Sulforaphane Pretreatment Research Articles

Bioactivated Glucoraphanin Improves Cell Survival, Upregulating Phospho-AKT, and Modulates Genes Involved in DNA Repair in an In Vitro Alzheimer's Disease Model: A Network-Transcriptomic Analysis.

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases, for which a definitive cure is still missing. Recently, natural compounds have been investigated for their possible neuroprotective role, including the bioactivated product of glucoraphanin (GRA), the sulforaphane (SFN), which is highly rich in cruciferous vegetables. It is known that SFN alleviates neuronal dysfunction, apoptosis, and oxidative stress in the brain. In the light of this evidence, the aim of this study was to investigate the molecular effects of SFN pre-treatment in differentiated SH-SY5Y neurons exposed to β-amyloid (Aβ). To this end, we first evaluated first cell viability via the Thiazolyl Blue Tetrazolium Bromide (MTT) assay, and then we analyzed the transcriptomic profiles by next-generation sequencing (NGS). Finally, we used a network analysis in order to understand which biological processes are affected, validating them by Western blot assay. SFN pre-treatment counteracted Aβ-induced loss of cell viability. The network-transcriptomic analysis revealed that SFN upregulates genes associated with DNA repair, such as ABRAXAS1, BRCA1, BRCA2, CDKN1A, FANCA, FANCD2, FANCE, NBN, and XPC. Finally, SFN also increased the phosphorylation of AKT, which is associated with DNA repair and cell survival. These data suggest that SFN is a natural compound that could be suitable in the prevention of AD, thanks to its neuroprotective role in increasing cell survival, potentially restoring DNA damage induced by Aβ exposure.

Sulforaphane substantially impedes testicular ferroptosis in adult rats exposed to di-2-ethylhexyl phthalate through activation of NRF-2/SLC7A11/GPX-4 trajectory.

Di-2-ethylhexyl phthalate (DEHP) is a common plasticizer with a deleterious impact on testicular functionality and male fertility. Growing evidence implicates ferroptosis as one of the plausible mechanisms for DEHP-induced testicular injury. Sulforaphane (SFN) is a natural isothiocyanate displaying beneficial effects on testicular injury in several animal models. Herein, we explored the potential protective effect of SFN on testicular ferroptosis and toxicity evoked by DEHP. Adult male Wistar rats were equally distributed into three groups (n = 6/group): (i) CON group; (ii) DEHP group, received DEHP (2g/kg PO) for 4weeks; and (iii) DEHP + SFN group, received SFN (10mg/kg, PO) 1week prior to DEHP then concurrently with DEHP for further 4weeks. Compared to CON group, exposure to DEHP caused testicular atrophy, deteriorated testicular architecture, testicular fibrosis, reduced sperm count and motility, higher sperm deformity, and declined serum testosterone level. All these abnormalities were ameliorated by SFN preconditioning. Additionally, pretreatment with SFN reversed the increased aromatase level and upregulated the steroidogenic markers in testes of DEHP-exposed rats. SFN pretreatment also counteracted DEHP-induced oxidative stress and boosted the total antioxidant capacity in testicular tissue via activation of the nuclear factor erythroid 2-related factor 2 (NRF-2) and its downstream target, hemeoxygenase-1 (HO-1). Moreover, SFN preconditioning mitigated DEHP-induced ferroptosis through up-surging SLC7A11, GPX-4, and GSH, while suppressing iron overload and ACSL4-induced lipid peroxidation in testicular tissue of rats. These findings may nominate SFN as a promising protective intervention to alleviate testicular ferroptosis associated with DEHP exposure through activation of NRF-2/SLC7A11/GPX-4 trajectory.

Glucose metabolite methylglyoxal induces vascular endothelial cell pyroptosis via NLRP3 inflammasome activation and oxidative stress in vitro and in vivo

Methylglyoxal (MGO), a reactive dicarbonyl metabolite of glucose, plays a prominent role in the pathogenesis of diabetes and vascular complications. Our previous studies have shown that MGO is associated with increased oxidative stress, inflammatory responses and apoptotic cell death in endothelial cells (ECs). Pyroptosis is a novel form of inflammatory caspase-1-dependent programmed cell death that is closely associated with the activation of the NOD-like receptor 3 (NLRP3) inflammasome. Recent studies have shown that sulforaphane (SFN) can inhibit pyroptosis, but the effects and underlying mechanisms by which SFN affects MGO-induced pyroptosis in endothelial cells have not been determined. Here, we found that SFN prevented MGO-induced pyroptosis by suppressing oxidative stress and inflammation in vitro and in vivo. Our results revealed that SFN dose-dependently prevented MGO-induced HUVEC pyroptosis, inhibited pyroptosis-associated biochemical changes, and attenuated MGO-induced morphological alterations in mitochondria. SFN pretreatment significantly suppressed MGO-induced ROS production and the inflammatory response by inhibiting the NLRP3 inflammasome (NLRP3, ASC, and caspase-1) signaling pathway by activating Nrf2/HO-1 signaling. Similar results were obtained in vivo, and we demonstrated that SFN prevented MGO-induced oxidative damage, inflammation and pyroptosis by reversing the MGO-induced downregulation of the NLRP3 signaling pathway through the upregulation of Nrf2. Additionally, an Nrf2 inhibitor (ML385) noticeably attenuated the protective effects of SFN on MGO-induced pyroptosis and ROS generation by inhibiting the Nrf2/HO-1 signaling pathway, and a ROS scavenger (NAC) and a permeability transition pore inhibitor (CsA) completely reversed these effects. Moreover, NLRP3 inhibitor (MCC950) and caspase-1 inhibitor (VX765) further reduced pyroptosis in endothelial cells that were pretreated with SFN. Collectively, these findings broaden our understanding of the mechanism by which SFN inhibits pyroptosis induced by MGO and suggests important implications for the potential use of SFN in the treatment of vascular diseases.

Sulforaphane-mediated immune regulation through inhibition of NF-kB and MAPK signaling pathways in human dendritic cells

Inflammation and immune responses are intricately intertwined processes crucial for maintaining homeostasis and combating against pathogens. These processes involve complex signaling pathways, notably the Nuclear Factor kappa-light-chain-enhancer of activated B-cells (NF-κB) and Mitogen-Activated Protein Kinase (MAPK) pathways, which play crucial roles. Sulforaphane (SFN), a nutraceutic, has emerged as a potential regulator of NF-κB and MAPK signaling pathways, exhibiting anti-inflammatory properties. However, limited knowledge exists regarding SFN’s effects on immune cell modulation. This study aimed to assess the immunomodulatory capacity of SFN pretreatment in human dendritic cells (DCs), followed by exposure to a chronic inflammatory environment induced by lipopolysaccharide. SFN pretreatment was found to inhibit the NF-κB and MAPK signaling pathways, resulting in phenotypic changes in DCs characterized by a slight reduction in the expression of surface markers, as well as a decrease of TNF-α/IL-10 ratio. Additionally, SFN pretreatment enhanced the proliferation of Treg-cells and promoted the production of IL-10 by B-cells before exposure to the chronic inflammatory environment. Furthermore, these changes in DCs were found to be influenced by the inhibition of NF-κB and MAPK pathways (specifically p38 MAPK and JNK), suggesting that these pathways may play a role in the regulation of the differentiation of adaptive immune responses (proliferation of T- and IL-10-producing regulatory-cells), prior to SFN pretreatment. Our findings suggest that SFN pretreatment may induce a regulatory response by inhibiting NF-κB and MAPK signaling pathways in an inflammatory environment. SFN could be considered a promising strategy for utilizing functional foods to protect against inflammation and develop immunoregulatory interventions.

Possible Prophylactic Effects of Sulforaphane on LPS-Induced Recognition Memory Impairment Mediated by Regulating Oxidative Stress and Neuroinflammatory Proteins in the Prefrontal Cortex Region of the Brain.

Alzheimer's disease (AD) presents a significant global health concern, characterized by neurodegeneration and cognitive decline. Neuroinflammation is a crucial factor in AD development and progression, yet effective pharmacotherapy remains elusive. Sulforaphane (SFN), derived from cruciferous vegetables and mainly from broccoli, has shown a promising effect via in vitro and in vivo studies as a potential treatment for AD. This study aims to investigate the possible prophylactic mechanisms of SFN against prefrontal cortex (PFC)-related recognition memory impairment induced by lipopolysaccharide (LPS) administration. Thirty-six Swiss (SWR/J) mice weighing 18-25 g were divided into three groups (n = 12 per group): a control group (vehicle), an LPS group (0.75 mg/kg of LPS), and an LPS + SFN group (25 mg/kg of SFN). The total duration of the study was 3 weeks, during which mice underwent treatments for the initial 2 weeks, with daily monitoring of body weight and temperature. Behavioral assessments via novel object recognition (NOR) and temporal order recognition (TOR) tasks were conducted in the final week of the study. Inflammatory markers (IL-6 and TNF), antioxidant enzymes (SOD, GSH, and CAT), and pro-oxidant (MDA) level, in addition to acetylcholine esterase (AChE) activity and active (caspase-3) and phosphorylated (AMPK) levels, were evaluated. Further, PFC neuronal degeneration, Aβ content, and microglial activation were also examined using H&E, Congo red staining, and Iba1 immunohistochemistry, respectively. SFN pretreatment significantly improved recognition memory performance during the NOR and TOR tests. Moreover, SFN was protected from neuroinflammation and oxidative stress as well as neurodegeneration, Aβ accumulation, and microglial hyperactivity. The obtained results suggested that SFN has a potential protective property to mitigate the behavioral and biochemical impairments induced by chronic LPS administration and suggested to be via an AMPK/caspase-3-dependent manner.

Facile preparation of biomimetic mineralized COFs based on magnetic silk fibroin and its effective extraction of sulforaphane from cruciferous vegetables

A novel biomimetic mineralized covalent organic framework (BM-COF) was prepared based on magnetic silk fibroin and a new sulforaphane pretreatment technology was constructed. First, metal coordination was performed on the surface of silk fibroin, and nanoparticles were deposited by in-situ mineralization after co-precipitation. Then, biomineralized COFs were prepared by in-situ self-assembly of a COF layer on Fe3O4@silk fibroin surface guided by interfacial directional growth technology. The BM-COFs had a multilayer structure, large specific surface area and pore volume, and superparamagnetic properties, which make them an ideal adsorbent. The adsorption of sulforaphane by BM-COFs is mainly multi-molecular layer adsorption and chemisorption, there might be electrostatic action, π-stacking and hydrogen bonding in the adsorption process. The composite material was successfully used for the pretreatment of sulforaphane in cruciferous vegetables. An extraction time of 30 min gave extraction efficiencies as high as 92%, and the recovery could reach more than 73%.

Sulforaphane prevents LPS-induced inflammation by regulating the Nrf2-mediated autophagy pathway in goat mammary epithelial cells and a mouse model of mastitis

BackgroundMastitis not only deteriorates the composition or quality of milk, but also damages the health and productivity of dairy goats. Sulforaphane (SFN) is a phytochemical isothiocyanate compound with various pharmacological effects such as anti-oxidant and anti-inflammatory. However, the effect of SFN on mastitis has yet to be elucidated. This study aimed to explore the anti-oxidant and anti-inflammatory effects and potential molecular mechanisms of SFN in lipopolysaccharide (LPS)-induced primary goat mammary epithelial cells (GMECs) and a mouse model of mastitis.ResultsIn vitro, SFN downregulated the mRNA expression of inflammatory factors (tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6), inhibited the protein expression of inflammatory mediators (cyclooxygenase-2 (COX2), and inducible nitric oxide synthase (iNOS)) while suppressing nuclear factor kappa-B (NF-κB) activation in LPS-induced GMECs. Additionally, SFN exhibited an antioxidant effect by increasing Nrf2 expression and nuclear translocation, up-regulating antioxidant enzymes expression, and decreasing LPS-induced reactive oxygen species (ROS) production in GMECs. Furthermore, SFN pretreatment promoted the autophagy pathway, which was dependent on the increased Nrf2 level, and contributed significantly to the improved LPS-induced oxidative stress and inflammatory response. In vivo, SFN effectively alleviated histopathological lesions, suppressed the expression of inflammatory factors, enhanced immunohistochemistry staining of Nrf2, and amplified of LC3 puncta LPS-induced mastitis in mice. Mechanically, the in vitro and in vivo study showed that the anti-inflammatory and anti-oxidative stress effects of SFN were mediated by the Nrf2-mediated autophagy pathway in GMECs and a mouse model of mastitis.ConclusionsThese results indicate that the natural compound SFN has a preventive effect on LPS-induced inflammation through by regulating the Nrf2-mediated autophagy pathway in primary goat mammary epithelial cells and a mouse model of mastitis, which may improve prevention strategies for mastitis in dairy goats.

The effect of sulforaphane on perinatal hypoxic-ischemic brain injury in rats.

Perinatal hypoxic-ischemic insult (HII) is one of the main devastating causes of morbidity and mortality in newborns. HII induces brain injury which evolves to neurological sequelae later in life. Hypothermia is the only therapeutic approach available capable of diminishing brain impairment after HII. Finding a novel therapeutic method to reduce the severity of brain injury and its consequences is critical in neonatology. The present paper aimed to evaluate the effect of sulforaphane (SFN) pre-treatment on glucose metabolism, neurodegeneration, and functional outcome at the acute, sub-acute, and sub-chronic time intervals in the experimental model of perinatal hypoxic-ischemic insult in rats. To estimate the effect of SFN on brain glucose uptake we have performed 18F-deoxyglucose (FDG) microCT/PET. The activity of FDG was determined in the hippocampus and sensorimotor cortex. Neurodegeneration was assessed by histological analysis of Nissl-stained brain sections. To investigate functional outcomes a battery of behavioral tests was employed. We have shown that although SFN possesses a protective effect on glucose uptake in the ischemic hippocampus 24 h and 1 week after HII, no effect has been observed in the motor cortex. We have further shown that the ischemic hippocampal formation tends to be thinner in HIE and SFN treatment tends to reverse this pattern. We have observed subtle chronic movement deficit after HII detected by ladder rung walking test with no protective effect of SFN. SFN should be thus considered as a potent neuroprotective drug with the capability to interfere with pathophysiological processes triggered by perinatal hypoxic-ischemic insult.

To each their own: Disparate abilities of Nrf2 activators to protect against various electrophilic and oxidative insults

Electrophilic and oxidative stress contribute to major diseases such as cancer and diabetes. Small‐molecule activators of the Nrf2 cytoprotective pathway show significant promise for prevention and amelioration of chronic diseases by combating these stressors through upregulation of detoxification and antioxidant enzymes. An understudied area is whether distinct small molecular Nrf2 activators can protect cells from all electrophilic and oxidative stressors, or whether certain molecules are better suited to protect against a particular type of stress.In this study, human keratinocyte HaCaT cells were preincubated for 24 h with one of four Nrf2 activators before different stressors were added for an additional 24 h and cell death was assessed. Three electrophiles were tested: sulforaphane (SFN), the subject of over 70 clinical trials, the multiple sclerosis drug Tecfidera (dimethyl fumarate, DMF), and bardoxolone methyl (BARD), in clinical trials for treatment of chronic kidney diseases and other conditions including COVID‐19. SFN, DMF, and BARD target C151 of the Keap1 protein, a negative regulator of Nrf2. Also tested was KI696, an inhibitor of the Keap1‐Nrf2 interaction. Three chemically distinct stressors—hydrogen peroxide, menadione (a quinone that rapidly generates ROS through one‐electron redox cycling), or chlorambucil (an alkylating chemotherapy agent)—were tested. Stressors were added to the cells at their LC50 concentration after pretreatment with a range of cytoprotective compound concentrations. Unexpectedly, SFN pretreatment showed little to no rescue for all stressors. However, DMF rescued the viability of cells stressed with hydrogen peroxide, menadione, and chlorambucil. BARD showed no rescue for hydrogen peroxide‐treated cells. KI696 improved the cell’s ability to survive chlorambucil. The ability of a given Nrf2 activator (or lack thereof) to protect against a given stressor points to a need to better understand the cytoprotective environment (specific enzymes upregulated, glutathione levels, etc.) that best protect against specific stressors.

Inhibiting nuclear factor erythroid 2 related factor 2-mediated autophagy in bovine mammary epithelial cells induces oxidative stress in response to exogenous fatty acids

BackgroundIn early lactation, bovine mammary epithelial cells undergo serious metabolic challenges and oxidative stress both of which could be alleviated by activation of autophagy. Nuclear factor erythroid 2 related factor 2 (NFE2L2), a master regulator of cellular redox homeostasis, plays an important role in the regulation of autophagy and oxidative stress. Thus, the objective of this study was to investigate the role of NFE2L2-mediated autophagy on oxidative stress of bovine mammary epithelial cells in response to exogenous free fatty acids (FFA).ResultsExogenous FFA induced linear and quadratic decreases in activities of glutathione peroxidase (GSH-Px), catalase (CAT), and superoxide dismutase (SOD), and increases in the contents of reactive oxygen species (ROS) and malondialdehyde (MDA). Protein abundance of LC3-phosphatidylethanolamine conjugate (LC3-II) and the number of autophagosomes and autolysosomes decreased in a dose-dependent manner, while protein abundance of p62 increased in cells challenged with FFA. Activation of autophagy via pre-treatment with Rap attenuated the FFA-induced ROS accumulation. Importantly, FFA inhibited protein abundance of NFE2L2 and the translocation of NFE2L2 into the nucleus. Knockdown of NFE2L2 by siRNA decreased protein abundance of LC3-II, while it increased protein abundance of p62. Furthermore, sulforaphane (SFN) pre-treatment attenuated the FFA-induced oxidative stress by activating NFE2L2-mediated autophagy.ConclusionsThe data suggested that NFE2L2-mediated autophagy is an important antioxidant mechanism in bovine mammary epithelial cells experiencing increased FFA loads.

Nuclear factor erythroid 2-related factor 2 protects bovine mammary epithelial cells against free fatty acid-induced mitochondrial dysfunction in vitro

Bovine mammary epithelial cells undergo an increase in metabolic rate, mitochondrial dysfunction, and oxidative stress after calving. Nuclear factor erythroid 2-related factor 2 (NFE2L2), a master regulator of cellular redox homeostasis, plays crucial roles in the regulation of mitochondrial function. The objective of this study was to investigate the role of NFE2L2 on mitochondrial function in bovine mammary epithelial cells under hyperlipidemic conditions. Three experiments were conducted as follows: (1) the immortalized bovine mammary epithelial cell line MAC-T was treated with various concentrations of free fatty acids (FFA; 0, 0.6, 1.2, or 2.4 mM) for 24 h to induce stress; (2) MAC-T cells were transfected with small interfering RNA targeting NFE2L2 (si-NFE2L2) and scrambled nontarget negative control (si-Control) for 48 h; and (3) MAC-T cells were pretreated with 10 μM sulforaphane (SFN), an activator of NFE2L2, for 24 h followed by treatment with 1.2 mM FFA for an additional 24 h. Results indicated that exogenous FFA challenge induced linear and quadratic increases in concentrations of mitochondrial reactive oxygen species (ROS). Compared with 0 mM FFA, mitochondrial membrane potential, mRNA abundance of oxidative phosphorylation complexes (CO I-V), protein abundance of nuclear respiratory factor 1 (NRF1), peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α), mitochondrial transcription factor A (TFAM), and NFE2L2 along with the contents of ATP, mitochondrial DNA (mtDNA), and total mitochondria were greater in the MAC-T challenged with 0.6 mM FFA group, but lower in the 1.2 and 2.4 mM FFA cultures. Knockdown of NFE2L2 via small interfering RNA led to greater mitochondrial ROS content and lower mitochondrial membrane potential along with contents of ATP, mtDNA, and total mitochondria. The SFN pretreatment upregulated protein abundance of NFE2L2 and attenuated the downregulation of NFE2L2 induced by FFA. Pretreatment with SFN attenuated the downregulation induced by FFA of PGC-1α, NRF1, and TFAM protein abundance along with contents of mtDNA and total mitochondria. Furthermore, SFN pretreatment attenuated the upregulation of mitochondrial ROS content, the downregulation of mitochondrial membrane potential, and the decreases in ATP, mtDNA, and mitochondrial content induced by FFA. Overall, data indicated that FFA inhibit NFE2L2, resulting in mitochondrial dysfunction and ROS production in bovine mammary epithelial cells. Thus, NFE2L2 may be a promising therapeutic target against metabolic challenge-driven mitochondrial dysfunction and oxidative stress in bovine mammary epithelial cells.

Epigenetic Modulation of TLR4 Expression by Sulforaphane Increases Anti-Inflammatory Capacity in Porcine Monocyte-Derived Dendritic Cells.

Simple SummaryEpigenetic modifications of the genes regulate the inflammation process that includes the DNA methylation and histone acetylation. Sulforaphane is well known for its immunomodulatory properties. Notably, the mechanism of its anti-inflammatory functions involving epigenetic modifications is unclear. This study highlighted the regulatory mechanism of sulforaphane in the innate immunity responses in an acute inflammatory state employ in vivo cell culture model. Porcine monocyte-derived dendritic cells were exposed to LPS with or without sulforaphane pre-treatment for these purposes. Epigenetics modulations of the important genes and regulatory factors were studies as well as the immune responses of the cells were vigorously studied over the period of time. This study deciphers the mechanism of SFN in restricting the excessive inflammatory reactions, thereby, exerting its protective and anti-inflammatory function though epigenetic mechanism.Inflammation is regulated by epigenetic modifications, including DNA methylation and histone acetylation. Sulforaphane (SFN), a histone deacetylase (HDAC) inhibitor, is also a potent immunomodulatory agent, but its anti-inflammatory functions through epigenetic modifications remain unclear. Therefore, this study aimed to investigate the epigenetic effects of SFN in maintaining the immunomodulatory homeostasis of innate immunity during acute inflammation. For this purpose, SFN-induced epigenetic changes and expression levels of immune-related genes in response to lipopolysaccharide (LPS) stimulation of monocyte-derived dendritic cells (moDCs) were analyzed. These results demonstrated that SFN inhibited HDAC activity and caused histone H3 and H4 acetylation. SFN treatment also induced DNA demethylation in the promoter region of the MHC-SLA1 gene, resulting in the upregulation of Toll-like receptor 4 (TLR4), MHC-SLA1, and inflammatory cytokines’ expression at 6 h of LPS stimulation. Moreover, the protein levels of cytokines in the cell culture supernatants were significantly inhibited by SFN pre-treatment followed by LPS stimulation in a time-dependent manner, suggesting that inhibition of HDAC activity and DNA methylation by SFN may restrict the excessive inflammatory cytokine availability in the extracellular environment. We postulate that SFN may exert a protective and anti-inflammatory function by epigenetically influencing signaling pathways in experimental conditions employing porcine moDCs.

Sulforaphane exposure impairs contractility and mitochondrial function in three-dimensional engineered heart tissue

Sulforaphane (SFN) is a phytochemical compound extracted from cruciferous plants, like broccoli or cauliflower. Its isothiocyanate group renders SFN reactive, thus allowing post-translational modification of cellular proteins to regulate their function with the potential for biological and therapeutic actions. SFN and stabilized variants recently received regulatory approval for clinical studies in humans for the treatment of neurological disorders and cancer. Potential unwanted side effects of SFN on heart function have not been investigated yet. The present study characterizes the impact of SFN on cardiomyocyte contractile function in cardiac preparations from neonatal rat, adult mouse and human induced-pluripotent stem cell-derived cardiomyocytes. This revealed a SFN-mediated negative inotropic effect, when administered either acutely or chronically, with an impairment of the Frank-Starling response to stretch activation. A direct effect of SFN on myofilament function was excluded in chemically permeabilized mouse trabeculae. However, SFN pretreatment increased lactate formation and enhanced the mitochondrial production of reactive oxygen species accompanied by a significant reduction in the mitochondrial membrane potential. Transmission electron microscopy revealed disturbed sarcomeric organization and inflated mitochondria with whorled membrane shape in response to SFN exposure. Interestingly, administration of the alternative energy source l-glutamine to the medium that bypasses the uptake route of pyruvate into the mitochondrial tricarboxylic acid cycle improved force development in SFN-treated EHTs, suggesting indeed mitochondrial dysfunction as a contributor of SFN-mediated contractile dysfunction. Taken together, the data from the present study suggest that SFN might impact negatively on cardiac contractility in patients with cardiovascular co-morbidities undergoing SFN supplementation therapy. Therefore, cardiac function should be monitored regularly to avoid the onset of cardiotoxic side effects.

Sulforaphane protects human umbilical vein endothelial cells from oxidative stress via the miR-34a/SIRT1 axis by upregulating nuclear factor erythroid-2-related factor 2.

Oxidative stress-induced vascular endothelial cell dysfunction serves an essential role in the initiation and development of atherosclerosis. Sulforaphane (SFN), a naturally occurring antioxidant, has previously demonstrated to exert protective effects on the endothelium against oxidative stress. However, further studies are required to determine its underlying molecular mechanism prior to clinical application. Accumulating evidence suggests that alterations in the microRNA (miRNA/miR)-34a/sirtuin-1 (SIRT1) axis occur with oxidative stress. Therefore, the present study aimed to investigate if SFN exerts a protective role against oxidative stress in vascular endothelial cells through regulation of the miR-34a/SIRT1 axis. Human umbilical vein endothelial cells (HUVECs) were treated with H2O2 in the presence or absence of SFN pretreatment. Cell viability and apoptosis were analyzed using CellTiter-Blue and flow cytometry, respectively. Reverse transcription-quantitative PCR and western blot analyses were performed to determine changes in the expression levels of miR-34a and SIRT1. The expression levels of miR-34a and SIRT1 were artificially regulated following transfection with miR-34a mimic and inhibitor or SIRT1expression plasmid and small interfering RNA, respectively. Subsequently, the effect of the expression changes of miR-34 and SIRT1 on oxidative stress-induced cell injury was investigated. Dual-luciferase reporter assay was used to confirm the targeted binding of miR-34a to SIRT1. SFN was found to ameliorate cellular damage caused by H2O2 and inhibited intracellular reactive oxygen species production. In addition, miR-34a upregulation was accompanied with reduced SIRT1 expression in HUVECs, following H2O2 treatment. miR-34a was revealed to directly target SIRT1 by binding to its 3'-untranslated region. Down-regulation of miR-34a and up-regulation of SIRT1 increased the survival of HUVECs under oxidative stress. Taken together, the results of the present study suggest that SFN may protect HUVECs from oxidative stress by inducing changes in the miR-34a/SIRT1 axis via upregulation of nuclear factor erythroid-2-related factor 2 expression.

Sulforaphane ameliorates cadmium induced hepatotoxicity through the up-regulation of /Nrf2/ARE pathway and the inactivation of NF-κB

This study aimed to assess the ameliorative effect and mechanisms of sulforaphane (SFN) on cadmium (Cd)-induced hepatotoxicity. Four in vitro free radical scavenging tests, HepG2 cell viability analysis, and cadmium chloride (CdCl2) indeced liver injured mice mode were employed. SFN scavenged the free radicals in vitro, and mitigated the oxidative damages in HepG2 cells incubated with SFN + CdCl2. In mice models, SFN pretreatment dose-dependently remarkably improved redox homeostasis, and attenuated the expressions of the key liver inflammatory factors (TNF-α, IL-6, IL-1β) by CdCl2. The pathological examinations were consistent with the above results. Moreover, both mRNA and protein expression of hepatic nuclear factor-erythroid 2-related factor 2 and hemeoxygenase-1 increased, while nuclear factor-kappa B reduced in Cd + SFN co-treated groups. Taken together, this study firstly demonstrated the anti-oxidant and anti-inflammatory effects of SFN against Cd induced liver damages, which associated with the modulation of intrinsic Nrf2/ARE and NF-κB pathway.

Sulforaphane modulates TGFβ2-induced conjunctival fibroblasts activation and fibrosis by inhibiting PI3K/Akt signaling.

To examine the effects of sulforaphane on fibrotic changes of transforming growth factor (TGFβ2) induced human conjunctival fibroblast (HConFs). HConFs were cultured and divided into control, TGFβ2 (1 ng/mL), sulforaphane and TGFβ2+sulforaphane groups. Cell viability and apoptosis were detected using the MTT and ApoTox-Glo Triplex assay. Cell migration was detected using scratch and Transwell assay. Real-time quantitative PCR method was used to evaluate mRNA expression of TGFβ2, matrix metalloproteinase-2 (MMP2), myosin light chain kinase (MYLK), integrin αV, integrin α5, fibronectin 1 and α-smooth muscle actin (α-SMA). The protein expression of α-SMA, p-PI3K, PI3K, p-Akt, and Akt were detected by Western blot. The proliferation of HConFs was significantly (P<0.05) suppressed by sulforaphane compared to control cells with the increase of the concentration and treatment time. Cell proliferation after 48h incubation was significantly reduced with 100 µmol/L sulforaphane treatment by 17.53% (P<0.05). The Transwell assay showed sulforaphane decreased cell migration by 18.73% compared with TGFβ2-induced HConF (P<0.05). TGFβ2-induced the increasing expression of fibronectin, type I collagen and α-SMA, and the phosphorylation of PI3K and Akt were all significantly suppressed by sulforaphane pretreatment. Sulforaphane inhibits proliferation, migration, and synthesis of the extracellular matrix in HConFs, and inhibiting the PI3K/Akt signaling pathway. Sulforaphane could be a potential therapeutic drug for prevention of scar formation in filtering bleb after trabeculectomy.

Sulforaphane modulates CX3CL1/CX3CR1 axis and inflammation in palmitic acid-induced cell injury in C2C12 skeletal muscle cells.

Studies have shown that sulforaphane (SFN) has potent anti-inflammatory and free radical scavenging effects on obesity and associated disorder such as diabetes, polycystic ovary syndrome, and metabolic syndrome. fractalkine (CX3CL1) and its receptor, CX3CR1, play an important role in muscle metabolism by improving insulin-sensitizing effects. Here, in this study we examined the SFN effect on CX3CL1 and its receptor, CX3CR1, in C2C12 myotubes in palmitic acid (PA)-induced oxidative stress and inflammation. The results showed that PA (750μM) evoked lipotoxicity as a reduction in cell viability, increased IL-6 and TNF-α expression, and enhanced reactive oxygen species (ROS). However, SFN pretreatment attenuated the levels of, IL-6 and TNF-α in C2C12 myotubes exposure to PA. Moreover, SFN pretreatment up-regulated nuclear factor erythroid related factor 2 (Nrf2) /heme oxygenase-1(HO-1) pathway protein in C2C12 cells as indicated by a decrease in ROS levels. Interestingly, PA also caused an increase in CX3CL1 and CX3CR1 expression that SFN abrogated it. We also found the protective effect of SFN agonist PA-induced lipotoxicity with promotes in UCP3 gene expression in C2C12 cells. Collectively, these findings suggest that SFN hampers the PA-induced inflammation in C2C12 cells by modulation of the Nrf2/HO-1 pathway and CX3CL1/CX3CR1 axis and may propose a new therapeutic approach to protect against obesity-associated disorders in skeletal musclecells.

Sulforaphane ameliorates serum starvation-induced muscle atrophy via activation of the Nrf2 pathway in cultured C2C12 cells.

Oxidative stress, an imbalance of redox homeostasis, contributes to the pathogenesis and progress of muscle atrophy. However, it is debated whether oxidative stress is a cause or consequence of muscle atrophy. In this study, we investigated the relationship between menadione-induced oxidative stress and serum starvation-induced muscle atrophy in C2C12 myotubes. We found that atrophic phenotypes including myotube diameter decrease, protein ubiquitination, and the expression of atrogenes were detected under oxidative stress as well as during serum starvation. Oxidative stress during serum starvation was assessed to confirm the correlation. Both intracellular reactive oxygen species (ROS) and protein oxidation were increased in atrophic myotubes. These results indicate that menadione-induced oxidative stress triggers muscle atrophy and vice versa. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key regulator of cellular response to oxidative stress and it is considered to have a cytoprotective role in the mitigation of muscle atrophy. Transcription of heme oxygenase-1 (HO-1) and NAD(P)H quinone dehydrogenase-1, target genes of Nrf2, was decreased during serum starvation, which is related to decreased nuclear translocation of Nrf2. Pre-treatment of sulforaphane (SFN), a known Nrf2 inducer, before serum starvation showed a protective effect via Nrf2/HO-1 upregulation. SFN can liberate Nrf2 from Keap1, enabling the nuclear translocation of Nrf2. Consequently, the expression of HO-1 increased and intracellular ROS was significantly reduced by SFN pre-treatment. These results demonstrate that oxidative stress mediates the pathophysiology of muscle atrophy, which can be improved via upregulation of the Nrf2-mediated antioxidant response.

Nrf-2 activator sulforaphane protects retinal cells from oxidative stress-induced retinal injury

• Retinal ischaemia-reperfusion (IR) injury induces oxidative stress and cell damage. • Sulforaphane activates detoxifying enzymes and suppresses inflammation in cell model. • Sulforaphane preserves retinal function in the rat model of retinal IR injury. • Protective effects originate from upregulation of Nrf2 and downregulation of NF-kB. Retinal ischaemia-reperfusion (IR) injury is implicated in many sight-threatening diseases. The reperfusion of tissue induces oxidative stress and cell damage. Sulforaphane, which is abundant in cruciferous vegetables, exerts anti-oxidative effects. We investigated the protective effect of sulforaphane against oxidative stress or IR injury in retinal cells. We utilized ARPE-19 cells and used tert -butyl hydroperoxide to induce oxidative stress. Cell survival improved after sulforaphane treatment. Sulforaphane attenuated reactive oxygen species production and diminished mitochondrial dysfunction. Furthermore, sulforaphane activated phase II detoxification enzymes and suppressed pro-inflammatory mediators. We transiently raised the intraocular pressure of rats to establish a model of retinal IR injury. High-dose sulforaphane pretreatment ameliorated b-wave decrement in electroretinogram, indicating that sulforaphane could preserve retinal function. Electrophoretic mobility shift assay revealed that these effects originated from upregulation of Nrf2 and downregulation of NF-kB pathway. Our results demonstrated that sulforaphane has a dose-dependent protective effect against oxidative stress and IR injury.

Sulforaphane reduces intracellular survival of Staphylococcus aureus in macrophages through inhibition of JNK and p38 MAPK‑induced inflammation.

Macrophages are active contributors to the innate immune defense system. As macrophage activation is clearly affected by the surrounding microenvironment, the present study investigated the effect of sulforaphane (SFN) on the bactericidal activity of macrophages and the underlying molecular mechanisms involved in this process. Human THP-1-derived macrophages, primary human peripheral blood mononuclear cell-derived macrophages, and primary mouse bone marrow derived-macrophages (BMDMs) pretreated with SFN or DMSO were utilized in a model of Staphylococcus aureus infection. The results suggested that SFN pretreatment of macrophages effectively repressed the intracellular survival of S. aureus through modulation of p38/JNK signaling and decreased S. aureus-induced caspases-3/7-dependent cell apoptosis, potentially through downregulation of microRNA (miR)-142-5p and miR-146a-5p. As SFN is a well-known activator of nuclear factor erythroid 2-related factor 2 (Nrf2), Nrf2−/− BMDMs were used to demonstrate that the SFN-mediated inhibitory effect was independent of Nrf2. Nevertheless, an increase in intracellular bacterial survival in Nrf2-deficient macrophages was observed. In addition, SFN pretreatment suppressed S. aureus-induced transcriptional expression of genes coding for the proinflammatory cytokines interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α), as well as for the M1 markers C-C motif chemokine receptor 7, IL-23 and inducible nitric oxide synthase (iNOS). Western blot analysis indicated that S. aureus challenge activated p38 mitogen-activated protein kinase (MAPK) (p38) and c-Jun N-terminal kinase (JNK) MAPK signaling pathways, while SFN pretreatment prevented p38 and JNK phosphorylation. Pretreatment with 2 specific inhibitors of p38 and JNK, SB203580 and SP600125, respectively, resulted in a decrease in S. aureus-induced proinflammatory gene expression levels compared with those observed in the SFN-pretreated macrophages. Furthermore, THP-1-derived macrophages pretreated with SB203580 or SP600125 prior to bacterial infection exhibited a significant inhibition in intracellular S. aureus survival. In conclusion, we hypothesize that concomitant targeting of the p38/JNK-inflammatory response and the S. aureus-induced apoptosis with SFN may be a promising therapeutic approach in S. aureus infection.