Protective effects of N -acetylcysteine on myocardial injury induced by acute fluctuations in blood glucose

Background: To investigate the effect of N -acetylcysteine (NAC) on oxidative stress and inflammation induced by acute fluctuations in blood glucose and the potential mechanism. Methods : Cannulated Wistar rats (n=6/group) were infused intravenously for 48 h with (1) saline (control), (2) 50% glucose intermittently (IHG), (3) 50% glucose continuously (PHG), (4) IHG plus NAC, or (5) PHG plus NAC. Levels of superoxide dismutase (SOD), nitric oxide (NO), nuclear factor-κB (NF-κB), inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF) in cardiac tissues were analyzed. We also evaluated apoptosis in the myocardium using a terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. Results : The IHG group had higher levels of oxidative and inflammatory markers in the myocardium than the PHG and control groups. Co-infusion with NAC prevented the elevation in inflammatory markers and NF-κB activity in the IHG + NAC and PHG + NAC groups. Apoptosis of myocardial cells was observed in the IHG group but was abolished by co-administration of NAC. Conclusions : Acute fluctuations in blood glucose levels induced more severe oxidative stress, inflammation and apoptosis in the myocardium than persistently high blood glucose levels. The antioxidant NAC may prevent apoptosis of myocardial cells caused by fluctuations in blood glucose levels in vivo , most likely because of its antioxidative and anti-inflammatory properties.


Background
Oxidative stress and inflammatory responses have been considered as a major hallmark for the pathogenesis and development of type 2 diabetes mellitus and is acknowledged to play a role in the development of diabeticcomplications [1][2][3] . Chronic inflammatory state is indicated by high plasma levels of numerous pro-inflammatory cytokines notably IL-1β, IL-6, CRP and IL-1β-dependent numerous other cytokines and chemokines 4 . N-acetyl cysteine (NAC) is an important potent antioxidant and anti-inflammatory molecule 5 that can suppress oxidative stress and decrease production of free radicals 6 . In our previous study [7][8][9] , we reported that acute blood glucose fluctuations induced myocardial apoptosis through oxidative stress and activation of nuclear factor-κB (NF-κB). We now wanted to determine whether NAC 3 could prevent the apoptosis of myocardial cells and to further investigate the different effects of acute fluctuations in blood glucose levels and persistent high blood glucose levels on the myocardium.
We used an established in vivo model of blood glucose fluctuation that involves intermittent, or persistent, infusion of high concentrations of glucose in Wistar rats. Co-infusion with NAC was designed to investigate the effects of NAC on apoptosis of myocardial cells and to explore potential mechanisms for the effect of fluctuations in blood glucose levels on myocardial tissues in vivo [7][8][9][10] . For the purpose of our study, we want to investigate the effect of N-acetylcysteine (NAC) on oxidative stress and inflammation induced by acute fluctuations in blood glucose and the potential mechanism. SYXK (Liaoning) 2008-0008). Wistar rats were adapted to the local animal environment (temperature: 24°C to 28°C, humidity: 60%) and no specific pathogenic conditions. Rats were free to drink water and diet.

Animal models and Surgical Procedures
The rats were maintained on a standard 12:12 h light-dark cycle and were provided with standard chow and water. Briefly, after acclimatization for 5 days, the rats were anesthetized with 10%chloral hydrate (0.35 mL/100g). Indwelling catheters 4 (PE-50; Cay Adams, Boston, MA, USA), extended with a segment of silastic tubing (length 3 cm, internal diameter 0.02 in; Care Express Products, Inc., New York, USA), were inserted into the internal jugular vein for infusion and into the carotid artery for blood sampling, as described previously [7][8][9][10] . Both catheters were closed at the end with a metal pin. Before the experiments the rats were permitted to recover from the surgery for 3-4 days.

Glucose concentrations in plasma were measured using a Beckman Glucose
Analyzer II (Beckman, Fullerton, CA, USA). The activities of superoxide dismutase (SOD) and nitric oxide (NO) in myocardium were determined using colorimetric kits.
Levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in myocardium were detected using enzyme linked immunosorbent assay (ELISA) kits. All of the assay kits were purchased from Jiancheng Institute of Biotechnology (Nanjing, China). 5

Immunohistochemistry for expression of NF-κB and inducible nitric oxide synthase (iNOS) in myocardium
Briefly, myocardium was fixed with 4% paraformaldehyde and embedded in paraffin wax. The samples were then cut into 4-μm-thick sections and placed on microscope slides, as described previously [7][8][9][10] . The expression of iNOS was also evaluated using immunohistochemistry. The primary antibody (monoclonal anti-iNOS) and rat biotinylated secondary antibody were purchased from Santa Cruz Biotechnology, Inc.

Measurement of apoptosis using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)
Briefly, myocardium paraffin sections were analyzed with a TUNEL assay, and myocardial apoptosis was measured using a kit purchased from Boster Biological Engineering Company (Wuhan, China), according to the manufacturer's instructions, as described previously. [7][8][9][10]

Statistical analysis
Data are presented as means ± standard deviation. Groups were compared using one-way analysis of variance and the Bonferroni-Dunn post hoc test was performed on raw data. Differences were considered to be significant at P < 0.05.

NO and SOD level in myocardium
The level of NO was significantly higher, while SOD activity was lower, in the IHG and PHG groups compared with the control group (p<0.05). There was also a significant difference in levels of NO and SOD activity between the IHG and PHG groups (p<0.05). NAC prevented the increase in oxidative markers in the IHG + NAC and PHG + NAC groups. (p<0.05, Table 1).

TNF-α and IL-6 levels in myocardium
Levels of TNF-α and IL-6 were markedly higher in the PHG group than in the control group (p<0.05). Levels of TNF-α and IL-6 were much higher in the IHG group than in the PHG and control groups (p<0.05). NAC prevented the production of inflammatory markers in the IHG + NAC and PHG + NAC groups. (p<0.05, Table 1).

Expression of NF-κB and iNOS in myocardium
As assessed by immunohistochemical staining, the relative expression of NF-κB and iNOS in myocardium was significantly increased in the group with fluctuating blood glucose levels compared with either the control group or the group with persistently high blood glucose levels (p<0.05). Co-infusion with NAC prevented the elevation of NF-κB and iNOS in the IHG + NAC and PHG + NAC groups.(p<0.05, Fig. 1 and Fig. 2

Apoptosis of myocardiocytes
Myocardial cell apoptosis was observed in the IHG group but no apoptosis was observed in the other groups (Fig. 3).

Discussion
It is estimated that more than 150 million people suffer from type 2 diabetes mellitus and its complications 11 and the number of patients with type 2 diabetes mellitus is still growing. Oxidative stress induced by hyperglycemia is an established risk factor for cardiovascular disease 12 . Oxidative stress may alter signal transduction pathways, such as NF-κB, c-Jun N-terminal kinase/stress-activated protein kinase, p38 mitogen-activated protein kinase and protein kinase C, leading to apoptosis 13 . 9 The glutathione (GSH) precursor NAC is recognized to be an important antioxidant 5,14 and is commonly administered as a safe way to increase levels of glutathione 15 . Hyperglycemia can cause increased levels of free oxygen radicals in diabetic patients 16 . Lee et al. 6 showed that NAC effectively reduced free oxygen radicals. SOD appears to be essential for maintaining cellular antioxidant capacity 17,18 and impaired SOD activity has been associated with developmental defects in mice as well as several chronic diseases in humans 19 . An earlier study showed that NAC increased SOD activity in immune cells of septic rats 20 . Levels of iNOS and free radicals such as NO, which are associated with cardiovascular diseases, are also increased in diabetes 21 . In the present study, we showed that myocardial levels of oxidative markers (NO, iNOS) were significantly higher, while SOD activity was lower, in the IHG group than in the PHG and control groups. NAC treatment increased SOD activity and decreased levels of NO and iNOS in the IHG + NAC group.
NF-κB is a redox-sensitive transcription factor 22 , activation of which has been identified as one of the most important causes of diabetes and its complications [23][24][25] .
Oxidative stress has been implicated in increased activity of transcription factors such as NF-Κb 26 and NF-κB has been identified as a key signal in apoptosis 27 . In present study, the level of NF-κB was increased in the IHG group compared with the other groups. Significant decreases in levels of NF-κB were detected in the groups treated with NAC.
Accumulating evidence suggests that diabetes has features in common with chronic inflammatory states. Circulating levels of IL-6 and TNF-α were both increased in diabetes 28,29 . Oxidative stressors may increase levels of IL-6, which has a strong relationship with NF-κB activity 30,31 . In the present study, levels of IL-6 and TNF-α were significant higher in the IHG group, which has fluctuating glucose levels, and were suppressed by NAC. This result is consistent with the findings of Khechaiet al. 32 Hsu et al. also showed that NAC prevented the release of inflammatory markers TNF-α and IL-6 33 . Previous studies have suggested that inflammatory cytokines, such as TNF-α and IL-6, can stimulate expression of SOD 34,35 , further enhancing oxidative 10 stress. This is a perilous circle. Gül et al. 36 showed thatreduction of oxidative injury by NAC was strongly correlated with GSH and TNF-α level.
Our earlier study suggested that apoptosis of myocardial cells was associated with oxidative stress and NF-κB activation. The antioxidant NAC has been shown to prevent the formation of malondialdehyde and to reduce cardiovascular events by decreasing apoptosis 37,38 . Most previous studies of the effect of NAC on apoptosis were, however, in vitro experiments [39][40][41][42] and few in vivo studies have been described.
The uniqueness of our study was that we In the present study, fluctuations in blood glucose levels induced more severe oxidative stress, chronic inflammation and apoptosis in the myocardium than persistently high blood glucose levels. The antioxidant NAC may prevent the apoptosis of myocardial cells induced by fluctuations in blood glucose levels in vivo.
This effect of NAC can likely be attributed to its anti-oxidative and anti-inflammatory properties.Although we did not use different doses of NAC and did not examine other possible mechanisms, our study has clearly demonstrated that heart injury can be minimized by the administration of NAC.

Conclusions
Acute fluctuations in blood glucose levels induced more severe oxidative stress, inflammation and apoptosis in the myocardium than persistently high blood glucose levels. The antioxidant NAC may prevent apoptosis of myocardial cells caused by fluctuations in blood glucose levels in vivo, most likely because of its antioxidative and anti-inflammatory properties.

Consent for publication
A written consent to publish the information and data of the participants was obtained.

Acknowledgments
we are grateful to Mr. Zuo Zhou for his outstanding statistics assistance.

Authors' contributions
WZ was responsible for the design conception of the experiments, collection, analysis and interpretation of the data, and drafting of the manuscript. GJ P was responsible for the interpretation of the data and drafting of the manuscript. SZ, YL and PH contributed to perform experiments. YZ L was principal investigator of the study, contributed to the analysis and interpretation of the results, and produced the final version of the manuscript. All authors read and approved the final manuscript.

Availability of data and materials
The datasets supporting the conclusions of this article are included within the article.

Ethics approval and consent to participate
All animal experiments were performed with approval from the animal ethics committee of Shengjing Hospital of China Medical University (2016PS026 K).