TIMELESS promotes reprogramming of glucose metabolism in oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC), the predominant malignancy of the oral cavity, is characterized by high incidence and low survival rates. Emerging evidence suggests a link between circadian rhythm disruptions and cancer development. The circadian gene TIMELESS, known for its specific expression in various tumors, has not been extensively studied in the context of OSCC. This study aims to explore the influence of TIMELESS on OSCC, focusing on cell growth and metabolic alterations. METHODS: We analyzed TIMELESS expression in OSCC using western blot, immunohistochemistry, qRT-PCR, and data from The Cancer Genome Atlas (TCGA) and the Cancer Cell Line Encyclopedia (CCLE). The role of TIMELESS in OSCC was examined through clone formation, MTS, cell cycle, and EdU assays, alongside subcutaneous tumor growth experiments in nude mice. We also assessed the metabolic impact of TIMELESS by measuring glucose uptake, lactate production, oxygen consumption, and medium pH, and investigated its effect on key metabolic proteins including silent information regulator 1 (SIRT1), hexokinase 2 (HK2), pyruvate kinase isozyme type M2 (PKM2), recombinant lactate dehydrogenase A (LDHA) and glucose transporter-1 (GLUT1). RESULTS: Elevated TIMELESS expression in OSCC tissues and cell lines was observed, correlating with reduced patient survival. TIMELESS overexpression enhanced OSCC cell proliferation, increased glycolytic activity (glucose uptake and lactate production), and suppressed oxidative phosphorylation (evidenced by reduced oxygen consumption and altered pH levels). Conversely, TIMELESS knockdown inhibited these cellular and metabolic processes, an effect mirrored by manipulating SIRT1 levels. Additionally, SIRT1 was positively associated with TIMELESS expression. The expression of SIRT1, HK2, PKM2, LDHA and GLUT1 increased with the overexpression of TIMELESS levels and decreased with the knockdown of TIMELESS. CONCLUSION: TIMELESS exacerbates OSCC progression by modulating cellular proliferation and metabolic pathways, specifically by enhancing glycolysis and reducing oxidative phosphorylation, largely mediated through the SIRT1 pathway. This highlights TIMELESS as a potential target for OSCC therapeutic strategies.

Abnormal cerebral metabolism and metabolic connectivity in individuals with heroin dependence: an integrated resting-state PET/fMRI study in large-scale networks.

BACKGROUND: Increasing evidence suggests that heroin addiction may be related to the dysfunction among the triple brain network (default mode network [DMN], salience network [SN] and executive control network [ECN]). However, the characteristics of glucose metabolism and metabolic connectivity among core regions of the triple brain network remain unknown. Therefore, we hypothesized that individuals with heroin dependence would show abnormal glucose metabolism and accompanied abnormal metabolic connectivity within the triple brain network. METHODS: Individuals with heroin dependence and healthy controls matched for age and sex underwent integrated positron emission tomography/magnetic resonance imaging (PET/MRI). Differences in glucose metabolism and metabolic connectivity among the DMN, SN and ECN were analyzed based on 18F-fluorodeoxyglucose PET and resting-state fMRI data. RESULTS: We included 36 individuals with heroin dependence and 30 matched healthy controls in our study. The heroin dependence group showed a significant reduction of glucose metabolism in the bilateral anterior insula (AI) and inferior parietal lobule (IPL), and a significantly decreased metabolic connectivity between the right AI and the left dorsolateral prefrontal cortex (DLPFC). The daily dose of methadone was negatively correlated with glucose metabolism of the right AI and right IPL. LIMITATIONS: The results revealed the glucose metabolism alterations and metabolic connectivity only within the triple brain network in individuals with heroin dependence; additional brain networks should be investigated in future studies. Although methadone is an opioid with a similar neurophysiological mechanism as heroin, the specific chronic effects of methadone on cerebral metabolism and metabolic connectivity should also be investigated in future studies. CONCLUSION: Our findings suggest that long-term opioid use might, to some extent, be associated with reduced synergistic ability between the SN and ECN, which may be associated with the dysfunction of cognitive control. In particular, the right AI, which showed hypometabolism and related reduction in SN-ECN metabolic connectivity, should receive increasing attention in future studies.

Cross-organ single-cell transcriptome profiling reveals macrophage and dendritic cell heterogeneity in zebrafish.

Tissue-resident macrophages (TRMs) and dendritic cells (DCs) are highly heterogeneous and essential for immunity, tissue regeneration, and homeostasis maintenance. Here, we comprehensively profile the heterogeneity of TRMs and DCs across adult zebrafish organs via single-cell RNA sequencing. We identify two macrophage subsets: pro-inflammatory macrophages with potent phagocytosis signatures and pro-remodeling macrophages with tissue regeneration signatures in barrier tissues, liver, and heart. In parallel, one conventional dendritic cell (cDC) population with prominent antigen presentation capacity and plasmacytoid dendritic cells (pDCs) featured by anti-virus properties are also observed in these organs. Remarkably, in addition to a single macrophage/microglia population with potent phagocytosis capacity, a pDC population and two distinct cDC populations are identified in the brain. Finally, we generate specific reporter lines for in vivo tracking of macrophage and DC subsets. Our study depicts the landscape of TRMs and DCs and creates valuable tools for in-depth study of these cells in zebrafish.

Dissecting the effect of ileal faecal diversion on the intestine using single-cell sequencing.

BACKGROUND: Although ileal faecal diversion is commonly used in clinical settings, complications accompany it. Elucidating the intestinal changes caused by ileal faecal diversion will help resolve postoperative complications and elucidate the pathogenic mechanisms of associated intestinal disorders, such as Crohn's disease (CD). Therefore, our study aimed to provide new insights into the effects of ileal faecal diversion on the intestine and the potential mechanisms. METHODS: Single-cell RNA sequencing was performed on proximal functional and paired distal defunctioned intestinal mucosae from three patients with ileal faecal diversion. We also performed in vitro cellular and animal experiments, tissue staining and analysed public datasets to validate our findings. RESULTS: We found that the epithelium in the defunctioned intestine tended to be immature, with defective mechanical and mucous barriers. However, the innate immune barrier in the defunctioned intestine was enhanced. Focusing on the changes in goblet cells, we demonstrated that mechanical stimulation promotes the differentiation and maturation of goblet cells through the TRPA1-ERK pathway, indicating that the absence of mechanical stimulation may be the main cause of defects in the goblet cells of the defunctioned intestine. Furthermore, we found obvious fibrosis with a pro-fibrotic microenvironment in the defunctioned intestine and identified that monocytes may be important targets for faecal diversion to alleviate CD. CONCLUSIONS: This study revealed the different transcription landscapes of various cell subsets and the potential underlying mechanisms within the defunctioned intestine, when compared to the functional intestine, based on the background of ileal faecal diversion. These findings provide novel insights for understanding the physiological and pathological roles of the faecal stream in the intestine.

Metaphocytes are IL-22BP-producing cells regulated by ETS transcription factor Spic and essential for zebrafish barrier immunity.

Metaphocytes are tissue-resident macrophage (TRM)/dendritic cell (DC)-like cells of non-hematopoietic origin in zebrafish barrier tissues. One remarkable property of metaphocytes is their ability to capture soluble antigens from the external environment via transepithelial protrusions, a unique function manifested by specialized subpopulations of the TRMs/DCs in mammal barrier tissues. Yet, how metaphocytes acquire myeloid-like cell properties from non-hematopoietic precursors and how they regulate barrier immunity remains unknown. Here, we show that metaphocytes are in situ generated from local progenitors guided by the ETS transcription factor Spic, the deficiency of which results in the absence of metaphocytes. We further document that metaphocytes are the major IL-22BP-producing cells, and the depletion of metaphocytes causes dysregulated barrier immunity that resembles the phenotype of IL-22BP-deficient mice. These findings reveal the ontogeny, development, and function of metaphocytes in zebrafish, which facilitates our understanding of the nature and function of the mammalian TRM/DC counterparts.

Molecular mechanisms of action of metformin: latest advances and therapeutic implications.

Metformin is among the most widely used antidiabetic drugs. Studies over the past few years have identified multiple novel molecular targets and pathways that metformin acts on to exert its beneficial effects in treating type 2 diabetes as well as other disorders involving dysregulated inflammation and redox homeostasis. In this mini-review, we discuss the latest cutting-edge research discoveries on novel molecular targets of metformin in glycemic control, cardiovascular protection, cancer intervention, anti-inflammation, antiaging, and weight control. Identification of these novel targets and pathways not only deepens our understanding of the molecular mechanisms by which metformin exerts diverse beneficial biological effects, but also provides opportunities for developing new mechanistically based drugs for human diseases.

Default mode network mechanisms of repeated transcranial magnetic stimulation in heroin addiction.

Repetitive transcranial magnetic stimulation (rTMS) over the left dorsolateral prefrontal cortex (DLPFC) has been shown to reduce cravings in heroin-dependent (HD) individuals, but the mechanisms underlying the anti-craving effects of rTMS are unknown. Abnormalities in the default mode network (DMN) are known to be consistent findings in HD individuals and are involved in cravings. We assessed the effect of rTMS on DMN activity and its relationship to the treatment response. Thirty HD individuals were included in this self-controlled study, and all HD participants received 10-Hz rTMS 7-session during a week. Data for cravings and withdrawal symptoms and resting-state functional magnetic resonance imaging data were collected before and after rTMS treatment. Thirty demographically matched healthy individuals who did not receive rTMS were included as controls. We focused on changes in coupling seeded from the medial prefrontal cortex (MPFC), posterior cingulate cortex (PCC), and bilateral inferior parietal lobe (IPL), which are the core regions of the DMN. The craving and withdrawal symptom score of HD individuals decreased significantly after rTMS treatment. The left IPL-left middle frontal gyrus coupling and the left IPL-right inferior occipital gyrus coupling decreased significantly, and the changes in the left IPL-left middle frontal gyrus coupling were positively correlated with changes in drug-cue induced cravings. rTMS could modulate the coupling between the DMN and executive control network (ECN). Alterations of the left IPL-left middle frontal gyrus coupling may play an important mechanistic role in reducing drug cue-induced cravings.

Triterpenoid CDDO-IM protects against lipopolysaccharide-induced inflammatory response and cytotoxicity in macrophages: The involvement of the NF-κB signaling pathway.

Lipopolysaccharide (LPS), also known as endotoxin, can trigger septic shock, a severe form of inflammation-mediated sepsis with a very high mortality rate. However, the precise mechanisms underlying this endotoxin remain to be defined and detoxification of LPS is yet to be established. Macrophages, a type of immune cells, initiate a key response responsible for the cascade of events leading to the surge in inflammatory cytokines and immunopathology of septic shock. This study was undertaken to determine whether the LPS-induced inflammation in macrophage cells could be ameliorated via CDDO-IM (2-cyano-3,12 dioxooleana-1,9 dien-28-oyl imidazoline), a novel triterpenoid compound. Data from this study show that gene expression levels of inflammatory cytokine genes such as interleukin-1 beta (IL-1ß), interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-α), and monocyte chemoattractant protein-1 (MCP-1) were considerably increased by treatment with LPS in macrophages differentiated from ML-1 monocytes. Interestingly, LPS-induced increase in expression of pro-inflammatory cytokine levels is reduced by CDDO-IM. In addition, endogenous upregulation of a series of antioxidant molecules by CDDO-IM provided protection against LPS-induced cytotoxicity in macrophages. LPS-mediated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) transcriptional activity was also noted to decrease upon treatment with CDDO-IM in macrophages suggesting the involvement of the NF-κB signaling. This study would contribute to improve our understanding of the detoxification of endotoxin LPS by the triterpenoid CDDO-IM.

Single-incision plus one-port laparoscopic gastrectomy versus conventional multi-port laparoscopy-assisted gastrectomy for gastric cancer: a retrospective study.

BACKGROUND: We compared short-term perioperative outcomes after single-incision plus one-port laparoscopic gastrectomy (SILG+1) and conventional multi-port laparoscopy-assisted gastrectomy (C-LAG) for gastric cancer. METHODS: The work was conducted between August 2017 and October 2019. A total of 90 patients with early or advanced gastric cancer were retrospectively analyzed: 43 patients of which underwent SILG+1, and 47 of which underwent C-LAG, respectively. These were divided into two groups: the total gastrectomy group (SILT+1 and C-LATG) and the distal gastrectomy group (SILD + 1 and C-LADG). The demographics, tumor characteristics, postoperative outcomes, and short-term complications of all enrolled patients were summarized and statistically analyzed. RESULTS: The mean incision length in SILT+1 group was 5.40 cm shorter than that in C-LATG group (3.15 ± 0.43 vs. 8.55 ± 2.72, P < 0.001). This comparison between the SILD + 1 and the C-LADG group produced comparable results. The SILT+1 group underwent a 56.32 min longer operation than the C-LATG group (273.03 ± 66.80 vs. 216.71 ± 82.61, P = 0.0205). SILG+1 group had better postoperative visual analog scale (VAS) and cosmetic score than those of the C-LATG group (P < 0.05). There were no significant differences in preoperative demographics or 30-day postoperative complication rates between the SILG+1 and C-LAG groups. Tumor-related index, including mass size, histological type, number of retrieved lymph nodes, pathological tumor-node-metastasis (TNM) stage, and proximal and distal edges were all equivalent between the SILG+1 and the C-LAG group. CONCLUSIONS: This retrospective study demonstrates the safety and feasibility of SILG+1 with D1+ or D2 lymphadenectomy for the treatment of early and advanced gastric cancers, compared with C-LAG.

Feasibility and preliminary experience of single-incision plus one-port laparoscopic total gastrectomy with Overlap esophagojejunostomy for gastric cancer: A study of 10 cases.

Background: This study aimed to explore the feasibility and safety of single-incision plus one-port laparoscopic total gastrectomy (SITG + 1) with Overlap esophagojejunostomy (SITG + 1-Overlap) and to share preliminary experiences. Methods: This retrospective study included 10 patients with gastric cancer located in the cardia or body who underwent SITG + 1-Overlap between August 2020 and October 2021.The demographics, tumor characteristics, postoperative outcomes, and short-term complications of all the enrolled patients were summarized and statistically analyzed. Data were expressed as mean ± standard deviation (SD) if they were normally distributed. Otherwise, Median (Quartile1, Quartile3) was used. Results: In the collective perioperative data of these 10 patients who underwent radical gastrectomy, the median of the length of transumbilical incision and blood loss were 3.0 cm and 100.0 ml respectively, and the mean operation time and 385.5 ± 51.6 min. Postoperative data indicated that the gastric tube was removed on 2.0 (2.0, 3.0) days, and the timing of first feeding, activity, flatus, and defecation was 1.5 (1.0, 2.0) days, 2.0 (2.0, 2.0) days, 3.0 (2.0, 3.0) days, and 3.8 ± 0.6 days, respectively. The timing of drainage tube removal was 4.6 ± 1.0 days after operation. The duration of hospital stay was 7.5 ± 1.2 days and the VAS pain scores for the 3 days following surgery were 3.0 (2.0, 3.3), 2.0 (2.0, 3.0), and 1.5 (1.0, 2.0) respectively. The mean number of retrieved lymph nodes was 30.7 ± 13.2. Most biochemical indicators gradually normalized with the recovery of the patients after surgery. No 30-day postoperative complications were noted. Conclusions: For the first time, our preliminary data indicate the feasibility and safety of Overlap esophagojejunostomy in SITG + 1 surgery. This modified Overlap procedure has the potential to simplify the reconstruction procedure and lower the technical challenge of SITG + 1 radical gastrectomy for cardia or upper gastric cancer in the early and advanced stages.

Macropinocytosis in Cancer-Associated Fibroblasts Is Dependent on CaMKK2/ARHGEF2 Signaling and Functions to Support Tumor and Stromal Cell Fitness.

Although pancreatic ductal adenocarcinoma (PDAC) cells are exposed to a nutrient-depleted tumor microenvironment, they can acquire nutrients via macropinocytosis, an endocytic form of protein scavenging that functions to support cancer metabolism. Here, we provide evidence that macropinocytosis is also operational in the pancreatic tumor stroma. We find that glutamine deficiency triggers macropinocytic uptake in pancreatic cancer-associated fibroblasts (CAF). Mechanistically, we decipher that stromal macropinocytosis is potentiated via the enhancement of cytosolic Ca2+ and dependent on ARHGEF2 and CaMKK2-AMPK signaling. We elucidate that macropinocytosis has a dual function in CAFs-it serves as a source of intracellular amino acids that sustain CAF cell fitness and function, and it provides secreted amino acids that promote tumor cell survival. Importantly, we demonstrate that stromal macropinocytosis supports PDAC tumor growth. These results highlight the functional role of macropinocytosis in the tumor stroma and provide a mechanistic understanding of how nutrient deficiency can control stromal protein scavenging. SIGNIFICANCE: Glutamine deprivation drives stromal macropinocytosis to support CAF cell fitness and provide amino acids that sustain PDAC cell survival. Selective disruption of macropinocytosis in CAFs suppresses PDAC tumor growth.This article is highlighted in the In This Issue feature, p. 1601.

Redox regulation of hormone sensitive lipase: Potential role in the mechanism of MEHP-induced stimulation of basal steroid synthesis in MA-10 Leydig cells.

Mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of di-(2-ethylhexyl) phthalate (DEHP), is a plasticizer with endocrine disruptor activity that has been shown to stimulate basal steroid biosynthesis in Leydig cells. The mechanism by which it does so is unknown. Using MA-10 mouse tumor Leydig cells, we assessed the effects of MEHP on reactive oxygen species (ROS) levels, and on the signal transduction pathways that mobilize cholesterol. Exposure to 0-300 µM MEHP stimulated basal progesterone production in a dose-dependent manner. Progesterone stimulation was correlated with increases in the phosphorylation of hormone-sensitive lipase (HSL; aka cholesteryl ester hydrolase), which is involved in the production of free cholesterol, and of steroidogenic acute regulatory (STAR) protein expression. Co-treating MA-10 cells with MEHP and the ROS scavenger N-acetyl cysteine (NAC) blocked the activation of HSL, blunted MEHP-induced STAR, and reduced basal progesterone formation. These observations suggest that ROS generation by MEHP leads to activation of HSL and increase in STAR which, together, result in increased free-cholesterol bioavailability and progesterone formation.

[Feasibility and preliminary technical experience of single incision plus one port laparoscopic total gastrectomy combined with π-shaped esophagojejunal anastomosis in surgical treatment of gastric cancer].

OBJECTIVE: To explore the feasibility, safety, and preliminary technical experience of single incision plus one port laparoscopic total gastrectomy combined with π-shaped esophagojejunal anastomosis (SILT-π) in the surgical treatment of gastric cancer. METHODS: Clinical data of 5 gastric cancer patients undergoing SILT-π operation at the Department of General Surgery, The Second Affiliated Hospital of the Army Medical University from August to October 2017 were retrospectively analyzed. A 2.5-3.0 cm incision around the umbilicus was made for placing the gloveport as the passage for the lens, and the instruments of the surgeon and the assistant. Another operative port was placed in the left upper quadrant with a 12-mm Trocar for the passage of the energy device, the endoscopic cutting closure, as well as the postoperative drainage tube. A D2 lymph node (LNs) dissection was regularly conducted. After the abdominal esophagus was routinely mobilized, a side-to-side esophagus-jejunum anastomosis was made through a gastric pre-pulling esophagojejunal π-shaped anastomosis. The transection was then performed with a ligation on the cardia (or esophagus above the upper margin of the tumor) using a sterilized hemp rope in order to better expose the abdominal esophagus. Throughout the course of reconstruction, the ligature rope was held by the assistant to hold down the esophagus to allow easier esophagojejunal anastomosis. A hole was then made on the posterior wall of the esophagus, between 2 cm and 3 cm above the ligature rope, and another hole was made at the anti-mesenteric border of the jejunum 40 cm distal to the Treitz ligament. A side-to-side esophagojejunal π-shaped anastomosis was performed through two holes. An entry hole was formed after the anastomosis. After checking the anastomosis, this entry hole was closed through an intestinal mesenteric hole pre-made on its opposite side. The resected esophagus and stomach, together with the afferent loop jejunum, were simultaneously transected above the level of the entry hole by a stapler from the Trocar of the left upper abdominal quadrant. After the gloveport was closed, a side-to-side jejunojejunostomy anastomosis applied with another two staples was performed between the afferent loop stump and the roux limb 30 cm below the esophagojejunal anastomosis. RESULTS: These five patients were all male, and aged (56.8±8.2) years with preoperative clinical stage cT2-4N0-2M0. All the 5 patients underwent SILT-π operation successfully. The average length of surgical incision was (2.9±0.2) cm. The average operation time was (396.0±36.1) minutes. The intraoperative blood loss was (140.0±66.7) ml. Postoperative pathology showed proximal and distal margins were (2.6±1.1) cm and (8.7±2.5) cm apart respectively, and the average number of retrieved lymph node was 25.8±7.2. Perioperative management was based on enhanced recovery following surgical (ERAS) principles. The average time to the first flatus was (2.6±0.5) days, and the average time to defecation was (3.6±0.5) days. The pain score on postoperative day 1 was 1-2, and the average postoperative hospital stay was (7.0±0.7) days. No perioperative complications occurred. CONCLUSIONS: SILT-π procedure is safe and feasible for patients with gastric cancer, and has positive short-term outcomes, satisfactory cosmetic abdominal incision, light postoperative abdominal pain and rapid postoperative recovery. Preliminary observations show that SILT-π procedure has good potential for clinical application in future.

STMN1 as a candidate gene associated with atypical meningioma progression.

OBJECTIVES: Meningiomas are the most common type of primary intracranial tumor. Atypical meningiomas are especially difficult to manage due to frequent disease recurrence. This study aimed to examine the role of stathmin (coded by the gene STMN1) as a factor in atypical meningioma recurrence. PATIENTS AND METHODS: A total of 59 sporadic atypical meningioma formalin-fixed paraffin-embedded (FFPE) samples were collected. The mRNA levels of the biomarker gene STMN1 were tested using quantitative RT-PCR. RESULTS: We observed significant up-regulation of STMN1 mRNA expression in recurrent tumors in comparison with primary tumors (p<0.05). Moreover, mRNA expression levels of STMN1 significantly correlated with Ki-67 score (r=0.93, p<0.01). Multivariate survival analyses indicated that high expression of STMN1, high Ki-67 score, and more advanced patient age at diagnosis (>60yrs) each act as independence prognostic factors for recurrence. Kaplan-Meier analysis revealed that STMN1 expression pattern could effectively predict prognosis of atypical meningioma in patients (p<0.01). CONCLUSIONS: Our study indicates for the first time that an increased risk of sporadic atypical meningioma recurrence can be found in cases with elevated expression of STMN1. These results suggest that STMN1 expression might serve as a biomarker for determining patient atypical meningioma prognosis.

Mitochondrial Electron Transport Chain-Derived Superoxide Exits Macrophages: Implications for Mononuclear Cell-Mediated Pathophysiological Processes.

The involvement of mitochondrial electron transport chain (METC)-derived superoxide anion radical in cell protooncogene activation, mitogenic responses, and cancerous growth has recently received much attention. In order for METC-derived superoxide to participate in any of the above processes, its exit from mitochondria would be a critical step. Detection of intracellular superoxide showed that mitochondrial respiration is the major source of cellular superoxide in unstimulated or resting monocytes/macrophages. However, direct evidence for the exit of superoxide from mitochondria is presently lacking. Here we show that METC-derived superoxide does exit from mitochondria in unstimulated monocytes/macrophages. Release of superoxide was first found to occur with substrate-supported mitochondria isolated from these cells. We also observed the presence of extracellular superoxide with the intact unstimulated/resting cells. Extracellular superoxide was markedly diminished (>90%) by the mitochondrial inhibitor, rotenone, or the uncoupler, carbonylcyanide p-(trifluromethy) phenylhydrazone. Furthermore, cells with a deficient METC exhibited significant reduction (>90%) in extracellular superoxide, demonstrating that with intact cells METC-derived superoxide not only exits from mitochondria, but can be released extracellularly. Superoxide anion radical released from mitochondria could react with exogenous nitric oxide, forming peroxynitrite. Mitochondria-derived extracellular superoxide could also oxidize low-density lipoprotein (LDL). These results thus resolve any uncertainty on the ability of superoxide to exit from mitochondria. This study for the first time also identifies mitochondria as the major source of extracellular superoxide in unstimulated resting monocytes/macrophages, which has implications for the involvement of these mononuclear cells in various pathophysiological situations.

Luteolin protects against vascular inflammation in mice and TNF-alpha-induced monocyte adhesion to endothelial cells via suppressing IΚBα/NF-κB signaling pathway.

Vascular inflammation plays a significant role in the pathogenesis of atherosclerosis. Luteolin, a naturally occurring flavonoid present in many medicinal plants and some commonly consumed fruits and vegetables, has received wide attention for its potential to improve vascular function in vitro. However, its effect in vivo and the molecular mechanism of luteolin at physiological concentrations remain unclear. Here, we report that luteolin as low as 0.5 µM significantly inhibited tumor necrosis factor (TNF)-α-induced adhesion of monocytes to human EA.hy 926 endothelial cells, a key event in triggering vascular inflammation. Luteolin potently suppressed TNF-α-induced expression of the chemokine monocyte chemotactic protein-1 (MCP-1) and adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), key mediators involved in enhancing endothelial cell-monocyte interaction. Furthermore, luteolin inhibited TNF-α-induced nuclear factor (NF)-κB transcriptional activity, IκBα degradation, expression of IκB kinase ß and subsequent NF-κB p65 nuclear translocation in endothelial cells, suggesting that luteolin can inhibit inflammation by suppressing NF-κB signaling. In an animal study, C57BL/6 mice were fed a diet containing 0% or 0.6% luteolin for 3 weeks, and luteolin supplementation greatly suppressed TNF-α-induced increase in circulating levels of MCP-1/JE, CXCL1/KC and sICAM-1 in C57BL/6 mice. Consistently, dietary intake of luteolin significantly reduced TNF-α-stimulated adhesion of monocytes to aortic endothelial cells ex vivo. Histology shows that luteolin treatment prevented the eruption of endothelial lining in the intima layer of the aorta and preserved elastin fibers' delicate organization as shown by Verhoeff-Van Gieson staining. Immunohistochemistry studies further show that luteolin treatment also reduced VCAM-1 and monocyte-derived F4/80-positive macrophages in the aorta of TNF-α-treated mice. In conclusion, luteolin protects against TNF-α-induced vascular inflammation in both in vitro and in vivo models. This anti-inflammatory effect of luteolin may be mediated via inhibition of the NF-κB-mediated pathway.

A novel mutation in the RPE65 gene causing Leber congenital amaurosis and its transcriptional expression in vitro.

The retinal pigment epithelium-specific 65 kDa protein is an isomerase encoded by the RPE65 gene (MIM 180069) that is responsible for an essential enzymatic step required for the function of the visual cycle. Mutations in the RPE65 gene cause not only subtype II of Leber congenital amaurosis (LCA) but also early-onset severe retinal dystrophy (EOSRD). This study aims to investigate a Chinese case diagnosed as EOSRD and to characterize the polymorphisms of the RPE65 gene. A seven-year-old girl with clinical symptoms of EOSRD and her parents were recruited into this study. Ophthalmologic examinations, including best-corrected visual acuity, slit-lamp, Optical coherence tomography (OCT), and fundus examination with dilated pupils, were performed to determine the clinical characteristics of the whole family. We amplified and sequenced the entire coding region and adjacent intronic sequences of the coding regions of the RPE65 gene for the whole family to explore the possible mutation. Our results demonstrate that the patient exhibited the typical clinically features of EOSRD. Her bilateral decimal visual acuity was 0.3 and 0.4 in the left and right eyes, respectively. Spectral-domain optical coherence tomography (SD-OCT) was used to assess the retinal stratification for the whole family. All together, we identified four mutations within the RPE65 gene (c.1056G>A, c.1243+2T>A, c.1338+20A>C and c.1590C>A) in the patient. Among the four mutations, c.1056G>A and c.1338+20A>C had been reported previously and another two were found for the first time in this study. Her mother also carried the novel mutation (c.1243+2T>A). Either a single or a compound heterozygous or a homozygous one mutation is expected to cause EOSRD because mutations of RPE65 gene usually cause an autosomal recessive disease. Therefore, we speculate that the c.1590C>A mutation together with the c.1243+2T>A mutation may cause the patient's phenotype.

Sulforaphane reduces vascular inflammation in mice and prevents TNF-α-induced monocyte adhesion to primary endothelial cells through interfering with the NF-κB pathway.

Sulforaphane, a naturally occurring isothiocyanate present in cruciferous vegetables, has received wide attention for its potential to improve vascular function in vitro. However, its effect in vivo and the molecular mechanism of sulforaphane at physiological concentrations remain unclear. Here, we report that a sulforaphane concentration as low as 0.5 µM significantly inhibited tumor necrosis factor-α (TNF-α)-induced adhesion of monocytes to human umbilical vein endothelial cells, a key event in the pathogenesis of atherosclerosis both in static and under flow conditions. Such physiological concentrations of sulforaphane also significantly suppressed TNF-α-induced production of monocyte chemotactic protein-1 and adhesion molecules including soluble vascular adhesion molecule-1 and soluble E-selectin, key mediators in the regulation of enhanced endothelial cell-monocyte interaction. Furthermore, sulforaphane inhibited TNF-α-induced nuclear factor (NF)-κB transcriptional activity, Inhibitor of NF-κB alpha (IκBα) degradation and subsequent NF-κB p65 nuclear translocation in endothelial cells, suggesting that sulforaphane can inhibit inflammation by suppressing NF-κB signaling. In an animal study, sulforaphane (300 ppm) in a mouse diet significantly abolished TNF-α-increased ex vivo monocyte adhesion and circulating adhesion molecules and chemokines in C57BL/6 mice. Histology showed that sulforaphane treatment significantly prevented the eruption of endothelial lining in the intima layer of the aorta and preserved elastin fibers' delicate organization, as shown by Verhoeff-van Gieson staining. Immunohistochemistry studies showed that sulforaphane treatment also reduced vascular adhesion molecule-1 and monocyte-derived F4/80-positive macrophages in the aorta of TNF-α-treated mice. In conclusion, sulforaphane at physiological concentrations protects against TNF-α-induced vascular endothelial inflammation, in both in vitro and in vivo models. This anti-inflammatory effect of sulforaphane may be, at least in part, associated with interfering with the NF-κB pathway.

Protective effects of extracts from Fructus rhodomyrti against oxidative DNA damage in vitro and in vivo.

OBJECTIVE: To evaluate the potential protective effects of extracts from Fructus rhodomyrti (FR) against oxidative DNA damage using a cellular system and the antioxidant ability on potassium bromate- (KBrO3-) mediated oxidative stress in rats. METHODS: The effects of FR on DNA damage induced by hydrogen peroxide (H2O2) were evaluated by comet assay in primary spleen lymphocytes cultures. The effects of FR on the activities of SOD, CAT, and GPx and the levels of GSH, hydroperoxides, and 8-OHdG were determined in the plasma and tissues of rats treated with KBrO3. RESULTS: FR was shown to effectively protect against DNA damage induced by H2O2 in vitro, and the maximum protective effect was observed when FR was diluted 20 times. Endogenous antioxidant status, namely, the activities of SOD, CAT, and GPx and the levels of GSH were significantly decreased in the plasma, the liver, and the kidney of the KBrO3-treated rats, while the pretreatment of FR prevented the decreases of these parameters. In addition, the pretreatment of FR was also able to prevent KBrO3-induced increases in the levels of hydroperoxides and 8-OHdG in the plasma, the liver, and the kidney in rats. CONCLUSIONS: Our findings suggested that FR might act as a chemopreventive agent with antioxidant properties offering effective protection against oxidative DNA damage in a concentration-dependent manner in vitro and in vivo.

Involvement of post-transcriptional regulation of FOXO1 by HuR in 5-FU-induced apoptosis in breast cancer cells.

The post-transcriptional control of specific mRNAs is a widespread mechanism of gene regulation, which contributes to numerous biological processes in a number of cell types. The Forkhead box O (FoxO) transcription factor FOXO1 is an important tumor suppressor involved in apoptosis, the cell cycle, DNA damage repair and oxidative stress. Bioinformatic prediction identified that the 3' untranslated region (UTR) of FOXO1 is enriched with binding motifs for the human ELAV/Hu protein (HuR), indicating that FOXO1 is a potential target of HuR. Luciferase reporter assays demonstrate that HuR specifically regulates FOXO1 expression through AU-rich elements (AREs) within the FOXO1 3' UTR. Immunoprecipitation studies confirmed that HuR associates with FOXO1 mRNA in MDA-MB-231 breast cancer cells and that HuR upregulates FOXO1 mRNA levels through increased mRNA stability. Using a HuR loss- and gain-of-function approach, we revealed that FOXO1 expression was correspondingly decreased or increased in MDA-MB-231 cells. Functional assays demonstrated that HuR and FOXO1 expression levels were markedly enhanced upon 5-fluorouracil (5-FU) stimulation in MDA-MB-231 cells. Knockdown of HuR apparently abrogated 5-FU-induced apoptosis detected by caspase-3 activities. Furthermore, in HuR knockdown cells, additional overexpression of FOXO1 moderately recovered 5-FU-induced apoptosis, which verified that HuR-modulated apoptosis upon 5-FU treatment was partially mediated by its post-transcriptional regulation of FOXO1. Therefore, modulating FOXO1 expression has been suggested to lead to the development of new therapeutic treatments for certain types of cancer.