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Oral Submucous Fibrosis is a potentially malignant disorder caused by habitual areca nut chewing, which contributes to the dispersion of active alkaloids into subepithelial tissues, stimulating excessive extracellular matrix deposition. Various treatment modalities are available; however, their efficacy in inhibiting fibrosis progression remains limited. Sulforaphane (SFN), an isothiocyanate found abundantly in cruciferous plants, is known to have effective antifibrotic properties. Objective: The present study investigated the antifibrotic effect of SFN via phosphatidylinositol 3 kinase (PI3K), Serine/Threonine Kinase 1 (AKT-1), mammalian target of rapamycin (mTOR) pathway in arecoline (AER) induced fibrosis in human gingival fibroblasts [HGFs]. Material and Methods: MTT assay determined the half-maximal inhibitory concentration of AER and SFN at 24h in the HGF cell line. Expression levels of transforming growth factor ß1 (TGFß1), collagen type 1 alpha 2 (COL1A2), hydroxyproline (HYP), PI3, AKT, mTOR, and nuclear factor erythroid 2related factor 2 (NRF2) were assessed post-AER and SFN treatment using qPCR and western blot analysis. Results: The findings of the study revealed that AER elicited a stimulatory effect, upregulating TGFß1, COL1A2, HYP, PI3K, AKT, and mTOR and downregulating NRF2 expression. Conversely, SFN treatment significantly upregulated NRF2, inhibiting TGFß1 mediated PI3/AKT/mTOR pathway. Conclusion: These observations suggest that SFN can be used as a promising synergistic antifibrotic agent to combat fibrogenesis via the non-Smad pathway (AU)
Fibrose submucosa oral é uma desordem potencialmente maligna causada pelo habito de mascar a noz da areca, o que contribui para a dispersão de alcalóides ativos nos tecidos subepiteliais, estimulando a deposição excessiva de matriz extracelular. Há várias modalidades terapêuticas, no entanto, com eficácia limitada no controle da progressão da fibrose. O sulforafano (SFN), isotiocianato encontrado abundantemente em plantas crucíferas, é conhecido por suas propriedades antifibróticas. Objetivo: Investigar os efeitos antifibróticos do SFN na via fosfatidilinositol3-quinase (PI3K), via quinase serina/treonina 1 (AKT-1), via do alvo da rapamicina em mamíferos (mTOR), na fibrose induzida por arecolina (AER) em fibroblastos gengivais de humanos (HGFs). Material e Métodos: A meia concentração inibitória mínima de AER e SFN em 24 horas nas células HGFs foi determinada por MTT. Os níveis de expressão de ß1 (TGFß1), colágeno tipo 1 alfa 2 (COL1A2), hidroxiprolina (HYP), PI3K, AKT, mTOR, fator nuclear eritroide 2 relacionado ao fator 2 (NRF2) foram analisados após tratamento com ERA e SFN através de qPCR e western blot. Resultados: O ERA apresentou efeito estimulatório aumentando a expressão de TGFß1, COL1A2, HYP, PI3K, AKT e mTOR e diminuindo a expressão de NRF2. Por outro lado, tratamento com SFN aumentou significativamente a expressão de NRF2, inibindo a liberação de TGFß1 mediada pela via PI3/AKT/mTOR. Conclusão: Esses achados sugerem que o SFN pode ser um agente antifibrótico promissor no combate à fibrogênese decorrente da via não-Smad (AU)
Subject(s)
Oral Submucous Fibrosis , Arecoline , NF-E2-Related Factor 2ABSTRACT
OBJECTIVE@#Acute liver failure (ALF) is characterized by severe liver dysfunction, rapid progression and high mortality and is difficult to treat. Studies have found that sulforaphane (SFN), a nuclear factor E2-related factor 2 (NRF2) agonist, has anti-inflammatory, antioxidant and anticancer effects, and has certain protective effects on neurodegenerative diseases, cancer and liver fibrosis. This paper aimed to explore the protective effect of SFN in ALF and it possible mechanisms of action.@*METHODS@#Lipopolysaccharide and D-galactosamine were used to induce liver injury in vitro and in vivo. NRF2 agonist SFN and histone deacetylase 6 (HDAC6) inhibitor ACY1215 were used to observe the protective effect and possible mechanisms of SFN in ALF, respectively. Cell viability, lactate dehydrogenase (LDH), Fe2+, glutathione (GSH) and malondialdehyde (MDA) were detected. The expression of HDAC6, NRF2, glutathione peroxidase 4 (GPX4), acyl-CoA synthetase long-chain family member 4 (ACSL4) and solute carrier family 7 member 11 (SLC7A11) were detected by Western blotting and immunofluorescence.@*RESULTS@#Our results show that NRF2 was activated by SFN. LDH, Fe2+, MDA and ACSL4 were downregulated, while GSH, GPX4 and SLC7A11 were upregulated by SFN in vitro and in vivo, indicating the inhibitory effect of SFN on ferroptosis. Additionally, HDAC6 expression was decreased in the SFN group, indicating that SFN could downregulate the expression of HDAC6 in ALF. After using the HDAC6 inhibitor, ACY1215, SFN further reduced HDAC6 expression and inhibited ferroptosis, indicating that SFN may inhibit ferroptosis by regulating HDAC6 activity.@*CONCLUSION@#SFN has a protective effect on ALF, and the mechanism may include reduction of ferroptosis through the regulation of HDAC6. Please cite this article as: Zhang YQ, Shi CX, Zhang DM, Zhang LY, Wang LW, Gong ZJ. Sulforaphane, an NRF2 agonist, alleviates ferroptosis in acute liver failure by regulating HDAC6 activity. J Integr Med. 2023; 21(5): 464-473.
Subject(s)
Humans , Ferroptosis , NF-E2-Related Factor 2/genetics , Liver Failure, Acute/drug therapy , Isothiocyanates/pharmacology , Glutathione , Histone Deacetylase 6ABSTRACT
Sulforaphane is a phytochemical with a variety of biological activities that exists widely in Cruciferae plants. This article summarizes the recent experimental studies of sulforaphane in the treatment of various types of liver injury in China and globally and reviews the role and mechanism of sulforaphane in protecting against liver injury. Based on the experimental animal models of liver injury, this article summarizes the therapeutic effect of sulforaphane on the models of chemical liver injury, drug-induced liver injury, alcoholic liver injury, immunological liver injury, and ischemia/reperfusion liver injury and analyzes the mechanism of action of sulforaphane in improving experimental liver injury, so as to provide a reference for in-depth research on sulforaphane in protecting against liver injury.
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Objective To investigate the effect of sulforaphane (SFN) on the proliferation and self-renewal of lung cancer stem cells and its regulatory mechanism. Methods MTT method was used to detect the effect of SFN on the proliferation of lung adenocarcinoma cell lines H460 and A549; tumor sphere formation experiment was used to detect the ability of tumor sphere formation; Western blot was applied to explore the expression of stemness-related proteins (such as β-catenin, Klf4, c-myc) in lung adenocarcinoma cells before and after SFN treatment; NGS sequencing was used to analyze the effect of SFN on the expression profile of tumor cell miRNAs. qRT-PCR verified the changes in the transcription level of key miRNAs by SFN. Western blot was used to detect the effect of SFN on the expression of DNMTs in tumor cells. We constructed miR-200c promoter-GFP plasmid, and applied IF, methylation PCR and DNA sequencing methods to detect the effect of SFN on the methylation level of tumor spheres and miRNA promoter. Results The miRNAs expression profile of lung adenocarcinoma tumor spheres changed significantly after SFN (5.0μmol/L) treatment, and miRNA-200c increased the most. Compared with the control group, the expression of β-catenin, Klf4, c-myc and Vimentin genes in H460 and A549 cells of SFN-S group decreased, and the protein expression levels of DNMT1 and DNMT3a were also significantly decreased. Compared with the control group, H460 and A549 cells stably expressing pEGFP-R200c plasmid in SFN-S group significantly reduced tumor sphere diameter, while tumor sphere fluorescence intensity increased, and GFP protein expression was up-regulated. There were 9 CpG-rich regions in the miR-200c promoter region in the above-mentioned pEGFP-R200c plasmid cell line, and the methylation levels were 88.9%, 44.4% and 38.8% in the control group, SFN-S group and 5-Aza-dC group, respectively. Conclusion SFN may downregulate the expression of stem-related genes in lung cancer stem cells by epigenetically decreasing the methylation level of miR-200c promoter and promoting the transcription of miR-200c.
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OBJECTIVE：To study the effects of sulforaphane on the prolifera tion and apoptosis of human renal tubular epithelial cells HK- 2 induced by high glucose ，and to investigate its mechanism primarily. METHODS ：HK-2 cells were divided into normal group ，high glucose group ，irbesartan group （positive control ，1 μmol/L），sulforaphane low ，medium and high concentration groups （10，20，40 μmol/L）. The cells in normal group were cultured in DMEM medium for 96 hours. T he cells in other groups were cultured in high glucose DMEM medium （containing 40 mmol/L glucose ）for 48 hours. After inducing cell injury，the cells were added with corresponding drugs for 48 hours. Survival rate and apoptotic rate of cells were detected. mRNA expression of cyclin D 1，caspase-3，Bcl-2 and Bax as well as protein expression of p-mTOR ，p-AMPK，p-Akt and p-PI 3K were also determined. In addition ，HK-2 cells were divided into normal group ，high glucose group ，sulforaphane high concentration group（40 μmol/L），acardicin group （AMPK agonist ，1 mmol/L），sulforaphane high concentration+compound C group （sulforaphane 40 μmol/L+AMPK inhibitor compound C 40 μmol/L），perifoxine group （Akt inhibitor ，19.95 μmol/L）、sulforaphane high concentration+SC 79 group（sulforaphane 40 μmol/L+Akt agonist SC79 4 μmol/L）. After cultured with the same method ， protein expression of p-mTOR ，p-AMPK，p-Akt and p-PI 3K were detected in HK- 2 cells. RESULTS ：Compared with normal group，survival rate of HK- 2 cells，mRNA expression of cyclin D 1 and Bcl- 2 as well as protein expression of p-AMPK were decreased significantly in high glucose group （P＜0.05）；apoptotic rate ，mRNA expression of caspase- 3 and Bax ，protein expression of p-mTOR ，p-Akt and p-PI 3K in HK- 2 cells were increased significantly （P＜0.05）. Compared with high glucose group，above indexes of sulforaphane low ，medium and high concentration groups ，irbesartan group were all improved significantly （P＜0.05）；the improvement of above indexes in sulforaphane medium and high concentration groups were significantly better those of sulforaphane low concentration group （P＜0.05）. There was no significant difference in above indexes between sulforaphane high concentration group and irbesartan group （P＞0.05）. Compared with sulforaphane high concentration group，there were no significant difference in the protein expression of p-AMPK ，p-mTOR in acardicin group and p-mTOR ，p-Akt and p-PI 3K in perifoxine group （P＞0.05）；the protein expression of p-AMPK in sulforaphane high concentration+compound C group was decreased significantly （P＜0.05），while the protein expression of p-mTOR was increased significantly （P＜0.05）；the protein expression of p-mTOR 、p-Akt、p-PI3K in sulforaphane high concentration+SC 79 group were increased significantly （P＜ 0.05）. CONCLUSIONS ：Sulforaphane can promote the proliferation of renal tubular epithelial cells and inhibit its apoptosis ；its mechanism may be associated with up-regulating the expression of p-AMPK and down-regulating the expression of p-mTOR ，p-Akt and p-PI 3K.
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Sulforaphane (SFN), a natural anti-tumor compound from cruciferous vegetables, has been reported to induce protective autophagy to cancer cells, which might impair the anti-tumor efficiency of SFN. However, the accurate function and mechanism of SFN inducing autophagy in cancers are still obscure, especially in esophageal squamous cell carcinoma (ESCC), one of malignancies with high incidence in North China. Here, we mainly explored the potential function of autophagy upon SFN treatment in ESCC and molecular mechanism. We demonstrated that SFN could inhibit cell proliferation and induce apoptosis by activating caspase pathway. Moreover, we found activation of NRF2 pathway by SFN was responsible for the induction of autophagy and also a disadvantage element to the anti-tumor effects of SFN on ESCC, indicating that SFN might induce protective autophagy in ESCC. We, therefore, investigated effects of autophagy inhibition on sensitivity of ESCC cells to SFN and found that chloroquine (CQ) could neutralize the activation of SFN on NRF2 and enhance the activation of SFN on caspase pathway, thus improved the anti-tumor efficiency of SFN on ESCC
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The organosulfur compound sulforaphane (SFN; C6H11NOS2) is a potent cytoprotective agent promoting antioxidant, anti-inflammatory, antiglycative, and antimicrobial effects in in vitro and in vivo experimental models. Mitochondria are the major site of adenosine triphosphate (ATP) production due to the work of the oxidative phosphorylation (OXPHOS) system. They are also the main site of reactive oxygen species (ROS) production in nucleated human cells. Mitochondrial impairment is central in several human diseases, including neurodegeneration and metabolic disorders. In this paper, we describe and discuss the effects and mechanisms of action by which SFN modulates mitochondrial function and dynamics in mammalian cells. Mitochondria-related pro-apoptotic effects promoted by SFN in tumor cells are also discussed. SFN may be considered a cytoprotective agent, at least in part, because of the effects this organosulfur agent induces in mitochondria. Nonetheless, there are certain points that should be addressed in further experiments, indicated here as future directions, which may help researchers in this field of research.
Subject(s)
Animals , Humans , Antioxidants/pharmacology , Apoptosis/drug effects , Brain/ultrastructure , Carbon Monoxide Poisoning/metabolism , Cytoprotection , Isothiocyanates/pharmacology , Membrane Potential, Mitochondrial/drug effects , Mitochondria/metabolism , SulfoxidesABSTRACT
The organosulfur compound sulforaphane (SFN; C6H11NOS2) is a potent cytoprotective agent promoting antioxidant, anti-inflammatory, antiglycative, and antimicrobial effects in in vitro and in vivo experimental models. Mitochondria are the major site of adenosine triphosphate (ATP) production due to the work of the oxidative phosphorylation (OXPHOS) system. They are also the main site of reactive oxygen species (ROS) production in nucleated human cells. Mitochondrial impairment is central in several human diseases, including neurodegeneration and metabolic disorders. In this paper, we describe and discuss the effects and mechanisms of action by which SFN modulates mitochondrial function and dynamics in mammalian cells. Mitochondria-related pro-apoptotic effects promoted by SFN in tumor cells are also discussed. SFN may be considered a cytoprotective agent, at least in part, because of the effects this organosulfur agent induces in mitochondria. Nonetheless, there are certain points that should be addressed in further experiments, indicated here as future directions, which may help researchers in this field of research.
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The intervention of sulforaphane can reduce the risk of diabetes and its complications. It is mainly achieved by regulating nuclear factor erythroid-2-related factor 2 (Nrf2) to inhibit endothelial cell activation and improve high density lipoprotein levels. In addition, sulforaphane can bring about a marked improvement on fatty liver disease by regulating lipid metabolism. Its important pathways include the regulation of peroxisome proliferators-activated receptors y (PPARy), hormone-sensitive triglyceride lipase (HSL) and AMP-activated protein kinase (AMPK) regulation promotes lipolysis. In summary, further exploration of the mechanism of action of plant-derived functional ingredients of sulforaphane may be the key to preventing and treating diabetes and fatty liver disease.
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Objective To observe the impacts of sulforaphane on proliferation, apoptosis and Wnt/β-catenin pathway of cultured retinoblastoma cells. Methods Retinoblastoma Y79 cells were cultured with varying concentrations of sulforaphane (10, 20, 40 mg/L) for 24 and 48 h. Cell proliferation and apoptosis were measured by CCK8 assay and flow cytometry. Apoptosis related proteins Bax and Bcl-2, as well as the expression levels of related proteins in Wnt/β-catenin signaling pathway were determined using Western Blot. Results Sulforaphane suppressed cell proliferation and stimulate apoptosis (12.47％ ± 2.24％, 24.63％ ± 3.44％, 57.49％ ± 5.41％ vs. 3.47％ ± 0.74％) in a dose- and time-dependent manner, respectively. Furthermore, 40 mg/L of sulforaphane increased the expression of Bax (0.57 ± 0.03 vs. 0.12 ± 0.01), but decreased the Bcl-2 (0.16 ± 0.01 vs. 0.81 ± 0.03) protein expression. The results of Western Blot displayed that sulforaphane decreased the expression levels of Wnt (0.19 ± 0.01 vs. 0.74 ± 0.02), β-catenin protein (0.22 ± 0.02 vs. 0.82 ± 0.03), indicating that sulforaphane can inhibit the activation of Wnt/β-catenin signaling pathway. Conclusions The sulforaphane could inhibit the proliferation and induce the apoptosis of retinoblastoma Y79 cells, probably regulating by the Wnt/β-catenin signaling pathway.
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Objective: To explore the effect and mechanism of sulforaphane on renal ischemia reperfusion injury (IRI) in mice to provide experimental evidence for effective intervention in renal IRI. Methods: Totally 24 male C57 mice were randomly divided into four groups: control group (Ctrl), IRI group, sulforaphane intervention IRI group (IRI + SFN), and sulforaphane alone group (SFN). SFN was injected into the mice at the dosage of 500 μg/kg 24 h before surgery. The renal IRI model was established by using the bilateral renal pedicles of mice with non-invasive vascular clamping for 45 min. After 24 h of renal blood perfusion, we sacrificed the mice and collected the whole blood and kidney tissue samples for pathological and biological examinations. The levels of TNF-α, IL-1β and IL-6 in murine serum were tested by ELISA. The expression levels of Nrf-2, keap-1, p-iκBα, IκBα and NFκB p-p65 in murine kidney were detected by Western blot. Results: Sulforaphane could improve renal function in mice with ischemia reperfusion and reduce apoptosis of renal tubular epithelial cells. Sulforaphane could also decrease the levels of TNF-α, IL-1β and IL-6 in murine serum and increase the expression of Nrf-2 in murine kidney tissue after ischemia reperfusion. Moreover, sulforaphane decreased the expression of p-iκBα in total protein and NFκB p-p65 in nucleus protein of renal tissue. Conclusion: Sulforaphane could inhibit phosphorylation, thus inhibiting the phosphorylation of NFκB p65 to translocation into the nucleus. Further it prevented the expressions of downstream inflammatory factors such as TNF-α, IL-1β and IL-6. Finally sulforaphane attenuated renal IRI in mice by Nrf-2 against inflammation.
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BACKGROUND: Oxidative stress is the hallmark of diabetic encephalopathy, which may be caused by hyperglycaemic toxicity. We aimed to discover pharmacologic targets to restore redox homeostasis. We identified the transcription factor Nrf2 as such a target. METHODS: HT22 cells were cultured in 25 or 50 mM D-glucose with various concentrations of sulforaphane (SFN) (from 1.25 to 5.0 µM). Cell viability was tested with the Cell Counting Kit-8 assay. Reactive oxygen species (ROS) production was detected with an inverted fluorescence microscope using the dichlorodihydrofluorescein-diacetate fluorescent probe. The expression of NF-E2-related factor 2 (Nrf2), haem oxygenase-1 (HO-1) and nuclear factor-κB (NF-κB) at the mRNA and protein levels was detected by reverse transcription quantitative polymerase chain reaction and western blotting. RESULT: We found that a high glucose concentration (50 mM) increased the generation of ROS, downregulated the expression of Nrf2/HO-1 and upregulated the expression of NF-κB. Moreover, HT22 cell viability significantly decreased after culture in high-glucose medium for 24, 48 and 72 h, whereas the activation of the Nrf2/HO-1 pathway using a pharmacological Nrf2 activator abrogated this high-glucose-induced toxicity. CONCLUSION: This study suggests that the activation of the Nrf2-ARE signalling pathway might be a therapeutic target for the treatment of diabetic encephalopathy.
Subject(s)
Animals , Mice , NF-E2-Related Factor 2/agonists , Neuroprotection , Glucose/toxicity , Hippocampus/drug effects , Time Factors , Cell Line , Blotting, Western , Fluorescent Antibody Technique , Electrophoresis, Gel, Pulsed-Field , Reactive Oxygen Species , Reverse Transcriptase Polymerase Chain Reaction , Hippocampus/cytologyABSTRACT
BACKGROUND: Multiple sclerosis (MS) is an immune-associated inflammatory disorder of the central nervous system and results in serious disability. Although many disease-modifying therapy drugs have been developed, these drugs have shown limited clinical efficacy and some adverse effects in previous studies, therefore, there has been reasonable need for less harmful and cost-effective therapeutics. Herein, we tested the anti-inflammatory effect of sulforaphane (SFN) in a mouse model of experimental autoimmune encephalomyelitis (EAE). METHODS: The EAE mice were randomly assigned into two experimental groups: the phosphate-buffered saline (PBS)-treated EAE group and SFN-treated EAE group. After EAE mice induction by auto-immunization against the myelin oligodendrocyte glycoprotein peptide, we evaluated EAE symptom scores and biochemical analyses such as infiltration of inflammatory cells and demyelination of the spinal cord. Furthermore, western blotting was performed using the spinal cords of EAE mice. RESULTS: In the behavioral study, the SFN-treated EAE mice showed favorable clinical scores compared with PBS-treated EAE mice at the 13th day (1.30 ± 0.15 vs. 1.90 ± 0.18; P = 0.043) and 14th day (1.80 ± 0.13 vs. 2.75 ± 0.17; P = 0.003). Additionally, the biochemical studies revealed that SFN treatment inhibited the inflammatory infiltration, demyelinating injury of the spinal cords, and the up-regulation of inducible nitric oxide synthase in the EAE mice. CONCLUSION: The SFN treatment showed anti-inflammatory and anti-oxidative effects in the EAE mice. Conclusively, this study suggests that SFN has neuroprotective effects via anti-inflammatory processing, so it could be a new therapeutic or nutritional supplement for MS.
Subject(s)
Animals , Mice , Blotting, Western , Central Nervous System , Demyelinating Diseases , Encephalomyelitis, Autoimmune, Experimental , Multiple Sclerosis , Myelin-Oligodendrocyte Glycoprotein , Neuromyelitis Optica , Neuroprotective Agents , Nitric Oxide Synthase Type II , Spinal Cord , Treatment Outcome , Up-RegulationABSTRACT
BACKGROUND: Autophagy is a highly balanced process in which lysosomes remove aged and damaged organelles and cellular proteins. Autophagy is essential to maintain homeostasis in the kidneys. METHODS: Using human renal tubule cells HK-2, we assessed the impact of high glucose (HG) on autophagy. We also evaluated the capability of sulforaphane (SFN) to protect the HK-2 cells from HG-induced apoptosis by modulating autophagy. RESULTS: SFN modulated autophagy and decreased apoptosis in the HK-2 cells that were cultured in 250 mM glucose medium for two days. The reactive oxygen species (ROS) levels increased, as expected, in the cells cultured in the 250 mM glucose medium. However, the SFN decreased the ROS levels in the HK-2 cells. The overexpression of heme oxygenase-1 (HO-1) by SFN decreased the expression of LC3 and beclin-1. LC3 and beclin-1 were involved in the downregulation of caspase-3 that was observed in the HG-induced cells. CONCLUSION: The activation of nuclear factor E2-related factor 2 (Nrf2)–HO–1 inhibited ROS expression and subsequently attenuated autophagy and cell apoptosis after HG injury was decreased. HG injury led to the activation of autophagy and HO-1 in order to combat oxidative stress and protect against cell apoptosis. Therefore, HO-1 activation can prevent ROS development and oxidative stress during HG injury, which considerably decreases autophagy and apoptosis.
Subject(s)
Humans , Apoptosis , Autophagy , Caspase 3 , Diabetic Nephropathies , Down-Regulation , Glucose , Heme Oxygenase-1 , Homeostasis , Kidney , Lysosomes , NF-E2-Related Factor 2 , Organelles , Oxidative Stress , Reactive Oxygen SpeciesABSTRACT
@#Objective: To investigate the effect of sulforaphane (SFN) on CD8+ T cells differentiation, phenotype and the secretion of intracellular cytokines, as well as to study the underlying molecular mechanism. Methods: In the in vitro culture experiment, the cells were categorized into control group, SNF 10 mmol/L group and SNF 20 mmol/L group according to the SNF concentration. The effect of SFN treatment on CD8+ T cells differentiation, phenotype and cytokine secretion were detected by flow cytometry, and the effect of mTOR siRNA on the expression of CD127 and LKRG1 in CD8+T cells was also detected by flow cytometry. Expression of Bcl-2 and Bcl-6 were analyzed by qRT-PCR. The effect of SFN on apoptosis of CD8+T cells was examined byAnnexin-V/PI staining. The protein expressions of p-mTOR, p-S6 and b-actin were detected by western blotting. Results: SFN significantly promoted the formation of memory precursor CD8+ T cells and decreased the expression level of PD-1 and Tim-3 in CD8+T cells(P<0.01); meanwhile, after the treatment of SFN, the expressions of anti-apoptosis genes Bcl-2 and Bcl-6 were significantly increased while the apoptosis of CD8+ T cells was significantly inhibited and the protein expressions of p-mTOR and p-S6 were also significantly inhibited(P<0.05 or P<0.01). Moreover, mTOR siRNA could significantly increasethe expression of CD127 and decrease the expression of LKRG1 (all P<0.01). Conclusion: Sulforaphone promotes the formation of memory precursor CD8+T cells possibly by inhibiting the p-mTOR signaling pathway, and this could obtain more T cells to provide new thoughts for clinical immunotherapy.
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Objective@#To investigate the effects of sulforaphane on lipopolysaccharide (LPS)- induced cardiac myocytes hypertrophy of rats and study its possible mechanism.@*Methods@#The hypertrophic primary cardiac cells of neuonatal rats were divided into control group, LPS-induced group, JAK2 inhibitor group, low-, middle-, and high-dose sulforaphane groups. The cells in all groups, except for control group, were intervened by LPS (1 mg/L). The low-, middle-, and high-dose sulforaphane groups were treated with 10, 20, 40 μg/ml sulforaphane respectively. The JAK2 inhibitor group was treated with 10 nmol/L of JAK2 inhibitor for 24 h. Computer photograph analysis system was used to determine the cardiomyocyte volume, BCA kit was used to analyze the total protein concentration, ELISA kit was used to observe the expression levels of inflammatory cytokine (including IL-6 and TNF-α), and the Western Blot technology was used to analyze the expression levels of the related proteins in JAK2/STAT3 signaling pathway.@*Results@#Compared with control group, the cell volume (1 635.71 ± 154.84 μm3, 1 450.06 ± 140.13 μm3, 1 194.51 ± 134.76 μm3 vs. 1 854.28 ± 181.37 μm3) and the levels of protein (20.14 ± 2.11 μg, 17.59 ± 1.64 μg, 14.27 ± 1.47 μg vs. 23.17 ± 2.56 μg) in low-, medium-, high- dose of sulforaphane groups were significantly decreased (P<0.05). The levels of IL-6 (1 410.08 ± 255.17 pg/ml, 1 065.61 ± 210.37 pg/ml, 790.09 ± 140.28 pg/ml vs. 1 804.07 ± 275.34 pg/ml) and TNF-α (164.16 ± 27.51 pg/ml, 130.13± 22.08 pg/ml, 92.27 ±12.16 pg/ml vs. 182.42 ± 30.37 pg/ml) in low-, medium-, high- dose of sulforaphane groups were significantly decreased (P<0.05). The expression levels of p-JAK2/JAK2 (0.39 ± 0.05, 0.27 ± 0.04, 0.21 ± 0.03 vs. 0.54 ± 0.07) and p-STAT3 (0.47 ± 0.05, 0.25 ± 0.03, 0.18 ± 0.03 vs. 0.53 ± 0.06) in low-, medium-, high- dose of sulforaphane groups were also significantly decreased (P<0.05).@*Conclusions@#Sulforaphane has a protective effect on LPS-induced cardiac hypertrophy, which is partially via inhibiting inflammatory response via suppressing the activation of JAK2/STAT3 signaling pathway.
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Objective To observe the effects of sulforaphane on the apoptosis in human gastric cancer HGC27 cells, and to study the possible mechanism of it. Methods The proliferation activity of HGC27 cells treated with sulforaphane was analyzed by CCK8 kit. Apoptosis rates were assessed by flow cytometry. After HGC27 cells were treated with sulforaphane, and the expression levels of Bcl-2, Bax, caspase-3, PI3K, Akt and p-Akt were measured by Western Blot. After HGC27 cells were treated with LY294002, sulforaphane and LY294002+sulforaphane, the expression levels of PI3K, Akt and p-Akt were also investigated by Western Blot. Results The proliferation of HGC27 cells was significantly suppressed by sulforaphane in dose-dependent and time-dependent manners. The apoptotic rates of HGC27 cells in 10, 20, 40 μg/ml of sulforaphane groups (10.29% ± 1.57%, 23.68% ± 1.69%, 35.29% ± 2.38% vs. 2.52% ± 0.74%) were greatly increased compared with the control group. The expression levels of Bax (18.92 ± 2.18, 34.06 ± 5.06, 44.08 ± 5.69 vs. 12.51 ± 2.15) in 10, 20, 40 μg/ml of sulforaphane groups were greatly increased compared with control group(P＜0.05). The expression levels of Bcl-2 (56.39 ± 5.27, 33.06 ± 4.26, 25.61 ± 4.01 vs. 78.25 ± 7.26), PI3K (51.06 ± 5.27, 42.06 ± 5.21, 23.08 ± 4.51 vs. 79.07 ± 8.12), p-Akt/Akt (58.62 ± 5.34, 35.24 ± 4.06, 14.52 ± 2.56 vs. 82.64 ± 8.25) in 10, 20, 40 μg/ml of sulforaphane groups were greatly decreased compared with control group (P＜0.05). The expression levels of PI3K (56.41 ± 5.36, 34.37 ± 4.52, 23.11 ± 3.05 vs. 81.24 ± 7.16), p-Akt/Akt (49.52 ± 5.84, 31.06 ± 4.09, 21.05 ± 2.28 vs. 77.52 ± 7.06) in the LY294002, sulforaphane and the LY294002+sulforaphane groups were greatly decreased compared with the control group (P＜0.05). Conclusions Sulforaphane may inhibit proliferation and promote apoptosis of HGC27 cells probably by inactivating PI3K/Akt signaling pathway.
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Objectives To evaluate the concentration differences of sulforaphene and sulforaphane at various ages and in different parts of Raphanus sativus L. var. caudatus with respect to their potential cancer preventive effect on HCT116 colon cancer cells. Methods FTIR–ATR and GC–MS were used to characterize the isothiocyanates in the plant extracts followed by HPLC for quantification. Antiproliferation and apoptosis induction were determined by using MTT assay and flow cytometry, respectively. Results The respective rank of anticancer activity of Raphanus sativus were as follows: vegetative (3 week) < older rosette (4 week) < early-bolting (5 week) < senescence (7 week) < late-bolting (6 week). The low to high concentration of sulforaphene and sulforaphane occurred in the same stage order. Conclusions The reproductive parts (flower, pod, and dry seed) of Raphanus sativus have the greatest isothiocyanate concentration, evidenced by a sulforaphene concentration higher than the sulforaphane. This result should inform the selection of the most appropriate harvesting stage and plant part for use as a potential chemopreventive agent.
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Objective: To investigate the effects of sulforaphane (SFN) on oxidative stress and Nrf2(Nuclear factor E2-related factor 2)/HO-1(heme oxygenase-1) signaling pathway in renal tubular epithelial cells (HK-2).Methods: HK-2 cells were cultured in vitro and divided into five groups, including the control group, H2O2 group, H2O2+10 μmol·L-1 SFN group, H2O2+20μmol·L-1 SFN group and H2O2+40 μmol·L-1 SFN group.The levels of MDA, NO, GSH and the activity of SOD were measured.The expression levels of Nrf2, HO-1 mRNAR and proteins were analyzed using RT-PCR and Western Blot methods.Results: The levels of MDA and NO in H2O2 group significantly increased when compared with those in the control group, while the levels of GSH and the activity of SOD significantly decreased(P<0.05).The levels of MDA and NO in the cells after the treatment of sulforaphane at different concentrations significantly reduced when compared with those in H2O2 group, while the levels of GSH and the activity of SOD significantly increased(P<0.05).The results of RT-PCR and Western Blot analysis showed that the expression levels of Nrf2, HO-1 mRNAR and proteins in the cells after the treatment of sulforaphane at different concentrations were significantly up-regulated when compared with those in H2O2 group (P<0.05).Conclusion: Sulforaphane can strengthen the antagonism effects on oxidative stress injury in HK-2 cells and the underlying mechanism might be associated with activating Nrf2/HO-1 signal transduction pathway.
ABSTRACT
Objective To investigate the role and mechanism of sulforaphane (SFN) in vascular calcification induced by oxidative stress.Methods The uremic vascular calcification model was established by treating rat vascular smooth muscle cells (RASMCs) with β-glycerophosphate.RASMCs were divided into 6 groups:normal control (NC) group,1 μmol/L SFN group,5 μmol/L SFN group,calcification group,1 μmol/L SFN+calcification group,5 μmol/L SFN+calcification group,and were all cultured for 72 h.Cell viability was measured by MTT.RASMCs calcification was visualized by Von Kossa staining.Calcium content was quantified by the microplate test,and mRNA level of FGF-23 was tested by real-time PCR.The expressions of OPN,Runx-2,Nrf-2 and Sirt-1 were evaluated by Western blotting.Confocal microscope was employed to observe mitochondria damage in RASMCs and the production of ROS in RASMCs was measured by reactive oxygen species assay.Results (1) SFN did not affect cell viability of the NC group,but both low dosage and high dosage increased the cell viability of calcification group (all P ＜ 0.05).(2) Compared with calcification group,SNF treatment decreased the calcium concentration,intracellular calcium deposition and the mRNA level of FGF-23 (all P ＜ 0.05).(3) Compared with calcification group,SNF treatment decreased the fluorensence intensity,mitochondria injury and the protein expressions of OPN and Runx-2,but increased the protein expressions of Nrf-2,Sirt-1 and cleaved caspase-3 (all P ＜ 0.05).Conclusion SNF can effectively protect RASMCs against vascular calcification induced by oxidative stress,since it prevents the ROS production and mitochondria dysfunction through Nrf-2 and Sirt-1.