Expression Of Signaling Molecules Research Articles

Epigenetic mechanisms of Müller glial reprogramming mediating retinal regeneration.

Retinal degenerative diseases, characterized by retinal neuronal death and severe vision loss, affect millions of people worldwide. One of the most promising treatment methods for retinal degenerative diseases is to reprogram non-neuronal cells into stem or progenitor cells, which then have the potential to re-differentiate to replace the dead neurons, thereby promoting retinal regeneration. Müller glia are the major glial cell type and play an important regulatory role in retinal metabolism and retinal cell regeneration. Müller glia can serve as a source of neurogenic progenitor cells in organisms with the ability to regenerate the nervous system. Current evidence points toward the reprogramming process of Müller glia, involving changes in the expression of pluripotent factors and other key signaling molecules that may be regulated by epigenetic mechanisms. This review summarizes recent knowledge of epigenetic modifications involved in the reprogramming process of Müller glia and the subsequent changes to gene expression and the outcomes. In living organisms, epigenetic mechanisms mainly include DNA methylation, histone modification, and microRNA-mediated miRNA degradation, all of which play a crucial role in the reprogramming process of Müller glia. The information presented in this review will improve the understanding of the mechanisms underlying the Müller glial reprogramming process and provide a research basis for the development of Müller glial reprogramming therapy for retinal degenerative diseases.

Fish SCD1 promotes SGIV infection via modulating the formation of lipid droplets and TBK1/MDA5-activated IFN signal pathway

Stearoyl CoA desaturase (SCD1), the rate-limiting enzyme for monounsaturated fatty acid production, involved in host metabolic regulation. Recent reports indicated that SCD1 played a crucial role in the process of viral infection. To investigate the function of SCD1 in teleost fish and the role it played in fish virus infection, EcSCD1 was cloned and characterized in the orange-spotted grouper, Epinephelus coioides. The open reading frame (ORF) of EcSCD1 consisted of 1005 nucleotides encoding a protein containing 334 amino acids with a predicted molecular weight of 38.33 kDa, which shared 74.05% and 60.48% identity with zebrafish and human, respectively. EcSCD1 possessed four transmembrane domains and three conserved His-rich regions. In healthy groupers, EcSCD1 was primarily expressed in the liver and brain. Subcellular localization assay revealed that EcSCD1 was mainly distributed in the cytoplasm with uniform distribution or dot-like aggregation forms, and EcSCD1 was co-localized with the endoplasmic reticulum (ER). Singapore grouper iridovirus (SGIV) as well as poly (dA: dT) stimulation induced the expression of EcSCD1. On the other hand, EcSCD1 overexpression increased SGIV infection and SCD1 enzymatic product oleic acid (OA) recovered SGIV infection suppressed by SCD1 inhibitor. In addition, EcSCD1 overexpression facilitated the formation of lipid droplets (LDs), which was crucial for SGIV replication. Furthermore, EcSCD1 overexpression down-regulated the expression of interferon signaling molecules and the activities of interferon (IFN)1/3 and interferon stimulated response element (ISRE) promotors. Moreover, EcSCD1 overexpression restricted TANK binding kinase 1 (TBK1)−/melanoma differentiation-associated protein 5 (MDA5)-, but not mitochondrial antiviral signaling protein (MAVS)-evoked interferon immune responses. Overall, our findings offered novel insights into the regulatory mechanism of EcSCD1 during virus infection.

Effect of HER2-targeted therapy on PDX and PDX-derived organoids generated from HER2-positive salivary duct carcinoma.

We previously established a patient-derived xenograft (PDX) model, patient-derived organoids (PDOs), and PDX-derived organoids (PDXOs) for salivary duct carcinoma (SDC). Using these models, this study examined the therapeutic effect of human epidermal growth factor receptor 2 (HER2) blockade on HER2-positive SDC. The therapeutic effect of lapatinib was assessed in SDC PDXOs with regards to cell growth, receptor/downstream signaling molecule expression, phosphorylation levels, and apoptosis. Effect of lapatinib treatment was evaluated in vivo in SDC PDX mice. The siRNA knockdown of HER2 and lapatinib suppressed cell proliferation in SDC PDXOs. Lapatinib inhibited the phosphorylation of HER2 and its downstream targets, and induced apoptosis in SDC PDXOs. Lapatinib also significantly reduced tumor volumes compared with that of the control in SDC PDX mice. For the first time, we demonstrated the efficacy of anti-HER2 therapy in HER2-positive SDC using preclinical models of SDC PDX and PDXO.

Effect of lupeol on insulin resistance in adipose tissue by modulating the expression of insulin and inflammatory signaling molecules in high-fat diet and sucrose-fed diabetic rats

The prevalence of obesity and diabetes has increased globally as a result of lifestyle modifications including eating a high-fat diet and not exercising. The target tissues to insulin action are the key pathophysiological event contributing to the development of type 2 diabetes mellitus. The treatments for type 2 diabetes that are now accessible are ineffectual due to their unfavourable side effects. Hence, search drugs, from plant sources. Lupeol is widely present in various medicinal plants. It has been reported to elicit multitude of bioactivities including antilipidemic and antihyperglycemic activity. However, specific effect of Lupeol on insulin signaling molecules of adipose tissue remains unclear. Hence, the study aimed to assess the beneficial role of Lupeol on the expression of insulin‑signaling molecules in adipose tissue of high fat diet and sucrose-induced type‑2 diabetic rats. The oral effective dose of lupeol at a dose of 25 mg per kg body weight per day orally for 30 days is observed. Lupeol, being a powerful antioxidant agent, normalises hyperglycemia, dyslipidaemia, and hyperinsulinemia while also suppressing oxidative stress by scavenging high levels of ROS. It also enhances insulin signalling in type 2 diabetic rats in adipose tissues. Lupeol administration decreased the levels of pro-inflammatory cytokines, which may be due to lupeol's possible antioxidant and anti-inflammatory activities, and thus it promotes insulin sensitivity via altering pro-inflammatory signalling molecules in adipose tissue such as IL-6, TNF alpha, and NFkB. As a result of the current findings, it is concluded that lupoel possesses potent anti-diabetic characteristics and could be used as a phytomedicine for the treatment of type 2 diabetes.

Dopamine receptor D4 (DRD4) negatively regulates UCP1- and ATP-dependent thermogenesis in 3T3-L1 adipocytes and C2C12 muscle cells.

The activation of beige fat and muscle tissues is an interesting and encouraging target for therapeutic intervention in obesity owing to their remarkable lipolytic activity and energy-consuming futile cycles. This study examined the effect of dopamine receptor D4 (DRD4) on lipid metabolisms as well as UCP1- and ATP-dependent thermogenesis in Drd4-silenced 3T3-L1 adipocytes and C2C12 muscle cells. Silencing of Drd4, followed by quantitative real-time PCR, immunoblot analysis, immunofluorescence, and staining methods, were applied to evaluate the effects of DRD4 on diverse target genes and proteins of both cells. The findings showed that DRD4 was expressed in the adipose and muscle tissues of normal and obese mice. Furthermore, the knockdown of Drd4 upregulated the expression of brown adipocyte-specific genes and proteins while downregulating lipogenesis and the adipogenesis marker proteins. Drd4 silencing also upregulated the expression of key signaling molecules involved in ATP-dependent thermogenesis in both cells. This was further elucidated by mechanistic studies showing that a Drd4 knockdown mediates UCP1-dependent thermogenesis via the cAMP/PKA/p38MAPK pathway in 3T3-L1 adipocytes and UCP1-independent thermogenesis via the cAMP/SLN/SERCA2a pathway in C2C12 muscle cells. In addition, siDrd4 also mediates myogenesis via the cAMP/PKA/ERK1/2/Cyclin D3 pathway in C2C12 muscle cells. Silencing of Drd4 promotes β3-AR-dependent browning in 3T3-L1 adipocytes and α1-AR/SERCA-based thermogenesis through an ATP-consuming futile process in C2C12 muscle cells. Understanding the novel functions of DRD4 on adipose and muscle tissues in terms of its ability to enhance energy expenditure and regulate whole-body energy metabolism will aid in developing novel obesity intervention techniques.

Acer truncatum leaves reduce lipid accumulation and improve liver and intestine health of grass carp (Ctenopharyngodon idella) fed with high-fat diet

The purpose of this study was to explore whether Acer truncatum leaves alleviated lipid accumulation, and improved liver and intestine health of grass carp fed with high-fat diet (HFD). Grass carp were fed with regular diet (RD), HFD, HFD contained 2% A. truncatum leaves (HFD_2AL) and 4% A. truncatum leaves (HFD_4AL), respectively, for 8 weeks. Growth performance, serum biochemical indexes, histopathology, expression of genes involved in lipid metabolism, inflammation and tight junctions, and changes in intestinal microbiota of grass carp were detected. Firstly, the results show that adding A. truncatum leaves in HFD reduced liver lipid accumulation of grass carp. The 2% and 4% of A. truncatum leaves reduced the levels of serum total cholesterol (TC) and the amount of lipid droplets in the liver, and down-regulated the expression of lipogenesis-related genes (SREBP-1, PPARγ, ACC1 and FAS), but up-regulated the expression of lipolysis-related genes (CPT-1, LPL and HSL) in the liver of grass carp fed with HFD (P < 0.05). Further, the supplement of A. truncatum leaves in HFD alleviated liver injury of grass carp. The levels of serum aspartate aminotransferase (AST) and alanine transaminase (ALT) were reduced in HFD_4AL group (P < 0.05). And the supplement of A. truncatum leaves reduced the mRNA expression of inflammatory signaling molecules (NF-κB p65 and p38 MAPK) and proinflammatory factors (IL-1β, IL-6, TLR-2 and TLR-4) in the liver of grass carp fed with HFD (P < 0.05). Moreover, the supplement of A. truncatum leaves in HFD enhanced intestine health of grass carp. The 2% and 4% A. truncatum leaves up-regulated the mRNA expression of tight junction proteins (ZO-1) and anti-inflammatory factor (TGFβ1), but suppressed the mRNA expression of proinflammatory factors (IL-1β, IL-6, IL-8, TLR-2, TLR-4 and TNFα) and inflammatory signaling molecules (NF-κB p65) in the intestine of grass carp fed with HFD (P < 0.05). In particular, A. truncatum leaves alleviated intestinal microbiota imbalance caused by HFD, which was characterized by inhibiting the proliferation of harmful bacteria, such as Mycobacterium, but promoting the proliferation of beneficial bacteria, such as Cetobacterium. In summary, the supplement of 2% and 4% of A. truncatum leaves in HFD reduced liver lipid accumulation and inflammation, alleviated intestinal inflammation and microbiota imbalance caused by HFD in grass carp.

Identification of Potential Therapeutic Targets for Plasmablastic Lymphoma Through Gene Expression Analysis: Insights into RAS and Wnt Signaling Pathways

Plasmablastic lymphoma (PBL) is a rare and aggressive B-cell lymphoma with overlapping characteristics with diffuse large B-cell lymphoma (DLBCL) and multiple myeloma. Hyperactive Wnt signaling derails homeostasis and promotes oncogenesis and chemoresistance in DLBCL and multiple myeloma. Evidence suggests active cross-talk between the Wnt and RAS pathways impacting metastasis in solid cancers in which combined targeted therapies show effective results. Recent genomic studies in PBL demonstrated a high frequency of mutations linked with the RAS signaling pathway. However, the role of RAS and Wnt signaling pathway molecule expression in PBL remained unknown. We examined the expression of Wnt and RAS pathway-related genes in a well-curated cohort of PBL. Because activated B cells are considered immediate precursors of plasmablasts in B cell development, we compared this data with activated B-cell type DLBCL (ABC-DLBCL) patients, employing NanoString transcriptome analysis (770 genes). Hierarchical clustering revealed distinctive differential gene expression between PBL and ABC-DLBCL. Gene set enrichment analysis labeled the RAS signaling pathway as the most enriched (37 genes) in PBL, including upregulating critical genes, such as NRAS, RAF1, SHC1, and SOS1. Wnt pathway genes were also enriched (n = 22) by gene set enrichment analysis. Molecules linked with Wnt signaling activation, such as ligands or targets (FZD3, FZD7, c-MYC, WNT5A, WNT5B, and WNT10B), were elevated in PBL. Our data also showed that, unlike ABC-DLBCL, the deranged Wnt signaling activity in PBL was not linked with hyperactive nuclear factor κB and B-cell receptor signaling. In divergence, Wnt signaling inhibitors (CXXC4, SFRP2, and DKK1) also showed overexpression in PBL. The high expression of RAS signaling molecules reported may indicate linkage with gain–in-function RAS mutations. In addition, high expression of Wnt and RAS signaling molecules may pave pathways to explore benefiting from combined targeted therapies, as reported in solid cancer, to improve prognosis in PBL patients.

Wnt5a/Ca2+ signaling regulates silica-induced ferroptosis in mouse macrophages by altering ER stress-mediated redox balance

Silicosis is a chronic pulmonary disease characterized by diffuse fibrosis of lung caused by the deposition of silica dust (SiO2). The inhaled silica-induced oxidative stress, ROS production and macrophage ferroptosis are key drivers of the pathological process of silicosis. However, mechanisms that involved in the silica-induced macrophage ferroptosis and its contributions to pathogenesis of silicosis remain elusive. In the present study, we showed that silica induced murine macrophage ferroptosis, accompanied by elevation of inflammatory responses, Wnt5a/Ca2+ signaling activation, and concurrent increase of endoplasmic reticulum (ER) stress and mitochondrial redox imbalance in vitro and vivo. Mechanistic study further demonstrated that Wnt5a/Ca2+ signaling played a key role in silica-induced macrophage ferroptosis by modulating ER stress and mitochondrial redox balance. The presence of Wnt5a/Ca2+ signaling ligand Wnt5a protein increased the silica-induced macrophage ferroptosis by activating ER-mediated immunoglobulin heavy chain binding protein (Bip)-C/EBP homology protein (Chop) signaling cascade, reducing the expression of negative regulators of ferroptosis, glutathione peroxidase 4 (Gpx4) and solute carrier family 7 member 11 (Slc7a11), subsequentially increasing lipid peroxidation. The pharmacologic inhibition of Wnt5a signaling or block of calcium flow exhibited an opposite effect to Wnt5a, resulted in the reduction of ferroptosis and the expression of Bip-Chop signaling molecules. These findings were further corroborated by the addition of ferroptosis activator Erastin or inhibitor ferrostatin-1. These results provide a mechanism by which silica activates Wnt5a/Ca2+ signaling and ER stress, sequentially leads to redox imbalance and ferroptosis in mouse macrophage cells.

Placenta-specific 8 facilitates the infection of duck hepatitis A virus type 1 by inhibiting the TLR7 MyD88-dependent signaling pathway

The placenta-specific 8 (PLAC8) gene, also known as ONZIN or C15, codes for a cysteine-rich peptide originally identified in mouse placental tissue and subsequently identified in a variety of epithelial tissues and immune cells. PLAC8 is also expressed in birds, such as ducks, where its functional roles remain unknown. Here, we aimed to determine the mRNA and protein expression profiles and the functional role of duck PLAC8 during the infection of duck hepatitis A virus type 1 (DHAV-1). We found that the duck PLAC8 is also a cysteine-rich polypeptide composed of 114 amino acid residues, with no signal peptide. Duck PLAC8 is highly expressed in the immune organs of young cherry valley ducks, including the thymus, bursa fabricius, and spleen. However, it has negligible expression level in liver, brain, kidney, and heart. Additionally, PLAC8 expression was considerably induced after DHAV-1 infection both in vitro and in vivo, especially in the immune organs of ducklings. This tissue expression distribution and induction upon infection suggest that PLAC8 might play a critical role in innate immunity. Our data showed that PLAC8 significantly suppressed the expression of Toll-like receptor 7 (TLR7), leading to decreased expression of downstream signaling molecules including myeloid differentiation primary response gene 88 (MyD88) and nuclear factor kappa-B (NF-κB). This ultimately resulted in low levels of type I interferon and interleukin 6 (IL-6). Additionally, PLAC8 positively regulated DHAV-1 replication levels. RNAi against PLAC8 in duck embryo fibroblasts considerably inhibited DHAV-1 propagation, while PLAC8 overexpression significantly facilitated DHAV-1 replication.

Abstract 2232: The identification of novel autophagy flux-related molecules closely linked to the acquired resistance to EGFR TKIs in NSCLC patients harboring EGFR mutations

Abstract Introduction: Non-small cell lung cancer (NSCLC) patients with activating EGFR mutations (mEGFR) well respond to EGFR TKIs, but the majority of patients eventually acquire drug resistance, leading to treatment failure. Recently, intracellular potential autophagy in addition to primary genetic alterations implicates the acquired drug resistance to targeted drugs in various carcinomas such as melanoma, pancreas, and breast cancer. We aimed to identify autophagy flux-related signal molecules which may be closely linked to acquired resistance in mEGFR NSCLC. Methods: We constructed the TMA composed of 579 surgically resected tumor tissues from lung adenocarcinoma patients and evaluated protein expression of autophagy flux-related signal molecules including CAGE, p-Beclin1, LC3B, p-AMPK, ULK1, p62, and ATG5 using immunohistochemistry (IHC). We established H1975OSI (166-folds) and PC9ERL (3,650-folds) resistant sub cell lines through the prolonged exposure of Osimertinib and Erlotinib to H1975 (L858R/T790M) and PC9 (Del19) cells, respectively. We also explored genes that induce autophagy flux activity using mRNA seq and qRT-PCR. Results: H-score of CAGE ( 95.6±7.9, 70±5.5), p-AMPK (73.4±7.8, 32±5.6) and p-Beclin1 (44.4±16.1, 19.7±5.5), ULK1( 57.4±4.9, 41.9±4.9) were significantly higher in mEGFR, compared to those of wild type EGFR NSCLC (P &amp;lt;0.001). According to EGFR mutation subtype, the expressions of CAGE (98±11.2, 70±5.5) and ULK1(60.7±5.0, 41.9±4.9) were significantly higher in Del19 (P&amp;lt;0.001), while the expressions of CAGE(93.2±5.6, 70±5.5,), p-AMPK(82.9±5.7, 32±5.6), and p-Beclin1(45.6±16.7, 19.7±5.5) were significantly higher in L858R (P&amp;lt;0.001). In advanced/metastatic mEGFR NSCLC patients treated with EGFR TKIs, p-Beclin1(13.0±10.9, 59±7.2) expression was significantly increased, when comparing before treatment and at treatment failure (P&amp;lt;0.018). As results of mRNA seq and qRT-PCR in H1975 parental cells and H1975OSI cells, we identified autophagy flux-related signal molecules including BECLIN1, GABAR1, NBR1, PINK1, ULK1 which were significantly increased more than 10 times. Conclusion: Taken together, our results strongly support the evidence that autophagy flux-related signal molecules as well as known genetic alterations are closely connected to the acquired resistance during period of EGFR TKI treatment in mEGFR NSCLC patients. Now, we are undergoing additional RNA profiling and function study of identified novel autophagy-related genes to examine a possibility as potential target to overcome EGFR TKI resistance. Citation Format: Jeong-Oh Kim, Jung-Young Shin, Min-Young Kim, Mi-Ran Lee, Seoree Kim, Hankyu Lee, Doyong Jeon, Seung Ryel Baek, Kim Joo Il, Kyoungjune Lee, Youn Soo Lee, Jin-Hyoung Kang. The identification of novel autophagy flux-related molecules closely linked to the acquired resistance to EGFR TKIs in NSCLC patients harboring EGFR mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2232.

Effects of Age and Suntan on the Expression of Second Messenger Signaling Pathways of Necroptosis in Skin Cells during Facelifting Surgery.

We studied the effects of age and suntan on the expression of necroptosis signaling molecules (RIPK1, RIPK3, and MLKL kinases) and first TNF receptor (TNFR1) in isolated skin cells from women undergoing facelift surgery. In women above 50 years, the expression of the TNFR1, kinases RIPK1, RIPK3, and MLKL, the phosphorylated forms of these kinases was significantly (p<0.05) increased in comparison with the corresponding parameters in women under 30 years. The expression of all necroptosis proteins and TNFR1 in women with suntan was significantly (p<0.05) higher than in those without tan. Cells from the surgical material were incubated with TNFα to determine the level of induced necroptosis. In women aged >50 years and women with suntan, the expression of phosphorylated forms of kinases was significantly increased, which attested to necroptosis activation. This study allowed identifying the targets on skin cells for prevention of necrosis and inflammation after facelift surgery.

Inhibition of protease activity and anti‐quorum sensing of the potential fraction of ethanolic extract from Sansevieria trifasciata Prain leaves against Pseudomonas aeruginosa

Sansevieria trifasciata is a plant that is commonly utilized in traditional medicine. The leaves of S. trifasciata show antibacterial properties against Pseudomonas aeruginosa. This bacterium is an opportunistic pathogen that can cause serious illness in humans and produce a variety of virulence factors responsible for bacterial pathogenesis with quorum sensing (QS) systems that mediate intracellular communication. Bacteria produce protease through a QS mechanism in which they express signaling molecules to become pathogens. Proteases are extracellular enzymes required for successful infection that mediate biofilm spread through QS and regulate a variety of cellular and physiological functions. This research aimed to evaluate the protease, and anti‐QS activities of the ethanolic extract from S. trifasciata leaves against P. aeruginosa and the expression of QS genes. An azocasein test was used to determine the protease activity in qualitative and quantitative methods. Using real‐time quantitative polymerase chain reaction, a study was conducted to investigate the effect of ethanolic extract from S. trifasciata leaves on selected QS‐regulatory genes at the transcriptional level. The results showed that the potential ethanolic extract from S. trifasciata leaves inhibited the protease enzyme activity by as much as 77.1%. The potential ethanolic extract from S. trifasciata leaves decreased the expressions of lasA, lasB, lasI, lasR, rhlI, and rhlR with 2‐ΔΔCt values of 0.81, 0.93, 0.76, 0.97, 0.90, and 0.55 respectively.

Porcine epidemic diarrhea virus (PEDV) ORF3 protein inhibits cellular type I interferon signaling through down-regulating proteins expression in RLRs-mediated pathway

Porcine epidemic diarrhea virus (PEDV) is an entero-pathogenic coronavirus, which belongs to the genus Alphacoronavirus in the family Coronaviridae, causing lethal watery diarrhea in piglets. Previous studies have shown that PEDV has developed an antagonistic mechanism by which it evades the antiviral activities of interferon (IFN), such as the sole accessory protein open reading frame 3 (ORF3) being found to inhibit IFN-β promoter activities, but how this mechanism used by PEDV ORF3 inhibits activation of the type I signaling pathway remains not fully understood. Thus, in this present study, we showed that PEDV ORF3 inhibited both polyinosine-polycytidylic acid (poly(I:C))- and IFNα2b-stimulated transcription of IFN-β and interferon-stimulated genes (ISGs) mRNAs. The expression levels of antiviral proteins in the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs)-mediated pathway was down-regulated in cells with over-expression of PEDV ORF3 protein, but global protein translation remained unchanged and the association of ORF3 with RLRs-related antiviral proteins was not detected, implying that ORF3 only specifically suppressed the expression of these signaling molecules. At the same time, we also found that the PEDV ORF3 protein inhibited interferon regulatory factor 3 (IRF3) phosphorylation and poly(I:C)-induced nuclear translocation of IRF3, which further supported the evidence that type I IFN production was abrogated by PEDV ORF3 through interfering with RLRs signaling. Furthermore, PEDV ORF3 counteracted transcription of IFN-β and ISGs mRNAs, which were triggered by over-expression of signal proteins in the RLRs-mediated pathway. However, to our surprise, PEDV ORF3 initially induced, but subsequently reduced the transcription of IFN-β and ISGs mRNAs to normal levels. Additionally, mRNA transcriptional levels of signaling molecules located at IFN-β upstream were not inhibited, but elevated by PEDV ORF3 protein. Collectively, these results demonstrate that inhibition of type I interferon signaling by PEDV ORF3 can be realized through down-regulating the expression of signal molecules in the RLRs-mediated pathway, but not via inhibiting their mRNAs transcription. This study points to a new mechanism evolved by PEDV through blockage of the RLRs-mediated pathway by ORF3 protein to circumvent the host's antiviral immunity.

Molecular Mechanisms Underlying TNFα-Induced Mitochondrial Biogenesis in Human Airway Smooth Muscle.

Proinflammatory cytokines such as TNFα mediate airway inflammation. Previously, we showed that TNFα increases mitochondrial biogenesis in human ASM (hASM) cells, which is associated with increased PGC1α expression. We hypothesized that TNFα induces CREB and ATF1 phosphorylation (pCREBS133 and pATF1S63), which transcriptionally co-activate PGC1α expression. Primary hASM cells were dissociated from bronchiolar tissue obtained from patients undergoing lung resection, cultured (one-three passages), and then differentiated by serum deprivation (48 h). hASM cells from the same patient were divided into two groups: TNFα (20 ng/mL) treated for 6 h and untreated controls. Mitochondria were labeled using MitoTracker green and imaged using 3D confocal microscopy to determine mitochondrial volume density. Mitochondrial biogenesis was assessed based on relative mitochondrial DNA (mtDNA) copy number determined by quantitative real-time PCR (qPCR). Gene and/or protein expression of pCREBS133, pATF1S63, PCG1α, and downstream signaling molecules (NRFs, TFAM) that regulate transcription and replication of the mitochondrial genome, were determined by qPCR and/or Western blot. TNFα increased mitochondrial volume density and mitochondrial biogenesis in hASM cells, which was associated with an increase in pCREBS133, pATF1S63 and PCG1α expression, with downstream transcriptional activation of NRF1, NRF2, and TFAM. We conclude that TNFα increases mitochondrial volume density in hASM cells via a pCREBS133/pATF1S63/PCG1α-mediated pathway.

Cold Storage Followed by Transplantation Induces Interferon-Gamma and STAT-1 in Kidney Grafts.

Cold storage (CS)-mediated inflammation, a reality of donor kidney processing and transplantation, can contribute to organ graft failure. However, the mechanisms by which this inflammation is perpetuated during and after CS remain unclear. Here, we examined the immunoregulatory roles of signal transducer and activator of transcription (STAT) family proteins, most notably STAT1 and STAT3, with our in vivo model of renal CS and transplant. Donor rat kidneys were exposed to 4 h or 18 h of CS, which was then followed by transplantation (CS + transplant). STAT total protein level and activity (phosphorylation) were evaluated via Western blot analysis and mRNA expression was tabulated using quantitative RT-PCR after organ harvest on day 1 or day 9 post-surgery. In vivo assays were further corroborated via similar analyses featuring in vitro models, specifically proximal tubular cells (human and rat) as well as macrophage cells (Raw 264.7). Strikingly, gene expression of IFN-γ (a pro-inflammatory cytokine inducer of STAT) and STAT1 were markedly increased after CS + transplant. STAT3 dephosphorylation was additionally observed after CS, a result suggestive of dysregulation of anti-inflammatory signaling as phosphorylated STAT3 acts as a transcription factor in the nucleus to increase the expression of anti-inflammatory signaling molecules. In vitro, IFN-γ gene expression as well as amplification of downstream STAT1 and inducible nitric oxide synthase (iNOS; a hallmark of ischemia reperfusion injury) was remarkably increased after CS + rewarming. Collectively, these results demonstrate that aberrant induction of STAT1 is sustained in vivo post-CS exposure and post-transplant. Thus, Jak/STAT signaling may be a viable therapeutic target during CS to mitigate poor graft outcomes when transplanting kidneys from deceased donors.

Inhibitory effect and related mechanism of decitabine combined with gemcitabine on proliferation of NK/T cell lymphoma cells.

Background: EBV-associated lymphoma is a neoplasm with a poor prognosis, highly aggressive, and progressive rapidly. There is no standard clinical treatment protocol. Decitabine and gemcitabine are known to have anticancer properties against cells of various cancer, respectively. However, the effect of the combination medication on NK/T cell lymphoma cells and potential mechanisms have not been thoroughly investigated. Methods: Human NK/T cell lymphoma cells NK92MI were treated with decitabine and gemcitabine alone or in combination. Experiments, including the Cell Counting Kit-8 and flow cytometry, were performed to investigate how the combination of decitabine and gemcitabine affects the biological behavior of NK92MI cells in vitro. mRNA sequencing, RT-PCR, and western blotting were used to detect changes in the related signal pathway, mRNA, and protein expressions. Results: Decitabine and gemcitabine significantly inhibited the viability and proliferation of NK92MI cells in a dose-dependent manner. The combination index was less than 1 after treating with two drugs, which was a significant synergistic effect. The decitabine concentration with the best synergistic effect was 4.046µM, and the gemcitabine concentration was 0.005µM. Flow cytometry showed that combining two drugs could significantly promote apoptosis and arrest the cell cycle at the S phase. In the combined DAC and GEM group, caspase3 protein levels were higher than in either group alone or the control group. The transcriptome sequence, KEGG, and PPI analysis showed that the differential genes after combined treatment were mainly enriched in signal pathways related to cell proliferation, adhesion, and migration compared with using alone and control groups. Based on the sequencing results, we further investigated the role of DAC and GEM in ferroptosis-related signaling molecules using RT-PCR and Western blot techniques. RT-PCR and western blotting showed that the expression levels of HMOX1 and EBV cleavage gene BRLF1 were higher in the group with combined DAC and GEM than in the group alone and the control group, while the protein and mRNA expression levels of SLC7A11 were lower than the others. In addition, the GPX4 protein expression level in the combination group was lower than in the drug-alone and control groups. In addition, the combination treatment increased the ROS level of NK92MI cells. Conclusion: Our current findings suggested that decitabine had an inhibitory effect on the proliferation of NK92MI cells when co-treated with gemcitabine. This combination may increase the expression of ferroptosis-related signaling molecules, thus inhibiting the proliferation of NK92MI cells. It also promoted apoptosis in NK/T cell lymphoma. For patients with NK/T cell lymphoma, this novel combination may provide clinical benefits.

Metadherin Regulates Inflammatory Breast Cancer Invasion and Metastasis.

Inflammatory breast cancer (IBC) is one of the most lethal subtypes of breast cancer (BC), accounting for approximately 1-5% of all cases of BC. Challenges in IBC include accurate and early diagnosis and the development of effective targeted therapies. Our previous studies identified the overexpression of metadherin (MTDH) in the plasma membrane of IBC cells, further confirmed in patient tissues. MTDH has been found to play a role in signaling pathways related to cancer. However, its mechanism of action in the progression of IBC remains unknown. To evaluate the function of MTDH, SUM-149 and SUM-190 IBC cells were edited with CRISPR/Cas9 vectors for in vitro characterization studies and used in mouse IBC xenografts. Our results demonstrate that the absence of MTDH significantly reduces IBC cell migration, proliferation, tumor spheroid formation, and the expression of NF-κB and STAT3 signaling molecules, which are crucial oncogenic pathways in IBC. Furthermore, IBC xenografts showed significant differences in tumor growth patterns, and lung tissue revealed epithelial-like cells in 43% of wild-type (WT) compared to 29% of CRISPR xenografts. Our study emphasizes the role of MTDH as a potential therapeutic target for the progression of IBC.

Immune-Enhancing Effects of Limosilactobacillus fermentum in BALB/c Mice Immunosuppressed by Cyclophosphamide.

This study evaluates the immune-enhancing effects of Limosilactobacillus fermentum on cyclophosphamide (CP)-induced immunosuppression in BALB/c mice. In vitro, the expressions of pro-inflammatory cytokines and MAPK signaling molecules in Raw264.7 cells were analyzed by ELISA and Western blot analysis. Moreover, cell proliferation, surface receptor expression, and cytotoxicity of NK-92 cells were examined by Cell Counting Kit-8, CytoTox96 assay, and flow cytometry, respectively. To investigate the immune-enhancing effects of selected L. fermentum strains in vivo, these strains were orally administered to BALB/c mice for 2 weeks, and CP was intraperitoneally injected. Then, liver, spleen, and whole blood were isolated from each animal. Administration of single L. fermentum strains or their mixture sustained the spleen weight, the counts of white blood cells compared to non-fed group. Splenocyte proliferation and NK cytotoxicity were significantly increased in all L. fermentum-fed groups. The frequency of B220+ cells was also significantly enhanced in splenocytes isolated from L. fermentum groups. In addition, the production of cytokines (TNF-α, IFN-γ) and antibodies was recovered in splenocyte supernatants isolated from L. fermentum groups. In conclusion, L. fermentum could be a suitable functional food additive for immune-enhancing effect.

Diallyl trisulfide inhibits monosodium urate-induced NLRP3 inflammasome activation via NOX3/4-dependent mitochondrial oxidative stress in RAW 264.7 and bone marrow-derived macrophages

BackgroundMonosodium urate (MSU) crystals are associated with gouty inflammatory diseases. MSU-associated inflammation is majorly triggered by NOD-like receptor protein 3 (NLRP3) inflammasome that promotes interleukin (IL)-1β secretion. Although diallyl trisulfide (DATS) is well-known polysulfide garlic compounds with anti-inflammatory effects, its action in MSU-induced inflammasome activation has not been known yet. PurposeThe objective of the current study was to investigate anti-inflammasome effects and mechanisms of DATS in RAW 264.7 and bone marrow-derived macrophages (BMDM). MethodsThe concentrations of IL-1β were analyzed with enzyme-linked immunosorbent assay. The MSU-induced mitochondrial damage and reactive oxygen species (ROS) production were detected by fluorescence microscope and flow cytometry. The protein expressions of NLRP3 signaling molecules, NADPH oxidase (NOX) 3/4 were assessed with Western blotting. ResultsDATS suppressed MSU-induced IL-1β and caspase-1 accompanied by decreased inflammasome complex formation in RAW 264.7 and BMDM. In addition, DATS restored mitochondrial damage. DATS downregulated NOX 3/4 that were upregulated by MSU as predicted by gene microarray and confirmed by Western blotting. ConclusionThis study first reports mechanistic finding that DATS alleviates MSU-induced NLRP3 inflammasome by mediating NOX3/4-dependent mitochondrial ROS production in macrophages in vitro and ex vivo, suggesting DATS could be effective therapeutic candidate for gouty inflammatory condition.

The novel hepatoprotective mechanisms of silibinin-phospholipid complex against d-GalN/LPS-induced acute liver injury

Background & AimsSilibinin-phospholipid complex (SPC) has been utilized to treat acute liver injury clinically. Nevertheless, the hepatoprotective mechanism of SPC remains to be further dissected in response to new insights into the pathogenesis of acute liver injury. Very recently, we have documented, for the first time, that M2-like macrophages exert the hepatoprotection against acute insult through inhibiting necroptosis-S100A9-necroinflammation. In the present work, we integrated this new finding into the mechanism of action of SPC, and attempted to dissect the hepatoprotective mechanism of SPC from this new perspective. MethodsSPC and corresponding controls were administered intragastrically into control mice subjected to d-GalN/LPS challenge. The hepatic damage was assessed, and the expression of necroptosis-S100A9-necroinflammation signaling molecules was detected. The correlation between SPC and macrophage activation was investigated. The expression of miR-223-3p and its regulation on macrophage activation were analyzed. The targeted inhibitory effects of miR-223-3p on necroptosis and necroinflammation signaling molecules were confirmed. ResultsSPC alleviated remarkably the hepatic damage triggered by d-GalN/LPS. The administration of SPC inhibited the expression of necroptosis-S100A9-necroinflammation signaling molecules. The levels of M2-like macrophage markers were increased significantly in SPC-treated mice or macrophages. miR-223-3p expression was enhanced in SPC-treated mice. miR-223-3p transfer led to up-regulated expression of M2-like macrophage markers. miR-223-3p directly targeted 3′ UTR of RIPK3 and NLRP3, and the expression of necroptosis and necroinflammation signaling molecules was inhibited in miR-223-3p-transferred hepatocytes and macrophages. ConclusionsSPC alleviates acute liver injury through up-regulating the expression of miR-223-3p. MiR-223-3p further promotes M2-like macrophage activation and the targeted inhibition of necroptosis and necroinflammation. Our findings provide novel insight into the hepatoprotective mechanism of SPC against acute liver injury.