https://orcid.org/0000-0002-4974-5850
Abstract
Hexavalent chromium (Cr(vi)), the most toxic valence state of chromium, is widely present in industrial effluents and wastes. Sulforaphane (SFN), rich in Brassica genus plants, bears multiple biological activity. Wistar rats were used to explore the protective role of SFN against the cardiotoxicity of chronic potassium dichromate (K2Cr2O7) exposure and reveal the potential molecular mechanism. The data showed that SFN alleviated hematological variations, oxidative stress, heart dysfunction and structure disorder, and cardiomyocyte apoptosis induced by K2Cr2O7. Moreover, SFN reduced p53, cleaved caspase-3, Bcl2-associated X protein, nuclear factor kappa-B, and interleukin-1β levels, and increased Sesn2, nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1, NAD(P)H quinone oxidoreductase-1, and phosphorylated adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) levels. This study demonstrates that SFN ameliorates Cr(vi)-induced cardiotoxicity via activation of the Sesn2/AMPK/Nrf2 signaling pathway. SFN may be a protector against Cr(vi)-induced heart injury and a novel therapy for chronic Cr(vi) exposure.
Hexavalent chromium (Cr(vi)), with the most toxic valence state of chromium, is widely present in industrial effluents and wastes.
Cr(vi) is widely present in industrial effluents and wastes threatening human and animal health. Chronic Cr(vi) exposure could induce tissue damage and several diseases. However, effective and safe therapeutic strategies are still under exploration. This study investigates the protective role of SFN against chronic Cr(vi) exposure-induced heart injury. Furthermore, this study demonstrates that SFN ameliorates Cr(vi)-induced cardiotoxicity via activation of the Sesn2/AMPK/Nrf2 signaling pathway. SFN may be a protector against Cr(vi)-induced heart injury and a novel therapy for chronic Cr(vi) exposure.
Introduction
Chromium is one of the most harmful substances, although chromium shows beneficial effects on the regulation of proper carbohydrate and lipid metabolism at supranutritional doses.1,2 Chromium is widely present in industrial effluents and wastes polluting drinking water and soil.3,4 As a frequently encountered metal pollutant in the environment, a number of diseases are directly related to chromium exposure. Chromium exists in the environment in two valence states as trivalent Cr(iii) and hexavalent Cr(vi). It is difficult for Cr(iii) to pass through cell membranes, which contributes to the low toxicity of Cr(iii). However, chronic Cr(vi) exposure could cause dermatitis, bronchitis, pulmonary congestion and oedema, gastrointestinal ulcers, tumors, and tissue damage.5–10
Occupational health risk assessment of hexavalent chromium compounds in 2015 from the National Institute of Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention (NIOHP, China CDC)11 reported that the average concentration range of Cr(vi) in the air of workplaces in China is from 0.001 mg m−3 (0.0004 ppm) to 5.58 mg m−3 (2.4 ppm)3. Humans are exposed to Cr(vi) through inhalation, dermal contact, and oral administration. And Cr(vi) mostly accumulates in the liver, kidneys, heart, blood, and endocrine glands. The heart is one of the target tissues of chronic Cr(vi) exposure. It has been reported that Cr(vi) could cause impairment in the cell membrane structure by aggravating lipid peroxidation in the heart.12In vivo and in vitro, Cr(vi) is also reported to induce cell impairment partly attributed to the generation of reactive oxygen species (ROS).13,14 However, the detailed mechanism of Cr(vi)-induced cardiotoxicity is still unclear to date.
Therapeutic strategies for chromium poisoning are eliminating the reabsorption of chromium, treating with the antidote as soon as possible, performing continuous venous hemofiltration, and preventing multiple organ failure. Unfortunately, multiple organ failure remains a major cause of death induced by chromium. Considering that chromium depresses the antioxidant defense system, antioxidant compounds could be proposed as potential protectors or dietary supplements which have a positive effect on oxidative stress induced injury and metabolic homeostasis disorders with low side effects.
Sestrins (Sesns) have been reported to play protective roles in many physiological and pathological processes. Sesn2, a member of the evolutionarily conserved stress-inducible protein family Sesns highly expressed in the heart,15 has an antioxidant function that suppresses reactive oxygen species (ROS) generation.16 In addition to its antioxidant activity, Sesn2 activates AMP-activated protein kinase (AMPK) and subsequently inhibits the mechanistic target of rapamycin complex 1.17 AMPK is also a crucial factor which affects the regulation of oxidative stress, energy homeostasis, cell growth, and apoptosis.18 In addition, Sesn2 could specifically target Keap1, causing Nrf2 to be freed from Keap1 under oxidative stress.19 Then Nrf2 is translocated to the nucleus, and promotes the transcriptional activation of genes controlled by the antioxidant response elements.20 And Nrf2 is a critical antioxidative gene in scavenging ROS and maintaining redox homeostasis.21 Thus, the pathways based on Sesn2 and Nrf2 may play a critical protective role in bodies or cells subjected to oxidative stress, e.g. Cr(vi)-exposed rats.
Sulforaphane (SFN) is a kind of glucosinolate-derived compound with a high content in cruciferous vegetables such as radish and kohlrabi.22 SFN has been reported to protect from tumors via inducing apoptosis, inhibiting nuclear factor kappa-B (NF-κB), and blocking cell proliferation.23–25 SFN has potential bioactivity in regulating the expression of genes to further protect the aerobic cells against oxidative stress, inflammation, DNA-damaging electrophiles, and radiation.26–29 It has also been confirmed that SFN could reduce the risks of developing malignancies30 and other chronic diseases such as cardiomyopathy, type 2 diabetes, and autistic spectrum disorder.31–33 Therefore, SFN may play a protective role in ameliorating heart injury induced by chronic Cr(vi) exposure.
Thus, we herein hypothesized that SFN might attenuate heart injury in rats chronically exposed to Cr(vi). This study, for the first time, concentrated on the cardioprotection of SFN against Cr(vi), and identified the positive effect of the Sesn2/AMPK/Nrf2 signaling pathway in this event to increase the knowledge of the protective role of SFN.
Materials and methods
Animals and treatments
Healthy male Wistar rats (170 ± 10 g body weight, 6–8 weeks age, n = 28) were obtained from the Experimental Animal Centre of Harbin Medical University (Harbin, China), and acclimated for a week before the start of the experiment, under the same laboratory conditions under which they were fed with standard pelleted rodent diet and water ad libitum. Before and during the experimental period, rats were housed under environmental conditions with a 12 h interval light/dark cycle, a minimum of 50% relative humidity, and a room temperature of 22 ± 2 °C. In addition, rats were provided with standard pelleted rodent diet and tap water.
Rats were randomly divided into 4 groups: control, SFN, potassium dichromate (K2Cr2O7), and K2Cr2O7 + SFN. All treatments lasted for 35 d. In the control group, rats were given saline solution via intraperitoneal injection and subcutaneous injection. In the SFN group, rats were given SFN (4 mg kg−1 body weight; purchased from Toronto Research Chemicals, Toronto, Canada) by subcutaneous injection 1 h after the intraperitoneal injection of saline solution. In the K2Cr2O7 group, rats were given K2Cr2O7 (4 mg kg−1 body weight; purchased from Tianjin Tianli Chemical Reagent, Tianjin, China) through intraperitoneal injection. The dose of K2Cr2O7 is 2.4 times as high as the average concentration range of Cr(vi) in the air of workplaces in China. In the K2Cr2O7 + SFN group, to explore whether SFN could alleviate the heart injury caused by Cr(vi) exposure, SFN (4 mg kg−1 body weight) was given to rats daily by subcutaneous injection 1 h after K2Cr2O7 treatment to avoid insoluble formation and guarantee the effects of Cr(vi) injection and SFN injection. Rats were individually housed in metabolic steel cages, which allows the measurement of food and water intake. Food intake and water intake were analyzed every week. The animal protocol was approved by the Ethical Committee for Animal Experiments (Northeast Agricultural University, Harbin, China). Rats were given ether anesthesia 24 h after the last treatment. Blood samples from the abdominal artery were taken with vacuum tubes containing a heparin sodium anticoagulant. Heart tissues were rapidly collected and homogenized in phosphate-buffered saline (PBS) (pH 7.4, w/v; 1 g tissue with 9 mL PBS) with an Ultra-Turrax T25 Homogenizer. After centrifugation at 10 000 × g for 10 min at 4 °C, the supernatant was separated for biochemical analysis.
Complete blood count and biochemical analysis
Some of the blood samples were used for complete blood count, which was automated by the use of the Bayer ADVIA 120 Blood Cell Analyzer (Bayer Corporation, Leverkusen, Germany). Red blood cell (RBC) count, white blood cell (WBC) count, and hemoglobin (HGB) concentration were determined. Other blood samples were centrifuged at 3000 × g for 10 min. The activities of lactic dehydrogenase (LDH), creatine kinase (CK), creatine kinase-MB (CK-MB), and alpha-hydroxybutyrate dehydrogenase (HBDH) in serum were determined using a Uni Cel DxC Synchron chemistry system (Beckman Coulter, Fulton, CA). LDH, CK, CK-MB, and HBDH assay kits were purchased from Jiancheng Bioengineering Institute (Nanjing, China). The determination was done according to the manufacturer's instructions.
Analysis of oxidative stress in the heart
Superoxide dismutase (SOD) activity, and glutathione (GSH) and malondialdehyde (MDA) content in the heart were determined according to the manufacturer's instructions. SOD activity, GSH and MDA assay kits were purchased from Jiancheng Bioengineering Institute (Nanjing, China).
Histopathology
Heart tissues from the rats were fixed in 4% paraformaldehyde overnight at 4 °C and then cut into blocks of 2 mm thickness. After being embedded in paraffin, sections (5 μm thickness) were cut on the coronal plane and stained with hematoxylin and eosin. Morphology was examined using a light microscope (BX-FM: Olympus Corp., Tokyo, Japan).
TUNEL staining assay
The TUNEL assay kit (Beyotime Biotechnology, Shanghai, China) was used to assess apoptosis in the hearts. Sections of heart tissues were placed in 50 μM TUNEL detection solution, then washed twice with PBS and incubated for 60 min at 37 °C in the dark. The sections were observed under a fluorescence microscope at an excitation wavelength range of 450–500 nm and an emission wavelength range of 515–565 nm.
Protein expression
Radioimmunoprecipitation assay (RIPA) buffer (Beyotime Biotechnology) was used to extract total protein in the hearts. Nuclear protein was extracted using a nuclear protein extraction kit (Beyotime Biotechnology). Protein expression was analyzed by western blot analysis. Equal aliquots of the protein samples (25–30 μg) were separated by SDS-PAGE gel electrophoresis and electrophoretically transferred to a polyvinylidene fluoride membrane, and then the membranes were probed with an appropriate combination of primary and horseradish peroxidase-conjugated secondary antibodies. The detailed information of primary antibodies is shown in Table 1. Proteins in the membranes were visualized using enhanced chemiluminescence kits. The protein bands were quantified by the average ratios of integral optic density following normalization to the expression of internal control GAPDH.
Detailed information of primary antibodies
| Protein | Company | Catalogue number |
|---|---|---|
| p53 | Cell Signaling Technology Inc. | #2524 |
| Bax | Cell Signaling Technology Inc. | #2772 |
| Bcl-2 | Santa Cruz Biotechnology | sc-492 |
| Bcl-xL | Cell Signaling Technology Inc. | #2764 |
| Cleaved caspase3 | Cell Signaling Technology Inc. | #9661 |
| NF-κB | Cell Signaling Technology Inc. | #8242 |
| IL-1β | Abcam | ab205924 |
| Nrf2 | Abcam | ab31163 |
| HO-1 | Cell Signaling Technology Inc. | #43966 |
| NQO1 | Abcam | ab2346 |
| Sesn2 | Proteintech Group Inc. | 10795-1-AP |
| p-AMPK | Cell Signaling Technology Inc. | #8208 |
| AMPK | Cell Signaling Technology Inc. | #8208 |
| GAPDH | Cell Signaling Technology Inc. | #5174 |
| LaminB | Cell Signaling Technology Inc. | #13435 |
| Protein | Company | Catalogue number |
|---|---|---|
| p53 | Cell Signaling Technology Inc. | #2524 |
| Bax | Cell Signaling Technology Inc. | #2772 |
| Bcl-2 | Santa Cruz Biotechnology | sc-492 |
| Bcl-xL | Cell Signaling Technology Inc. | #2764 |
| Cleaved caspase3 | Cell Signaling Technology Inc. | #9661 |
| NF-κB | Cell Signaling Technology Inc. | #8242 |
| IL-1β | Abcam | ab205924 |
| Nrf2 | Abcam | ab31163 |
| HO-1 | Cell Signaling Technology Inc. | #43966 |
| NQO1 | Abcam | ab2346 |
| Sesn2 | Proteintech Group Inc. | 10795-1-AP |
| p-AMPK | Cell Signaling Technology Inc. | #8208 |
| AMPK | Cell Signaling Technology Inc. | #8208 |
| GAPDH | Cell Signaling Technology Inc. | #5174 |
| LaminB | Cell Signaling Technology Inc. | #13435 |
Detailed information of primary antibodies
| Protein | Company | Catalogue number |
|---|---|---|
| p53 | Cell Signaling Technology Inc. | #2524 |
| Bax | Cell Signaling Technology Inc. | #2772 |
| Bcl-2 | Santa Cruz Biotechnology | sc-492 |
| Bcl-xL | Cell Signaling Technology Inc. | #2764 |
| Cleaved caspase3 | Cell Signaling Technology Inc. | #9661 |
| NF-κB | Cell Signaling Technology Inc. | #8242 |
| IL-1β | Abcam | ab205924 |
| Nrf2 | Abcam | ab31163 |
| HO-1 | Cell Signaling Technology Inc. | #43966 |
| NQO1 | Abcam | ab2346 |
| Sesn2 | Proteintech Group Inc. | 10795-1-AP |
| p-AMPK | Cell Signaling Technology Inc. | #8208 |
| AMPK | Cell Signaling Technology Inc. | #8208 |
| GAPDH | Cell Signaling Technology Inc. | #5174 |
| LaminB | Cell Signaling Technology Inc. | #13435 |
| Protein | Company | Catalogue number |
|---|---|---|
| p53 | Cell Signaling Technology Inc. | #2524 |
| Bax | Cell Signaling Technology Inc. | #2772 |
| Bcl-2 | Santa Cruz Biotechnology | sc-492 |
| Bcl-xL | Cell Signaling Technology Inc. | #2764 |
| Cleaved caspase3 | Cell Signaling Technology Inc. | #9661 |
| NF-κB | Cell Signaling Technology Inc. | #8242 |
| IL-1β | Abcam | ab205924 |
| Nrf2 | Abcam | ab31163 |
| HO-1 | Cell Signaling Technology Inc. | #43966 |
| NQO1 | Abcam | ab2346 |
| Sesn2 | Proteintech Group Inc. | 10795-1-AP |
| p-AMPK | Cell Signaling Technology Inc. | #8208 |
| AMPK | Cell Signaling Technology Inc. | #8208 |
| GAPDH | Cell Signaling Technology Inc. | #5174 |
| LaminB | Cell Signaling Technology Inc. | #13435 |
Statistical analysis
Data represent the mean ± SEM. Statistical analyses undertaken by SPSS 19.0 software (SPSS, Chicago, IL, USA) were performed by one-way or two-way analysis of variance with Tukey's test for post hoc comparisons and two-way Student's t-test when comparing between two groups. A two-tailed P < 0.05 was considered to be significant.
Results
SFN attenuated Cr(vi)-induced physiological disturbance
From week 3, the food intake of rats in the K2Cr2O7 group was significantly decreased compared to the control group (P < 0.05, Fig. 1A). And the water intake of these rats was significantly decreased consistently (P < 0.05, Fig. 1B). From week 4, the body weight of rats in the K2Cr2O7 group was significantly decreased (P < 0.05, Fig. 1C). However, the food intake, water intake and body weight of rats in the K2Cr2O7 + SFN group were significantly increased compared with those of the K2Cr2O7 group (P < 0.05, Fig. 1). In addition, there were also no significant changes in food intake, water intake, and body weight between the control group and the SFN group.
Effect of SFN on Cr(vi)-induced physiological disturbance. Food intake (A), water intake (B), and body weights (C) were analyzed. Values are mean ± SEM (n = 7). * Significantly different from the corresponding control group, P < 0.05; # significantly different from the corresponding K2Cr2O7 group, P < 0.05.
SFN attenuated Cr(vi)-induced changes in the blood
In the K2Cr2O7 group, RBC count and HGB concentration were significantly reduced, while the WBC count was significantly increased compared with the control group (P < 0.05, Fig. 2). However, SFN significantly reversed the changes induced by K2Cr2O7 in the blood. No significant difference was observed between the control group and the SFN group (P < 0.05, Fig. 2).
Effect of SFN on Cr(vi)-induced changes in the blood. RBC count (A), WBC count (B), and HGB concentration (C) were determined. Values are mean ± SEM (n = 7). * Significantly different from the corresponding control group, P < 0.05; # significantly different from the corresponding K2Cr2O7 group, P < 0.05.
SFN attenuated Cr(vi)-induced heart dysfunction
K2Cr2O7 treatment significantly increased LDH, HBDH, CK, and CK-MB activities in serum compared with the control (P < 0.05, Fig. 3). However, SFN significantly decreased LDH, HBDH, CK, and CK-MB activities increased by K2Cr2O7 (P < 0.05, Fig. 3).
Effect of SFN on Cr(vi)-induced heart dysfunction. LDH (A), HBDH (B), CK (C), and CK-MB (D) activities in the plasma of all samples were determined. Values are mean ± SEM (n = 7). * Significantly different from the corresponding control group, P < 0.05; # significantly different from the corresponding K2Cr2O7 group, P < 0.05.
SFN attenuated Cr(vi)-induced heart injury
In the K2Cr2O7 group, myocardial fiber fracture, myocyte necrosis, myocardial swelling, and diffuse hemorrhage were significantly observed. While in the K2Cr2O7 + SFN group, slight cytosolic dissolution and hemorrhage were noticed. No obvious lesions in the hearts from the control group and SFN group were observed (Fig. 4).
Histopathological variation in cardiac tissues and the protective role of SFN against K2Cr2O7. Paraffin sections of cardiac tissues from the control group (A), K2Cr2O7 group (B), K2Cr2O7 + SFN group (C), and SFN group (D) were stained with hematoxylin and eosin (magnification 200×). Bar = 100 μm. Arrowhead: atrophy of cardiac muscle fibers; thin arrow: cytosolic dissolution; thick arrow: coagulation necrosis; star: hemorrhage.
SFN attenuated Cr(vi)-induced apoptosis
As shown by TUNEL, the level of apoptotic heart cells was significantly raised compared with the control (P < 0.05). However, the stimulatory effect on apoptosis by K2Cr2O7 was attenuated in the K2Cr2O7 + SFN-treated group, indicating that SFN significantly attenuated K2Cr2O7-induced apoptosis (P < 0.05, Fig. 5A and B).
Effect of SFN on Cr(vi)-induced apoptosis and cytotoxicity. Cardiocyte apoptosis was determined using the TUNEL assay. Representative images of tissues from rats in the 4 groups (A) are shown, and the ratio of apoptosis cells (B) was used with the images of 10 independent experiments (mean ± SEM, n = 4). * Significantly different from the corresponding control group, P < 0.05; # significantly different from the corresponding K2Cr2O7 group, P < 0.05.
SFN attenuated Cr(vi)-induced oxidative stress in the heart
In the rat heart, a significant increase of MDA concentration in the K2Cr2O7-treated group was observed compared to the control group (P < 0.05, Fig. 6A). In addition, K2Cr2O7 significantly decreased the GSH concentration and SOD activity in the heart (P < 0.05, Fig. 6B and C), whereas cotreatment with SFN significantly reversed all these changes induced by K2Cr2O7, and further inhibited oxidative stress-induced by K2Cr2O7 (P < 0.05, Fig. 6).
Effect of SFN on Cr(vi)-induced oxidative stress. MDA content (A), GSH content (B), and SOD activity (C) in rat hearts were determined (mean ± SEM, n = 7). * Significantly different from the corresponding control group, P < 0.05; # significantly different from the corresponding K2Cr2O7 group, P < 0.05.
Effect of SFN and Cr(vi) on the apoptosis-related pathway in the heart
Additionally, protein levels of apoptosis-related proteins in the heart, including B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra large (Bcl-xL), Bax, cleaved caspase3, and p53 were detected by the western blot assay. K2Cr2O7 significantly increased Bax, cleaved caspase3, and p53 protein levels, and decreased Bcl-2 and Bcl-xL protein levels compared to the control group. However, SFN significantly attenuated these alterations induced by K2Cr2O7 (P < 0.05, Fig. 7A and B), suggesting that SFN attenuated apoptosis induced by K2Cr2O7via regulating an apoptosis-related pathway.
Effect of SFN and Cr(vi) on protein levels in rat hearts. Protein levels in the hearts were determined by western blot analysis. Anti-GAPDH and anti-LaminB antibodies were used as a loading control. Blots in images were run under the same conditions. Representative images shown in (A), (C), (E), quantitative data (B), (D), (F), and phosphorylated rate (G) were performed with images of 4 independent experiments (mean ± SEM, n = 4). * Significantly different from the corresponding control group, P < 0.05; # significantly different from the corresponding K2Cr2O7 group, P < 0.05.
Effect of SFN and Cr(vi) on protein levels of inflammatory factors in the heart
K2Cr2O7 administration significantly raised protein levels of nuclear NF-κB and interleukin 1β (IL-1β) in the heart (P < 0.05), suggesting the occurrence of an inflammatory response. In contrast, SFN significantly attenuated the aggravation of K2Cr2O7 on the inflammatory response (P < 0.05, Fig. 7C and D).
Effect of SFN and Cr(vi) on the Nrf2 signaling pathway in the heart
The Nrf2 signaling pathway is reported to play an important role in resisting oxidative stress. Western blot analysis confirmed that K2Cr2O7 significantly suppressed the Nrf2 protein level in the nucleus and p-AMPK rate compared with the control group (P < 0.05), while SFN significantly alleviated these effects of K2Cr2O7 (P < 0.05). In addition, K2Cr2O7 significantly reduced Sesn2, NQO1, and HO-1 protein levels in the heart consistently. However, SFN significantly reversed these alterations induced by K2Cr2O7 (P < 0.05, Fig. 7E–G).
Discussion
Natural products have been of great significance due to their enormous potential in protecting organisms.34–39 In this study, SFN inhibits oxidative stress, inflammation, and apoptosis, further ameliorating heart damage and physiological disorders, ultimately protecting the heart from Cr(vi) poisoning. The results confirm the protection of SFN against Cr(vi) exposure, and provide a new perspective to understand the mechanism of SFN to protect against heart injury in patients exposed to Cr(vi).
Complete blood count is one of the most commonly ordered blood tests in medicine providing an overview of an individual's general health status as well as information on infection, inflammation and inflammatory disease, deficiencies in the immune system, bone marrow disease, and other health-related conditions.40,41 It is reported that Cr(vi) could enter into red blood cells easily, and cause structural disorder, dysmorphosis, and fragmentation of red blood cells.42 In addition, Cr(vi) oxidizes Fe2+ in the lumen of the small intestines and perturbs iron absorption, which may continuously cause iron deficiency and hemoglobin synthesis disorders.43 In this study, SFN efficiently reversed changes induced by Cr(vi) exposure in the blood, including the reduction of RBC count and HGB concentration, and increase of WBC count, exhibiting that SFN efficiently protects an individual's general health status damaged by Cr(vi) exposure. In addition, the ability of SFN to reduce WBC count raised by Cr(vi) hints that the inhibition of inflammation may play an important role in the protection of SFN against Cr(vi).
Apoptosis is known to directly mediate tissue injury, including in the liver, lungs, heart, and other tissues.44–48 The results of TUNEL analysis suggest that SFN attenuates Cr(vi)-induced apoptosis in rat heart. This event is mostly attributed to the maintenance of membrane integrity and apoptosis. It has been indicated that tissue injury caused by Cr(vi) is mainly attributed to oxidative stress.49,50 Our data also confirm serious oxidative stress in the impaired hearts exposed to Cr(vi), and indicate that SFN regulates oxidative stress to attenuate Cr(vi)-induced heart injury. Thus, SFN protects rat hearts against Cr(vi) exposure potentially through oxidative stress and apoptosis.
To further investigate the mechanism of the regulatory effects of SFN induced by Cr(vi), the expression of proteins was analyzed. Oxidative stress stimulates Nrf2 activation, promoting Nrf2 to dissociate from Keap1 and translocate into the nucleus.51,52 However, excessive and long-term oxidative stress causes Nrf2 depletion, disrupting the homeostasis between the expression and degradation of Nrf2.35 In this study, long-term Cr(vi) exposure inhibits the Nrf2 signaling pathway in the heart confirming that Nrf2 signaling pathways are associated with Cr(vi)-induced heart injury. And it has also been shown that the activation of the Nrf2 signaling pathway is involved in the protective role of SFN against Cr(vi) exposure, indicating that SFN attenuates Cr(vi)-induced oxidative stress associated with the Nrf2 signaling pathway to protect the rat heart.
The results of complete blood count show the significance of inflammation induced by Cr(vi) in the protection of SFN. Activation of NF-κB plays a pivotal role in the regulation of inflammatory responses by governing the expression of chemokines and leukocyte adhesion molecules.53 It is shown that SFN attenuated the inflammatory response by inhibiting nuclear NF-κB p65 (the RelA subunit of NF-κB), and NF-κB upstream gene IL-1β which were enhanced by Cr(vi), coinciding with the results of complete blood count. These suggest that SFN attenuates Cr(vi)-induced heart impairment associated with IL-1β/NF-κB-mediated inflammation.
Apoptosis signaling pathways involve p53, the Bcl-2 protein family, and cleaved caspase 3.54,55 The tumor suppressor protein p53 influences apoptosis and can modulate levels of the Bcl-2 protein family, which regulates caspase 3 activation.56 The results indicate that SFN alleviates Cr(vi)-induced apoptosis associated with p53/Bcl-2/caspase3 signaling pathways.
Inflammation and apoptosis triggered by oxidative stress are causes of many, perhaps even most, chronic diseases particularly tissue dysfunction and injury.57–59 Oxidative stress originating from ROS can be identified in most of the key steps in the pathophysiology of atherosclerosis and the consequential clinical manifestations of cardiovascular disease including lipid metabolism, myocardial injury, fibrosis and failure.60,61 Furthermore, current information indicates the complex interplay/crosstalk mechanisms between Nrf2 and NF-κB pathways wherein NF-κB is inhibited by Nrf2 activation and may directly repress Nrf2 signaling.62–64 Moreover, it is clear that Nrf2 could bind with the Bcl-2 gene antioxidant response element to upregulate anti-apoptotic protein Bcl-2 and prevent cellular apoptosis with implications in antioxidant protection.65 Upregulation of IL-1β protein levels and NF-κB translocation are recognized as the reasons that inflammation causes apoptosis.66–68 Hence, it can be concluded that SFN attenuates Cr(vi)-induced cardiotoxicity depending on the Nrf2-mediated oxidative stress-inflammation-apoptosis axis.
Sesn2, a member of Sestrins which belong to the stress responsive protein family and have been found to be expressed universally in mammals,69 plays an important role in the heart during myocardial I/R injury.70 It has been shown that Sesn2 can suppress reactive oxygen species arising from oxidative stress through its antioxidant function.71 DNA damage, oxidative stress, glucose starvation, and amino acid starvation have been reported to affect Sesn2 expression.72 Previous studies have shown that Sesn2 directly activates AMPK and indirectly activates Nrf2.73,74 These results suggest that Sesn2 and its downstream proteins including AMPK and Nrf2 are correlated with the event that SFN protects against Cr(vi)-induced cardiotoxicity. Moreover, these also indicate that SFN may attenuate Cr(vi)-induced cardiotoxicity via Sesn2-mediated AMPK or/and Nrf2 activation. In addition, Nrf2 could be triggered by both Sesn2 and phosphorylation of AMPK.13,74 Thus, all these data confirm that Cr(vi)-induced inflammation and apoptosis are indirectly abolished by Sesn2 regulated by SFN, and AMPK and Nrf2 are the essential proteins in the regulatory effects of Sesn2 on inflammation and apoptosis. Therefore, SFN attenuates Cr(vi)-induced cardiotoxicity attributed to the excessive oxidative stress via activation of the Sesn2/AMPK/Nrf2 signaling pathway (Fig. 8).
The mechanism via which SFN attenuates cardiotoxicity induced by Cr(vi) exposure. SFN ameliorates K2Cr2O7-induced heart injury via activation of the Sesn2/AMPK/Nrf2 signaling pathway.
SFN effectively attenuates chronic Cr(vi) exposure induced heart injury via alleviating oxidative stress and apoptosis potentially associated with the activation of the Sesn2/AMPK/Nrf2 signaling pathway. Although we report direct correlations between the cardioprotection of SFN against Cr(vi) and Sesn2 activation, further studies on the detailed molecular mechanisms will still be needed.
Conclusion
In conclusion, this study highlights the protection of SFN against chronic Cr(vi) poisoning, and demonstrates that SFN attenuates Cr(vi) induced heart injury, at least in part, through the Sesn2/AMPK/Nrf2 signaling pathway. SFN supplements may offer a novel and safe therapy to attenuate Cr(vi) accumulation and protect human health against Cr(vi) exposure.
Conflicts of interest
There are no conflicts of interest to declare.
Acknowledgements
This work was funded by the National Natural Science Foundation of China (31972754 and 31660726) and the Natural Science Foundation of Inner Mongolia (2018LH03016).
References
