Induced Inflammatory and Oxidative Markers in Cerebral Microvasculature by Mentally Depressive Stress.

Background: Cerebrovascular disease (CVD) is recognized as the leading cause of permanent disability worldwide. Depressive disorders are associated with increased incidence of CVD. The goal of this study was to establish a chronic restraint stress (CRS) model for mice and examine the effect of stress on cerebrovascular inflammation and oxidative stress responses. Methods: A total of forty 6-week-old male C57BL/6J mice were randomly divided into the CRS and control groups. In the CRS group (n = 20), mice were placed in a well-ventilated Plexiglas tube for 6 hours per day for 28 consecutive days. On day 29, open field tests (OFT) and sucrose preference tests (SPT) were performed to assess depressive-like behaviors for the two groups (n = 10/group). Macrophage infiltration into the brain tissue upon stress was analyzed by measuring expression of macrophage marker (CD68) with immunofluorescence in both the CRS and control groups (n = 10/group). Cerebral microvasculature was isolated from the CRS and controls (n = 10/group). mRNA and protein expressions of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), vascular cell adhesion molecule-1 (VCAM-1), and macrophage chemoattractant protein-1 (MCP-1) in the brain vessels were measured by real-time PCR and Western blot (n = 10/group). Reactive oxygen species (ROS), hydrogen peroxide (H2O2), and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activities were quantified by ELISA to study the oxidative profile of the brain vessels (n = 10/group). Additionally, mRNA and protein expressions of NOX subunits (gp91phox, p47phox, p67phox, and p22phox) in the cerebrovascular endothelium were analyzed by real-time PCR and Western blot (n = 10/group). Results: CRS decreased the total distances (p < 0.05) and the time spent in the center zone in OFT (p < 0.001) and sucrose preference test ratio in SPT (p < 0.01). Positive ratio of CD68+ was increased with CRS in the entire region of the brain (p < 0.001), reflecting increased macrophage infiltration. CRS increased the expression of inflammatory factors and oxidative stress in the cerebral microvasculature, including TNF-α (p < 0.001), IL-1ß (p < 0.05), IL-6 (p < 0.05), VCAM-1 (p < 0.01), MCP-1 (p < 0.01), ROS (p < 0.001), and H2O2 (p < 0.001). NADPH oxidase (NOX) was activated by CRS (p < 0.01), and mRNA and protein expressions of NOX subunits (gp91phox, p47phox, p67phox, and p22phox) in brain microvasculature were found to be increased. Conclusions: To our knowledge, this is the first study to demonstrate that CRS induces depressive stress and causes inflammatory and oxidative stress responses in the brain microvasculature.

Ethyl 6-Hydroxyfulvene-1-Carboxylate: A Reagent Discriminating Primary Amines from Secondary Amines.

A highly chemo-selective reaction was observed when ethyl 6-hydroxyfulvene-1-carboxylate 1 was treated with different nucleophiles such as primary amines, secondary amines, alcohols, and thiols. Among them, only primary amines are reactive toward 1 to afford the condensation products 3, which exhibit good stability under both weakly acidic and basic conditions. The condensation process proved to be reversible between different primary amines. On the basis of this observation, the chemical selectivity of typical primary aromatic amines was evaluated quantitatively by determining equilibrium constants of the condensation reactions with aniline as a reference. Moreover, the primary amines of 3 can be readily released upon treatment with aqueous ammonia, making 6-hydroxyfulvene-1-carboxylate 1 a promising protecting reagent for primary amines.

Regioselective addition of phosphites to acyl cyclopropanes and following rearrangements: a facile access to enol phosphates.

An inorganic base-promoted domino reaction with organophosphites and acyl cyclopropane derivatives is developed and proved to provide an efficient access to functionalized enol phosphates. Unlike the well-known Perkow reaction, which employs trialkyl phosphite as the nucleophile, dialkyl phosphite is the key to the success of our transformation. This method is compatible with a series of dialkyl phosphites and acyl cyclopropanes possessing electron-withdrawing substituents, and an array of functionalized enol phosphates are successfully prepared. Based on the results of isotope-labeling and control experiments, this transformation is presumably initiated by the deprotonation of dialkyl phosphite and the following nucleophilic addition/anion shift/ring opening sequence leads to the formation of enol phosphates. Both the strain release of a three-membered ring and the formation of a relatively stable anion are indispensable driving forces for this process.

Triflic Acid-Catalyzed Cycloisomerization Reactions of Donor-Acceptor Cyclopropanes: Access to Alkyl 5-Arylfuran-2-carboxylates.

A direct synthetic strategy starting from alkyl 1-alkoxy-2-aroylcyclopropanecarboxylates was developed for the construction of alkyl 5-arylfuran-2-carboxylates. These donor-acceptor cyclopropanes smoothly undergo a simple ring-opening reaction or/and cycloisomerization reaction in the presence of acid at room temperature, which greatly depends on the properties of the acid used in the experiment. Alkyl 5-arylfuran-2-carboxylates were afforded in high yields in triflic acid, whereas alkyl 2,5-dioxo-5-phenylpentanoate became the major product in other protic acids and Lewis acids.

Construction of 2-Pyrone Skeleton via Domino Sequence between 2-Acyl-1-Chlorocyclopropanecarboxylate and Amines.

A base-promoted domino reaction of 2-acyl-1-chlorocyclopropaneformic esters with amines is described. In the presence of inorganic bases like Cs2CO3 or Mg(OEt)2, the reaction proceeded smoothly in acetonitrile to afford 2-pyrone derivatives in modest to excellent yields (up to 97%). This reaction provides a straightforward and transition metal-free protocol to efficiently construct 2-pyrone skeleton. A possible mechanistic process involving 1,2-elimination of hydrogen chloride, aza-Michael addition, ring-opening, and intramolecular lactonization was suggested to rationalize the formation of the target 2-pyrone derivatives.

Synthesis of functionalized fulvenes: [3 + 2] annulation of ethyl α-chlorocyclopropaneformates with 1,3-dicarbonyl compounds.

A base-promoted [3 + 2] annulation reaction of ethyl α-chlorocyclopropaneformates with 1,3-dicarbonyl compounds is described. This method provides an efficient and straightforward route to acidic multi-substituted fulvenes with distinctive properties.

Forkhead Box 1(FoxO1) mediates psychological stress-induced neuroinflammation.

OBJECTIVES:  Neuroinflammation plays a key role in cerebrovascular disease (CVD). Neuropsychiatric disorders appear to share an epidemiological association with inflammation, but the mechanisms are unclear. Forkhead box 1 (FoxO1) regulates inflammatory signaling in diabetes and cardiovascular diseases, but its role in psychological stress-induced neuroinflammation remains unknown. Therefore, we investigated the potential involvement of FoxO1 in repeated social defeat stress (RSDS)-induced neuroinflammation. METHODS:  6-week-old male C57BL/6 J mice were randomly divided into RSDS or control groups. In the RSDS group, mice (18-22 g) were individually subjected to social defeat by an 8-week-old CD-1 mouse (28-32 g) for 10 min daily for 10 consecutive days. At 24 h after this 10-day process, corticosterone (CORT), epinephrine (EPI), hydrogen peroxide, and inflammatory factors (TNF-α, IL-6, IL-1ß, and VCAM-1) from serum and brain tissues were assayed using ELISA, real-time PCR, and Western blot. Iba-1 was determined by immunofluorescence (IF), and FoxO1 siRNA was transfected into BV2 cells to further analyze the expression of inflammatory factors. RESULTS: RSDS significantly increased the levels of TNF-α, IL-6, IL-1ß, and VCAM-1 in the serum; it also increased both mRNA and protein expression of these in the brain. FoxO1 was significantly increased after stress, while its knockdown significantly suppressed stress-induced inflammation. Immunofluorescence demonstrated the activation of microglia in the setting of RSDS. CONCLUSION: RSDS induced a measurable inflammatory response in the blood and brain, and FoxO1 was demonstrated in vitro to aggravate stress-induced inflammation.

miRNA-31 increases radiosensitivity through targeting STK40 in colorectal cancer cells.

OBJECTIVE: To propose and verify that miRNA-31 increases the radiosensitivity of colorectal cancer and explore its potential mechanism. METHOD: A bioinformatics analysis was performed to confirm that the expression of miRNA-31 was higher in colorectal cancer than in normal colorectal tissue. The expression of miRNA-31 was detected to verify the change in its expression in a radiotherapy-resistant cell line. Methylation was detected to explore the cause of the decrease in miRNA-31 expression. Overexpression or inhibition of miRNA-31 further confirmed the change in its expression in colorectal cancer cell lines. Bioinformatics methods were used to screen the downstream target genes and for experimental verification. A luciferase assay was performed to determine the miRNA-31 binding site in STK40. Overexpression or inhibition of STK40 in colorectal cancer cell lines further confirmed the change in STK40 expression in vitro. RESULTS: The bioinformatics results showed higher expression of miRNA-31 in tumors than in normal tissue, and miRNA-31 mainly participated in the pathway related to cell replication. Next, we observed the same phenomenon: miRNA-31 was expressed at higher levels in colorectal tumors than in normal colorectal tissue and its expression decreased in radiation-resistant cell lines after radiation, implying that miRNA-31 increased the radiosensitivity of colorectal cancer cell lines. No significant change in upstream methylation was observed. miRNA-31 regulated the radiosensitivity of colorectal cancer cell lines by inhibiting STK40. Notably, miRNA-31 played a role by binding to the 3' untranslated region of SK40. STK40 negatively regulated the radiosensitivity of colorectal cancer cells. CONCLUSIONS: miRNA-31 increases the radiosensitivity of colorectal cancer cells by targeting STK40; miRNA-31 and STK40 are expected to become potential biomarkers for increasing the sensitivity of tumor radiotherapy in clinical treatment.

Filtered air intervention reduces inflammation and hypothalamus-pituitary-adrenal axis activation in adult male and female rats after PM 2.5 exposure.

Previous studies have indicated that particulate matter 2.5 (PM2.5) exposure stimulates systemic inflammation and activates the hypothalamus-pituitary-adrenal (HPA) axis, both of which are associated with stroke incidence and mortality. However, whether filtered air (FA) intervention modulates inflammation and HPA axis activation is still largely unknown. For FA group and PM2.5 group, adult Sprague-Dawley male and female rats were exposed to FA or PM2.5 for 6 months, respectively. For PM2.5 + 15 days FA group, the rats were achieved by receiving 15 days FA after PM2.5 exposure for 6 months. The immune cells and inflammatory biomarker levels in the blood and brain were analyzed by flow cytometry, ELISA, and qRT-PCR. To assess HPA axis activation, the levels of hormones in the blood were also analyzed by ELISA. FA intervention increased the percentage of CD4 T cells and T cells in the blood, which had decreased after PM2.5 exposure in both male and female rats. The ELISA and qRT-PCR results showed that FA intervention significantly reduced the levels of inflammatory biomarkers in the peripheral blood, and alleviated neuroinflammation in the cortex, hippocampus, and striatum. In addition, FA intervention also inhibited the inflammation in the hypothalamus and pituitary and adrenal glands, and decreased the levels of HPA axis hormones. Our results indicate that FA intervention exerts a protective effect on the brain by decreasing inflammation and HPA axis activation after PM2.5 exposure in both male and female rats.

PEX5, a novel target of microRNA-31-5p, increases radioresistance in hepatocellular carcinoma by activating Wnt/ß-catenin signaling and homologous recombination.

Rationale: Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide, with high recurrence and metastasis rates. Although radiation is an effective treatment for tumors, it is often limited by intrinsic radioresistance in HCC. The contributions of dysregulated microRNAs, including miR-31-5p, to HCC progression have been recently reported. However, the role of miR-31-5p in the radiation response of HCC is unknown. In this study, we aimed to investigate the impact of miR-31-5p on HCC radiosensitivity. Methods: miR-31-5p expression in HCC tissues, paired adjacent tissues, and HCC cell lines was measured using quantitative real-time polymerase chain reaction and in situ hybridization. Bioinformatic analyses, gain- and loss-of-function experiments, and luciferase reporter assays were performed to validate peroxisomal biogenesis factor 5 (PEX5) as a direct target of miR-31-5p. The biofunctions of PEX5 and miR-31-5p in HCC were determined by Transwell, wound-healing, and Cell Counting Kit-8 (CCK8) assays. A colony formation assay was used to evaluate the radiosensitivity of HCC cells. The interaction among PEX5, ß-catenin, Rac1, and JNK-2 was confirmed by coimmunoprecipitation. A xenograft tumor model was established to validate the effects of miR-31-5p and PEX5 on HCC progression and radiosensitivity in vivo.Results: Low expression of miR-31-5p in HCC specimens, as observed in this study, predicted a poor clinical outcome. However, the expression pattern of PEX5, as a direct target of miR-31-5p, was opposite that of miR-31-5p, and high PEX5 expression indicated poor prognosis in HCC patients. Ectopic expression of PEX5 increased the proliferation, migration, and invasion abilities and enhanced the radioresistance of HCC cells in vitro and in vivo; however, these phenotypes were inhibited by miR-31-5p. Mechanistically, PEX5 stabilized cytoplasmic ß-catenin and facilitated ß-catenin nuclear translocation to activate Wnt/ß-catenin signaling. Moreover, upon radiation exposure, PEX5 reduced excessive reactive oxygen species (ROS) accumulation and activated the homologous recombination (HR) pathway, which protected HCC cells from radiation-induced damage. Conclusions: Our findings demonstrated a novel role for PEX5 as a miR-31-5p target and a mediator of the Wnt/ß-catenin signaling and HR pathways, providing new insights into studying HCC radiation responses and implicating PEX5 and miR-31-5p as potential therapeutic targets in HCC.

Neuroinflammation caused by mental stress: the effect of chronic restraint stress and acute repeated social defeat stress in mice.

Objectives: Cerebrovascular disease (CVD) is the leading cause of permanent disability worldwide. Inflammation has been reported to play an important role in the progression of CVD. Neuropsychiatric disorders such as depression are associated with increased incidence of CVD epidemiologically, although the mechanisms underlying this association are not clear. In this study, we assessed the effect of the acute repeated social defeat stress (RSDS) and chronic restraint stress (CRS) on neuroinflammation in mice. Methods: A total of 40 6-week-old male C57BL/6J mice were divided into RSDS, CRS, and corresponding control groups. In the RSDS group, male C57BL/6J mice were repeatedly subjected to bouts of social defeat by a larger CD-1 mouse for 10 min daily for 10 consecutive days. In the CRS group, the mice were exposed to restraint stress for 6 h per day for 28 consecutive days. Depressive behavior was evaluated by conducting sucrose preference test over 24 h. Peripheral blood serum and brain tissues were collected for measurement of corticosterone (CORT), epinephrine (EPI), and inflammatory factors (TNF-α and IL-6) using ELISA or real-time PCR 24 h after the sucrose preference test. Results: Both RSDS and CRS decreased the sucrose preference ratio. The acute stress increased serum CORT and EPI, while the chronic stress did not significantly influence them. Both stress models induced an inflammatory response in peripheral serum and the brain. Conclusions: RSDS and CRS are two effective models of depressive behavior, and both models cause neuroinflammation, which may be responsible for the increased risk of CVD seen in patients with depression.

A mini review: garlic extract and vascular diseases.

Vascular diseases refer to medical conditions that narrow blood vessels. Narrowed cardiac or cerebral arteries can lead to myocardial infarction or ischemic stroke. Risk factors including atherosclerosis, hypertension, and diabetes may induce either cardiovascular or cerebral complications. Based on current research, garlic favorably affects atherosclerosis, hypertension and diabetes, and helps decrease the risk of myocardial infarction and ischemic stroke. Garlic has been utilized for hundreds of years as a natural health remedy. New research is emerging regarding its effectiveness in treating common diseases, including atherosclerosis, hypertension, and diabetes. The underlying mechanisms by which garlic, and its byproducts, can alter pathophysiology have begun to be elucidated by these studies. Garlic is a prominent topic for future research regarding its potential as an adjuvant to conventional pharmacotherapy for these common health conditions. In this mini-review, we discuss the current state of the literature regarding garlic and its effects in patients with vascular disease. Specifically, we decided to briefly discuss the key points regarding the mechanisms underlying garlic's anti-hypertensive, anti-hyperlipidemic, and hypoglycemic effects. This allows the readers to understand each process while keeping the paper concise. These mechanisms can be further explored in the original articles, at the reader's discretion.

A potential role of microRNAs in protein accumulation in cellular senescence analyzed by bioinformatics.

Cellular senescence is an important protective mechanism against cell proliferation and has critical roles in aging and aging-related disease. Recently, one interesting observation is that the protein abundance is higher in senescent cells than that in young cells. So far, some factors were presented to interpret this observation, such as active protein synthesis linked with autophagy, mTOR, and oxidative stress. Here, applying bioinformatic analysis of microRNA profiles in young cells and aging cells, we revealed that globally senescent cells show lower miRNA abundance than that in young cells, suggesting that the repression of protein synthesis by miRNA in senescent cells could be largely attenuated. This finding provides clues that protein accumulation in cellular senescence could be associated with lower miRNA abundance in aging cells.

Genetic alteration and misexpression of Polycomb group genes in hepatocellular carcinoma.

Although the abnormal expression of Polycomb-group (PcG) proteins is closely associated with carcinogenesis and the clinicopathological features of hepatocellular carcinoma (HCC), the genetic mutation profile of PcG genes has not been well established. In this study of human HCC specimens, we firstly discovered a highly conserved mutation site, G553C, in the Polycomb Repressive Complex 2 (PRC2) gene enhancer of zeste homolog 2 (EZH2). This site also harbors a single nucleotide polymorphism (SNP), rs2302427, which plays an important antagonistic role in HCC. Kaplan-Meier survival curves showed that the tumor-free and overall survival of patients with EZH2 G553C were superior to those without the mutation. The G allele frequencies in patients and healthy subjects were 0.2% and 0.122%, respectively, with significant differences in distribution. The individuals carrying the GG and the GC genotypes at rs2302427 showed 3.083-fold and 1.827-fold higher risks of HCC, respectively, compared with individuals carrying the wild-type allele. Furthermore, Immunohistochemical staining revealed that the expression levels of CBX8 (in 53/123 samples) and BMI1 (in 60/130 samples) were markedly increased in human HCC specimens. Importantly, the overall and tumor-free survival rates were significantly reduced in the group of patients who simultaneously expressed PRC1 and PRC2. These results argue that a combination of PRC1 and PRC2 expression has a significant predictive/prognostic value for HCC patients. Taken together, our results indicate the abnormal expression and genetic mutation of PcG members are two independent events; cumulative genetic and epigenetic alterations act synergistically in liver carcinogenesis.