Endogenous ornithine decarboxylase/polyamine system mediated the antagonist role of insulin/PEG-CMCS preconditioning against heart ischemia/reperfusion injury in diabetes mellitus.

INTRODUCTION: Insulin has shown antioxidation and cytoprotective effects to decrease heart ischemia/reperfusion injury (HI/RI) in diabetes mellitus (DM), but the role of insulin/poly(ethylene glycol)-carboxymethyl chitosan (PEG-CMCS) on HI/RI in DM is not known. This research explored whether insulin/PEG-CMCS revealed a protective effect on HI/RI in DM through ornithine decarboxylase (ODC)/polyamine systems.
MATERIALS AND METHODS: Diabetes was induced via streptozotocin (STZ) in Sprague Dawley (SD) rats, which suffered from HI via blocking the left circumflex artery for 45 minutes, followed by 2 hours of reperfusion. α-Difluoromethylornithine-ethylglyoxal bis (guanylhydrazone) (DFMO-EGBG) and insulin/PEG-CMCS were administered to diabetic rats to explore their roles on severity of HI/RI.
RESULTS: Insulin could be fleetly and efficiently loaded via the nanocarrier PEG-CMCS at pH =6, showing efficient loading and stable release. In addition, insulin/PEG-CMCS showed significant hypoglycemic activity in diabetic rats. On the other hand, ischemia/reperfusion obviously augmented the contents of creatine kinase (CK), lactic dehydrogenase (LDH), putrescine (Pu), myocardial infarct size, and NF-κB and spermidine/spermine N'-acetyltransferase (SSAT) expressions and decreased the levels of spermine (Sp), polyamine pools (PAs), heart rate (HR), coronary blood flow (CF), left ventricular developed pressure (LVDP), and ODC expression, compared with Sham. Administration of insulin and insulin/PEG-CMCS both reduced the contents of CK, LDH, Pu, myocardial infarct size, and NF-κB and SSAT expressions and increased the levels of Sp, PAs, HR, CF, LVDP, and ODC expression, while insulin/PEG-CMCS significantly indicated the protective results, and DFMO-EGBG showed the opposite effects.
CONCLUSION: The research showed that insulin/PEG-CMCS could play a protective effect on HR/RI in diabetic rats via its antioxidative, antiapoptotic, and anti-inflammatory roles and modulating ODC/polyamine systems.

Introduction

Heart ischemia/reperfusion injury (HI/RI) is an important pathophysiological phenomenon, involving apoptosis, necrosis, oxidative stress, and inflammatory reaction.1–6 In addition, diabetes mellitus (DM) aggravates the sensibility of HI/RI and exacerbates myocardial damage.7 In experimental research studies, administration of different pharmacological agents, such as insulin, during HI/RI has shown promising results decreasing myocardial infarct size. Many studies have shown that insulin may decrease myocardial damage and improve heart functions, especially may improve myocardial metabolism on HI/RI.8 On the other hand, insulin may also ameliorate HI/RI in DM.9

Polyamine including putrescine (Pu), spermidine (Spd), and spermine (Sp) is widely distributed in prokaryotic and eukaryotic tissues and cells and plays an important role in the growth, development, and apoptosis of tissues and cells.10 Ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) are two key enzymes involved in polyamine anabolism, which can be activated by various stress events, resulting in increased polyamine synthesis.11–13 Some studies have also shown that polyamine, especially Sp, has many biological functions such as antioxidation, scavenging free radicals, regulating intracellular calcium, and inhibiting the opening of mitochondrial permeability transport pore.14–16 In addition, the report has proven that the activities of ODC and the polyamine pools (PAs) are decreased, especially Sp is significantly reduced on HI/RI; exogenous Sp exerts its anti-HI/RI effects by inhibiting apoptosis and necrosis.17 On the other hand, ischemic preconditioning can attenuate HI/RI in rats by upregulating the ODC/polyamine systems.18 Numerous documents have corroborated that insulin can induce ODC activity in various responsive cells.19,20 Despite insulin can decrease HI/RI in DM and modulate ODC, the half-life of insulin is exceedingly short in blood circulation, and it is very difficult to administer the insulin dosage clinically because of the continual occurrence of hypoglycemia.21,22

In this study, we report poly(ethylene glycol)-carboxymethyl chitosan (PEG-CMCS) as a potential insulin nanocarrier. The designed PEG-CMCS is composed of PEG and CMCS (Figure S1). At pH =6, PEG-CMCS is combined with insulin to form nanoparticles. In this research, loading of insulin in the nanoparticles, release of insulin from the nanoparticles, and the anti-HI/RI effect of the nanoparticles are observed by using insulin as control. Moreover, we measure the hypothesis that insulin or insulin/PEG-CMCS administered prior to ischemia provides cardioprotection by regulating ODC/polyamine systems. The primary measure/outcome is to investigate the potential mechanisms between ODC/polyamine systems and HI/RI underlying the effects of insulin or insulin/PEG-CMCS.

Materials and methods

Materials

Methoxypolyethylene glycols (mPEG, molecular weight = 2,000 Da) was supplied from Aladdin (Shanghai, People’s Republic of China). CMCS (Mn =10,000) was bought from Qingdao Hong Hai Biological Technology Co., Ltd (Qingdao, China). Insulin and streptozotocin (STZ) were bought from Sigma-Aldrich Co. (St. Louis, MO, USA). H9C2 cells were purchased from Shanghai Bioleaf Biotech Co., Ltd (Shanghai, China). α-Difluoromethylornithine (DFMO) and ethylglyoxal bis (guanylhydrazone) (EGBG) were purchased from Sigma-Aldrich Co. Other reagents were purchased from Sigma-Aldrich Co. and Aladdin.

Synthesis of PEG-CMCS

About 0.5 g of PEG was mixed with 0.1 g of CMCS dissolved in deionized water and regulated pH to 6.5. Then, 1 mL of sodium cyanoborohydride was slowly added into the reaction solution and stirred. The mixture was stirred at 37°C for 12 hours and dialyzed for 48 hours. The PEG-CMCS nanoparticles was gained by freeze drying. The final product was measured by 1H nuclear magnetic resonance (1H NMR).

Cytotoxicity evaluation

The method and protocol of the cytotoxicity evaluation of PEG-CMCS (for H9C2 cells) is based on our previous works.23–28

Encapsulation evaluation

About 20 mg of PEG-CMCS dissolved in deionized water was mixed with 20 mg of insulin at 37°C and pH =6, and 0.5 mg/mL CaCl2 solution was slowly added into the reaction solution until an apparent milky light color occurs. At last, the insulin/PEG-CMCS nanoparticles were produced. The PEG-CMCS-encapsulated insulin was observed by high-performance liquid chromatography (HPLC) and transmission electron microscopy (TEM).

Insulin release in vitro

Release of insulin from PEG-CMCS was observed through dialysis method (molecular weight cutoff [MWCO] = 3,500 Da) at 37°C, with 5 mL of insulin-encapsulated PEG-CMCS against PBS at pH 7.4, 6.8, and 1.2. The insulin/PEG-CMCS compounds were prepared by loading insulin with PEG-CMCS. After a fixed time interval, 3 mL of the release media was extracted and supplemented with 3 mL of fresh release media. The quantity of insulin released was observed through HPLC, and the circular two-dimensional chromatogram of the released insulin was measured at 4°C.

Hypoglycemic effect

Male Sprague Dawley (SD) rats (150–210 g) were obtained from Jiaxing University Medical College in China. The procedures and care of the SD rats were approved by the Institutional Ethics Committee of Jiaxing University Medical College in China. The expedition conformed to the guidelines for the care and use of laboratory animals published by the US National Institutes of Health (NIH Publication updated in 2011).

SD rats were injected with STZ only once at a dose of 50 mg/kg via abdomen. After 3 days, diabetes was assessed by measuring blood glucose levels by using the glucose oxidase-peroxidase (GOD-POD) method.29 Animals with blood glucose levels >16.7 mmol/L were included in the research. Thirty diabetic rats were randomly divided into three groups (n=10 in each group): 1) insulin group (subcutaneous injection): 30 U/kg as a single abdominal subcutaneous injection; 2) insulin group (intragastric administration): 30 U/kg as a single intragastric administration; and 3) insulin/PEG-CMCS group (intragastric administration): 30 U/kg as a single intragastric administration. At a certain time interval, blood glucose levels were measured by using the GOD-POD method.29

Surgical procedure of HI/RI

In this experiment, 60 diabetic rats were divided into six groups (each group =10 rats): 1) Sham group; 2) I/R group: diabetic rats were anesthetized with 1% pentobarbital sodium (50 mg/kg) by intraperitoneal injection; the thoracic cavity was opened, and the left circumflex artery was exposed and ligated for 45 minutes of ischemia, followed by 2 hours of reperfusion; 3) insulin group: diabetic rats were administered insulin once a day (30 U/kg) by abdominal subcutaneous injection for 6 hours prior to I/R process; 4) insulin/PEG-CMCS group: diabetic rats were administered insulin/PEG-CMCS once a day (30 U/kg) by intragastric administration for 6 hours prior to I/R process; 5) insulin+DFMO-EGBG group: diabetic rats were administered insulin once a day (30 U/kg) by abdominal subcutaneous injection for 6 hours and then 45 minutes of ischemia, and DFMO (2 mmol/L) and EGBG (1 mmol/L) were poured into heart before 2 hours of reperfusion; and 6) insulin/PEG-CMCS+DFMO-EGBG group: diabetic rats were administered insulin/PEG-CMCS once a day (30 U/kg) by intragastric administration for 6 hours and then 45 minutes of ischemia, and DFMO (2 mmol/L) and EGBG (1 mmol/L) were poured into heart before 2 hours of reperfusion.

Blood sample was collected from the abdominal aorta and centrifuged at 3,600×g for 20 minutes to acquire the sera. Myocardiums were collected and stored at −80°C until further analysis.

Infarct size evaluation

The method has been described previously.30–32 The hearts were stained by slowly infusing 1 mL of Evan’s blue (3% wt/vol) through the aorta, followed by perfusion using Krebs bicarbonate buffer to rinse off unbound stains. The hearts were then rapidly extracted from the perfusion apparatus and stored overnight at −20°C. Later, they were cut into 2 mm slices and stained with 1% 2,3,5-triphenyltetrazolium chloride (TTC) under phosphate buffer (PB) for 15 minutes at 37°C, and then fixed by 10% formalin. Within viable myocardiums, TTC was converted by dehydrogenase enzymes, a red pigment which stained tissue dark red. Nonviable infracted myocardiums that did not observe TTC stain remained pale in color. The slices were photographed, and the myocardial infarct sizes were obtained as percentage of total areas, which are calculated by using Adobe Photoshop.

Creatine kinase (CK) and lactic dehydrogenase (LDH) activities

The activities of CK and LDH in the serum were observed to estimate the myocardium injury by using the double antibody sandwich enzyme-linked immunosorbent assay kits.

Heart rate (HR), coronary blood flow (CF), and left ventricular developed pressure (LVDP) measurement

The HR, CF, and LVDP were measured by using the PowerLab physiological system.

Pu, Spd, Sp, and PAs contents

The contents of Pu, Spd, Sp, and PAs in myocardial tissues were measured by the HPLC method.

NF-κB expression, cell apoptosis, and spermidine/spermine N′-acetyltransferase (SSAT) and ODC expressions

The method had been depicted previously.6 The hearts were extracted, fixed in 4% paraformaldehyde for 4 hours, dehydrated in 30% sucrose overnight at 4°C, embedded in tissue freezing medium, and then cut into 5 μm frozen sections. NF-κB translocation from the cytoplasm to the nucleus in areas at risk was observed through immunofluorescence by mouse NF-κB p65.

Myocardial sections (sized 5 μm) were stained by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling on the basis of manufacturer’s instruction and observed by light microscopy. The apoptotic indices were calculated as the percentages of stained cells, namely number of apoptosis cells×100/total number of nucleated cells. The method had been depicted previously.3

SSAT and ODC expressions were measured by Western blot according to previous method.18,33 Briefly, myocardial tissues were homogenized in protein lysate buffer. The homogenate was resolved on polyacrylamide SDS gels and electrophoretically transferred to polyvinylidene difluoride membranes. The membranes were blocked by 3% bovine serum albumin (BSA), incubated with primary antibodies against active SSAT, ODC, and subsequently with alkaline phosphatase-conjugated secondary antibodies. These were finally developed by adding 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium. Blots were stained by anti-β-actin antibody, and the contents of proteins were normalized with respect to β-actin and density.

Statistical analysis

All data were accomplished in triplicate unless otherwise noted. They were expressed as mean ± SD. Statistical analyses were executed by ANOVA by using post hoc testing, and data were analyzed by using SPSS (version 19.0; IBM Corporation, Armonk, NY, USA). A p-value <0.01 denoted statistical significance.

Results

Synthesis route of PEG-CMCS is described in the Supplementary materials (Figure S1). In brief, synthetic product needed two steps: 1) reaction of mPEG with succinic anhydride and modification of aldehyde and 2) mPEG-CHO and CMCS produced the final product mPEG-CMCS with the condition of pH =6.5 and sodium cyanoborohydride as a reductant.

Synthesis and characterization of PEG-CMCS

PEG-CMCS comprised PEG and CMCS (Figure S1). In our research, the molecular weight of PEG was 2,000 Da, and the Mn of CMCS was 10,000. The process involved in its synthesis is shown in Figure S1. The 1H NMR spectrum of PEG-CMCS is shown in Figure S2A. The “a” refers CMCS, and δ=4.7 was the solvent peak (D2O), and δ=1.92 is the proton peak of residual acetyl amino (–NHCOCH3) from CMCS (Figure S2Aa). δ=3.66–3.95 is the proton peak of sugar rings from CMCS, and δ=3.5–3.6 is the proton peak of carboxymethyl from CMCS (Figure S2Aa). δ=3.62 seems to be a strong peak in PEG-CMCS, and the peak is the repetitive unit (–CH2–CH2–O–) of PEG (Figure S2Ab). δ=3.30 seems to be the proton peak of terminal methyl from PEG (Figure S2Ab). The characteristic proton peak of both PEG and CMCS was measured, authenticating that the synthesis proceeded in a controlled manner and was successful.

Cellular viability

Cellular toxicity on the basis of PEG-CMCS in H9C2 cells was observed after 24 hours of culturing, and the results are shown in Figure 1A. PEG-CMCS showed low cellular toxicity even at a content as high as 200 μg/mL.

Figure 1

Cell viability, cumulative release, blood glucose, and circular 2D chromatogram.

Notes: Cellular viability of H9C2 cells cultured with different concentrations of PEG-CMCS (A); cumulative release profile of insulin from insulin/PEG-CMCS in different pH (B); blood concentration of free insulin (subcutaneous injection), free insulin (intragastric administration), and insulin/PEG-CMCS (intragastric administration) (C); circular 2D chromatogram of insulin (standard solution) and insulin/PEG-CMCS (release solution) (D).

Abbreviation: PEG-CMCS, poly(ethylene glycol)-carboxymethyl chitosan.

Encapsulating capacity

Insulin was discovered to be efficiently loaded in PEG-CMCS at pH =6. It was added to PEG-CMCS (mass ratio =1:5) and dialyzed (MWCO =3,500 Da) against PB. The dialysis of free insulin as a control was also conducted at pH =6 in PB. To observe the loading efficiency of insulin in PEG-CMCS, the quantity of insulin in the dialysate was observed by HPLC and deducted from the total quantity of added insulin. The loading efficiency of insulin was found to be 18.7%, expressed as the mass ratios of loaded insulin to the nanoparticles.

Characterization of PEG-CMCS

PEG-CMCS and insulin/PEG-CMCS were observed by TEM, and the images are shown in Figure S2B. PEG-CMCS shows an orbicular structure, and the diameter is ~29 nm (Figure S2Ba). Insulin/PEG-CMCS also shows an orbicular structure, and the diameter is ~68 nm (Figure S2Bb).

In vitro release

The release of insulin from PEG-CMCS was observed by the dialysis method (MWCO =3,500 Da) at 37°C, with 5 mL of insulin-encapsulated PEG-CMCS. The cumulative release rates of insulin from insulin/PEG-CMCS are shown in Figure 1B. At pH =7.4, ~11.27% of insulin was released from insulin/PEG-CMCS after 1.5 hours, indicative of an initial burst release of insulin. Approximately 75.78% of insulin was released after 40 hours. At pH =6.8, ~12.39% of insulin was released from insulin/PEG-CMCS after 2 hours, indicative of an initial burst release of insulin. Approximately 35.31% of insulin was released after 40 hours. At pH =1.2, ~2.08% of insulin was released from insulin/PEG-CMCS after 2 hours. Approximately 14.81% of the insulin was released after 40 hours.

Blood glucose

To observe the blood glucose level in a fixed time of diabetic rats administered insulin/PEG-CMCS, and to compare with that of the insulin group, the results revealed that diabetic rats injected with insulin (abdominal subcutaneous injection) maintained blood glucose levels in normal change for about 1 hour, after which glycemia slowly enhanced. Diabetic rats administered with insulin (intragastric administration) showed that blood sugar lowering was not obvious; it could be related to the digestive enzymes such as protease and trypsin of gastrointestinal tract, and digestive enzymes were involved in the degradation and digestion of insulin. By contrast, insulin/PEG-CMCS (intragastric administration) reduced blood glucose levels to 5.6 mmol/L at 1.5 hours after administration. Over the following 6 hours, blood glucose level increased to 5.3 mmol/L and was then maintained at ~4.7–6.1 mmol/L for 6 hours (Figure 1C).

Circular 2D chromatogram

The wavelengths of insulin from insulin and insulin/PEG-CMCS solutions both showed negative peaks at 208 and 222 nm, and they were the characteristic peaks corresponding to alpha helix and beta folding, respectively. The results showed that the structure of insulin did not change obviously before and after release, and the biological activity of insulin remained unchanged (Figure 1D).

Myocardial infarct size

As shown in Figure 2, no infarct-risk myocardium tissues were surveyed in the Sham group with a mean infarct size of 5.4%±1.4%. The I/R group led to myocardial infarction with a mean infarct size of 43.5%±2.2%, which was remarkably higher than the Sham group (p<0.01). Administration of insulin (p<0.01) decreased the infarct size (28.8%±4.4%) compared to the I/R group; administration of insulin/PEG-CMCS (p<0.01) significantly decreased the infarct size (21.6%±3.6%) compared to the I/R group, whereas administration of insulin+DFMO-EGBG further enhanced the infarct size (34.3%±2.9%), which was greater than the insulin group (p<0.01); and administration of insulin/PEG-CMCS+DFMO-EGBG further enhanced the infarct size (24.5%±1.8%), which was greater than the insulin/PEG-CMCS group (p<0.01).

Figure 2

Myocardial infarct size.

Notes: (A) Representative myocardial cross sections of TTC-stained hearts were collected from Sham group, I/R group, insulin group, insulin/PEG-CMCS group, insulin+DFMO-EGBG, and insulin/PEG-CMCS+DFMO-EGBG group. (B) Quantitative infarct size is expressed as mean ± SD. A significant increase relative to the Sham group is denoted by “*” (p<0.01), a significant decrease relative to the I/R group is denoted by “**” (p<0.01), a significant decrease relative to the I/R group is denoted by “***” (p<0.01), a significant increase relative to the insulin group is denoted by “#” (p<0.01), and a significant increase relative to the insulin/PEG-CMCS group is denoted by “##” (p<0.01).

Abbreviations: TTC, 2,3,5-triphenyltetrazolium chloride; PEG-CMCS, poly(ethylene glycol)-carboxymethyl chitosan; DFMO, α-difluoromethylornithine; EGBG, ethylglyoxal bis (guanylhydrazone).

CK and LDH activities

Activities of CK and LDH were observed after reperfusion for 2 hours (Figure 3A), and it was found to be higher in the I/R group than in the Sham group (p<0.01; CK: Sham group 518.94±13.79 U/L, I/R group 1,451.14±11.26 U/L; LDH: Sham group 152.72±10.24 U/L, I/R group 398.83±13.99 U/L). Administration of insulin reduced CK (1,024.57±15.39 U/L) and LDH (311.27±11.63 U/L) compared with that in the I/R group (p<0.01). Administration of insulin/PEG-CMCS significantly reduced CK (708.29±12.87 U/L) and LDH (247.34±12.98 U/L) compared with that in the I/R group (p<0.01). Administration of insulin+DFMO-EGBG increased CK (1,235.49±14.58 U/L) and LDH (345.65±15.04 U/L) compared with that in the insulin group (p<0.01). Administration of insulin/PEG-CMCS+DFMO-EGBG increased CK (889.79±13.34 U/L) and LDH (273.41±14.23 U/L) compared with that in the insulin/PEG-CMCS group (p<0.01).

Figure 3

CK, LDH, HR, CF, LVDP, Pu, Spd, Sp, and PAs levels.

Notes: The CK, LDH activities and HR, CF, LVDP, Pu, Spd, Sp, PAs levels of Sham group, I/R group, insulin group, insulin/PEG-CMCS group, insulin+DFMO-EGBG, and insulin/PEG-CMCS+DFMO-EGBG group were measured after 2 hours of reperfusion. Results are expressed as mean ± SD. (A) A significant increase relative to the Sham group is denoted by “*” (p<0.01), a significant decrease relative to the I/R group is denoted by “**” (p<0.01), a significant decrease relative to the I/R group is denoted by “***” (p<0.01), a significant increase relative to the insulin group is denoted by “#” (p<0.01), and a significant increase relative to the insulin/PEG-CMCS group is denoted by “##” (p<0.01) (CK and LDH). (B) A significant decrease relative to the Sham group is denoted by “*” (p<0.01), a significant increase relative to the I/R group is denoted by “**” (p<0.01), a significant increase relative to the I/R group is denoted by “***” (p<0.01), a significant decrease relative to the insulin group is denoted by “#” (p<0.01), and a significant decrease relative to the insulin/PEG-CMCS group is denoted by “##” (p<0.01). (C) A significant increase relative to the Sham group is denoted by “*” (p<0.01), a significant decrease relative to the I/R group is denoted by “**” (p<0.01), a significant decrease relative to the I/R group is denoted by “***” (p<0.01), a significant decrease relative to the insulin group is denoted by “#” (p<0.01), and a significant decrease relative to the insulin/PEG-CMCS group is denoted by “##” (p<0.01) (Pu); a significant increase relative to the Sham group is denoted by “*” (p<0.01), a significant increase relative to the I/R group is denoted by “**” (p<0.01), a significant increase relative to the I/R group is denoted by “***” (p<0.01), a significant decrease relative to the insulin group is denoted by “#” (p<0.01), and a significant decrease relative to the insulin/PEG-CMCS group is denoted by “##” (p<0.01) (Spd); a significant decrease relative to the Sham group is denoted by “*” (p<0.01), a significant increase relative to the I/R group is denoted by “**” (p<0.01), a significant increase relative to the I/R group is denoted by “***” (p<0.01), a significant decrease relative to the insulin group is denoted by “#” (p<0.01), and a significant decrease relative to the insulin/PEG-CMCS group is denoted by “##” (p<0.01) (Sp); a significant decrease relative to the Sham group is denoted by “*” (p<0.01), a significant increase relative to the I/R group is denoted by “**” (p<0.01), a significant increase relative to the I/R group is denoted by “***” (p<0.01), a significant decrease relative to the insulin group is denoted by “#” (p<0.01), and a significant decrease relative to the insulin/PEG-CMCS group is denoted by “##” (p<0.01) (PAs). (D) A significant decrease relative to the Sham group is denoted by “*” (p<0.01), a significant increase relative to the I/R group is denoted by “**” (p<0.01), a significant increase relative to the I/R group is denoted by “***” (p<0.01), a significant decrease relative to the insulin group is denoted by “#” (p<0.01), and a significant decrease relative to the insulin/PEG-CMCS group is denoted by “##” (p<0.01). (E) A significant decrease relative to the Sham group is denoted by “*” (p<0.01), a significant increase relative to the I/R group is denoted by “**” (p<0.01), a significant increase relative to the I/R group is denoted by “***” (p<0.01), a significant decrease relative to the insulin group is denoted by “#” (p<0.01), and a significant decrease relative to the insulin/PEG-CMCS group is denoted by “##” (p<0.01).

Abbreviations: CK, creatine kinase; LDH, lactic dehydrogenase; HR, heart rate; CF, coronary blood flow; LVDP, left ventricular developed pressure; Pu, putrescine; Spd, spermidine; Sp, spermine; PAs, polyamine pools; PEG-CMCS, poly(ethylene glycol)-carboxymethyl chitosan; DFMO, α-difluoromethylornithine; EGBG, ethylglyoxal bis (guanylhydrazone).

HR, CF, and LVDP

The level of HR was observed after reperfusion for 2 hours (Figure 3B), and it was found to be lower in the I/R group than in the Sham group (p<0.01; HR: Sham group 210±11 beats/min, I/R group 162±13 beats/min). Administration of insulin enhanced HR (174±9 beats/min) compared with that in the I/R group (p<0.01). Administration of insulin/PEG-CMCS significantly enhanced HR (189±14 beats/min) compared with that in the I/R group (p<0.01). Administration of insulin+DFMO-EGBG reduced HR (169±8 beats/min) compared with that in the insulin group (p<0.01). Administration of insulin/PEG-CMCS+DFMO-EGBG reduced HR (180±12 beats/min) compared with that in the insulin/PEG-CMCS group (p<0.01).

The level of CF was observed after reperfusion for 2 hours (Figure 3D), and it was found to be lower in the I/R group than in the Sham group (p<0.01; CF: Sham group 12.4±0.5 mL/min, I/R group 8.5±0.7 mL/min). Administration of insulin enhanced CF (9.3±0.6 mL/min) compared with that in the I/R group (p<0.01). Administration of insulin/PEG-CMCS significantly enhanced CF (10.9±0.9 mL/min) compared with that in the I/R group (p<0.01). Administration of insulin+DFMO-EGBG reduced CF (8.9±0.8 mL/min) compared with that in the insulin group (p<0.01). Administration of insulin/PEG-CMCS+DFMO-EGBG reduced CF (9.7±0.4 mL/min) compared with that in the insulin/PEG-CMCS group (p<0.01).

The level of LVDP was observed after reperfusion for 2 hours (Figure 3E), and it was found to be lower in the I/R group than in the Sham group (p<0.01; LVDP: Sham group 90±9 mmHg, I/R group 25±7 mmHg). Administration of insulin enhanced LVDP (48±6 mmHg) compared with that in the I/R group (p<0.01). Administration of insulin/PEG-CMCS significantly enhanced LVDP (71±8 mmHg) compared with that in the I/R group (p<0.01). Administration of insulin+DFMO-EGBG reduced LVDP (37±4 mmHg) compared with that in the insulin group (p<0.01). Administration of insulin/PEG-CMCS+DFMO-EGBG reduced LVDP (57±6 mmHg) compared with that in the insulin/PEG-CMCS group (p<0.01).

Pu, Spd, Sp, and PAs

The level of Pu was observed after reperfusion for 2 hours (Figure 3C), and it was found to be higher in the I/R group than in the Sham group (p<0.01; Pu: Sham group 15.31±5.29 nmol/g, I/R group 31.08±5.08 nmol/g). Administration of insulin reduced Pu (29.11±6.54 nmol/g) compared with that in the I/R group (p<0.01). Administration of insulin/PEG-CMCS significantly reduced Pu (22.96±4.31 nmol/g) compared with that in the I/R group (p<0.01). Administration of insulin+DFMO-EGBG reduced Pu (12.16±5.18 nmol/g) compared with that in the insulin group (p<0.01). Administration of insulin/PEG-CMCS+DFMO-EGBG reduced Pu (7.02±5.61 nmol/g) compared with that in the insulin/PEG-CMCS group (p<0.01).

The level of Spd was observed after reperfusion for 2 hours (Figure 3C), and it was found to be higher in the I/R group than in the Sham group (p<0.01; Spd: Sham group 283.46±4.89 nmol/g, I/R group 357.12±5.77 nmol/g). Administration of insulin enhanced Spd (388.85±6.12 nmol/g) compared with that in the I/R group (p<0.01). Administration of insulin/PEG-CMCS significantly enhanced Spd (428.86±5.58 nmol/g) compared with that in the I/R group (p<0.01). Administration of insulin+DFMO-EGBG reduced Spd (201.58±6.71 nmol/g) compared with that in the insulin group (p<0.01). Administration of insulin/PEG-CMCS+DFMO-EGBG reduced Spd (247.68±5.34 nmol/g) compared with that in the insulin/PEG-CMCS group (p<0.01).

The level of Sp was observed after reperfusion for 2 hours (Figure 3C), and it was found to be lower in the I/R group than in the Sham group (p<0.01; Sp: Sham group 301.17±8.24 nmol/g, I/R group 225.53±6.43 nmol/g). Administration of insulin enhanced Sp (284.55±7.01 nmol/g) compared with that in the I/R group (p<0.01). Administration of insulin/PEG-CMCS significantly enhanced Sp (330.16±6.52 nmol/g) compared with that in the I/R group (p<0.01). Administration of insulin+DFMO-EGBG reduced Sp (178.51±5.29 nmol/g) compared with that in the insulin group (p<0.01). Administration of insulin/PEG-CMCS+DFMO-EGBG reduced Sp (199.81±6.13 nmol/g) compared with that in the insulin/PEG-CMCS group (p<0.01).

The level of PAs was observed after reperfusion for 2 hours (Figure 3C), and it was found to be lower in the I/R group than in the Sham group (p<0.01; PAs: Sham group 675.41±7.29 nmol/g, I/R group 481.23±5.34 nmol/g). Administration of insulin enhanced PAs (538.83±4.99 nmol/g) compared with that in the I/R group (p<0.01). Administration of insulin/PEG-CMCS significantly enhanced PAs (618.76±6.81 nmol/g) compared with that in the I/R group (p<0.01). Administration of insulin+DFMO-EGBG reduced PAs (386.13±4.43 nmol/g) compared with that in the insulin group (p<0.01). Administration of insulin/PEG-CMCS+DFMO-EGBG reduced PAs (438.35±5.89 nmol/g) compared with that in the insulin/PEG-CMCS group (p<0.01).

NF-κB expression, cell apoptosis, and SSAT and ODC expressions

Expression of NF-κB was observed after reperfusion for 2 hours (Figure 4A), and it was found to be higher in the I/R group than in the Sham group. Administration of insulin reduced NF-κB expression compared with that in the I/R group. Administration of insulin/PEG-CMCS significantly reduced NF-κB expression compared with that in the I/R group. Administration of insulin+ DFMO-EGBG further enhanced NF-κB expression compared with that in the insulin group. Administration of insulin/PEG-CMCS+DFMO-EGBG further enhanced NF-κB expression compared with that in the insulin/PEG-CMCS group.

Figure 4

NF-κB expression, cell apoptosis, and SSAT and ODC expressions.

Notes: Expression of nuclear NF-κB in myocardial tissue from rats subjected to Sham group, I/R group, insulin group, insulin/PEG-CMCS group, insulin+DFMO-EGBG, and insulin/PEG-CMCS+DFMO-EGBG group after 2 hours of reperfusion. (A) Immunofluorescence assessment of NF-κB nuclear translocation by I/R. Representative photomicrographs of sections stained for NF-κB translocation from the cytoplasm to the nucleus are described (×400). Green fluorescence shows the presence of NF-κB; DAPI counterstaining (blue) shows total nuclei. The green fluorescence embedded in the blue fluorescence represents NF-κB nuclear translocation. (B) Cell apoptosis was measured after 2 hours of reperfusion. Results are expressed as mean ± SD. A significant increase relative to the Sham group is denoted by “*” (p<0.01), a significant decrease relative to the I/R group is denoted by “**” (p<0.01), a significant increase relative to the insulin group is denoted by “#” (p<0.01), and a significant increase relative to the insulin/PEG-CMCS group is denoted by “##” (p<0.01). (C) SSAT and ODC expressions were measured by Western blot after 2 hours of reperfusion. Results are expressed as mean ± SD. A significant increase relative to the Sham group is denoted by “*” (p<0.01), a significant decrease relative to the I/R group is denoted by “**” (p<0.01), a significant decrease relative to the I/R group is denoted by “***” (p<0.01), a significant increase relative to the insulin group is denoted by “#” (p<0.01), and a significant increase relative to the insulin/PEG-CMCS group is denoted by “##” (p<0.01) (SSAT). A significant decrease relative to the Sham group is denoted by “*” (p<0.01), a significant increase relative to the I/R group is denoted by “**” (p<0.01), a significant increase relative to the I/R group is denoted by “***” (p<0.01), a significant decrease relative to the insulin group is denoted by “#” (p<0.01), and a significant decrease relative to the insulin/PEG-CMCS group is denoted by “##” (p<0.01) (ODC).

Abbreviations: SSAT, spermidine/spermine N′-acetyltransferase; ODC, ornithine decarboxylase; PEG-CMCS, poly(ethylene glycol)-carboxymethyl chitosan; DFMO, α-difluoromethylornithine; EGBG, ethylglyoxal bis (guanylhydrazone).

Cell apoptosis was observed after reperfusion for 2 hours (Figure 4B), and it was found to be higher in the I/R group than in the Sham group (p<0.01). Administration of insulin reduced cell apoptosis compared with that in the I/R group (p<0.01). Administration of insulin/PEG-CMCS significantly reduced cell apoptosis compared with that in the I/R group (p<0.01). Administration of insulin+DFMO-EGBG further enhanced cell apoptosis compared with that in the insulin group (p<0.01). Administration of insulin/PEG-CMCS+DFMO-EGBG further enhanced cell apoptosis compared with that in the insulin/PEG-CMCS group (p<0.01).

Expression of SSAT was observed after reperfusion for 2 hours (Figure 4C), and it was found to be higher in the I/R group than in the Sham group (p<0.01). Administration of insulin reduced SSAT expression compared with that in the I/R group (p<0.01). Administration of insulin/PEG-CMCS significantly reduced SSAT expression compared with that in the I/R group (p<0.01). Administration of insulin+DFMO-EGBG further enhanced SSAT expression compared with that in the insulin group (p<0.01). Administration of insulin/PEG-CMCS+DFMO-EGBG further enhanced SSAT expression compared with that in the insulin/PEG-CMCS group (p<0.01).

Expression of ODC was observed after reperfusion for 2 hours (Figure 4C), and it was found to be lower in the I/R group than in the Sham group (p<0.01). Administration of insulin enhanced ODC expression compared with that in the I/R group (p<0.01). Administration of insulin/PEG-CMCS significantly enhanced ODC expression compared with that in the I/R group (p<0.01). Administration of insulin+DFMO-EGBG reduced ODC expression compared with that in the insulin group (p<0.01). Administration of insulin/PEG-CMCS+DFMO-EGBG reduced ODC expression compared with that in the insulin/PEG-CMCS group (p<0.01).

Discussion

In experimental research studies, administration of different pharmacological agents on HI/RI had corroborated promising results reducing myocardial damage.2 Many experimental research studies confirmed that insulin had a cardioprotective effect on HI/RI and that insulin also showed a protective role in HI/RI in DM.8,9 However, the half-life of insulin was exceedingly short in the blood circulation and was very difficult to administer the insulin dosage clinically because of the continual occurrence of hypoglycemia.21,22 Hence, we designed a delivery system (PEG-CMCS) for insulin to prolong the pharmacological actions and bioactivities.

Experimentally, ODC and SAMDC were the rate-limiting enzymes for polyamine synthesis.18 ODC catalyzed the formation of Pu from ornithine, SAMDC catalyzed the formation of Spd from Pu and generated Sp from Spd.18 SSAT was the rate-limiting enzyme for polyamine decomposition, and it catalyzed Sp to be inverse-transformed into Spd and catalyzed Spd to be inverse-transformed into Pu.10 Some studies showed that polyamine played an important role in many pathophysiological processes,18 and ischemic area increased Pu, decreased Sp and Spd in the focal cerebral ischemia of rats, and polyamine levels decreased damaged chromatin stability, reduced the calcium buffering capacity of mitochondria, increased tissue susceptibility to oxidative stress in the focal cerebral ischemia of rats, and exogenous Sp could restore intracellular PAs and protect ischemic injury of the brain.34 Excessive activation of polyamine decomposition catabolism enzymes (mainly SSAT) in transgenic rats, and intracellular PA depletion (mainly Sp reduced) induced pancreatitis in rats.35 In this study, we found that insulin and insulin/PEG-CMCS both downregulated SSAT expression on HI/RI in DM compared with the I/R group; these results hinted that insulin and insulin/PEG-CMCS inhibited polyamine decomposition catabolism on HI/RI in DM.

Some studies showed that ODC expression, Sp, total PAs reduced and Pu increased on HI/RI.18 In this study, we also found that ODC expression, Sp, total PAs reduced and Pu increased on HI/RI in DM. We speculated that Pu in the total PAs was relatively low, and 70% of its content in cells was related to catabolism of polyamine. In addition, the results of evaluation of cardiac function, myocardial infarction area, and myocardial cell apoptosis rate showed that insulin and insulin/PEG-CMCS both attenuated HI/RI in DM, and this was consistent with the results reported in the literature.18 On the other hand, we found that insulin and insulin/PEG-CMCS both upregulated ODC expression and increased total PA and Sp levels compared with the I/R group; these results suggested that insulin and insulin/PEG-CMCS could upregulate the ODC/polyamine system on HI/RI in DM. However, we found that the Pu levels in insulin and insulin/PEG-CMCS groups were lower than that in the I/R group, and the results showed that insulin and insulin/PEG-CMCS inhibited polyamine decomposition catabolism to make Sp inverse into Spd and Sp reversed into Pu to reduce. Rate-limiting enzyme inhibitor of polyamine synthetic metabolism depleted intracellular PAs and abolished the effect which insulin and insulin/PEG-CMCS decreased myocardial infarction size and myocardial cell apoptosis on HI/RI in DM. It suggested that endogenous polyamines could be involved in cardioprotection mediated by insulin and insulin/PEG-CMCS on HI/RI in DM.

Previous literature showed that intracellular polyamine levels were important for cell survival, and too high or too low levels could cause cytotoxicity.36 In the present study, insulin and insulin/PEG-CMCS promoted the moderate increase in myocardial polyamine concentration to protect myocardium, and depletion of intracellular PAs led to myocardial injury on HI/RI in DM.

Conclusion

Synthesized PEG-CMCS could significantly improve the bioactivity and short half-life of insulin; in clinic this could decrease the frequency of the patients with insulin injections and financial burden. In addition, it showed that insulin and insulin/PEG-CMCS both attenuated HI/RI in DM, and insulin/PEG-CMCS significantly decreased HI/RI through modulating endogenous ODC/polyamine systems.

Synthesis of mPEG-CMCS.

Characterization of CMCS, mPEG-CMCS, and insulin/mPEG-CMCS.

Note: 1H NMR spectra of CMCS (Aa); 1H NMR spectra of mPEG-CMCS (Ab); TEM image of mPEG-CMCS (Ba); and TEM image of insulin/mPEG-CMCS (Bb).

Abbreviations: PEG-CMCS, poly(ethylene glycol)-carboxymethyl chitosan; 1H NMR, 1H nuclear magnetic resonance; TEM, transmission electron microscopy.