Basic Study on Inhibitory Effect of Sulforaphane on Inflammatory Response and Alleviation of Airway Remodeling in COPD Rats / 广州中医药大学学报

Objective To investigate the ameliorative effect of sulforaphane on inflammatory response and airway remodeling in rats with chronic obstructive pulmonary disease(COPD).Methods Seventy-five SD rats were randomly divided into the normal group,the model group,and the low-,medium-,and high-dose groups of sulforaphane,with 15 rats in each group.Except for the normal group,the COPD model was prepared in the remaining group using aroma smoke inhalation combined with intratracheal droplet lipopolysaccharide(LPS)method.After the successful modelling,the rats were administered the drug by gavage for 28 days.At the end of the administration,the general conditions of the rats in each group were observed,and the lung function[forced vital capacity(FVC),peak expiratory flow-rate(PEF),forceful expiratory volume in 1 second(FEV1)]was examined,and the pathological changes of the lung tissues were observed by hematoxylin-eosin(HE)staining method,and the indexes of airway remodeling(thickness of the bronchial wall,thickness of the smooth muscle)were measured;the enzyme-linked immunosorbent assay(ELISA)was used to examine the lung function of the rats.The levels of inflammatory factors[tumor necrosis factor α(TNF-α),interleukin 1β(IL-1β)]were detected in lung tissue by enzyme-linked immunosorbent assay(ELISA),and changes in the protein expressions of Toll-like receptor 4(TLR4),myeloid differentiation factor 88(MyD88),and nuclear transcription factor κB(NF-κB)were detected in lung tissue by Western Blot.Results(1)The rats in the model group had dry and lack of glossy fur,obvious coughing and nose scratching,shortness of breath,slow movement,and preferred to arch their backs and lie curled up;the rats in the low-,medium-and high-dose groups of sulforaphane showed significant improvement in shortness of breath,coughing,and other abnormal manifestations.(2)HE staining showed that the airway wall and smooth muscle of rats in the model group were thickened,the airway epithelium was damaged,and alveolar destruction,fusion,and massive infiltration of inflammatory cells were seen;the histopathological changes in the lungs of rats in the low-,medium-and high-dose groups of sulforaphane improved to varying degrees,with the airway wall becoming thinner,the degree of alveolar destruction being reduced,and the infiltration of inflammatory cells being reduced.(3)Compared with the normal group,FVC,PEF and FEV1 were significantly reduced in the model group(P＜0.05),and the levels of TNF-α and IL-1β,bronchial wall thickness,smooth muscle thickness,and the expression levels of TLR4,MyD88 and NF-κB were significantly increased in the model group(P＜0.05);and in comparison with the model group,the levels of FVC,PEF,and FEV1 were significantly increased in the rats in the sulforaphane low-,medium-,and high-dose groups(P＜0.05),and the levels of TNF-α,IL-1β,bronchial wall thickness,smooth muscle thickness,and the expression levels of TLR4,MyD88,and NF-κB were significantly decreased(P＜0.05)compared with the model group.Conclusion Sulforaphane helps to inhibit the inflammatory response,attenuate airway remodeling,and improve the pathological injury and lung function of lung tissue in rats with COPD,and its mechanism may be related to the inhibition of TLR4,MyD88,and NF-κB protein expressions.

Sulforaphane regulates macrophage glycolysis and inhibits the progression of diabetic nephropathy by modulating the ALOX5/NF-κB signaling pathway / 安徽医科大学学报

Objective @# To investigate the effects of sulforaphane (SFN) in regulating the macrophage glycolysis via the arachidonate 5-lipoxygenase (ALOX5) /nuclear factor kappa B (NF-κB) signaling pathway on the progression of diabetic nephropathy (DN) . @*Methods @#Bioinformatics analysis was used to identify the target genes of SFN in the treatment of DN . Human proximal tubular epithelial cell line (HK-2 cells) was induced with 30 mmol/L high glucose (HG) to create an in vitro model of DN . HK-2 cells were divided into the following groups : normal glucose (NG) group , HG group , HG + SFN (3 mmol/L) group , HG + ALOX5 group , HG + SFN (3 mmol/L) + ALOX5 group , HG-treated macrophages + HK-2 group , HG + SFN (3 mmol/L) treated macrophages s + HK-2 group , HG + ALOX5 transfection treated macrophages + HK-2 group , HG + SFN (3 mmol/L) + ALOX5 transfection treated macrophages + HK-2 group . CCK-8 assay was used to detect cell viability , Terminal deoxynucleotidyl transferase- mediated dUTP nick-end labeling (TUNEL) method was used to detect cell apoptosis; glucose and lactate levels in the cells were measured using assay kits; Western blot was performed to detect the expression of ALOX5 , NF-κB , and glycolysis-related proteins hexokinase-2 ( HK2 ) , pyruvate kinase M2 ( PKM2 ) , glucose transporter 1 (GLUT1) in each group . Diabetic nephropathy (DN) mouse models were established using streptozotocin (STZ) and treated with SFN (0. 5 mg/kg) . Various biochemical parameters were measured in the mice , and kidney tissue pathology was examined using H&E staining. Western blot was used to detect the expression of glycolysis-related proteins (HK2 , PKM2 , GLUT1) in kidney macrophages . @*Results @# Bioinformatics analysis revealed ALOX5 as the target gene of SFN in treating DN . Compared to the HG group , SFN treatment enhanced HK-2 cell viability and in- hibited apoptosis (P < 0. 05) ; concurrently , SFN treatment suppressed HG-induced macrophage glycolysis-related protein and attenuated macrophage-mediated HK-2 cellular injury ( P < 0. 05) . Western blot results showed that SFN inhibited the expression of ALOX5 and NF-κB ( P < 0. 05) . The mouse experiment results showed that SFN treatment improved kidney function and pathological changes in the kidney of DN mice , and inhibited the related protein expression of acrophage glycolysis in kidney tissue (P < 0. 05) . @*Conclusion @#SFN improves the progression of DN by inhibiting the expression of macrophage glycolysis-related protein through the ALOX5/NF-κB signaling pathway .

Molecular mechanism of sulforaphane promoting the differentiation of bone marrow stem cells into osteoblasts / 局解手术学杂志

Objective To investigate the molecular mechanism of sulforaphane(Sul)promoting bone marrow stem cells(BMSCs)differentiating into osteoblasts.Methods BMSCs were divided into the control group(without any treatment),induction group(induction of osteogenic differentiation),and induction+Sul group(induction of osteogenic differentiation with the addition of 40 μmol/L of Sul).The adenovirus-shRNA-Mock,-shRNA-TET1,-shRNA-TET2,and-shRNA-TET3 were transfected into BMSCs as the shRNA-Mock group,shRNA-TET1 group,shRNA-TET2 group,and shRNA-TET3 group.BMSCs were cultured in cell culture medium containing osteogenic differentiation induction medium and 40 μmol/L of Sul,and then transfected with adenovirus-shRNA-TET1,-shRNA-TET2,-shRNA-TET3,and-shRNA-Mock as the induction+Sul+shRNA-TET1 group,induction+Sul+shRNA-TET2 group,induction+Sul+shRNA-TET3 group,and induction +Sul+shRNA-Mock group.The mRNA and protein expression levels of Runx2 after BMSCs differentiated into osteoblasts were determined by qPCR and Western blot.The DNA content of Runx2 promoter region bound to Histone H3 after BMSCs differentiated into osteoblasts was determined by chromatin immunocoprecipitation(ChIP).The methylation level of Runx2 promoter region of BMSCs differentiated into osteoblasts was determined by HpaⅡenzyme and MspⅠenzyme digestion combined with qPCR.The degree of BMSCs differentiated into osteoblasts was determined by alizarin red staining.Results Compared with the induction group,the mRNA and protein expression levels of Runx2 in the induction+Sul group were significantly increased(P<0.05);the content of DNA in the Runx2 promoter region bound to Histone H3 was increased(P<0.05),the methylation level of Runx2 promoter region was reduced(P<0.05),and the alizarin red staining score was elevated(P<0.05).Compared with the induction+Sul group,the content of DNA in the Runx2 promoter region bound to Histone H3 in the induction+Sul+shRNA-TET1 group was decreased(P<0.05),the methylation level of Runx2 promoter region was increased(P<0.05),and the alizarin red staining score was decreased(P<0.05).While there was no significant change among the induction+Sul+shRNA-TET2 group,induction+Sul+shRNA-TET3 group,induction+Sul+shRNA-Mock group(P>0.05).Conclusion Sul can promote the differentiation of BMSCs into osteoblasts through promoting DNA demethylation of Runx2 promoter region by TET1.

The Pharmaceutical Properties of Sulforaphane and Its Role in Tumor and Neurodegenerative Diseases / 生物化学与生物物理进展

Sulforaphane is a naturally occurring active substance derived from cruciferous vegetables with potent antioxidant and anticancer properties. Researches have shown that sulforaphane has good bioavailability and can be absorbed by the small intestine through passive transport, followed by excretion in the form of urine via the hydrophobic acid pathway. In addition, since sulforaphane is easy to be absorbed and metabolized, wrapping sulforaphane with nanomaterials can improve its bioavailability and stability, prolong its action time in human body, and better utilize its therapeutic effect. In terms of mechanism of action, sulforaphane can activate Nrf2 and HSF1 signaling pathways, induce the expression of phase II detoxification enzymes HO-1, NADPH, GST and HSP, thus regulating the concentration of oxidative stress ROS in vivo; inhibit NF-κB signaling pathway, thus suppressing the expression of inflammatory factors TNF-α, IL-1 and IL-6; regulate epigenetic modifications, thus inhibiting HDAC and DNMT, and increasing the concentration of histone H3 and H4. By regulating the expression levels of the above factors, sulforaphane can affect the occurrence and development of cancer, neurodegenerative diseases and other diseases. In recent years, several phase I/II clinical trials have shown that sulforaphane has good drug-generating properties. For example, researchers have found that patients with skin cancer have not shown any health problems and their corresponding functional problems have improved greatly after long-term use of sulforaphane. This suggests that in the future sulforaphane has a very high medicinal potential for the treatment of cancer and neurodegenerative diseases. In this paper, we review the pharmacokinetics, target of action and safety of sulforaphane and its research progress in tumor and neurodegenerative diseases to provide a reference for the future application of sulforaphane in the treatment of tumor and neurodegenerative diseases.

Therapeutic potential of sulforaphane: modulation of NRF2 mediated PI3/AKT/mTOR pathway in oral fibrosis / Potencial terapêutico do sulforafano: modulação do NRF-2 mediado o pela via PI3/AKT/mTOR na fibrose oral

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)

Progress in the role of sulforaphane in the treatment of malignant tumors / 中国医师杂志

Sulforaphane (SFN) is an isothiocyanate rich in cruciferous vegetables, and is one of the plant active substances with strong anticancer activity. SFN can protect cells from environmental carcinogens and induce growth arrest and apoptosis of various cancer cells. This article reviews the research progress of SFN in tumor prevention, treatment, and its role in tumor stem cells, explores the feasibility of SFN in clinical application for cancer prevention, and provides new strategies to reduce the development and recurrence of malignant tumors and improve patient survival.

The effects of sulforaphane on proliferation , migration and invasion of human renal carcinoma cells and its mechanism / 安徽医科大学学报

Objective@#To investigate the effects of sulforaphane (SFN) on proliferation , migration and invasion of human renal carcinoma cells and its mechanism.@*Methods@#The cultured human renal carcinoma cells 786⁃O were divided into control group (0 μmol/L) and SFN group (5 , 10 , 20 μmol/L) . The activated proliferation of cells was detected by CCK⁃8 ; the effect of SFN on migration of 786⁃O cells was detected by scratch healing assay and Transwell cell migration assay; the effect of SFN on the invasion ability of 786⁃O cells was detected by Transwell cell invasion ability assay; Western blot and qRT⁃PCR were used to detect the effects of SFN on the expression of epithelial⁃mesenchymal transition (EMT) Ⅳrelated proteins and mRNA. The effect of SFN on the expression of NF⁃κB signaling pathway was detected by Western blot. @*Results@#After SFN treatment for 24 , 48 and 72 h , the proliferation activity of 786⁃O cells decreased with the increase of SFN concentration ; compared with the control group , the cell migration and invasion ability of SFN⁃treated group were significantly reduced ; with the increase of SFN concentration , the mRNA and protein expression levels of E ⁃cadherin in 786⁃O cells increased , while the mRNA and protein expression levels of N ⁃cadherin and Vimentin decreased ; the levels of NF⁃κB signaling pathway related protein phosphorylated p65 and phosphorylated IκBα decreased with the increase of SFN concentration. @*Conclusion@#SFN may inhibit the proliferation , migration and invasion of human renal carcinoma cells by regulating the EMT process of renal carcinoma through inhibition of NF⁃κB signaling pathway.

Sulforaphane, an NRF2 agonist, alleviates ferroptosis in acute liver failure by regulating HDAC6 activity / 中西医结合学报

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.

Research advances in the protective effect of sulforaphane against liver injury and related mechanisms / 临床肝胆病杂志

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.

Effects of Sulforaphane on the Proliferation and Apoptosis of Human Renal Tubular Epithelial Cells Induced by High Glucose and Its Mechanism / 中国药房

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.

Inhibiting autophagy enhances sulforaphane-induced apoptosis

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

Sulforaphane Suppressed Stemness Characters of Lung Adenocarcinoma Stem Cells by Down-regulating Methylation of miR-200c Promoter / 肿瘤防治研究

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.

Research progress in regulatory effect of sulforaphane on diabetes and fatty liver disease / 中国药理学与毒理学杂志

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.

Effects of sulforaphane on brain mitochondria: mechanistic view and future directions / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

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.

Effects of sulforaphane on brain mitochondria: mechanistic view and future directions / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

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.

Sulforaphane attenuates renal ischemia reperfusion injury in mice by Nrf-2 against inflammation / 西安交通大学学报(医学版)

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.

Impacts of sulforaphane on the Wnt/β-catenin pathway of cultured retinoblastoma cells / 国际中医中药杂志

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.

The Anti-Inflammatory Effect of Sulforaphane in Mice with Experimental Autoimmune Encephalomyelitis

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.

Nrf2-Heme oxygenase-1 modulates autophagy and inhibits apoptosis triggered by elevated glucose levels in renal tubule cells

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.

Neuroprotective effects of an Nrf2 agonist on high glucose-induced damage in HT22 cells

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.