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SFN, a dietary phytochemical, is a significant member of isothiocyanates present in cruciferous vegetables at high levels in broccoli. It is a well-known activator of the Nrf2/ARE antioxidant pathway. Long since, the therapeutic effects of SFN have been widely studied in several different diseases. Other than the antioxidant effect, SFN also exhibits an anti-inflammatory effect through suppression of various mechanisms, including inflammasome activation. Considerably, SFN has been demonstrated to inhibit multiple inflammasomes, including NLRP3 inflammasome. NLRP3 inflammasome induces secretion of pro-inflammatory cytokines and promotes inflammatory cell death. The release of pro-inflammatory cytokines enhances the inflammatory response, in turn leading to tissue damage. These self-propelling inflammatory responses would need modulation with exogenous therapeutic agents to suppress them. SFN is a promising candidate molecule for the mitigation of NLRP3 inflammasome activation, which has been related to the pathogenesis of numerous disorders. In this review, we have provided fundamental knowledge about Sulforaphane, elaborated its characteristics, and evidentially focused on its mechanisms of action with regard to its anti-inflammatory, anti-oxidative, and neuroprotective features. Thereafter, we have summarized both in vitro and in vivo studies regarding SFN effect on NLRP3 inflammasome activation.

NLRP3 inflammasome is a part of the innate immune system and responsible for the rapid identification and eradication of pathogenic microbes, metabolic stress products, reactive oxygen species, and other exogenous agents. NLRP3 inflammasome is overactivated in several neurodegenerative, cardiac, pulmonary, and metabolic diseases. Therefore, suppression of inflammasome activation is of utmost clinical importance. Melatonin is a ubiquitous hormone mainly produced in the pineal gland with circadian rhythm regulatory, antioxidant, and immunomodulatory functions. Melatonin is a natural product and safer than most chemicals to use for medicinal purposes. Many in vitro and in vivo studies have proved that melatonin alleviates NLRP3 inflammasome activity via various intracellular signaling pathways. In this review, the effect of melatonin on the NLRP3 inflammasome in the context of diseases will be discussed.

Mesenchymal stem cells (MSCs) maintain the musculoskeletal system by differentiating into multiple lineages, including osteoblasts and adipocytes. Mechanical signals, including strain and low-intensity vibration (LIV), are important regulators of MSC differentiation via control exerted through the cell structure. Lamin A/C is a protein vital to the nuclear architecture that supports chromatin organization and differentiation and contributes to the mechanical integrity of the nucleus. We investigated whether lamin A/C and mechanoresponsiveness are functionally coupled during adipogenesis in MSCs. siRNA depletion of lamin A/C increased the nuclear area, height, and volume and decreased the circularity and stiffness. Lamin A/C depletion significantly decreased markers of adipogenesis (adiponectin, cellular lipid content) as did LIV treatment despite depletion of lamin A/C. Phosphorylation of focal adhesions in response to mechanical challenge was also preserved during loss of lamin A/C. RNA-seq showed no major adipogenic transcriptome changes resulting from LIV treatment, suggesting that LIV regulation of adipogenesis may not occur at the transcriptional level. We observed that during both lamin A/C depletion and LIV, interferon signaling was downregulated, suggesting potentially shared regulatory mechanism elements that could regulate protein translation. We conclude that the mechanoregulation of adipogenesis and the mechanical activation of focal adhesions function independently from those of lamin A/C.

The NLRP3 inflammasome is a multiprotein complex that activates caspase-1 and triggers the release of the proinflammatory cytokines IL-1 beta and IL-18 in response to diverse signals. Although inflammasome activation plays critical roles against various pathogens in host defense, overactivation of inflammasome contributes to the pathogenesis of inflammatory diseases, including acute CNS injuries and chronic neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. In the current study, we demonstrated that Sulforaphane (SFN), a dietary natural product, inhibits NLRP3 inflammasome mediated IL-1 beta and IL-18 secretion and pyroptosis in murine microglial cells. SFN decreased the secretion of IL-1 beta and IL-18, and their mRNA levels in LPS primed microglia triggered by ATP. SFN suppressed the overexpression of cleaved caspase-1 and NLRP3 protein expressions as measured by caspase activity assay and western blot, respectively. SFN also prevented caspase-1 dependent pyroptotic cell death in microglia. Our data indicate that SFN suppresses NLRP3 inflammasome via the inhibition of NF-kappa B nuclear translocation and Nrf2 mediated miRNAs expression modulation in murine microglia.

Mesenchymal stem cells (MSC) maintain the musculoskeletal system by differentiating into multiple cell types including osteocytes and adipocytes. Mechanical signals, including strain and low intensity vibration (LIV), are important regulators of MSC differentiation. Lamin A/C is a vital protein for nuclear architecture that supports chromatin organization, as well as mechanical integrity and mechano-sensitivity of the nucleus in MSCs. Here, we investigated whether Lamin A/C and mechano-responsiveness are functionally coupled during adipogenesis. Lamin depletion in MSCs using siRNA increased nuclear area, height and volume and decreased circularity and stiffness, while phosphorylation of focal adhesions and dynamic substrate strain in response to LIV remained intact. Lamin A/C depletion decelerates adipogenesis as reflected by delayed appearance of key biomarkers (e.g., adiponectin/ADIPOQ). Based on RNA-seq data, reduced Lamin A/C levels decrease the activation of the adipocyte transcriptome that is normally observed in response to adipogenic cues mediating differentiation of MSCs. Mechanical stimulation via daily LIV application reduced the expression levels of ADIPOQ in both control and Lamin A/C depleted cells. Yet, treatment with LIV did not induce major transcriptome changes in either control or Lamin A/C depleted MSCs, suggesting that the biological effects of LIV on adipogenesis may not occur at the transcriptional level. We conclude that while Lamin A/C activation is essential for normal adipogenesis, it is dispensible for activation of focal adhesions by dynamic vibration induced mechanical signals.

Inflammation is a crucial component of various stress-induced responses that contributes to the pathogenesis of major depressive disorder (MDD). Depressive-like behavior (DLB) is characterized by decreased mobility and depressive behavior that occurs in systemic infection induced by Lipopolysaccharide (LPS) in experimental animals and is considered as a model of exacerbation of MDD. We assessed the effects of melatonin on behavioral changes and inflammatory cytokine expression in hippocampus of mice in LPS-induced DLB, as well as its effects on NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome activation, oxidative stress and pyroptotic cell death in murine microglia in vitro. Intraperitoneal 5 mg/kg dose of LPS was used to mimic depressive-like behaviors and melatonin was given at a dose of 500 mg/kg for 4 times with 6 h intervals, starting at 2 h before LPS administration. Behavioral assessment was carried out at 24 h post-LPS injection by tail suspension and forced swimming tests. Additionally, hippocampal cytokine and NLRP3 protein levels were estimated. Melatonin increased mobility time of LPS-induced DLB mice and suppressed NLRP3 expression and interleukin-113 (IL-113) cleavage in the hippocampus. Immunofluorescence staining of hippocampal tissue showed that NLRP3 is mainly expressed in ionized calcium-binding adapter molecule 1 (lba1) -positive microglia. Our results show that melatonin prevents LPS and Adenosine triphosphate (ATP) induced NLRP3 inflammasome activation in murine microglia in vitro, evidenced by inhibition of NLRP3 expression, Apoptosis-associated speck-like protein containing a CARD (ASC) speck formation, caspase-1 cleavage and interleukin-1 beta (IL-1 beta) maturation and secretion. Additionally, melatonin inhibits pyroptosis, production of mitochondrial and cytosolic reactive oxygen species (ROS) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappa B) signaling. The beneficial effects of melatonin on NLRP3 inflammasome activation were associated with nuclear factor erythroid 2-related factor 2 (Nrf2) and Silent information regulator 2 homolog 1 (SIRT1) activation, which were reversed by Nrf2 siRNA and SIRT1 inhibitor treatment.

Ethyl pyruvate is a molecule with anti-inflammatory and pro-metabolic effects. Ethyl pyruvate has been shown to ameliorate the clinical and pathological findings of neurodegenerative diseases such as Alzheimer’s and Parkinson’s Diseases in rodents. Its anti-inflammatory and neuroprotective effects are widely investigated in animal and cellular models. Our study aimed to investigate the mechanism of the impact of Ethyl pyruvate on NLRP3 inflammasome activation in the N9 microglial cell line. Our results indicated that ethyl pyruvate significantly suppressed LPS and ATP-induced NLRP3 inflammasome activation, decreased active caspase-1 level, secretion of IL-1β and IL-18 cytokines, and reduced the level of pyroptotic cell death resulting from inflammasome activation. Furthermore, ethyl pyruvate reduced the formation of total and mitochondrial ROS and suppressed inflammasome-induced HMGB1 upregulation and nuclear NF-κB translocation and reversed the inflammasome activation-induced miRNA expression profile for miR-223 in N9 cells. Our study suggests that ethyl pyruvate effectively suppresses the NLRP3 inflammasome activation in microglial cells regulation by miR-223 and NF-κB/HMGB1 axis.

Background The NLRP3 inflammasome formation and following cytokine secretion is a crucial step in innate immune responses. Internal and external factors may trigger inflammasome activation and result in inflammatory cytokine secretion. Inflammasome formation and activity play critical roles in several disease pathologies such as cardiovascular, metabolic, renal, digestive, and CNS diseases. Underlying pathways are not yet clear, but phytochemicals as alternative therapies have been extensively used for suppression of inflammatory responses. Purpose In this review, we aimed to summarize in vivo and in vitro effects on NLRP3 inflammasome activation of selected phytochemicals. Method Three phytochemicals; Sulforaphane, Curcumin, and Resveratrol were selected, and studies were reviewed to clarify their intracellular signaling mechanism in NLRP3 inflammasome activity. PubMed and Scopus databases are used for the search. For sulforaphane, 8 articles, for curcumin, 25 articles, and for resveratrol, 41 articles were included in the review. Conclusion In vitro and in vivo studies pointed out that the selected phytochemicals have inhibitory properties on NLRP3 inflammasome activity. However, neither the mechanism is clear, nor the study designs and doses are standardized.

βcatenin acts as a primary intracellular signal transducer for mechanical and Wnt signaling pathways to control cell function and fate. Regulation of βcatenin in the cytoplasm has been well studied but βcatenin nuclear trafficking and function remains unclear. In a previous study we showed that, in mesenchymal stem cells (MSC), mechanical blockade of adipogenesis relied on inhibition of βcatenin destruction complex element GSK3β (glycogen synthase kinase 3β) to increase nuclear βcatenin as well as the function of Linker of Cytoskeleton and Nucleoskeleton (LINC) complexes, suggesting that these two mechanisms may be linked. Here we show that shortly after inactivation of GSK3β due to either low intensity vibration (LIV), substrate strain or pharmacologic inhibition, βcatenin associates with the nucleoskeleton, defined as the insoluble nuclear fraction that provides structure to the integrated nuclear envelope, nuclear lamina and chromatin. Co-depleting LINC elements Sun-1 and Sun-2 interfered with both nucleoskeletal association and nuclear entry of βcatenin, resulting in decreased nuclear βcatenin levels. Our findings reveal that the insoluble structural nucleoskeleton actively participates in βcatenin dynamics. As the cytoskeleton transmits applied mechanical force to the nuclear surface to influence the nucleoskeleton and its LINC mediated interaction, our results suggest a pathway by which LINC mediated connectivity may play a role in signaling pathways that depend on nuclear access of βcatenin.

Ozcivici, Engin/0000-0003-4464-0475, PubMed: 25349533, Introduction: Mechanical loads can regulate cell proliferation and differentiation at various stages of development and homeostasis. However, the extension of this regulatory effect of mechanical loads on cancer cells is largely unknown. Increased physical compliance is one of the key features of cancer cells, which may hamper the transmission of mechanical loads to these cells within tumor microenvironment. Here we tested whether brief daily application of an external low magnitude mechanical stimulus (LMMS), would impede the growth of MDA-MB-231 aggressive type breast cancer cells in vitro for 3 wks of growth. Methods: The signal was applied in oscillatory form at 90 Hz and 0.15 g, a regimen that would induce mechanical loads on MDA-MB-231 cells via inertial properties of cells rather than matrix deformations. Experimental cells were exposed to LMMS 15 min/day, 5 days/week in ambient conditions while control cells were sham loaded. Cell proliferation, viability, cycle, apoptosis, morphology and migration were tested via Trypan Blue dye exclusion, MTT, PI, Annexin V, Calcein-AM and phalloidin stains and scratch wound assays. Results: Compared to sham controls, daily application of LMMS reduced the number and viability of cancerous MDA-MB-231 cells significantly after first week in the culture, while non-cancerous MCF10A cells were found to be unaffected. Flow cytomety analyses suggested that the observed decrease for the cancer cells in the LMMS group was due to a cell cycle arrest rather than apoptosis. LMMS further reduced cancer cell circularity and increased cytoskeletal actin in MDA-MB-231 cells. Conclusion: Combined, results suggest that direct application of mechanical loads negatively regulate the proliferation of aggressive type cancer cells. If confirmed, this non-invasive approach may be integrated to the efforts for the prevention and/or treatment of cancer., Scientific and Technological Research Council of TurkeyTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [111T577, 111M604], Financial support by The Scientific and Technological Research Council of Turkey (111T577 and 111M604) is gratefully acknowledged. Expert technical help from Drs. Ozden Yalcin-Ozuysal, Esra Erdal and Izmir Institute of Technology, Biotechnology and Bioengineering Research and Application Center is much appreciated.