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Basic Science Aspects

Genistein Protects Against Burn-Induced Myocardial Injury via Notch1-Mediated Suppression of Oxidative/Nitrative Stress

Fang, Zhuoqun; Wu, Gaofeng; Zhang, Dongliang; Wang, Kejia; Deng, Xudong; Liu, Mengdong; Han, Juntao; Hu, Dahai; Yang, Xuekang

Author Information
doi: 10.1097/SHK.0000000000001464

Abstract

INTRODUCTION

Severe burn injury results in multiple organ dysfunction, which is the leading cause of death in intensive care units (1, 2). Among the multiple complications, myocardial injury is a major contributor to mortality, particularly in individuals with pre-existing cardiac pathology (3–6). Numerous experimental studies have been performed to identify the molecular mechanisms involved burn-induced myocardial injury with the end goal of creating novel therapeutic interventions and agents to reduce the incidence of life-threatening complications (3–8). However, after decades of lab studies and clinical practices, there is still a lack of effective therapies to increase myocardial resistance to burn injury. Thus, to identify novel drug targets that can at least minimize the extent of myocardial damage induced by burn injury is urgently needed.

Amounting evidence has shown that oxidative/nitrative stress plays an important role in burn-induced myocardial injury. Agents that can prevent myocardium from oxidative/nitrative stress might be promising therapeutics to ameliorate myocardial injury postburn (6–11). Genistein (Gen; 4′,5,7-Trihydroxy isoflavone), a natural biologically active flavonoid found in soy, has been shown to possess multiple pharmacological actions, including anticancer, anti-inflammatory, anti-oxidative, and antinitrative effects (12–18). Increasing evidence has indicated that it also possesses cardioprotective properties in cardiac hypertrophy, myocardial infarction, and myocardial ischemia–reperfusion (MI/R) injury (15, 16, 19–21). However, little work has been done on its possible use as a therapeutic drug in the treatment of burn-induced myocardial injury.

As a family of highly conserved trans-membrane receptors, Notch pathway has been identified as key regulators of cell fate, differentiation, proliferation, and apoptosis (22). In mammals, four Notch receptors (Notch1–4) and five Notch ligands (Delta-like1, 3, 4, and Jagged1, 2) have been identified. The binding of Notch ligand to its receptor triggers the γ-secretase-mediated proteolytic cleavage of the Notch intracellular domain (NICD), which then translocates into the nucleus to form a transcription-activating complex. This complex mediates transcription of downstream target genes such as Hes1, Hey1, and cyclin D (22–24). Jagged1 and Notch1 are expressed in the adult heart and functions to protect cardiac tissue under various pathophysiological conditions, including alcoholic cardiomyopathy, myocardial infarction, cardiac hypertrophy, and MI/R injury (22, 25–28). More recent studies reveal that Notch1 possesses important properties in suppressing oxidative stress in hepatocytes and endothelial cells (29–31). Notably, we recently found that Notch1 protects against MI/R injury via reducing oxidative/nitrative stress (32).

Several studies have reported that Gen could induce apoptosis by modulating the Notch signaling pathway in multiple types of cancer cells (33, 34). However, whether the Notch1 signaling pathway plays a role in the Gen-induced cardioprotective effects, especially following burn injury, remains to be determined. Therefore, this study was designed to evaluate the cardioprotective effect of Gen treatment on myocardial injury postburn and to investigate its potential effects on oxidative/nitrative stress. The involvement of Notch1 signaling in mediating the protective mechanisms of Gen was also evaluated.

MATERIALS AND METHODS

Materials

Gen (purity 95%) was purchased from Xi’an QingYue Biotechnology Co, Ltd (Xi’an, Shaanxi, China). Notch1 and Hes1 antibodies were purchased from Santa Cruz Company (Santa Cruz, Calif). Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) kits were obtained from Roche (Mannheim, Germany). Caspase-3 colorimetric assay kit was purchased from Chemicon (Temecula, Calif). The kits for the measurement of superoxidase dismutase (SOD) activity, total nitric oxide (NO) and nitrotyrosine content were purchased from Institute of Jiancheng Bioengineering (Nanjing, Jiangsu, China). The inducible NO synthase (iNOS), gp91phox, and glyceraldehydes-3-phosphate antibodies were purchased from Cell Signaling Biotechnology (Boston, Mass). The rabbit antigoat, goat antirabbit, and goat antimouse secondary antibodies were purchased from Zhongshan Company (Beijing, China).

Animals

All animal-related procedures were approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health. The male wild-type C57BL/6 mice of 6 to 8 weeks of age were purchased from the Center of Experimental Animals of the Fourth Military Medical University (Xi’an, China). Conditional RBP-J allele (RBP-J floxed) mice were a gift from Dr Hua Han (Department of Medical Genetics and Developmental Biology, The Fourth Military Medical University). Myh6-Cre mice were obtained from Model Animal Research Center of Nanjing University (Nanjing, China). Cardio-specific RBP-J knockout mice were generated with the Cre/loxP system by breeding RBP-J floxed mice with Myh6-Cre mice. When mice reached 6 weeks postnatal, Tamoxifen (50 mg/kg) was administered by intraperitoneal injections once a day for 5 days. Animals were housed in a temperature- and humidity-controlled room with a 12/12-h light–dark cycle, and fed with formula diet with 10% kcal% fat (D12450B, Opensource diets). A 2-week adaptation time was allowed before any further procedure. Mice at the end of experiments were sacrificed in a CO2 chamber, followed by cervical dislocation. The whole procedure was according to the Institutional Animal Care and Use Committee.

Burn procedure

Animals were anesthetized lightly with pentobarbital sodium (50 mg/kg) and buprenorphine (0.1 mg/kg). The mice were then placed in a prefabricated template with a rectangular opening that exposed the dorsal and ventral skin surface, while protecting the remaining skin from burn exposure. The template limits the burn area to a predetermined 40% total burn surface area. The exposed skin surface was immersed in 95°C to 98°C water for 8 s. Sham-burn control animals were treated identically to those in the burn group, except that they were immersed in 25°C water. Following exposure to water, animals were immediately infused with 1 mL Ringer's lactate solution according to Parkland's formula, and received a subcutaneous injection of 0.5 mL normal saline with 0.1 mg/kg of buprenorphine (Sigma, St. Louis, Mo) for pain control. Access to formula diet with 10% kcal% fat (D12450B, Opensource diets) and water was allowed ad libitum. Animals were sacrificed at various time points.

Determination of cardiac function, myocardial necrosis, and apoptosis

Left ventricular ejection fraction (LVEF), a major cardiac function index, was obtained with transthoracic echocardiography. Myocardial necrosis was evaluated by serum lactate dehydrogenase (LDH) and creatine kinase (CK) levels. All assays were performed by a chemistry autoanalyzer (Vitros 750, Johnson & Johnson, Rochester, NY).

TUNEL assay was performed using In Situ Cell Death Detection Kit according to the manufacturer's instructions. To identify apoptosis in cardiac myocytes, tissue sections were further stained using a-sarcomeric actin antibodies (1:50; Sigma). Hoechst 33342 (10 μM; Sigma-Aldrich) staining was used to count the total number of nuclei. Images were acquired with a confocal fluorescence microscope (Olympus FV1000). TUNEL-positive nuclei of cardiac myocytes were counted. Quantification of TUNEL-positive cardiac myocyte numbers was performed on five randomly selected fields (400×) per mouse in each experimental group. Index of apoptosis was calculated as the percentage of apoptotic myocyte nuclei per total number of nuclei.

Cardiac caspase-3 activity was performed by using a caspase-3 colorimetric assay kit following the manufacturer's instructions. In brief, myocardial tissue was homogenized in ice-cold lysis buffer for 30 s. The homogenates were centrifuged, supernatants were collected, and protein concentrations were measured by the bicinchoninic acid method. To each well of a 96-well plate, supernatant containing 200 μg of protein was loaded and incubated with 25 μg caspase-3 substrate N-acetyl-Asp-Glu-Val-Asp(DEVD)-p-nitroanilide at 37°C for 1.5 h. The optical density was measured at 405 nm with a SpectraMax-Plus microplate spectrophotometer. The activity of caspase-3 in tissue samples was calculated using a standard curve and expressed as fold increase over the mean value of sham MI.

Quantification of superoxide anions production

Myocardial superoxide anions (O2) content was determined by lucigenin-enhanced luminescence (32). Samples were weighed, cut into uniform cubes (0.5 mm3), and transferred into a polypropylene tube containing 1 mL PBS and lucigenin (Sigma, 0.25 mmol/L). The tube was placed in a FB12-Berthold luminometer (Berthold Technologies, Bad Wildbad, Germany). The RLU emitted was recorded and integrated over 30 s intervals for 5 min. Activity was normalized with dry tissue weights.

Determination of total NO and nitrotyrosine content in cardiac tissues

Cardiac tissues were rinsed and homogenized. The tissue NO and its in vivo metabolites NO2 and NO3 (collectively termed NOx) were determined by nitrate reductase kits. Nitrotyrosine in the cardiac tissues, a footprint of in vivo peroxynitrite (ONOO) formation and an index for nitrative stress, was quantified using a competitive enzyme-linked immunosorbent assay kit. Cardiac tissues were harvested and fixed in paraformaldehyde, cut into sections 4μm to 5 μm thick, and stained with an antibody against nitrotyrosine. Immunostaining was done with Vectastain ABC kit, and slides were analyzed by light microscopy (35).

Western blot analyses

The myocardial tissues were homogenized in NP40 lysis buffer (1% NP-40, 0.15 M NaCl, 50 mM Tris, pH 8.0) containing 1% protease inhibitor cocktail (Roche). The lysates were centrifuged for 15 min at 12,000 g, and the resulting supernatant was transferred to a new tube and stored at −70°C. The protein concentrations were determined using a Bradford protein assay. Equal amount of proteins was separated by 10% SDS polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membranes were blocked for 1 h in Tris-buffered saline and Tween 20 (TBST, pH 7.6) containing 5% nonfat dry milk and then incubated overnight at 4°C with antibodies against NICD1, Hes1, iNOS, gp91phox, and glyceraldehyde-3-phosphate, followed by washes with TBST. The membranes were then probed with appropriate secondary antibodies (1: 5,000 dilution) at room temperature for 90 min and washed with TBST. The protein bands were detected using a BioRad imaging system (Bio-Rad, Hercules, Calif) and quantified using the Quantity One software package (West Berkeley, Calif).

Determination of SOD activity

The SOD activity was determined with a spectrophotometric assay kit, following the manufacturer's instruction. In brief, 30 μL of supernatant were added to the reaction buffer containing xanthine, xanthine oxidase, and hydroxylamine. After 40 min of incubation at 37°C, accumulation of nitrite was quantified by the Griess reaction. Tissue antioxidant capacity is inversely related to the concentration of nitrate. Results were normalized against the mean value of control and expressed as fold changes.

Statistical analysis

Data were analyzed using GraphPad Prism-5 statistical software (La Jolla, Calif). All values are presented as the mean ± the standard error of the mean (SEM). One-way ANOVA was conducted across all groups first. Post hoc tests were then performed using Bonferroni correction, and all two-group comparisons were made. A difference of P < 0.05 was considered to be statistically significant.

RESULTS

Gen ameliorated burn-induced myocardial injury

To investigate the potential cardioprotective effects of Gen in burn model, mice were treated with different does of Gen (0.25 mg/kg, 0.5 mg/kg, or 1 mg/kg) via intraperitoneal injection immediately after burn injury. Burn injury caused significant cardiac dysfunction as manifested by decrease of LVEF compared with the untreated sham group. In comparison with that of the burn group, Gen (0.5 mg/kg) significantly alleviated cardiac dysfunction at 8 and 12 h, Gen (1 mg/kg) significantly alleviated cardiac dysfunction at 4, 8, and 12 h (Fig. 1A). As illustrated in Figure 1B and C, burn resulted in significant myocardial necrosis at 12 h, as evidenced by higher levels of LDH and CK. Gen (0.5 mg/kg or 1 mg/kg) significantly attenuated serum LDH levels, and Gen (0.25 mg/kg, 0.5 mg/kg, or 1 mg/kg) significantly attenuated serum CK levels. Furthermore, we determined myocardial apoptosis post-burn using caspase-3 activity and TUNEL staining. As shown in Figure 1D, burn injury induced a significant rise in caspase-3 activity. Treatment with Gen (0.25 mg/kg, 0.5 mg/kg, or 1 mg/kg) substantially reduced this burn-induced increase of myocardial caspase-3 activity. As shown in Figure 1E, burn injury caused significant number of TUNEL-positive cells, while Gen (1 mg/kg) significantly reduced TUNEL-positive staining. These results demonstrated that Gen significantly alleviated burn-induced myocardial injury.

F1
Fig. 1:
Gen ameliorated burn-induced myocardial injury.

Gen significantly reduced O2 overproduction in myocardium postburn

O2 is the most important ROS in organisms. Malondialdehyde (MDA) is the major metabolites of lipid oxidation and plays a key role in oxidative stress. As illustrated in Figure 2A and B, burn significantly increased O2 production and MDA content. Gen (0.25 mg/kg, 0.5 mg/kg, or 1 mg/kg) substantially reduced this burn-induced increase of O2 production and MDA content. It is well known that ROS are mainly derived from NADPH oxidase in the heart, thus we detected the expression of gp91phox which is a major component of NADPH oxidase. As a result, gp91phox expression was markedly enhanced in myocardium postburn. More importantly, Gen (0.25 mg/kg, 0.5 mg/kg, or 1 mg/kg) treatment reduced burn-induced increase of gp91phox (Fig. 2C). In addition, antioxidant capacity was determined by the activity of SOD (O2 scavenger) in cardiac tissue. Burn-induced reduction in SOD activity was significantly preserved after Gen (0.5 mg/kg or 1 mg/kg) treatment (Fig. 2D). These results indicate that Gen reduces O2 production via downregulation of gp91phox and upregulation of SOD activity.

F2
Fig. 2:
Gen significantly reduced burn-induced oxidative stress in myocardium postburn.

Gen decreased NOx production through downregulation of iNOS expression

Low concentration of NO released by eNOS or NO donors has been shown to exert cardioprotective effect, whereas excess NO generation induced by iNOS is responsible for ONOO synthesis, a major source of cellular nitrative stress (36). We proceeded to explore whether Gen could change NO production. Therefore, NOx production (NO's contents and its in vivo metabolites NO2 and NO3) and iNOS expression were determined in myocardium postburn. As indicated in Figure 3, burn significantly increased NOx production and iNOS expression. Moreover, both the overproduction of NOx production and the increase of iNOS expression induced by burn injury were ameliorated by Gen (0.25 mg/kg, 0.5 mg/kg, or 1 mg/kg). These results suggest that Gen can reduce NOx production by inhibiting iNOS expression in myocardium postburn.

F3
Fig. 3:
Gen decreased NOx production and iNOS expression in myocardium postburn.

Gen depressed ONOO overproduction, while ONOO scavenger preferentially attenuated cardiac injury postburn

It is well known that NO and O2 react at a diffusion-limited rate to form the highly cytotoxic molecule ONOO, and overproduction of ONOO with resultant nitrative stress is an important contributor to myocardial apoptosis (37). We quantified nitrotyrosine content (a footprint of in vivo ONOO formation and an index for nitrative stress) to determine Gen's effects in response to burn injury. As shown in Figure 4A, burn injury resulted in significant ONOO production as evidenced by more than 4-fold increase in nitrotyrosine content. More importantly, treatment of Gen (0.25 mg/kg, 0.5 mg/kg, or 1 mg/kg) mitigated burn-induced increase of ONOO formation. These results suggest that the protection of Gen against burn injury is probably attributed to its inhibition of the ONOO production.

F4
Fig. 4:
Gen depressed peroxynitrite overproduction in myocardium postburn.

To obtain more evidence to support this conclusion, an additional series of experiments was performed. Mice were subjected to burn injury as described above and treated with either uric acid (an ONOO scavenger, 5 mg/kg intraperitoneal injection after burn injury), or uric acid plus Gen (1 mg/kg). As summarized in Figure 4B, treatment with uric acid significantly reduced burn-induced ONOO production. Treatment with Gen had no additional effect. Uric acid treatment also markedly attenuated myocardial injury as determined by lower levels of LDH, CK, and caspase-3 activity. Treatment with Gen afforded no additional cardioprotection (Fig. 4, C–F). These results demonstrated that scavenging ONOO preferentially protected hearts against burn injury and that decreased production of ONOO plays a critical role in the cardioprotection of Gen.

Gen activated myocardial Notch1 pathway postburn

Gen has been implicated in regulating Notch 1 pathway in several models. Our present study determined expressions of NICD1 (the activated form of Notch1) and Hes1 (one main downstream effector of Notch1) in cardiac tissues by western blots. As a result, we found that Gen (0.25 mg/kg, 0.5 mg/kg, 1 mg/kg, or 5 mg/kg) significantly increased NICD1 expression, and Gen (0.5 mg/kg, 1 mg/kg, or 5 mg/kg) significantly increased Hes1 expression in cardiac tissues of sham-burn mice (Supplemental Fig.1, Supplemental Digital Content 1, https://links.lww.com/SHK/A960). Importantly, we found that both NICD1 and Hes1 were increased by burn injury, indicating endogenous activation of cardiac Notch1 signaling by burn injury. Notably, Gen administration further enhanced the protein expression of NICD1 and Hes1 compared with the burn group (Fig. 5). These data indicated that Gen enhanced the activation of the Notch1 pathways in burn-induced myocardial injury.

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Fig. 5:
Gen activated myocardial Notch1 pathway postburn.

Gen ameliorated burn-induced myocardial injury via Notch1 signaling

The cardioprotective effect of Notch1 has been well demonstrated. To investigate whether Notch1 plays a critical role in Gen's cardioprotection, conditional Notch-RBP-J knockout mice were introduced. Compared with the wild-type and RBP-J+/− groups, the LVEF values in the RBP-J−/− group were decreased. Gen significantly increased LVEF in RBP-J+/− mice. In sharp contrast, Gen did not change LVEF in the RBP-J−/− mice (Fig. 6A). In addition, myocardial necrosis, as evidenced by serum levels of LDH and CK, and myocardial apoptosis in the RBP-J−/− group were increased compared with the wild-type and RBP-J+/− groups. Myocardial necrosis and apoptosis in the Gen RBP-J+/− group were significantly reduced compared with the RBP-J+/− group. Similarly, there was no apparent difference in myocardial necrosis or apoptosis between the RBP-J−/− group and Gen RBP-J−/− group (Fig. 6, B–E). These data indicated that Gen protected against burn-induced myocardial injury via Notch1 signaling.

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Fig. 6:
Notch1 knockout abolished Gen-induced protection on myocardial injury.

Gen reduced burn-induced oxidative/nitrative stress through Notch1 pathway

We further determined oxidative/nitrative stress indicator in conditional RBP-J-knockout mice following burn injury. As summarized in Figure 7, compared with the wild-type and RBP-J+/− groups, oxidative stress indicator, as evidenced by O2 production, MDA content, and gp91phox expression were increased, whereas SOD activity was decreased in the RBP-J−/− group. Oxidative stress in the Gen RBP-J+/− group were significantly reduced compared with the RBP-J+/− group. Importantly, there was no apparent difference in oxidative stress between the RBP-J−/− group and Gen RBP-J−/− group. As shown in Figure 8, nitrative stress indicator, as evidenced by NO production, iNOS expression, and ONOO production in the RBP-J−/− group was increased compared with the wild-type and RBP-J+/− groups. Moreover, Gen significantly increased nitrative stress in RBP-J+/−. In sharp contrast, Gen did not change nitrative stress in the RBP-J−/− group. These data indicated that the protective effects of Gen against burn-induced oxidative/nitrative stress were mediated through the Notch1 pathway.

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Fig. 7:
Notch1 knockout abolished Gen-induced protection on oxidative stress.
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Fig. 8:
Notch1 knockout abolished Gen-induced protection on nitrative stress.

DISCUSSION

Several important observations were made in our present study. First, we observed for the first time that Gen attenuates oxidative/nitrative stress and inhibits burn-induced myocardial injury. Second, Gen can activate cardiac Notch1 signaling in myocardium postburn. Third, we provided direct evidence that the anti-oxidative/antinitrative effects of Gen in burn-induced myocardial injury are partly mediated by cardiac Notch1 signaling.

Gen possesses a variety of pharmacological and biological properties and potentially mediates cardiovascular protection (15, 16, 19–21). The present study explored its role in burn-induced myocardial injury. In our preliminary experiment, we first evaluate the effects of Gen on sham-burn animals, mice were treated with vehicle or different does of Gen (0.1 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 1 mg/kg, or 5 mg/kg) via intraperitoneal injection. We found that Gen did not induce any cardioprotective effects in mice (Supplemental Fig.2, Supplemental Digital Content 2, https://links.lww.com/SHK/A961) succedent experiment of the burn-induced myocardial injury model, we found that Gen (0.25 mg/ kg, 0.5 mg/kg, or 1 mg/kg) treatment significantly alleviate d burn-induced myocardial injury, as evidenced by increased LVEF, reduced serum levels of LDH and CK, and decreased myocardial apoptosis. The effects of 0.1 mg/kg and 5 mg/kg Gen were also explored in preliminary experiment. The results demonstrated that 0.1 mg/kg Gen had almost no effect on improving the LVEF levels during the burn procedure, whereas the effect of 5 mg/kg Gen on improving the LVEF levels during the burn procedure was not better than the effect of 1 mg/kg Gen (data not shown). As a result, 0.25 μM, 0.5 μM, and 1 μM Gen were selected for this study. To the best of our knowledge, this is the first demonstration of the cardioprotective effects of Gen in burn injury.

ROS have long been recognized to cause oxidative stress and act as the major mediators of myocardial injury postburn (6–8, 38). Here, we provide that Gen significantly reduced burn-induced O2 overproduction, thus attenuates oxidative injury. This finding is consistent with previous study which revealed that Gen abolished depolarization-induced O2 formation in human endothelial cells (39). NADPH oxidase is the most important source for O2 production under pathological conditions (40). In the present study, we have demonstrated that treatment with Gen significantly reduced gp91phox (a critical component of NADPH oxidase) expression in myocardium postburn. On the other hand, SOD, O2 scavenger, has been well demonstrated to reduce O2-dependent damages by transforming O2 into H2O2(41). We have demonstrated that treatment with Gen significantly improved SOD activity in myocardium postburn. On the basis of these observations, it is suggested that the reduction of gp91phox expression and the improvement of SOD activity should be responsible for suppression of O2 afforded by Gen in myocardium post-burn.

High and sustained release of NO produced from iNOS may be detrimental in pathological cardiac injury (9, 10, 32, 36). The previous study has reported that burn injury increased myocardial iNOS expression, and inhibition of iNOS significantly attenuated burn-mediated cardiac injury (9). Similarly, our present study found that burn significantly increased NO production and iNOS expression. Importantly, both the overproduction of NO metabolites and the increase of iNOS expression induced by burn injury were ameliorated by Gen. This finding is consistent with the previous study which revealed that Gen prevents isoproterenol-induced cardiac hypertrophy through inhibition of iNOS (15).

It is noteworthy that cellular concentrations of NO and O2 are well balanced under physiological conditions, whereas pathological insult may stimulate the reaction between NO and O2, hence the formation of ONOO(42, 43), a strong biological oxidant and nitrating species. The myocardial cytotoxicity of ONOO involves oxidative damage to proteins, lipids, DNA, activation of metalloproteinases, and nitration of tyrosine residues (37, 44, 45). This study suggests that Gen treatment markedly reduced burn-induced ONOO formation by inhibition of NADPH oxidase-derived O2 and iNOS-derived NO. In addition, scavenging ONOO preferentially protected hearts against burn injury, whereas treatment with Gen had no additional effect, which further supported that Gen afforded cardioprotection via reducing ONOO-caused oxidative/nitrative stress.

Notch signaling is highly relevant for proper myocardial function and response to injury (22, 25–27, 46). Our previous study has reported that Notch1 signaling protects against MI/R injury partly via reducing oxidative/nitrative stress (32). Subsequent study found that knockout mice of the transcription factor RBP-J aggravated post-burn myocardial injury, and increased intracellular ROS production, which suggested that the deficiency of Notch signaling aggravated postburn myocardial injury through increased ROS levels (38). In the present study, we showed an increased NICD1 and Hes1 expression in the acute stage after exposure to severe burn, suggesting endogenous activation of cardiac Notch1 signal during burn injury. Complete Notch deficiency increased burn-induced oxidative/nitrative stress, and potentiated myocardial injury using the Notch RBP-J knockout mice. These data further explored the role of the Notch 1 pathway in burn-induced myocardial injury, and further confirmed the cardioprotective effect of the Notch 1 pathway postburn. Moreover, Gen has been implicated in regulating Notch 1 pathway in several models (33, 34). In this study, Gen markedly activated myocardial Notch1 pathway post-burn, as evidenced by increased Notch1 ICD and Hes1 expression, indicating that the cardioprotective effect of Gen might be involved in Notch 1 pathway regulation. To further elucidate the role of Notch signaling in Gen-induced cardioprotection postburn, conditional Notch-RBP-J knockout mice were introduced. As expected, we found that Gen treatment did not demonstrate cardioprotective effects in RBP-J−/− mice subjected to burn injury. These data provided strong evidence that the Notch1 pathway plays an important role in mediating the cardioprotective effects of Gen.

The current study demonstrated, for the first time, that Gen exerts a profound cardioprotective effect following burn-induced myocardial injury. This protection might result from Notch signaling pathway-mediated anti-oxidative and antinitrative effects. Our findings suggest that Gen may be a promising candidate for the treatment of post-burn myocardial injury.

Acknowledgments

The authors thank all other members of our laboratory for their insight and technical support.

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Keywords:

Burn injury; genistein; myocardial injury; notch1 signaling; oxidative/nitrative stress

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