A novel combinatorial approach using sulforaphane- and withaferin A-rich extracts for prevention of estrogen receptor-negative breast cancer through epigenetic and gut microbial mechanisms.

Estrogen receptor-negative [ER(-)] mammary cancer is the most aggressive type of breast cancer (BC) with higher rate of metastasis and recurrence. In recent years, dietary prevention of BC with epigenetically active phytochemicals has received increased attention due to its feasibility, effectiveness, and ease of implementation. In this regard, combinatorial phytochemical intervention enables more efficacious BC inhibition by simultaneously targeting multiple tumorigenic pathways. We, therefore, focused on investigation of the effect of sulforaphane (SFN)-rich broccoli sprouts (BSp) and withaferin A (WA)-rich Ashwagandha (Ash) combination on BC prevention in estrogen receptor-negative [ER(-)] mammary cancer using transgenic mice. Our results indicated that combinatorial BSp + Ash treatment significantly reduced tumor incidence and tumor growth (~ 75%) as well as delayed (~ 21%) tumor latency when compared to the control treatment and combinatorial BSp + Ash treatment was statistically more effective in suppressing BC compared to single BSp or Ash intervention. At the molecular level, the BSp and Ash combination upregulated tumor suppressors (p53, p57) along with apoptosis associated proteins (BAX, PUMA) and BAX:BCL-2 ratio. Furthermore, our result indicated an expressional decline of epigenetic machinery HDAC1 and DNMT3A in mammary tumor tissue because of combinatorial treatment. Interestingly, we have reported multiple synergistic interactions between BSp and Ash that have impacted both tumor phenotype and molecular expression due to combinatorial BSp and Ash treatment. Our RNA-seq analysis results also demonstrated a transcriptome-wide expressional reshuffling of genes associated with multiple cell-signaling pathways, transcription factor activity and epigenetic regulations due to combined BSp and Ash administration. In addition, we discovered an alteration of gut microbial composition change because of combinatorial treatment. Overall, combinatorial BSp and Ash supplementation can prevent ER(-) BC through enhanced tumor suppression, apoptosis induction and transcriptome-wide reshuffling of gene expression possibly influencing multiple cell signaling pathways, epigenetic regulation and reshaping gut microbiota.

Reversal of high-glucose-induced transcriptional and epigenetic memories through NRF2 pathway activation.

Diabetes complications such as nephropathy, retinopathy, or cardiovascular disease arise from vascular dysfunction. In this context, it has been observed that past hyperglycemic events can induce long-lasting alterations, a phenomenon termed "metabolic memory." In this study, we evaluated the genome-wide gene expression and chromatin accessibility alterations caused by transient high-glucose exposure in human endothelial cells (ECs) in vitro. We found that cells exposed to high glucose exhibited substantial gene expression changes in pathways known to be impaired in diabetes, many of which persist after glucose normalization. Chromatin accessibility analysis also revealed that transient hyperglycemia induces persistent alterations, mainly in non-promoter regions identified as enhancers with neighboring genes showing lasting alterations. Notably, activation of the NRF2 pathway through NRF2 overexpression or supplementation with the plant-derived compound sulforaphane, effectively reverses the glucose-induced transcriptional and chromatin accessibility memories in ECs. These findings underscore the enduring impact of transient hyperglycemia on ECs' transcriptomic and chromatin accessibility profiles, emphasizing the potential utility of pharmacological NRF2 pathway activation in mitigating and reversing the high-glucose-induced transcriptional and epigenetic alterations.

Two new and effective food-extracted immunomodulatory agents exhibit anti-inflammatory response activity in the hACE2 acute lung injury murine model of COVID-19.

Objective: The coronavirus disease 2019 (COVID-19) spread rapidly and claimed millions of lives worldwide. Acute respiratory distress syndrome (ARDS) is the major cause of COVID-19-associated deaths. Due to the limitations of current drugs, developing effective therapeutic options that can be used rapidly and safely in clinics for treating severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections is necessary. This study aims to investigate the effects of two food-extracted immunomodulatory agents, ajoene-enriched garlic extract (AGE) and cruciferous vegetables-extracted sulforaphane (SFN), on anti-inflammatory and immune responses in a SARS-CoV-2 acute lung injury mouse model. Methods: In this study, we established a mouse model to mimic the SARS-CoV-2 infection acute lung injury model via intratracheal injection of polyinosinic:polycytidylic acid (poly[I:C]) and SARS-CoV-2 recombinant spike protein (SP). After the different agents treatment, lung sections, bronchoalveolar lavage fluid (BALF) and fresh faeces were harvested. Then, H&E staining was used to examine symptoms of interstitial pneumonia. Flow cytometry was used to examine the change of immune cell populations. Multiplex cytokines assay was used to examine the inflammatory cytokines.16S rDNA high-throughput sequencing was used to examine the change of gut microbiome. Results: Our results showed that AGE and SFN significantly suppressed the symptoms of interstitial pneumonia, effectively inhibited the production of inflammatory cytokines, decreased the percentage of inflammatory cell populations, and elevated T cell populations in the mouse model. Furthermore, we also observed that the gut microbiome of genus Paramuribaculum were enriched in the AGE-treated group. Conclusion: Here, for the first time, we observed that these two novel, safe, and relatively inexpensive immunomodulatory agents exhibited the same effects on anti-inflammatory and immune responses as neutralizing monoclonal antibodies (mAbs) against interleukin 6 receptor (IL-6R), which have been suggested for treating COVID-19 patients. Our results revealed the therapeutic ability of these two immunomodulatory agents in a mouse model of SARS-CoV-2 acute lung injury by promoting anti-inflammatory and immune responses. These results suggest that AGE and SFN are promising candidates for the COVID-19 treatment.

Sulforaphane decreases oxidative stress and inhibits NLRP3 inflammasome activation in a mouse model of ulcerative colitis.

Excessive oxidative stress and NLRP3 inflammasome activation are considered the main drivers of inflammatory bowel disease (IBD), and inhibition of inflammasomes ameliorates clinical symptoms and morphological manifestations of IBD. Herein, we examined the roles of NLRP3 activation in IBD and modulation of NLRP3 by sulforaphane (SFN), a compound with multiple pharmacological activities that is extracted from cruciferous plants. To simulate human IBD, we established a mouse colitis model by administering dextran sodium sulfate in the drinking water. SFN (25, 50 mg·kg-1·d-1, ig) or the positive control sulfasalazine (500 mg/kg, ig) was administered to colitis-affected mice for 7 days. Model mice displayed pathological alterations in colon tissue as well as classic symptoms of colitis beyond substantial tissue inflammation. Expression of NLRP3, ASC, and caspase-1 was significantly elevated in the colonic epithelium. The expression of NLRP3 inflammasomes led to activation of downstream proteins and increases in the cytokines IL-18 and IL-1ß. SFN administration either fully or partially reversed these changes, thus restoring IL-18 and IL-1ß, substantially inhibiting NLRP3 activation, and decreasing inflammation. SFN alleviated the inflammation induced by LPS and NLRP3 agonists in RAW264.7 cells by decreasing the levels of reactive oxygen species. In summary, our results revealed the pathological roles of oxidative stress and NLRP3 in colitis, and indicated that SFN might serve as a natural NLRP3 inhibitor, thereby providing a new strategy for alternative colitis treatment.

CC Chemokine Family Members' Modulation as a Novel Approach for Treating Central Nervous System and Peripheral Nervous System Injury-A Review of Clinical and Experimental Findings.

Despite significant progress in modern medicine and pharmacology, damage to the nervous system with various etiologies still poses a challenge to doctors and scientists. Injuries lead to neuroimmunological changes in the central nervous system (CNS), which may result in both secondary damage and the development of tactile and thermal hypersensitivity. In our review, based on the analysis of many experimental and clinical studies, we indicate that the mechanisms occurring both at the level of the brain after direct damage and at the level of the spinal cord after peripheral nerve damage have a common immunological basis. This suggests that there are opportunities for similar pharmacological therapeutic interventions in the damage of various etiologies. Experimental data indicate that after CNS/PNS damage, the levels of 16 among the 28 CC-family chemokines, i.e., CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL11, CCL12, CCL17, CCL19, CCL20, CCL21, and CCL22, increase in the brain and/or spinal cord and have strong proinflammatory and/or pronociceptive effects. According to the available literature data, further investigation is still needed for understanding the role of the remaining chemokines, especially six of them which were found in humans but not in mice/rats, i.e., CCL13, CCL14, CCL15, CCL16, CCL18, and CCL23. Over the past several years, the results of studies in which available pharmacological tools were used indicated that blocking individual receptors, e.g., CCR1 (J113863 and BX513), CCR2 (RS504393, CCX872, INCB3344, and AZ889), CCR3 (SB328437), CCR4 (C021 and AZD-2098), and CCR5 (maraviroc, AZD-5672, and TAK-220), has beneficial effects after damage to both the CNS and PNS. Recently, experimental data have proved that blockades exerted by double antagonists CCR1/3 (UCB 35625) and CCR2/5 (cenicriviroc) have very good anti-inflammatory and antinociceptive effects. In addition, both single (J113863, RS504393, SB328437, C021, and maraviroc) and dual (cenicriviroc) chemokine receptor antagonists enhanced the analgesic effect of opioid drugs. This review will display the evidence that a multidirectional strategy based on the modulation of neuronal-glial-immune interactions can significantly improve the health of patients after CNS and PNS damage by changing the activity of chemokines belonging to the CC family. Moreover, in the case of pain, the combined administration of such antagonists with opioid drugs could reduce therapeutic doses and minimize the risk of complications.

Chiral Thianthrenes.

The absolute configuration and stability of two thianthrene chiral sulfoxides has been determined by means of X-ray single-crystal structure determinations. The analyses and configurations allow verification that the diastereomeric sulfoxides are stable in solution and are not interconverting, which has been suggested in some studies of sulfoxides. The two thianthrene sulfoxides have slightly different Rf values, which allowed their separation using flash chromatography on silica. The spots run back-to-back, which posed a challenge for their separation. The pure, separated compounds in solution remain as separate, single spots on a Thin Layer Chromatography (TLC) plate.

The Experimental and In Silico-Based Evaluation of NRF2 Modulators, Sulforaphane and Brusatol, on the Transcriptome of Immortalized Bovine Mammary Alveolar Cells.

Changes during the production cycle of dairy cattle can leave these animals susceptible to oxidative stress and reduced antioxidant health. In particular, the periparturient period, when dairy cows must rapidly adapt to the sudden metabolic demands of lactation, is a period when the production of damaging free radicals can overwhelm the natural antioxidant systems, potentially leading to tissue damage and reduced milk production. Central to the protection against free radical damage and antioxidant defense is the transcription factor NRF2, which activates an array of genes associated with antioxidant functions and cell survival. The objective of this study was to evaluate the effect that two natural NRF2 modulators, the NRF2 agonist sulforaphane (SFN) and the antagonist brusatol (BRU), have on the transcriptome of immortalized bovine mammary alveolar cells (MACT) using both the RT-qPCR of putative NRF2 target genes, as well as RNA sequencing approaches. The treatment of cells with SFN resulted in the activation of many putative NRF2 target genes and the upregulation of genes associated with pathways involved in cell survival, metabolism, and antioxidant function while suppressing the expression of genes related to cellular senescence and DNA repair. In contrast, the treatment of cells with BRU resulted in the upregulation of genes associated with inflammation, cellular stress, and apoptosis while suppressing the transcription of genes involved in various metabolic processes. The analysis also revealed several novel putative NRF2 target genes in bovine. In conclusion, these data indicate that the treatment of cells with SFN and BRU may be effective at modulating the NRF2 transcriptional network, but additional effects associated with cellular stress and metabolism may complicate the effectiveness of these compounds to improve antioxidant health in dairy cattle via nutrigenomic approaches.

Comparative analysis of isothiocyanates in eight cruciferous vegetables and evaluation of the hepatoprotective effects of 4-(methylsulfinyl)-3-butenyl isothiocyanate (sulforaphene) from daikon radish (Raphanus sativus L.) sprouts.

The contributions of cruciferous vegetables to human health are widely recognised, particularly at the molecular level, where their isothiocyanates play a significant role. However, compared to the well-studied isothiocyanate 4-(methylsulfinyl)butyl isothiocyanate (sulforaphane) produced from broccoli sprouts, less is known about the pharmacological effects of other isothiocyanates and the stage of vegetables preferable to obtain their benefits. We analysed the quantity and quality of isothiocyanates produced in both the sprouts and mature stages of eight cruciferous vegetables using gas chromatography-mass spectrometry (GC-MS). Additionally, we investigated the hepatoprotective effects of isothiocyanates in a mouse model of acute hepatitis induced by carbon tetrachloride (CCl4). Furthermore, we explored the detoxification enzyme-inducing activities of crude sprout extracts in normal rats. Among the eight cruciferous vegetables, daikon radish (Raphanus sativus L.) sprouts produced the highest amount of isothiocyanates, with 4-(methylsulfinyl)-3-butenyl isothiocyanate (sulforaphene) being the dominant compound. The amount of sulforaphene in daikon radish sprouts was approximately 30 times that of sulforaphane in broccoli sprouts. Sulforaphene demonstrated hepatoprotective effects similar to sulforaphane in ameliorating CCl4-induced hepatic injury in mice. A crude extract of 3-day-old daikon radish sprouts upregulated the detoxifying enzyme glutathione S-transferase (GST) in the liver, whereas the crude extract of broccoli sprouts showed limited upregulation. This study highlights that daikon radish sprouts and sulforaphene have the potential to serve as functional food materials with hepatoprotective effects. Furthermore, daikon radish sprouts may exhibit more potent hepatoprotective effects compared to broccoli sprouts.

The Effect of Broccoli Glucoraphanin Supplementation on Ameliorating High-Fat-Diet-Induced Obesity through the Gut Microbiome and Metabolome Interface.

SCOPE: Obesity and its metabolic comorbidities pose a major global challenge for public health. Glucoraphanin (GRN) is a natural bioactive compound enriched in broccoli that is known to have potential health benefits against various human chronic diseases. METHODS AND RESULTS: This study investigats the effects of broccoli GRN supplementation on body weight, metabolic parameters, gut microbiome and metabolome associated with obesity. The study is conducted on an obese-related C57BL/6J mouse model through the treatment of normal control diet, high-fat diet (HFD)and GRN-supplemented HFD (HFD-GRN) to determine the metabolic protection of GRN. The results shows that GRN treatment alleviates obesity-related traits leading to improved glucose metabolism in HFD-fed animals. Mechanically, the study noticed that GRN significantly shifts the gut microbial diversity and composition to an eubiosis status. GRN supplement also significantly alters plasma metabolite profiles. Further integrated analysis reveal a complex interaction between the gut microbes and host metabolism that may contribute to GRN-induced beneficial effects against HFD. CONCLUSION: These results indicate that beneficial effects of broccoli GRN on reversing HFD-induced adverse metabolic parameters may be attributed to its impacts on reprogramming microbial community and metabolites. Identification of the mechanistic functions of GRN further warrants it as a dietary candidate for obesity prevention.

Sulforaphane Inhibits Adhesion and Migration of Cisplatin- and Gemcitabine-Resistant Bladder Cancer Cells In Vitro.

Only 20% of patients with muscle-invasive bladder carcinoma respond to cisplatin-based chemotherapy. Since the natural phytochemical sulforaphane (SFN) exhibits antitumor properties, its influence on the adhesive and migratory properties of cisplatin- and gemcitabine-sensitive and cisplatin- and gemcitabine-resistant RT4, RT112, T24, and TCCSUP bladder cancer cells was evaluated. Mechanisms behind the SFN influence were explored by assessing levels of the integrin adhesion receptors ß1 (total and activated) and ß4 and their functional relevance. To evaluate cell differentiation processes, E- and N-cadherin, vimentin and cytokeratin (CK) 8/18 expression were examined. SFN down-regulated bladder cancer cell adhesion with cell line and resistance-specific differences. Different responses to SFN were reflected in integrin expression that depended on the cell line and presence of resistance. Chemotactic movement of RT112, T24, and TCCSUP (RT4 did not migrate) was markedly blocked by SFN in both chemo-sensitive and chemo-resistant cells. Integrin-blocking studies indicated ß1 and ß4 as chemotaxis regulators. N-cadherin was diminished by SFN, particularly in sensitive and resistant T24 and RT112 cells, whereas E-cadherin was increased in RT112 cells (not detectable in RT4 and TCCSup cells). Alterations in vimentin and CK8/18 were also apparent, though not the same in all cell lines. SFN exposure resulted in translocation of E-cadherin (RT112), N-cadherin (RT112, T24), and vimentin (T24). SFN down-regulated adhesion and migration in chemo-sensitive and chemo-resistant bladder cancer cells by acting on integrin ß1 and ß4 expression and inducing the mesenchymal-epithelial translocation of cadherins and vimentin. SFN does, therefore, possess potential to improve bladder cancer therapy.

Optimizing combination therapy in prostate cancer: mechanistic insights into the synergistic effects of Paclitaxel and Sulforaphane-induced apoptosis.

BACKGROUND: Combination therapies in cancer treatment have demonstrated synergistic or additive outcomes while also reducing the development of drug resistance compared to monotherapy. This study explores the potential of combining the chemotherapeutic agent Paclitaxel (PTX) with Sulforaphane (SFN), a natural compound primarily found in cruciferous vegetables, to enhance treatment efficacy in prostate cancer. METHODS: Two prostate cancer cell lines, PC-3 and LNCaP, were treated with varying concentrations of PTX, SFN, and their combination. Cell viability was assessed using the thiazolyl blue tetrazolium bromide (MTT) assay to determine the EC50 values. Western blot analysis was conducted to evaluate the expression of Bax, Bcl2, and Caspase-3 activation proteins in response to individual and combined treatments of PTX and SFN. Fluorescent microscopy was employed to observe morphological changes indicative of apoptotic stress in cell nuclei. Flow cytometry analysis was utilized to assess alterations in cell cycle phases, such as redistribution and arrest. Statistical analyses, including Student's t-tests and one-way analysis of variance with Tukey's correction, were performed to determine significant differences between mono- and combination treatments. RESULTS: The impact of PTX, SFN, and their combination on cell viability reduction was evaluated in a dose-dependent manner. The combined treatment enhanced PTX's effects and decreased the EC50 values of both drugs compared to individual treatments. PTX and SFN treatments differentially regulated the expression of Bax and Bcl2 proteins in PC-3 and LNCaP cell lines, favoring apoptosis over cell survival. Our data indicated that combination therapy significantly increased Bax protein expression and the Bax/Bcl2 ratio compared to PTX or SFN alone. Flow cytometry analysis revealed alterations in cell cycle phases, including S-phase arrest and an increased population of apoptotic cells. Notably, the combination treatments did not have a discernible impact on necrotic cells. Signs of apoptotic cell death were confirmed through Caspase-3 cleavage, and morphological changes in cell nuclei were assessed via western blot and fluorescent microscopy. CONCLUSION: This combination therapy of PTX and SFN has the potential to improve prostate cancer treatment by minimizing side effects while maintaining efficacy. Mechanistic investigations revealed that SFN enhances PTX efficacy by promoting apoptosis, activating caspase-3, inducing nuclear morphology changes, modulating the cell cycle, and altering Bax and Bcl2 protein expression. These findings offer valuable insights into the synergistic effects of PTX and SFN, supporting the optimization of combination therapy and providing efficient therapeutic strategies in preclinical research.

Sulforaphane-Enriched Extracts from Broccoli Exhibit Antimicrobial Activity against Plant Pathogens, Promising a Natural Antimicrobial Agent for Crop Protection.

Sulforaphane (SFN) is one of the hydrolysates of glucosinolates (GSLs), primarily derived from Brassica vegetables like broccoli. In clinical therapy, SFN has been proven to display antimicrobial, anticancer, antioxidant, and anti-inflammatory properties. However, the antimicrobial effects and mechanism of SFN against plant pathogens need to be further elucidated, which limits its application in agriculture. In this study, the genetic factors involved in SFN biosynthesis in 33 B. oleracea varieties were explored. The finding showed that besides the genetic background of different B. oleracea varieties, myrosinase and ESP genes play important roles in affecting SFN content. Subsequently, the molecular identification cards of these 33 B. oleracea varieties were constructed to rapidly assess their SFN biosynthetic ability. Furthermore, an optimized protocol for SFN extraction using low-cost broccoli curds was established, yielding SFN-enriched extracts (SFN-ee) containing up to 628.44 µg/g DW of SFN. The antimicrobial activity assay confirmed that SFN-ee obtained here remarkably inhibit the proliferation of nine tested microorganisms including four plant pathogens by destroying their membrane integrity. Additionally, the data demonstrated that exogenous application of SFN-ee could also induce ROS accumulation in broccoli leaves. These results indicated that SFN-ee should play a dual role in defense against plant pathogens by directly killing pathogenic cells and activating the ROS signaling pathway. These findings provide new evidence for the antimicrobial effect and mechanism of SFN against plant pathogens, and suggest that SFN-ee can be used as a natural plant antimicrobial agent for crop protection and food preservation.

Novel Meta-Diamide Compounds Containing Sulfide Derivatives Were Designed and Synthesized as Potential Pesticides.

The meta-diamide (m-diamide) insecticide, Broflanilide, was characterized by its high efficiency, low toxicity and lack of cross-resistance with traditional GABA receptors. In accordance with the principles of drug molecular design, easily derivable sulfur with diverse bioactivities was introduced while leading with the parent Broflanilide. Twelve novel m-diamide target compounds containing sulfide derivatives were synthesized through exploration guided by the literature. Their structures were confirmed by melting points, 1H NMR, 13C NMR and HRMS. Insecticidal activity assessments revealed that most target compounds A-D exhibited 100% lethality against Plutella xylostella (P. xylostella) and Aphis craccivora Koch (A. craccivora) at 500 mg·L-1. Notably, for P. xylostella, compounds C-2, C-3, C-4 and D-2 demonstrated 60.00-100.00% insecticidal activity even at a concentration as low as 0.625 mg·L-1. As determined by structure-activity relationship (SAR) analysis, compounds with R1 = CH3 and R2 = Br (B-1, C-2 and D-2) and sulfoxide compound C-3 contained 100.00% lethality against A. craccivora at 500 mg·L-1, surpassing the lethality when leading with the parent Broflanilide in terms of efficacy. Consequently, it can be inferred that the sulfoxide compound (C-3) requires further investigation as a potential active molecule for new insecticides. These explorations provide valuable references for future research on the synthesis and insecticidal activities of sulfide-containing m-diamide compounds.

Balanced activation of Nrf-2/ARE mediates the protective effect of sulforaphane on keratoconus in the cell mechanical microenvironment.

Keratoconus (KC) is a progressive degenerative disease that usually occurs bilaterally and is characterized by corneal thinning and apical protrusion of the cornea. Oxidative stress is an indicator of the accumulation of reactive oxygen species (ROS), and KC keratocytes exhibit increased ROS production compared with that of normal keratocytes. Therefore, oxidative stress in KC keratocytes may play a major role in the development and progression of KC. Here, we investigated the protective effect of sulforaphane (SF) antioxidants using a hydrogel-simulated model of the cell mechanical microenvironment of KC. The stiffness of the KC matrix microenvironment in vitro was 16.70 kPa and the stiffness of the normal matrix microenvironment was 34.88 kPa. Human keratocytes (HKs) were cultured for 24 h before observation or drug treatment with H2O2 in the presence or absence of SF. The levels of oxidative stress, nuclear factor E2-related factor 2 (Nrf-2) and antioxidant response element (ARE) were detected. The high-stress state of HKs in the mechanical microenvironment of KC cells compensates for the activation of the Nrf-2/ARE signaling pathway. H2O2 leads to increased oxidative stress and decreased levels of antioxidant proteins in KC. In summary, SF can reduce endogenous and exogenous oxidative stress and increase the antioxidant capacity of cells.

Sulforaphane's role in Redefining autophagic Responses in depression associated with polycystic ovarian syndrome: Unveiling the SIRT1/AMPK/LKB1 pathway connection.

Polycystic ovarian syndrome (PCOS) has been associated with depression and suicidal ideations in females. Studies have highlighted the role of autophagic deficiency in depression pathogenesis. Sulforaphane (SFN) is a natural product that improved autophagic deficiency and showed antidepressant activity in depressed patients. Herein, the study aimed to evaluate the impact of using SFN on depression-associated with PCOS via hippocampal energy sensors and cellular bioenergetics. PCOS was induced by administering letrozole (1 mg/kg, p. o.) for 21 days, followed by SFN treatment (0.5 mg/kg, i. p.) for one week. Two days before euthanasia, PCOS rats showed anhedonic behavior in the sucrose preference test and increased immobility time in the forced swimming test. Depressed rats showed a reduction in nuclear SIRT1 and an elevated cytoplasmic one. This was associated with a reduction in phosphorylation of energy sensors, liver kinase B1 (LKB1), and adenosine monophosphate kinase (AMPK), along with an imbalance of autophagic markers such as Beclin-1, microtubule-associated protein I/II light chain 3, autophagy enzyme 7 and selective autophagy receptor P62. Additionally, Nrf2 and KEAP1 levels were decreased. These abnormalities were alleviated by SFN treatment, as evidenced by the nuclear translocation of SIRT1 and the repression of downstream proteins, including FOXO1, NF-κB, and TNF-α production. These changes were reflected in improved behavioral performance in the sucrose preference test (SPT) and forced swimming test (FST). The antidepressant effects of SFN were counteracted by an autophagic inhibitor, 3-methyladenine. Eventually, SFN, as a nutraceutical, has a promising antidepressant effect via restoring autophagic-related depression in the PCOS rat model.

Comment on Magner et al. Sulforaphane Treatment in Children with Autism: A Prospective Randomized Double-Blind Study. Nutrients 2023, 15, 718.

I read your recent interesting double-blind study by Magner et al. [...].

Versatile ferrous oxidation-xylenol orange assay for high-throughput screening of lipoxygenase activity.

Lipoxygenases (LOXs) catalyze dioxygenation of polyunsaturated fatty acids (PUFAs) into fatty acid hydroperoxides (FAHPs), which can be further transformed into a number of value-added compounds. LOXs have garnered interest as biocatalysts for various industrial applications. Therefore, a high-throughput LOX activity assay is essential to evaluate their performance under different conditions. This study aimed to enhance the suitability of the ferrous-oxidized xylenol orange (FOX) assay for screening LOX activity across a wide pH range with different PUFAs. The narrow linear detection range of the standard FOX assay restricts its utility in screening LOX activity. To address this, the concentration of perchloric acid in the xylenol orange reagent was adjusted. The modified assay exhibited a fivefold expansion in the linear detection range for hydroperoxides and accommodated samples with pH values ranging from 3 to 10. The assay could quantify various hydroperoxide species, indicating its applicability in assessing LOX substrate preferences. Due to sensitivity to pH, buffer types, and hydroperoxide species, the assay required calibration using the respective standard compound diluted in the same buffer as the measured sample. The use of correction factors is suggested when financial constraints limit the use of FAHP standard compounds in routine LOX substrate preference analysis. FAHP quantification by the modified FOX assay aligned well with results obtained using the commonly used conjugated diene method, while offering a quicker and broader sample pH range assessment. Thus, the modified FOX assay can be used as a reliable high-throughput screening method for determining LOX activity. KEY POINTS: • Modifying perchloric acid level in FOX reagent expands its linear detection range • The modified FOX assay is applicable for screening LOX activity in a wide pH range • The modified FOX assay effectively assesses substrate specificity of LOX.

Sulforaphane inhibits the migration and invasion of BPDE-induced lung adenocarcinoma cells by regulating NLRP12.

This study aims to explore the impact and underlying mechanism of sulforaphane (SFN) intervention on the migration and invasion of lung adenocarcinoma induced by 7, 8-dihydroxy-9, 10-epoxy-benzo (a) pyrene (BPDE). Human lung adenocarcinoma A549 cells were exposed to varying concentrations of BPDE (0.25, 0.50, and 1.00 µM) and subsequently treated with 5 µM SFN. Cell viability was determined using CCK8 assay, while migration and invasion were assessed using Transwell assays. Lentivirus transfection was employed to establish NLRP12 overexpressing A549 cells. ELISA was utilized to quantify IL-33, CXCL12, and CXCL13 levels in the supernatant, while quantitative real-time PCR (qRT-PCR) and Western Blot were used to analyze the expression of NLRP12 and key factors associated with canonical and non-canonical NF-κB pathways. Results indicated an increase in migratory and invasive capabilities, concurrent with heightened expression of IL-33, CXCL12, CXCL13, and factors associated with both canonical and non-canonical NF-κB pathways. Moreover, mRNA and protein levels of NLRP12 were decreased in BPDE-stimulated A549 cells. Subsequent SFN intervention attenuated BPDE-induced migration and invasion of A549 cells. Lentivirus-mediated NLRP12 overexpression not only reversed the observed phenotype in BPDE-induced cells but also led to a reduction in the expression of critical factors associated with both canonical and non-canonical NF-κB pathways. Collectively, we found that SFN could inhibit BPDE-induced migration and invasion of A549 cells by upregulating NLRP12, thereby influencing both canonical and non-canonical NF-κB pathways.

Pharmacological effects of methysticin and L-sulforaphane through the Nrf2/ARE signaling pathway in MLO-Y4 osteocytes: in vitro study.

BACKGROUND: Oxidative stress plays a crucial role in the pathogenesis of many skeletal diseases by inducing osteocyte death. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulator of various antioxidant gene expressions through antioxidant response element (ARE) against cellular oxidative stress and can be induced by various stimulants, including the phytochemicals methysticin (MET) and L-sulforaphane (SFN). This study aimed to establish an osteocyte in vitro model to investigate the pharmacological effects of MET and SFN on the Nrf2/ARE pathway. METHODS: MLO-Y4 murine osteocytes and the stably transduced MLO-Y4-SIN-lenti-ARE reporter gene cell line were used. MET and SFN were used as Nrf2 inducers. The cytotoxicity of MET, SFN, and hydrogen peroxide (H2O2) was evaluated using the CytoTox-Glo™ Assay. Time- and dose-dependent ARE induction was examined by Monoluciferase Assay. The mRNA and protein expressions of Nrf2 target markers, such as heme-oxygenase 1 (Ho-1), NADPH quinone dehydrogenase 1 (Nqo1), and thioredoxin reductase 1 (Txnrd1), were detected by RT-qPCR, Western Blot, and immunofluorescence staining, respectively. Osteogenesis markers, osteopontin, and osteocalcin were compared with and without treatment by immunofluorescence staining. RESULTS: The experimental data showed that MET and SFN induced ARE activity in a time- and dose-dependent manner and increased the mRNA and protein expression of antioxidant markers compared to vehicle-treated controls. The protein expression of osteopontin and osteocalcin in the samples treated with SFN were significantly higher than without treatment, and the number of cell death treated with SFN was significantly lower than without treatment under H2O2-induced stress conditions. CONCLUSIONS: Nrf2 inducers MET and SFN increased the mRNA expression of antioxidant genes through the Nrf2/ARE pathway in osteocytes. Notably, SFN increased the protein expression of osteocyte-associated osteogenic markers and suppressed cell death under H2O2-induced stress condition. Thus, Nrf2 stimulators can exert stress-relieving and osteogenic effects on osteocytes.

Antinociceptive effects of Raphanus sativus sprouts involve the opioid and 5-HT1A serotonin receptors, cAMP/cGMP pathways, and the central activity of sulforaphane.

Raphanus sativus L. cv. Sango, commonly known as red radish, is widely consumed around the world as a vegetable, but its benefit in pain relief is not sufficiently investigated. This study aimed to evaluate the antinociceptive effects of R. sativus and a possible mechanism of action. An aqueous extract of R. sativus sprouts (AERSS) was investigated by parenteral (10, 30, and 100 mg kg-1, i.p.) and enteral (500 mg kg-1, p.o.) administration in the neurogenic and inflammatory phases of the formalin test, where gastric damage was also evaluated as a possible adverse effect. Ketorolac (5 mg kg-1, i.p.) was used as the reference drug. Endogenous opioid and 5-HT1A serotonin receptors, as well as the cAMP/NO-cGMP pathways, were explored in the study of a possible mechanism of action by using their corresponding antagonists: naloxone, 1 mg kg-1, i.p., WAY100635, 1 mg kg-1, i.p., and enzymatic activators or inhibitors, respectively. Sulforaphane (SFN), a known bioactive metabolite, was analyzed using electroencephalography (EEG) to evidence its central involvement. A significant and dose-dependent antinociceptive activity was observed with the AERSS resembling the antinociceptive effect of the reference drug, with an equivalent significant response with a dose of 500 mg kg-1, p.o. without causing gastric damage. The participation of the endogenous opioid and 5-HT1A serotonin receptors at central and peripheral levels was also observed, with a differential participation of cAMP/NO-cGMP. SFN as one metabolite produced significant changes in the EEG analysis, reinforcing its effects on the CNS. Our preclinical evidence supports the benefits of consuming Raphanus sativus cv. Sango sprouts for pain relief.