results in organ failure Since in the present study EGCG preparation was shown to suppress HSP 90 in diabetic mice more persistently than diabetes alone, therefore, EGCG-induced cardiotoxicity could be attributed to its ability to decrease HSP 90, and hence maladapted stress defense responses.

The present decrease in HSP 90 after EGCG administration in diabetic mice confirms our previous report in the kidney 19 and is consistent with the previous finding which elucidated that this proteo-stress marker was markedly down-regulated in mice receiving high doses of green tea polyphenols in diet Furthermore, in response to excessive oxidative stress-induced by high doses of EGCG, HSP90 was downregulated in liver and kidney It has been anticipated that oxidative stress and the pro-inflammatory cytokine TNF-α could contribute to diabetic cardiomyopathy through the stimulation of the intrinsic and extrinsic pathways of programmed cell death, respectively Additionally, Kim et al.

suggested that the decreased HSP 90 expression in type 2 diabetic kidneys may mediate apoptosis; hence affording an additional clarification for increased apoptosis associated by EGCG administration in STZ mice. Accordingly, in the present study, the excessive cardiac toxicity produced by the polyphenol under hyperglycemic conditions could been linked to the present exaggerated oxidative stress and inflammation responses, as well as the reduction of HSP 90 in the heart to jeopardize cardiomyocytes function leading to CK-MB and troponin-I elevation as previously anticipated.

Therefore, the current investigation elaborated that parentral application of EGCG preparation Teavigo ® exhibited cardiotoxicity in the presence of diabetes although no toxicity was observed in normal animals receiving the same dose and route of administration.

This deleterious effect could be attributed to EGCG-induced apoptosis triggered by oxidative stress, inflammation and HSP 90 suppression which eventually leads to cardiac damage evidenced by elevation of myocardial biomarkers.

In spite of its well-known favorable effects, EGCG administration to diabetic mice revealed increased cardiotoxicity. Considering the rise of global diabetes, EGCG administration may impact cardiovascular health accelerating the progression of the disease-associated complications.

Mediation of oxidative stress, inflammation, and apoptosis in the cardiomyopathy-induced by EGCG preparation in diabetes can be considered especially for parentral application of EGCG which exhibits higher bioavailability and thus its related toxicity.

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