BoMyrosinase plays an essential role in sulforaphane accumulation in response to selenite treatment in broccoli

Sulforaphane, a naturally specialized metabolite, plays significant roles in human disease prevention and plant defense. Myrosinase(MY) is a key gene responsible for the catalysis of sulforaphane formation, but the molecular mechanisms through which MY regulates sulforaphane biosynthesis in plants remains largely unknown. Here, we discovered that the change of sulforaphane content in broccoli sprouts caused by exogenous selenite treatments is positively related to BoMY expression. BoMY overexpression in the Arabidopsis thaliana tgg1 mutants could dramatically increase myrosinase activity and sulforaphane content in the rosette leaves of 35S::BoMY/tgg1 and rescue its phenotypes.Moreover, an obvious increase of myrosinase activity and sulforaphane content was displayed in transgenic BoMY-overexpressed broccoli lines.In addition, a 2 033 bp promoter fragment of BoMY was isolated. Yeast one-hybrid(Y1H) library screening experiment uncovered that one bHLH transcription factor, BoFAMA, could directly bind to BoMY promoter to activate its expression, which was further evidenced by Y1H assay and dual-luciferase reporter assay. BoFAMA is a selenite-responsive transcription factor that is highly expressed in broccoli leaves;its protein is solely localized to nucleus. Additionally, genetic evidence suggested that the knockdown of FAMA gene in Arabidopsis thaliana could significantly decrease sulforaphane yield by inhibiting the expression of myrosinase genes. Interestingly, exogenous selenite supply could partially restore the low level of sulforaphane content in transgenic Arabidopsis FAMA-silencing plants. Our findings uncover a novel function of FAMAMY module in the regulation of selenite-mediated sulforaphane synthesis and provide a new insights into the molecular mechanism by which selenite regulates the accumulation of sulforaphane in plants.

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

Background Mastitis not only deteriorates the composition or quality of milk,but also damages the health and pro-ductivity of dairy goats.Sulforaphane(SFN)is a phytochemical isothiocyanate compound with various pharmacologi-cal 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.Results In vitro,SFN downregulated the mRNA expression of inflammatory factors(tumor necrosis factor-α(TNF-α),interleukin(IL)-1βand 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)produc-tion 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.Conclusions These results indicate that the natural compound SFN has a preventive effect on LPS-induced inflam-mation 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.

Sulforaphane protects liver injury induced by intestinal ischemia reperfusion through Nrf2-ARE pathway

AIM: To investigate the effect of sulforaphane (SFN) on regulation of NF-E2-related factor-2 (Nrf2)-antiox-idant response element (ARE) pathway in liver injury induced by intestinal ischemia/reperfusion (I/R). METHODS: Rats were divided randomly into four ex-perimental groups: control, SFN control, intestinal I/R and SFN pretreatment groups (n = 8 in each group). The intestinal I/R model was established by clamping the superior mesenteric artery for 1 h and 2 h reperfu-sion. In the SFN pretreatment group, surgery was performed as in the intestinal I/R group, with intraperitoneal administration of 3 mg/kg SFN 1 h before the op-eration. Intestine and liver histology was investigated. Serum levels of aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were measured. Liver tissue superoxide dismutase (SOD), myeloperoxidase (MPO), glutathione (GSH) and glutathione peroxidase (GSH-Px) activity were assayed. The liver transcription factor Nrf2 and heme oxygenase-1 (HO-1) were determined by immunohistochemical analysis and Western blotting analysis.RESULTS: Intestinal I/R induced intestinal and liver injury, characterized by histological changes as well as a signif icant increase in serum AST and ALT levels (AST: 260.13 ± 40.17 U/L vs 186.00 ± 24.21 U/L, P < 0.01; ALT: 139.63 ± 11.35 U/L vs 48.38 ± 10.73 U/L, P < 0.01), all of which were reduced by pretreatment with SFN, respectively (AST: 260.13 ± 40.17 U/L vs 216.63 ± 22.65 U/L, P < 0.05; ALT: 139.63 ± 11.35 U/L vs 97.63 ± 15.56 U/L, P < 0.01). The activity of SOD in the liver tissue decreased after intestinal I/R (P < 0.01), which was enhanced by SFN pretreatment (P < 0.05). In ad-dition, compared with the control group, SFN markedly reduced liver tissue MPO activity (P < 0.05) and elevat-ed liver tissue GSH and GSH-Px activity (P < 0.05, P < 0.05), which was in parallel with the increased level of liver Nrf2 and HO-1 expression.CONCLUSION: SFN pretreatment attenuates liver injury induced by intestinal I/R in rats, attributable to the antioxidant effect through Nrf2-ARE pathway.

Factors Influencing Glucoraphanin and Sulforaphane Formation in Brassica Plants:A Review

Sulforaphane is a type of sulfur-containing isothiocyanates hydrolyzed from glucosinolates by myrosinase found in Brassica plants. Sulforaphane is a naturally occurring inducer of phase II enzymes in human and animal bodies to detoxify cancer-causing chemicals. Glucoraphanin is the precursor of sulforaphane and its content is greatly influenced by plant species and genotype, plant organs, pre-harvest factors, and post-harvest processing, thus sulforaphane formation is also directly influenced. Here, we review the formation mechanism of sulforaphane and the factors influencing sulforaphane formation. In the end, the future directions are also discussed.

Sulforaphane-rich broccoli sprout extract improves hepatic abnormalities in male subjects

AIM: To evaluate effects of dietary supplementation of sulforaphane(SF)-rich broccoli sprout(BS) extract on hepatic abnormalities in Japanese male participants.METHODS: In a randomized,placebo-controlled,double blind trial,male participants with fatty liver received either BS capsules containing glucoraphanin [GR; a precursor of SF(n = 24)] or placebo(n = 28) for 2 mo. Liver function markers,serum levels of aspartate and alanine aminotransferases(AST and ALT,respectively) and γ-glutamyl transpeptidase(γ-GTP) and an oxidative stress marker,urinary levels of 8-hydroxydeoxyguanosine(8-OHd G),were measured and compared in participants before and after the trial period. In an animal model,chronic liver failure was induced in Sprague-Dawley rats by successive intraperitoneal injection with N-nitrosodimethylamine(NDMA) for 4 wk. Concomitantly,rats received AIN-76 diets supplemented with or without BS extract. Thereafter,rats were sacrificed,and their sera and livers were collected to measure serum liver function markers and hepatic levels of thiobarbituric acid reactive substances(TBARS) levels and hepatic glutathione S-transferase(GST) activity,a prototypical phase 2 antioxidant enzyme.RESULTS: Dietary supplementation with BS extract containing SF precursor GR for 2 mo significantly decreased serum levels of liver function markers,ALT [median(interquartile range),before: 54.0(34.5-79.0) vs after supplementation: 48.5(33.3-65.3) IU/L,P < 0.05] and γ-GTP [before: 51.5(40.8-91.3) vs after: 50.0(37.8-85.3) IU/L,P < 0.05],as well as the alkali phosphatase activity. Placebo showed no significant effects on the markers. The urinary level of 8-OHd G,an established oxidative stress marker,was significantly reduced in participants who had received BS capsules but not the placebo [before: 6.66(5.51-9.03) vs after: 5.49(4.89-6.66) ng/mg-creatinine,P < 0.05]. The reduction of urinary 8-OHd G was significantly correlated with decreased levels of both ALT and γ-GTP [?8-OHd G and ?ALT: Spearman r(r) 0.514 and P = 0.012,?8-OHd G and ?γ-GTP: r = 0.496 and P = 0.016]. Intake of BS extract prevented NDMA-induced chronic liver failure in rats,which was attributable to the suppression of the increase in TBARS through induction of hepatic phase 2 antioxidant enzymes including hepatic GST(86.6 ± 95.2 vs 107.8 ± 7.7 IU/g,P < 0.01).CONCLUSION: Dietary supplementation with BS extract containing the SF precursor GR is likely to be highly effective in improving liver function through reduction of oxidative stress.

Sulforaphane as a Promising Natural Molecule for Cancer Prevention and Treatment

Tumorigenicity-inhibiting compounds have been identifled in our daily diet.For example,isothiocyanates(ITCs)found in cruciferous vegetables were reported to have potent cancer=prevention activities.The best characterized ITC is sulforaphane(SF).SF can simultaneously modulate multiple cellular targets involved in carcinogenesis,including(1)modulating carcinogen-metabolizing enzymes and blocking the action of mutagens;(2)inhibition of cell proliferation and induction of apoptosis;and(3)inhibition of neo-angiogenesis and metastasis.SF targets cancer stem cells through modulation of nuclear factor kappa B(NF-κB),Sonic hedgehog(SHH),epithelial-mesenchymal transition,and Wnt/βcatenin pathways.Conventional chemotherapy/SF combination was tested in several studies and resulted in favorable outcomes.With its favorable toxicological profile,SF is a promising agent in cancer prevention and/or therapy.In this article,we discuss the human metabolism of SF and its effects on cancer prevention,treatment,and targeting cancer stem cells,as well as providing a brief review of recent human clinical trials on SF.

Sulforaphene and sulforaphane in commonly consumed cruciferous plants contributed to antiproliferation in HCT116 colon cancer cells

Objective: To analyze two isothiocyanates(sulforaphene and sulforaphane) and their antiproliferative effect of 11 indigenous cruciferous vegetables.Methods: Phytoconstituents identification was conducted by high performance liquid chromatography and gas chromatography-mass spectrometer techniques. The antiproliferation was evaluated in colon cancer cell line HCT116 by MTT assay.Results: Isothiocyanate identification by high performance liquid chromatography showed that broccoli, cabbage, "Khi-Hood"(Raphanus sativus L. var. caudatus Alef) and Chinese radish contained isothiocyanates sulforaphane. Sulforaphene and sulforaphane in broccoli, cabbage and "Khi-Hood" were characterized by the gas chromatography-mass spectrometer analysis. Antiproliferation screening by MTT assay found that the potent plants which possessed IC_(50) below 50 mg/m L were cabbage and "Khi-Hood", while the others had low antiproliferation with IC_(50) higher than 50 mg/m L. Difference in antiproliferation was probably due to difference existed phytochemical constituents in each plant. "Khi-Hood" possessed the highest antiproliferation against HCT116 with the lowest IC_(50)at(9.42 ± 0.46) mg/m L. The IC_(50) of chemotherapeutic drug(mitomycin C)was(19.12 ± 1.00) mg/m L, while both melphalan and 5-fluorouracil possessed the IC_(50) value higher than 50 mg/m L.Conclusions: Commonly consumed cruciferous vegetables exerted varied antiproliferation and isothiocyanate contents. High isothiocyanate content in "Khi-Hood" was contributed to high antiproliferation. Among 11 plants studied, "Khi-Hood" could be an alternative chemopreventive diet.

Sulforaphane attenuates dextran sodium sulphate induced intestinal inflammation via IL-10/STAT3 signaling mediated macrophage phenotype switching

Innate immunity,particularly macrophages,is critical for intestinal homeostasis.Sulforaphane,a dietary isothiocyanate from cruciferous vegetables,has been reported to protect against intestinal inflammation.However,the role of macrophages in sulforaphane mediated intestinal inflammation and the underlying molecular mechanisms have not been studied yet.In this study,sulforaphane effectively attenuated dextran sodium sulphate(DSS)induced intestinal inflammation in murine model.Of note,sulforaphane skewed the switching from classically(M1)to alternatively(M2)activated phenotype both in intestinal and bone marrow-derived macrophages(BMDMs).The expression levels of M1 associated maker genes induced by DSS or lipopolysaccharide(LPS)plus interferon gamma-γ(IFN-γ)were suppressed by sulforaphane while M2 marker gene expression levels were improved.This resulted in alteration of inflammatory mediators,particularly interleukin-10(IL-10),both in colon tissues and culture medium of BMDMs.Subsequently,IL-10 was found to mediate the sulforaphane induced M2 phenotype switching of BMDMs through the activation of STAT3 signaling.This was confirmed by immunofluorescence analysis with increased number of p-STAT3-positive cells in the colon sections.Moreover,anti-IL-10 neutralizing antibody significantly interfered M2 phenotyping of BMDMs induced by sulforaphane with reduced STAT3 phosphorylation.Findings here introduced a potential utilization of sulforaphane for intestinal inflammation treatment with macrophages as the therapeutic targets.

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

AIM:To examine the effects of sulforaphane on fibrotic changes of transforming growth factor(TGFβ2)induced human conjunctival fibroblast(HCon Fs).METHODS:HCon Fs were cultured and divided into control,TGFβ2(1 ng/m L),sulforaphane and TGFβ2+sulforaphane groups.Cell viability and apoptosis were detected using the MTT and Apo Tox-Glo Triplex assay.Cell migration was detected using scratch and Transwell assay.Real-time quantitative PCR method was used to evaluate m RNA expression of TGFβ2,matrix metalloproteinase-2(MMP2),myosin light chain kinase(MYLK),integrinαV,integrinα5,fibronectin 1 andα-smooth muscle actin(α-SMA).The protein expression ofα-SMA,p-PI3 K,PI3 K,p-Akt,and Akt were detected by Western blot.RESULTS:The proliferation of HCon Fs was significantly(P<0.05)suppressed by sulforaphane compared to control cells with the increase of the concentration and treatment time.Cell proliferation after 48 h incubation was significantly reduced with 100μmol/L sulforaphane treatment by 17.53%(P<0.05).The Transwell assay showed sulforaphane decreased cell migration by 18.73%compared with TGFβ2-induced HCon F(P<0.05).TGFβ2-induced the increasing expression of fibronectin,type I collagen andα-SMA,and the phosphorylation of PI3 K and Akt were all significantly suppressed by sulforaphane pretreatment.CONCLUSION:Sulforaphane inhibits proliferation,migration,and synthesis of the extracellular matrix in HCon Fs,and inhibiting the PI3 K/Akt signaling pathway.Sulforaphane could be a potential therapeutic drug for prevention of scar formation in filtering bleb after trabeculectomy.

Antiglycative activity of sulforaphane: a new avenue to counteract neurodegeneration?

Neurodegeneration is a key aspect of a large number of diseases that come under the umbrella of＂neurodegenerative diseases＂with the most notable being Parkinson＇s,Alzheimer＇s,and Huntington＇s diseases（AD,PD,HD）.They are all incurable and debilitating conditions that result in progressive degeneration and/or death of neurons and are the leading cause of disability in the elderly. The incidence of these diseases is on the rise and yet there is a paucity of effective therapies to treat them.

Inhibition of TGF-β2-induced migration and epithelial-mesenchymal transition in ARPE-19 by sulforaphane

AIM: To investigate the effects of sulforaphane(SFN) on transforming growth factor(TGF)-β2 stimulated migration and epithelial-mesenchymal transition(EMT) in ARPE-19 cells.METHODS: ARPE-19 cells were cultured in the presence or absence of SFN or TGF-β2. SFN toxicity was assessed by performing a lactate dehydrogenase assay(LDH) and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium(MTS) assays, and cell migration was evaluated by Transwell migration assay. Actin stress fiber formation in ARPE-19 cells was determined using immunofluorescence analysis. Immunoblotting analysis was used to determine fibronectin and α-smooth muscle actin expressions along with the degree of Smad and Akt phosphorylation.RESULTS: SFN inhibited ARPE-19 migration. Additionally, SFN attenuated TGF-β2-induced appearance of actin stress fibers as well as fibronectin and α-smooth muscle actin expressions in these cells. SFN also hindered the TGF-β2-stimulated phosphorylation of Smad2, Smad3, and Akt. SFN showed no cytotoxicity towards ARPE-19 cells. CONCLUSION: SFN inhibits TGF-β2-stimulated migration and EMT in ARPE-19 cells, probably by preventing the establishment of actin stress fibers and Akt and Smad2/3 signaling.

Sulforaphane enhances Nrf2-mediated antioxidant responses of skeletal muscle induced by exhaustive exercise in HIIT mice

Nuclear factor erythroid-derived 2-like 2(Nrf2)is the master regulator of antioxidant defenses.High-intensity interval training(HIIT)has been proposed as a time-efficient training program and has become a substantial component of modern training program In the present study,we evaluated the effects of sulforaphane(SFN),a dietary isothiocyanate derived from cruciferous vegetables and a potent Nrf2 activator,on Nrf2-mediated antioxidant defense responses of skeletal muscle induced by exhaustive exercise in HIIT mice.Male C57 BL/6 J mice were randomly allocated into control group,HIIT group,and HIIT pretreated with SFN(HIIT+SFN)group.On the third day after completion of a 6-weeks HIIT protocol,an exhaustive treadmill test was conducted in all mice.Mice were intraperitoneally injected with SFN(HIIT+SFN group)or PBS(HIIT and control mice)4 times in 3 days prior to the exhaustive treadmill test.The results indicated that the 6-weeks HIIT protocol did not increase the antioxidative capacity of skeletal muscle during exhaustive exercise.Importantly,SFN treatment improved anti oxidative capacity of skeletal muscle in response to the acute exhaustive exercise by increasing mRNA and nucleoprotein expression of Nrf2 and these genes involved in antioxidant generation and decreasing blood creatine kinase(CK)and 4-hydroxy-2-nonenal(4-HNE)-modified protein levels in the HIIT mice.

Low-Dose of the Sulforaphane Precursor Glucoraphanin as a Dietary Supplement Induces Chemoprotective Enzymes in Humans

Broccoli sprout (BS) supplements have been marketed for over a decade for the promising health beneficial effects of sulforaphane (SFN), which induces Nrf2 signaling and downstream chemoprotective genes, including phase 2 enzymes. Most commercially available BS supplements encapsulate heat-processed BS containing glucoraphanin (GR), which is hydrolyzed to SFN by the intestinal microbiota. However, the absorption behavior of SFN following the intake of such BS supplements is still unclear. Additionally, the GR dose (around 30 mg) recommended by many manufacturers of BS supplements is relatively lower than the effective dose determined in previous intervention studies. The aims of this study were to assess the effects of a single administration of a typical BS supplement containing lower doses of GR (30 or 60 mg from 3 or 6 capsules, respectively) on SFN absorption, and also to assess the serum activities of phase 2 enzymes as possible surrogate markers of the beneficial effects of SFN. Urinary excreted isothiocyanates and dithiocarbamates showed that the SFN absorption following administration of BS supplement was prolonged and varied among individuals, which conforms to the well-known characteristics of intestinal microbiota-mediated SFN absorption. The amount of SFN absorbed increased dose-dependently but not linear fashion (9.27 μmol and 13.5 μmol for 3 and 6 capsules, respectively). There was no significant difference in SFN bioavailability and the number of capsules consumed. Serum activities of phase 2 enzymes glutathione S-transferase (GST) and NAD(P)H: quinone oxidoreductase 1 (NQO1), which have been reported to display “chemoprotected states” in organs such as the liver, were dose-dependently and synchronously elevated (p < 0.05) following BS supplement intake. This suggests that a low dose of GR (30 mg) exerts chemoprotective effects in humans. In conclusion, our findings will be useful in future clinical studies investigating the chemoprotective effects of SFN, and for the development of BS supplement products.

Role of Sulforaphane on Histone Deacetylase Activity in Solid Ehrlich Carcinoma

Histone acylation is one in every of the posttranslational modification that incorporates a role within the regulation of sequence expression. This study was aimed to ameliorate neoplasm cell model Solid Ehrlich malignant neoplastic disease with sulforaphane extracted from cabbage alone and together with immune suppressant drug (MTX) by study the activity of simple protein deacetylase accelerator (HDAC). In this study, sulforaphane was extracted from cabbage leaves and evaluated victimization GC-MS and ultraviolet illumination spectrophotometry. 60 white male rats were divided into six equal groups. Group I: management ordinarily. The remaining mice were subjected to Ehrlich neoplasm cells. Group II: Tumor-bearing group. Group III: immune suppressant drug “MTX” treated group. Group IV, V treated with SFN before and when Paul Ehrlich cells implantation group VI (Methotrexate and sulforaphane-treated group). This result showed that sulforaphane was extracted from cabbage (Brassica oleracea) in concentration of 833 μg/g leave. HDAC was attenuated when treatment with sulforaphane after treatment with methotrexate or sulforaphane alone. The desoxyribonucleic acid injury was attenuated in neoplasm tissue of tumor-bearing mice when treatment with immune suppressant drug and hyperbolic considerably in tumor tissue of tumor-bearing mice treated with sulforaphane before and after carcinogenesis and together with methotrexate treatment after carcinogenesis.

In-vitro pharmacological evaluation of Sulforaphane from Brassica oleracea

Objectives: Sulforaphane has numerous Pharmacological and therapeutic effects like anti-oxidant, anti-cancer, anti-diabetic, anti-arthritic, anti-ulcer, anti-viral. The present study was designed to evaluate the anti-arthritic activity of Sulforaphane by in-vitro screening methods. Methods: The anti-arthritic activity of Sulforaphane was investigated by protein denaturation assay by using Bovine serum albumin (BSA) and egg albumin. Sulforaphane used in various concentration (10, 50, 100, 250, 500 μg/ml) against both the methods and Diclofenac sodium was used as reference standard. Results: The anti-arthiritic activity sulforaphane was directly proportional to inhibition of denaturation of albumin. Sulforaphane shows concentration dependent inhibition activity in both bovine serum albumin and egg albumin assay. The maximum inhibition was observed in the concentration of 500 μg/ml with 92.8% inhibition for BSA and 96.3% for egg albumin respectively. Conclusion: Sulforaphane have significant anti-arthritic activity accessed by BSA denaturation and egg albumin denaturation assay. Based on present finding future direction also planned to conform the activity by using well established in-vivo methods.

Induction of Phase II Enzymes Glutathione-S-Transferase and NADPH: Quinone Oxydoreductase 1 with Novel Sulforaphane Derivatives in Human Keratinocytes: Evaluation of the Intracellular GSH Level

Phase II enzymes including NADPH: Quinone Oxydoreductase 1 (NQO1) and Glutathione-S-Transferase (GST) represents a major and natural cellular protection system against deleterious environmental factors which cause skin damages. Sulforaphane is one of the most popular isothiocyanates found in cruciferous vegetables and known for its cytoprotective effects by inducing Phase II enzymes. Five novel sulforaphane derivatives were synthetized and tested for their activity on NQO1 and GST induction as well as for their effect on total GSH intracellular level using colorimetric assays on human keratinocytes cell line (HaCat). As sulforaphane and the synthetized components showed variable toxicity after their evaluation by means of in vitro cytotoxicity (MTT test), cells were treated at a concentration of 5 μM during 48 hours. The results showed that the addition products of sulforaphane decreased cytotoxity but none of those derivatives had a better effect than referenced sulforaphane on Phase II enzymes. It seems that the isothiacyanate function remains important for the sulforaphane activity.

Sulforaphane,an NRF2 agonist,alleviates ferroptosis in acute liver failure by regulating HDAC6 activity

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.

NRF2 signaling cascade in amyotrophic lateral sclerosis:bridging the gap between promise and reality

Amyotrophic lateral sclerosis is a very disabling disease due to the degeneration of motor neurons.Symptoms include muscle weakness and atrophy,spasticity,and progressive paralysis.Currently,there is no treatment to reverse damage to motor neurons and cure amyotrophic lateral sclerosis.The only two treatments actually approved,riluzole and edaravone,have shown mitigated beneficial effects.The difficulty to find a cure lies in the complexity and multifaceted pattern of amyotrophic lateral sclerosis pathogenesis.Among mechanisms,abnormal RNA metabolism,nucleocytoplasmic transport defects,accumulation of unfolded protein,and mitochondrial dysfunction would in fine induce oxidative damage and vice versa.A potent therapeutic strategy will be to find molecules that break this vicious circle.Sharpening the nuclear factor erythroid-2 related factor 2 signaling may fulfill this objective since nuclear factor erythroid-2 related factor 2 has a multitarget profile controlling antioxidant defense,mitochondrial functioning,and inflammation.We here discuss the interest of developing nuclear factor erythroid-2 related factor 2-based therapy in regard to the pathophysiological mechanisms and we provide a general overview of the attempted clinical assays in amyotrophic lateral sclerosis.

Sulforaphane抑制热灭活大肠杆菌诱导的HeLa细胞凋亡

目的研究Sulforaphane(SFN)对细胞凋亡的抑制作用及其在脓毒症中对细胞的保护作用。方法将HeLa细胞分为4组:对照组、热灭活大肠杆菌(Escherichia coli,E.coli)处理组、SFN处理组(SFN组)和热灭活E.coli+SFN组。酶联免疫反应检测炎症因子白细胞介素(interleukin,IL)-1β和肿瘤坏死因子(tumor necrosis factor,TNF)-α;流式细胞术(annexin V/propidium iodide双染)检测细胞凋亡;免疫荧光检测细胞内活性氧(reactive oxygen species,ROS)含量及线粒体膜电位的变化;Western blot检测活化的caspase 3、bax和bcl-2水平。结果热灭活E.coli处理后,细胞产生的炎症因子IL-1β[(121.13±8.44)pg/mL比(36.67±5.82)pg/mL,P<0.05]和TNF-α[(123.83±7.32)pg/mL比(20.63±1.91)pg/mL,P<0.05]明显升高、凋亡细胞数明显增加[(32.49±3.96)%比(12.52±1.37)%,P<0.05]、活化的caspase 3[(2.28±0.19)比(1.01±0.19),P<0.05)]和BAX[(4.81±0.80)比(1.04±0.25),P<0.05)]水平明显升高,而bcl-2[(0.27±0.08)比(0.96±0.18),P<0.05)]水平明显降低。用SFN处理后(包括SFN组、热灭活E.coli+SFN组),IL-1β[(45.41±13.16)pg/mL比(121.13±8.44)pg/mL,P<0.05],TNF-α[(30.3±6.0)pg/mL比(123.8±7.3)pg/mL,P<0.05]水平明显降低、凋亡细胞明显减少[(11.34±2.05)%比(32.49±3.96)%,P<0.05]、活化的caspase3[(1.01±0.07)比(2.282±0.19),P<0.05)]、BAX[(2.15±0.14)比(4.81±0.80),P<0.05)]明显减少,而bcl-2[(1.34±0.18)比(0.27±0.08),P<0.05)]水平明显增加。与此相对应,与灭活E.coli处理的细胞相比较,加入SFN(包括SFN组和热灭活E.coli+SFN组),ROS水平明显下降[(1.27±0.17)比(6.30±1.50),P<0.05)],而线粒体膜电位明显上升[(1.18±0.10)比(0.14±0.01),P<0.05)]。结论SFN可有效抑制热灭活E.coli诱导的HeLa细胞调亡、稳定线粒体膜电位、降低ROS、抑制炎性因子的产生。因此,SFN可能作为脓毒血症时保护细胞的药物。

Inhibiting autophagy enhances sulforaphane-induced apoptosis via targeting NRF2 in esophageal squamous cell carcinoma

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 in vitro and in vivo. Our study provides a preclinical rationale for development of SFN and its analogs to the future treatment of ESCC.