Novel role of LLGL2 silencing in autophagy: reversing epithelial-mesenchymal transition in prostate cancer.

PURPOSE: Prostate cancer (PCa) is a major urological disease that is associated with significant morbidity and mortality in men. LLGL2 is the mammalian homolog of Lgl. It acts as a tumor suppressor in breast and hepatic cancer. However, the role of LLGL2 and the underlying mechanisms in PCa have not yet been elucidated. Here, we investigate the role of LLGL2 in the regulation of epithelial-mesenchymal transition (EMT) in PCa through autophagy in vitro and in vivo. METHODS: PC3 cells were transfected with siLLGL2 or plasmid LLGL2 and autophagy was examined. Invasion, migration, and wound healing were assessed in PC3 cells under autophagy regulation. Tumor growth was evaluated using a shLLGL2 xenograft mouse model. RESULTS: In patients with PCa, LLGL2 levels were higher with defective autophagy and increased EMT. Our results showed that the knockdown of LLGL2 induced autophagy flux by upregulating Vps34 and ATG14L. LLGL2 knockdown inhibits EMT by upregulating E-cadherin and downregulating fibronectin and α-SMA. The pharmacological activation of autophagy by rapamycin suppressed EMT, and these effects were reversed by 3-methyladenine treatment. Interestingly, in a shLLGL2 xenograft mouse model, tumor size and EMT were decreased, which were improved by autophagy induction and worsened by autophagy inhibition. CONCLUSION: Defective expression of LLGL2 leads to attenuation of EMT due to the upregulation of autophagy flux in PCa. Our results suggest that LLGL2 is a novel target for alleviating PCa via the regulation of autophagy.

Mixed Medicinal Mushroom Mycelia Attenuates Alzheimer's Disease Pathologies In Vitro and In Vivo.

Alzheimer's disease (AD) is characterized by memory impairment and existence of amyloid-ß (Aß) plaques and neuroinflammation. Due to the pivotal role of oxidative damage in AD, natural antioxidative agents, such as polyphenol-rich fungi, have garnered scientific scrutiny. Here, the aqueous extract of mixed medicinal mushroom mycelia (MMMM)-Phellinus linteus, Ganoderma lucidum, and Inonotus obliquus-cultivated on a barley medium was assessed for its anti-AD effects. Neuron-like PC12 cells, which were subjected to Zn2+, an Aß aggregator, were employed as an in vitro AD model. The cells pretreated with or without MMMM were assayed for Aß immunofluorescence, cell viability, reactive oxygen species (ROS), apoptosis, and antioxidant enzyme activity. Then, 5XFAD mice were administered with 30 mg/kg/day MMMM for 8 weeks and underwent memory function tests and histologic analyses. In vitro results demonstrated that the cells pretreated with MMMM exhibited attenuation in Aß immunofluorescence, ROS accumulation, and apoptosis, and incrementation in cell viability and antioxidant enzyme activity. In vivo results revealed that 5XFAD mice administered with MMMM showed attenuation in memory impairment and histologic deterioration such as Aß plaque accumulation and neuroinflammation. MMMM might mitigate AD-associated memory impairment and cerebral pathologies, including Aß plaque accumulation and neuroinflammation, by impeding Aß-induced neurotoxicity.

Neuroprotective Effects of Water Extract from Brown Algae Petalonia binghamiae in an Experimental Model of Focal Cerebral Ischemia In Vitro and In Vivo.

Focal cerebral ischemia (fCI) can result in brain injury and sensorimotor deficits. Brown algae are currently garnering scientific attention as potential therapeutic candidates for fCI. This study investigated the therapeutic effects of the hot water extract of Petalonia binghamiae (wPB), a brown alga, in in vitro and in vivo models of fCI. The neuroprotective efficacy of wPB was evaluated in an in vitro excitotoxicity model established using HT-22 cells challenged with glutamate. Afterward, C57/BL6 mice were administered wPB for 7 days (10 or 100 mg/kg, intragastric) and subjected to middle cerebral artery occlusion and reperfusion (MCAO/R) operation, which was used as an in vivo fCI model. wPB co-incubation significantly inhibited cell death, oxidative stress, and apoptosis, as well as stimulated the expression of heme oxygenase-1 (HO-1), an antioxidant enzyme, and the nuclear translocation of its upstream regulator, nuclear factor erythroid 2-related factor 2 (Nrf2) in HT-22 cells challenged with glutamate-induced excitotoxicity. Pretreatment with either dose of wPB significantly attenuated infarction volume, neuronal death, and sensorimotor deficits in an in vivo fCI model. Furthermore, the attenuation of oxidative stress and apoptosis in the ischemic lesion accompanied the wPB-associated protection. This study suggests that wPB can counteract fCI via an antioxidative effect, upregulating the Nrf2/HO-1 pathway.

Perilla frutescens Leaf Extract Attenuates Vascular Dementia-Associated Memory Deficits, Neuronal Damages, and Microglial Activation.

Vascular dementia (VaD) is characterized by a time-dependent memory deficit and essentially combined with evidence of neuroinflammation. Thus, polyphenol-rich natural plants, which possess anti-inflammatory properties, have received much scientific attention. This study investigated whether Perilla frutescens leaf extract (PFL) exerts therapeutic efficacy against VaD. Sprague Dawley rats were divided into five groups: SO, sham-operated and vehicle treatment; OP, operated and vehicle treatment; PFL-L, operated and low-dose (30 mg/kg) PFL treatment; PFL-M, operated and medium-dose (60 mg/kg) PFL treatment; and PFL-H, operated and high-dose (90 mg/kg) PFL treatment. Two-vessel occlusion and hypovolemia (2VO/H) were employed as a surgical model of VaD, and PFL was given orally perioperatively for 23 days. The rats underwent the Y-maze, Barnes maze, and passive avoidance tests and their brains were subjected to histologic studies. The OP group showed VaD-associated memory deficits, hippocampal neuronal death, and microglial activation; however, the PFL-treated groups showed significant attenuations in all of the above parameters. Using lipopolysaccharide (LPS)-stimulated BV-2 cells, a murine microglial cell line, we measured PFL-mediated changes on the production of nitric oxide (NO), TNF-α, and IL-6, and the activities of their upstream MAP kinases (MAPKs)/NFκB/inducible NO synthase (iNOS). The LPS-induced upregulations of NO, TNF-α, and IL-6 production and MAPKs/NFκB/iNOS activities were globally and significantly reversed by 12-h pretreatment of PFL. This suggests that PFL can counteract VaD-associated structural and functional deterioration through the attenuation of neuroinflammation.

Regioselective Synthesis of 6-O-Acetyl Dieckol and Its Selective Cytotoxicity against Non-Small-Cell Lung Cancer Cells.

Dieckol, a phlorotannin from Ecklonia cava, has shown potential for use as an anticancer agent that selectively kills cancer cells. However, it is necessary to amplify its potency without damaging its inherent safety in order to develop it as a competitive chemotherapeutic. Here, we explored the controlled O-acylations of dieckol. Acyl groups could be consistently introduced to the 6-O position of dieckol with a high regioselectivity, which was confirmed by NOESY, HMBC and HSQC spectroscopies. In cytotoxicity studies on the newly synthesized 6-O-acetyl, 6-O-benzoyl dieckols and previously synthesized 6-O-alkyl dieckols against A549 vs. normal cells, all of the derivatives showed low cytotoxicity in normal cells with an IC50 of 481-719 µM, and highly structure-dependent cytotoxicity in A549 cells with an IC50 of 7.02 (acetyl)-842.26 (benzyl) µM. The selectivity index also showed a large structure dependency in the range of 0.67 (benzyl)-68.58 (acetyl). An analysis of the structure-activity relationship indicated that the activity was dramatically reduced in the presence of a benzene ring and was highly increased in the presence of small polar substituents. Conclusions: Controlled mono-O-modifications of dieckol could be a powerful tool to enhance the anticancer activity of dieckol, thus contributing to the development strategy for dieckol-based chemotherapeutics.

Effects and Mechanism of Particulate Matter on Tendon Healing Based on Integrated Analysis of DNA Methylation and RNA Sequencing Data in a Rat Model.

Exposure to particulate matter (PM) has been linked with the severity of various diseases. To date, there is no study on the relationship between PM exposure and tendon healing. Open Achilles tenotomy of 20 rats was performed. The animals were divided into two groups according to exposure to PM: a PM group and a non-PM group. After 6 weeks of PM exposure, the harvest and investigations of lungs, blood samples, and Achilles tendons were performed. Compared to the non-PM group, the white blood cell count and tumor necrosis factor-alpha expression in the PM group were significantly higher. The Achilles tendons in PM group showed significantly increased inflammatory outcomes. A TEM analysis showed reduced collagen fibrils in the PM group. A biomechanical analysis demonstrated that the load to failure value was lower in the PM group. An upregulation of the gene encoding cyclic AMP response element-binding protein (CREB) was detected in the PM group by an integrated analysis of DNA methylation and RNA sequencing data, as confirmed via a Western blot analysis showing significantly elevated levels of phosphorylated CREB. In summary, PM exposure caused a deleterious effect on tendon healing. The molecular data indicate that the action mechanism of PM may be associated with upregulated CREB signaling.

Water Extract of Mixed Mushroom Mycelia Grown on a Solid Barley Medium Is Protective against Experimental Focal Cerebral Ischemia.

Although the individual consumption of medicinal mushrooms, including Phellinus linteus (PL), Ganoderma lucidum (GL), and Inonotus obliquus (IO), is known to be neuroprotective, the associated mechanisms underlying their therapeutic synergism on focal cerebral ischemia (fCI) have yet to be elucidated. This study aimed to demonstrate the neuroprotective effects of mixed mushroom mycelia (MMM) against experimental fCI. The water-fractions, ethanolic-fractions, and ethyl acetate-fractions of the MMM (PL, GL, and IO) grown in a barley medium using solid-state fermentation techniques were prepared and their protective effects against glutamate-induced excitotoxicity were compared in PC-12 cells. After the identification of the water extracts of MMM (wMMM) as the most suitable form, which possessed the lowest toxicity and highest efficacy, further analyses for evaluating the anti-apoptotic effects of wMMM, including Hoechst 33258-based nuclear staining, fluorescence-activated cell sorting, and reactive oxygen species (ROS) detection assays, were performed. Rats were subjected to a 90 min middle cerebral artery occlusion and reperfusion, after which a wMMM treatment resulted in significant dose-dependent improvements across a number of parameters. Furthermore, measurements of intracellular ROS and levels of antioxidant enzymes revealed a wMMM-mediated ROS attenuation and antioxidant enzyme upregulation. We suggest that wMMM is neuroprotective against fCI through its anti-apoptotic and anti-oxidative effects.

Protective Effect of Cholic Acid-Coated Poly Lactic-Co-Glycolic Acid (PLGA) Nanoparticles Loaded with Erythropoietin on Experimental Stroke.

Stroke is a major cause of adult mortality and morbidity worldwide. However, the treatment of stroke using the vast majority of possible drug candidates, including erythropoietin (EPO), remains problematic because of the presence of the blood-brain barrier (BBB), resulting in scarce penetration onto the brain. To overcome this, we synthesized a novel EPO delivery system, namely the cholic acid-coated poly lactic-co-glycolic acid (PLGA) nanoparticles loaded with EPO (EPO-CA-NPs), with the aim of enabling efficient penetration of EPO-CA-NPs across the BBB. The therapeutic efficacy of EPO-CA-NPs on an animal model of stroke was compared with that of EPO. The experimental stroke model was produced by subjecting rats to the middle carotid artery occlusion and reperfusion (MCAO/R) technique. The results indicated that EPO-CA-NPs reduced the extent of the infarct volume and cellular apoptosis to a greater extent than EPO alone at postoperative day (POD) 1. Furthermore, EPO-CA-NPs showed better performance on sensorimotor functions than EPO alone at POD 1, 3, 5, and 7. Taken together, EPO-CA-NPs, a newly synthesized brain-targeted EPO-delivery system, has stronger therapeutic effects on stroke than EPO alone, by enabling efficient EPO delivery into the brain.

Impaired autophagy promotes bile acid-induced hepatic injury and accumulation of ubiquitinated proteins.

Chronic exposure to hydrophobic bile acids such as chenodeoxycholic acid (CDCA) and cholic acid (CA) in the liver during cholestasis causes hepatotoxicity and inflammatory response. However, the detailed mechanisms regarding the role of autophagy in cholestatic hepatotoxicity remain largely unknown. Here we determined autophagic clearance in livers of bile duct-ligated mice, in which bile acids accumulate, and in human hepatoma HepG2 cells treated with CDCA and CA. The accumulation of bile acids caused defective autophagic clearance, shown by the accumulation of insoluble p62 and ubiquitinated proteins and cell death accompanied by caspase-3 processing. Hepatocytes exposed to bile acids also showed the accumulation of autophagosomes via suppressed autophagy flux. Treatment of CDCA markedly suppressed Beclin-1 expression, which exhibits a higher cytotoxicity than CA. Moreover, pharmacological or genetic inhibition of autophagy enhanced bile acid-induced cell death. Finally, in vivo, bile duct ligation led to aberrant accumulation of p62 and ubiquitinated proteins in the liver. Our data demonstrate that inhibited autophagy is an essential component of liver injury during cholestasis.

Protective Effects of Indole-3-Carbinol-Loaded Poly(lactic-co-glycolic acid) Nanoparticles Against Glutamate-Induced Neurotoxicity.

Indole-3-carbinol (I3C) has anti-oxidant and anti-inflammatory properties. Nonetheless, the potential of I3C to treat neurodegenerative diseases remains unclear because of its poor ability to penetrate the blood-brain barrier (BBB). Because polymer-based drug delivery systems stabilized by surfactants have been intensively utilized as a strategy to cross the blood-brain barrier, we prepared I3C-loaded poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) that were stabilized by Tween 80 (T80) (I3C-PLGA-T80-NPs) and examined their neuroprotective potential in vitro. We prepared I3C-PLGA-T80-NPs with an oil-in-water (o/w) emulsion solvent evaporation technique and confirmed their successful synthesis with both transmission electron microscopy and Fourier transform-infrared spectroscopy. I3C-PLGA-T80-NPs were then used to treat PC12 neuronal cells injured by glutamate excitotoxicity (GE) and examined the resulting survival rates compared with PC12 cells treated with I3C only. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay revealed higher survival rates in I3C-PLGA-T80-NPs-treated cells after GE injury compared with those treated with I3C only. Furthermore, I3C-PLGA-T80-NPs decreased the levels of reactive oxygen species (ROS) and apoptosis-related enzymes (Caspase-3 and -8) in GE-damaged neuronal cells. Taken together, I3C-PLGA-T80-NPs might possess neuroprotective effects against GE through ROS scavenging and subsequent apoptosis blockage.

An arthroscopic safety zone for the medial compartment of the hip joint.

The purpose of this study was to investigate the safety zone without any neurovascular injury to the medial compartment of hip joint through an anatomical method and describe the relationship of the extra-articular anatomic structures from the surface of the hip joint. Thirty-two fresh specimens from 17 adult Korean cadavers (8 males and 9 females, age range 54-79 years at death) were used for this study. For the measurements, the most superolateral point of the pubic symphysis (PS) and prominent point of the anterior superior iliac spine (ASIS) were identified before dissection. The line connecting the PS and ASIS was defined as a reference line and the PS was a starting point for measurements. All 19 variables measured in this study were related to the femoral head, neck, and surrounding neurovascular structures. The variables were measured according to the x- and y-coordinates in relation to the reference line. The femoral head was generally located 39.5-71.0 mm on the x-coordinate and located 33.5-34.6 mm on the y-coordinate. The junction of the femoral neck and body was located at 52.8 mm on the x-coordinate, and 65.3 mm on the y-coordinate. The junction of the femoral head and neck was located at 47.1 mm on the x-coordinate, and 51.4 mm on the y-coordinate. The location of the medial compartment of the hip joint was located from 38.0 to 43.0 % on the x-coordinate and located from 5.1 to 6.5 cm. These results of this study provide detailed anatomy for arthroscopic hip surgeons.

Protective effects of poly(lactic-co-glycolic acid) nanoparticles loaded with erythropoietin stabilized by sodium cholate against glutamate-induced neurotoxicity.

The final aim of this study was to confirm the neuroprotective effects of recombinant human erythropoietin (rhEPO)-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles stabilized by sodium cholate (rhEPO-Ch-NP) and compare their effects with those of rhEPO using an in vitro model of cerebral ischemia. Glutamate-induced excitotoxic damage on SH-SY5Y cells, a human neuroblastoma cell line, with or without rhEPO-Ch-NPs was quantitatively evaluated. The rhEPO-Ch-NPs were carefully prepared using a water-in-oil-in-water (w/o/w) emulsion solvent evaporation technique with PLGA, sodium cholate hydrate, and ethyl acetate. The rhEPO-Ch-NPs were fully characterized by both transmission electron microscopy (TEM) and differential scanning calorimetry (DSC). In addition, significant intracellular uptake of these particles was monitored by confocal microscopy. Notably, the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and nuclear changes observed by 4',6-diamidino-2-phenylindole (DAPI) staining in SH-SY5Y cells demonstrated that rhEPO-Ch-NPs were safer at any concentration investigated and rescued more neuronal cells, while preserving normocytic features against glutamate-induced excitotoxic damages compared to rhEPO.

Neuroprotective effect of estradiol-loaded poly(lactic-co-glycolic acid) nanoparticles on glutamate-induced excitotoxic neuronal death.

Different concentrations of estradiol (E2)-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (E2-PLGA-NPs) were synthesized using the emulsion-diffusion method. Transmission electron microscopy results showed that the average particle size of E2-PLGA-NPs was 98 ± 1.9 nm when stabilized with polyvinyl alcohol and 103 ± 4.9 nm when stabilized with Tween-80. Fourier transform-infrared spectroscopy with diamond attenuated total reflectance was used to identify the presence or absence of E2 molecules in PLGA nanocapsules. Cell proliferation was assessed after treating SH-SY5Y neuroblastoma cells with 1 nM-1 µM of E2 and E2-PLGA-NPs. The neuroprotective efficacy against glutamate-induced excitotoxicity was also investigated in SH-SY5Y neuroblastoma cells. Neuroprotection was greater in E2-PLGA-NP-treated cells than in cells treated with the same concentration of E2. Furthermore, E2- and E2-PLGA-NP-treated cells expressed more p-ERK1/2 and p-CREB than cells treated with glutamate only. Moreover, the expression of p-ERK1/2 was higher than that of p-CREB. In this study, p-ERK1/2 had a greater influence on the neuroprotective effect of E2 and E2-PLGA-NPs than p-CREB.

Fabrication of atelocollagen-coated bioabsorbable suture and the evaluation of its regenerative efficacy in Achilles tendon healing using a rat experimental model.

Recently, the incidence of Achilles tendon ruptures (ATRs) has become more common, and repair surgery using a bioabsorbable suture is generally preferred, particularly in the case of healthy patients. Sutures composed of poly(lactic-co-glycolic acid) (PLGA) are commonly used in ATR surgeries. Nevertheless, owing to the inherent limitations of PLGA, novel bioabsorbable sutures that can accelerate Achilles tendon healing are sought. Recently, several studies have demonstrated the beneficial effects of atelocollagen on tendon healing. In this study, poly(3,4-dihydroxy-L-phenylalanine) (pDOPA), a hydrophilic biomimetic material, was used to modify the hydrophobic surface of a PLGA suture (Vicryl, VC) for the stable coating of atelocollagen on its surface. The main objective was to fabricate an atelocollagen-coated VC suture and evaluate its performance in the healing of Achilles tendon using a rat model of open repair for ATR. Structural analyses of the surface-modified suture indicated that the collagen was successfully coated on the VC/pDOPA suture. Postoperative in vivo biomechanical analysis, histological evaluation, ultrastructural/morphological analyses, and western blotting confirmed that the tendons in the VC/pDOPA/Col group exhibit superior healing than those in the VC and VC/pDOPA groups after 1 and 6 weeks following the surgery. The this study suggests that atelocollagen-coated PLGA/pDOPA sutures are preferable for future medical applications, especially in the repair of ATR.

All-trans-retinoic acid rescues neurons after global ischemia by attenuating neuroinflammatory reactions.

Retinoic acid (RA) plays an important role in the developing mammalian nervous system. Based on this concept, some studies have demonstrated the beneficial effects of RA administration on neurogenesis in neuropathological diseases. Some investigations have revealed the anti-inflammatory effects of RA treatment in multiple systems, in addition to its role in neurogenesis. To date, however, the neuroprotective efficacy of RA after cerebral ischemia, especially in the context of its anti-inflammatory effects, has been poorly demonstrated. Additionally, to the best of our knowledge, experiments of the therapeutic efficacy of RA treatment in a transient global ischemic model in the Mongolian gerbil have been lacking worldwide. Here, we studied the neuroprotective effects and neurobehavioral outcomes of intraperitoneally administered all-trans-RA (ATRA; a synthetic form of RA) on brains with transient global ischemia that was induced with the bilateral common carotid artery occlusion and reperfusion (BCCAO/R) model in the gerbil. In order to identify whether these neuroprotective mechanisms were due to the anti-inflammatory effects of ATRA, in vivo hippocampal expression of proinflammatory cytokines including tissue necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) after ATRA injection and in vitro levels of release of nitric oxide, TNF-α and IL-6 from lipopolysaccharide (LPS)-stimulated BV2 microglial cells after ATRA treatment were evaluated. The results showed that ATRA can protect pyramidal neurons in the hippocampal CA1 region against BCCAO-induced neuronal apoptosis and significantly reduce the extent of astrocytosis and microglial activation. In addition, the ischemia-induced neurobehavioral changes were normalized by ATRA injection. Consistent with these phenotypic data, we observed the diminishing effects of ATRA treatment on the production of proinflammatory mediators (e.g., TNF-α and IL-6) in hippocampal homogenates and LPS-stimulated BV2 cells, and these effects were dose-dependent. These results suggest a beneficial role of ATRA in the attenuation of global cerebral ischemia due to its anti-inflammatory properties, resulting in, at least partly, the inhibition of microglial secretion of variable proinflammatory cytokines.

An Atelocollagen Injection Enhances the Healing of Nonoperatively Treated Achilles Tendon Tears: An Experimental Study in Rats.

Background: There is growing interest in nonoperative treatment for the management of Achilles tendon ruptures (ATRs). However, nonoperative treatment is limited by the risk of tendon reruptures and low satisfaction rates. Recently, atelocollagen injections have been reported to have beneficial effects on tendon healing. Purpose: To evaluate the beneficial effects of injected atelocollagen on Achilles tendon healing and investigate the mechanism of atelocollagen on tendon healing. Study Design: Controlled laboratory study. Methods: Percutaneous tenotomy of the right Achilles tendon in 66 rats was performed. The animals were equally divided into the noninjection group (NG) and the collagen injection group (CG). At 1, 3, and 6 weeks, the Achilles functional index, cross-sectional area, load to failure, stiffness, stress, and the modified Bonar score were assessed. Transmission electron microscopy, western blotting, and immunohistochemistry were also performed. Results: The Achilles functional index (-6.8 vs -43.0, respectively; P = .040), load to failure (42.1 vs 27.0 N, respectively; P = .049), and stiffness (18.8 vs 10.3 N/mm, respectively; P = .049) were higher in the CG than those in the NG at 3 weeks. There were no significant differences in histological scores between the 2 groups. Transmission electron microscopy analysis showed that the mean diameter of collagen fibrils in the CG was greater than that in the NG at 3 weeks (117.2 vs 72.6 nm, respectively; P < .001) and 6 weeks (202.1 vs 144.0 nm, respectively; P < .001). Western blot analysis showed that the expression of collagen type I in the CG was higher than that in the NG at 1 week (P = .005) and 6 weeks (P = .001). Conclusion: An atelocollagen injection had beneficial effects on the healing of nonoperatively treated Achilles tendon injuries. The Achilles tendon of CG rats exhibited better functional, biomechanical, and morphological outcomes compared with NG rats. The molecular data indicated that the mechanism of atelocollagen injections may be associated with an increased amount of collagen type I. Clinical Relevance: An atelocollagen injection might be a good adjuvant option for the nonoperative treatment of ATRs.

Rubus fruticosus leaf extract inhibits vascular dementia-induced memory impairment and neuronal loss by attenuating neuroinflammation.

Vascular dementia (VaD) is characterized by progressive memory impairment, which is associated with microglia-mediated neuroinflammation. Polyphenol-rich natural plants, which possess anti-inflammatory activities, have attracted scientific interest worldwide. This study investigated whether Rubus fruticosus leaf extract (RFLE) can attenuate VaD. Sprague-Dawley rats were separated into five groups: SO, sham-operated and treated with vehicle; OP, operated and treated with vehicle; RFLE-L, operated and treated with low dose (30 mg/kg) of RFLE; RFLE-M, operated and treated with medium dose (60 mg/kg) of RFLE; and RFLE-H, operated and treated with high dose (90 mg/kg) of RFLE. Bilateral common carotid artery and hypotension were used as a modeling procedure, and the RFLE were intraorally administered for 5 days (preoperative 2 and postoperative 3 days). The rats then underwent memory tests including the novel object recognition, Y-maze, Barnes maze, and passive avoidance tests, and neuronal viability and neuroinflammation were quantified in their hippocampi. The results showed that the OP group exhibited VaD-associated memory deficits, neuronal death, and microglial activation in hippocampi, while the RFLE-treated groups showed significant attenuation in all above parameters. Next, using BV-2 microglial cells challenged with lipopolysaccharide (LPS), we evaluated the effects of RFLE in dynamics of proinflammatory mediators and the upstream signaling pathway. RFLE pretreatment significantly inhibited the LPS-induced release of nitric oxide, TNF-α, and IL-6 and upregulation of the MAPKs/NF-κB/iNOS pathway. Collectively, we suggest that RFLE can attenuate the histologic alterations and memory deficits accompanied by VaD, and these roles are, partly due to the attenuation of microglial activation.

Activation of Nod1 and Nod2 induces innate immune responses of prostate epithelial cells.

BACKGROUND: Nod1 and Nod2 are cytosolic receptors which are responsible for sensing bacterial peptidoglycan derivatives. In this study, we determined whether Nod1 and Nod2 are involved in the innate immune responses of prostate epithelial cells. METHODS: The expression of Nod1 and Nod2 was examined by RT-PCR and immunohistochemistry. ELISA was performed to determine the production of cytokines/chemokines. Activation of NF-κB and MAPK was examined using western blot analysis. RESULTS: The Nod1 gene was distinctly expressed in all tested cells including DU145, PC3, and TRAMP-C2 cells, whereas Nod2 expression was weak. Both Nod1 and Nod2 proteins were expressed in normal mouse prostate epithelia with difference of expression levels. Tri-DAP (Nod1 agonist), but not MDP (Nod2), increased the production of IL-8 (or KC) and IL-6 in prostate epithelial cells. Tri-DAP and MDP could upregulate the gene expression of COX-2 and activate NF-κB and MAPK. In addition, Tri-DAP and MDP synergized with TLR agonists to induce the production of IL-8/KC or IL-6 in PC3 and TRAMP-C2 cells. We finally showed that Nod1 and Nod2 were also expressed in a wide range of prostate lesions including prostate intraepithelial neoplasm (PIN), phyllodes-like tumor, and adenocarcinoma in TRAMP (transgenic adenocarcinoma of the mouse prostate) mice, even though the expression level of Nod1 and Nod2 was different. CONCLUSION: These results indicate that Nod1 and Nod2 may play important roles in the innate immune response of prostate epithelial cells and the development and progression of prostate cancer.

Sestrin2 protects against cholestatic liver injury by inhibiting endoplasmic reticulum stress and NLRP3 inflammasome-mediated pyroptosis.

Chronic exposure to bile acid in the liver due to impaired bile flow induces cholestatic liver disease, resulting in hepatotoxicity and liver fibrosis. Sestrin2, a highly conserved, stress-inducible protein, has been implicated in cellular responses to multiple stress conditions and the maintenance of cellular homeostasis. However, its role in cholestatic liver injury is not fully understood. In this study, we investigated the role of hepatic Sestrin2 in cholestatic liver injury and its underlying mechanisms using in vivo and in vitro approaches. Hepatic Sestrin2 expression was upregulated by activating transcription factor 4 (ATF4) and CCAAT/enhancer-binding protein-ß (C/EBP-ß) after treatment with bile acids and correlated with endoplasmic reticulum (ER) stress responses. Bile-duct ligation (BDL)-induced hepatocellular apoptosis and liver fibrosis were exacerbated in Sestrin2-knockout (Sesn2-/-) mice. Moreover, Sestrin2 deficiency enhanced cholestasis-induced hepatic ER stress, whereas Sestrin2 overexpression ameliorated bile acid-induced ER stress. Notably, the mammalian target of rapamycin (mTOR) inhibitor rapamycin and the AMP-activated protein kinase (AMPK) activator AICAR reversed bile acid-induced ER stress in Sestrin2-deficient cells. Furthermore, Sestrin2 deficiency promoted cholestasis-induced hepatic pyroptosis by activating NLRP3 inflammasomes. Thus, our study provides evidence for the biological significance of Sestrin2 and its relationship with cholestatic liver injury, suggesting the potential role of Sestrin2 in regulating ER stress and inflammasome activation during cholestatic liver injury.

Nicotine alleviates alcohol-induced memory and long-term potentiation impairment.

Alcohol and nicotine are routinely abused together. There is one plausible explanation that comorbidity can alleviate alcohol-induced cognitive impairment. However, the mechanism involved is not known. The aim of this report was to evaluate the interactive effects of alcohol coadministration with nicotine on hippocampal memory and long-term potentiation (LTP). C57BL/6 mice were distributed into 4 treatment groups: control, alcohol, nicotine, and alcohol plus nicotine. All mice received tap water or alcohol solution and saline or nicotine. In water maze test, the alcohol group showed significant decreases in hippocampus function and acquisition training than control, nicotine, and combined treatment groups. The alcohol group also showed a significantly shorter latency in entering the foot-shock compartment than control, nicotine, and combined treatment groups in a passive avoidance test. Theta burst stimulation was adopted to induce concrete LTP in CA1 field recording using hippocampal slice. Alcohol alone administration failed to maintain LTP. Nicotine alone administration did not alter hippocampal LTP. There were no negative effects of alcohol on hippocampal LTP in mice administrated with nicotine. The current study successfully demonstrated beneficial effects of nicotine on alcohol induced memory impairment accompanied by hippocampal LTP impairment after one week of co-administration and one-day withdrawal.