In Silico Evaluation of Natural Compounds for an Acidic Extracellular Environment in Human Breast Cancer.

The survival rates for breast cancer (BC) have improved in recent years, but resistance, metastasis, and recurrence still remain major therapeutic challenges for BC. The acidic tumor microenvironment (TME) has attracted attention because of its association with tumorigenesis, metastasis, drug resistance, and immune surveillance. In this study, we evaluated natural compounds from traditional herbal medicine used to treat cancer that selectively target genes regulated by extracellular acidosis. We integrated four transcriptomic data including BC prognostic data from The Cancer Genome Atlas database, gene expression profiles of MCF-7 cells treated with 102 natural compounds, patterns of gene profiles by acidic condition, and single-cell RNA-sequencing from BC patient samples. Bruceine D (BD) was predicted as having the highest therapeutic potential, having an information gain (IG) score of 0.24, to regulate reprogrammed genes driven by acidosis affecting the survival of BC patients. BD showed the highest IG on EMT (IG score: 0.11) and invasion (IG score: 0.1) compared to the other phenotypes with the CancerSEA database. BD also demonstrated therapeutic potential by interfering with the tumor cell-TME interactions by reducing the amyloid beta precursor protein and CD44 expression. Therefore, BD is a potential candidate to target the acidic TME induced metastatic process in BC.

The effect of Apium graveolens L., Levisticum officinale and Calendula officinalis L. on cell viability, membrane integrity, steroidogenesis, and intercellular communication in mice Leydig cells in vitro.

Several plants have the potential to protect essential reproductive processes such as spermatogenesis or steroidogenesis, however, effective concentrations and main mechanisms of action are still unknown. This in vitro study was aimed to assess the effects of Apium graveolens L., Levisticum officinale, and Calendula officinalis L. extracts on the structural integrity, functional activity and gap junctional intercellular communication (GJIC) in mice Leydig cells. TM3 cells were grown in the presence of experimental extracts (37.5; 75; 150 and 300 µg/ml) for 24 h. For the present study, high-performance liquid chromatography analysis was used to quantify flavonoids or phenolic acids. Subsequently, Leydig cell viability was assessed by alamarBlue assay, while the cell membrane integrity was detected by 5-carboxyfluorescein diacetate-acetoxymethyl ester. The level of steroid hormones production was determined by enzyme-linked immunosorbent assay. Additionally, GJIC was assessed by scalpel loading/dye transfer assay. According to our results, Apium graveolens L. significantly increased the viability and cell membrane integrity at 75 µg/ml (109.0±4.3%) followed by a decline at 300 µg/ml (89.4±2.3%). In case of Levisticum officinale and Calendula officinalis L. was observed significant decrease at 150 µg/ml (88.8±11.66%; 87.4±6.0%) and 300 µg/ml (86.2±9.3%; 84.1±4.6%). Furthermore, Apium graveolens L. significantly increased the progesterone and testosterone production (75 and 150 µg/ml) however, Levisticum officinale and Calendula officinalis L. significantly reduced steroid hormones synthesis at 150 and 300 µg/ml. Finally, the disturbance of GJIC was significantly affected at 300 µg/ml of Levisticum officinale (82.5±7.7%) and Calendula officinalis L. (79.8±7.0%). The balanced concentration ratio may support the Leydig cell function, steroidogenesis as well as all essential parameters that may significantly improve reproductive functions.

Leonotis Leonurus improves the crosstalk between peripheral tissues both in vivo and in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Unravelling the anti-diabetic mechanism of action of L. leonurus at adipose, liver, muscle and pancreatic level. AIMS: To investigate the mechanism of action of an organic extract of L. leonurus and marrubiin at the gene level in adipose, liver and muscle tissues of an obese rat model and in a co-culture model. MATERIALS AND METHODS: Obese Wistar rats were fed a cafeteria diet for eight weeks, treated with an extract of L. Leonurus, marrubiin, sulfonylurea and aspirin for two weeks and the level of gene expression of selected markers were investigated across different tissues. The effects mediated by the different treatments were investigated in co-culture cell models involving 3T3-L1 (fat), Chang (liver), C2C12 (muscle) and INS-1 (pancreatic) cells under both normal and hyperglycemic conditions. RESULTS: L. leonurus extract mediated a significant increase in PPAR gamma, glucokinase, FAS and UCP2 gene expression in adipose tissue, whilst the opposite was observed in the liver. At the muscle level, a significant increase in FAS gene expression was observed relative to the obese control rats. Furthermore, the extract as well as marrubiin, modulated improvements in the adipokine profile. The co-culture models showed that the effect mediated by the extract was dependent on, the tissue type as well as the glycemic conditions. CONCLUSIONS: L. Leonurus extract as well as marrubiin exhibit anti-diabetic properties where the mechanism of action is mainly at the adipose tissue level. The increase in expression of the genes of interest mentioned above potentially play a protective role towards the liver and possibly towards the muscle tissues as well.

Baicalin suppresses type 2 immunity through breaking off the interplay between mast cell and airway epithelial cell.

ETHNOPHARMACOLOGICAL RELEVANCE: The traditional Japanese herbal medicine Shin'iseihaito was reported to ameliorate the airway type 2 inflammatory response in clinical and experimental studies. Airway type 2 inflammatory diseases, including bronchial asthma and eosinophilic chronic rhinosinusitis (ECRS), often coexist and interact with each other. However, it is still unclear how Shin'iseihaito exerts its pharmacological effects on cells involved in airway mucosa. AIM OF THE STUDY: This study aims to examine the direct effect of baicalin, a representative bioactive compound of Shin'iseihaito, on type 2 immune responses in human airway epithelial cells and mast cells. MATERIAL AND METHODS: We measured the plasma pharmacokinetics of flavonoids derived from Shin'iseihaito and investigated the effects of baicalin on type 2 immune responses in human airway epithelial cells and human mast cells. RESULTS: Baicalin, wogonin, and wogonoside were detected in the plasma. The maximum plasma concentration of baicalin was highest at 1610 ng/ml (3.6 µM). In the normal human bronchial epithelial cells treated with baicalin, with or without stimulation by IFN-γ, the IL-33 expression was significantly downregulated. However, baicalin treatment did not affect the levels of thymic stromal lymphopoietin and IL-25. We noted that IL-33-dependent expression of tryptase mRNA in mast cells was significantly inhibited by baicalin. Also, the expression of IL-5 in mast cells enhanced by stimulation with TSLP plus IL-1ß was significantly downregulated by baicalin treatment. Moreover, the enhancement of IL-13 expression in mast cells by IL-33 simulation was also significantly inhibited by baicalin. CONCLUSIONS: Our results prove that by breaking off the vicious circle of mast cells and airway epithelial cells, baicalin may be an effective alternative therapeutic option for the treatment of type 2 inflammatory diseases, such as ECRS and comorbid asthma.

Cardiomyocyte-derived small extracellular vesicles can signal eNOS activation in cardiac microvascular endothelial cells to protect against Ischemia/Reperfusion injury.

Rationale: The crosstalk between cardiac microvascular endothelial cells (CMECs) and cardiomyocytes (CMs) has emerged as a key component in the development of, and protection against, cardiac diseases. For example, activation of endothelial nitric oxide synthase (eNOS) in CMECs, by therapeutic strategies such as ischemic preconditioning, plays a critical role in the protection against myocardial ischemia/reperfusion (I/R) injury. However, much less is known about the signals produced by CMs that are able to regulate CMEC biology. Here we uncovered one such mechanism using Tongxinluo (TXL), a traditional Chinese medicine, that alleviates myocardial ischemia/reperfusion (I/R) injury by activating CMEC eNOS. The aim of our study is to identify the signals produced by CMs that can regulate CMEC biology during I/R. Methods:Ex vivo, in vivo, and in vitro settings of ischemia-reperfusion were used in our study, with the protective signaling pathways activated in CMECs identified using genetic inhibition (p70s6k1 siRNA, miR-145-5p mimics, etc.), chemical inhibitors (the eNOS inhibitor, L-NNA, and the small extracellular vesicles (sEVs) inhibitor, GW4869) and Western blot analyses. TritonX-100 at a dose of 0.125% was utilized to inactivate the eNOS activity in endothelium to investigate the role of CMEC-derived eNOS in TXL-induced cardioprotection. Results: We found that while CMEC-derived eNOS activity was required for the cardioprotection of TXL, activation of eNOS in CMECs by TXL did not occur directly. Instead, eNOS activation in CMECs required a crosstalk between CMs and CMECs through the uptake of CM-derived sEVs. We further demonstrate that TXL induced CM-sEVs contain increased levels of Long Intergenic Non-Protein Coding RNA, Regulator Of Reprogramming (Linc-ROR). Upon uptake into CMECs, linc-ROR downregulates its target miR-145-5p leading to activation of the eNOS pathway by facilitating the expression of p70s6k1 in these cells. The activation of CMEC-derived eNOS works to increase survival in both the CMECs and the CMs themselves. Conclusions: These data uncover a mechanism by which the crosstalk between CMs and CMECs leads to the increased survival of the heart after I/R injury and point to a new therapeutic target for the blunting of myocardial I/R injury.

Human Mesenchymal Stromal Cell Secretome Promotes the Immunoregulatory Phenotype and Phagocytosis Activity in Human Macrophages.

Human mesenchymal stromal/stem cells (hMSCs) show great promise in cell therapy due to their immunomodulatory properties. The overall immunomodulatory response of hMSCs resembles the resolution of inflammation, in which lipid mediators and regulatory macrophages (Mregs) play key roles. We investigated the effect of hMSC cell-cell contact and secretome on macrophages polarized and activated toward Mreg phenotype. Moreover, we studied the effect of supplemented polyunsaturated fatty acids (PUFAs): docosahexaenoic acid (DHA) and arachidonic acid, the precursors of lipid mediators, on hMSC immunomodulation. Our results show that unlike hMSC cell-cell contact, the hMSC secretome markedly increased the CD206 expression in both Mreg-polarized and Mreg-activated macrophages. Moreover, the secretome enhanced the expression of programmed death-ligand 1 on Mreg-polarized macrophages and Mer receptor tyrosine kinase on Mreg-activated macrophages. Remarkably, these changes were translated into improved Candida albicans phagocytosis activity of macrophages. Taken together, these results demonstrate that the hMSC secretome promotes the immunoregulatory and proresolving phenotype of Mregs. Intriguingly, DHA supplementation to hMSCs resulted in a more potentiated immunomodulation with increased CD163 expression and decreased gene expression of matrix metalloproteinase 2 in Mreg-polarized macrophages. These findings highlight the potential of PUFA supplementations as an easy and safe method to improve the hMSC therapeutic potential.

Mating-induced increase in Kiss1 mRNA expression in the anteroventral periventricular nucleus prior to an increase in LH and testosterone release in male rats.

Kisspeptin has an indispensable role in gonadotropin-releasing hormone/gonadotropin secretion in mammals. In rodents, kisspeptin neurons are located in distinct brain regions, namely the anteroventral periventricular nucleus-periventricular nucleus continuum (AVPV/PeN), arcuate nucleus (ARC), and medial amygdala (MeA). Among them, the physiological role of AVPV/PeN kisspeptin neurons in males has not been clarified yet. The present study aims to investigate the acute effects of the olfactory and/or mating stimulus with a female rat on hypothalamic and MeA Kiss1 mRNA expression, plasma luteinizing hormone (LH) and testosterone levels in male rats. Intact male rats were exposed to the following stimuli: exposure to clean bedding; exposure to female-soiled bedding as a female-olfactory stimulus; exposure to female-soiled bedding and mating stimulus with a female rat. The mating stimulus significantly increased the number of the AVPV/PeN Kiss1 mRNA-expressing cells in males within 5 minutes after the exposure, and significantly increased LH and testosterone levels, followed by an increase in male sexual behavior. Whereas, the males exposed to female-soiled bedding showed a moderate increase in LH levels and no significant change in testosterone levels and the number of the AVPV/PeN Kiss1 mRNA-expressing cells. Importantly, none of the stimuli affected the number of Kiss1 mRNA-expressing cells in the ARC and MeA. These results suggest that the mating-induced increase in AVPV/PeN Kiss1 mRNA expression may be, at least partly, involved in stimulating LH and testosterone release, and might consequently ensure male mating behavior. This study would be the first report suggesting that the AVPV/PeN kisspeptin neurons in males may play a physiological role in ensuring male reproductive performance.

Emerging functions and clinical prospects of connexins and pannexins in melanoma.

Cellular communication through gap junctions and hemichannels formed by connexins and through channels made by pannexins allows for metabolic cooperation and control of cellular activity and signalling. These channel proteins have been described to be tumour suppressors that regulate features such as cell death, proliferation and differentiation. However, they display cancer type-dependent and stage-dependent functions and may facilitate tumour progression through junctional and non-junctional pathways. The accumulated knowledge and emerging strategies to target connexins and pannexins are providing novel clinical opportunities for the treatment of cancer. Here, we provide an updated overview of the role of connexins and pannexins in malignant melanoma. We discuss how targeting of these channel proteins may be used to potentiate antitumour effects in therapeutic settings, including through improved immune-mediated tumour elimination.

GPR30-Expressing Gastric Chief Cells Do Not Dedifferentiate But Are Eliminated via PDK-Dependent Cell Competition During Development of Metaplasia.

BACKGROUND & AIMS: Gastric chief cells, a mature cell type that secretes digestive enzymes, have been proposed to be the origin of metaplasia and cancer through dedifferentiation or transdifferentiation. However, studies supporting this claim have had technical limitations, including issues with the specificity of chief cell markers and the toxicity of drugs used. We therefore sought to identify genes expressed specifically in chief cells and establish a model to trace these cells. METHODS: We performed transcriptome analysis of Mist1-CreERT-traced cells, with or without chief cell depletion. Gpr30-rtTA mice were generated and crossed to TetO-Cre mice, and lineage tracing was performed after crosses to R26-TdTomato mice. Additional lineage tracing experiments were performed using Mist1-CreERT, Kitl-CreERT, Tff1-Cre, and Tff2-Cre mice crossed to reporter mice. Mice were given high-dose tamoxifen or DMP-777 or were infected with Helicobacter pylori to induce gastric metaplasia. We studied mice that expressed mutant forms of Ras in gastric cells, using TetO-KrasG12D, LSL-KrasG12D, and LSL-HrasG12V mice. We analyzed stomach tissues from GPR30-knockout mice. Mice were given dichloroacetate to inhibit pyruvate dehydrogenase kinase (PDK)-dependent cell competition. RESULTS: We identified GPR30, the G-protein-coupled form of the estrogen receptor, as a cell-specific marker of chief cells in gastric epithelium of mice. Gpr30-rtTA mice crossed to TetO-Cre;R26-TdTomato mice had specific expression of GPR30 in chief cells, with no expression noted in isthmus stem cells or lineage tracing of glands. Expression of mutant Kras in GPR30+ chief cells did not lead to the development of metaplasia or dysplasia but, instead, led to a reduction in labeled numbers of chief cells and a compensatory expansion of neck lineage, which was derived from upper Kitl+ clones. Administration of high-dose tamoxifen, DMP-777, or H pylori decreased the number of labeled chief cells. Chief cells were eliminated from epithelia via GPR30- and PDK-dependent cell competition after metaplastic stimuli, whereas loss of GRP30 or inhibition of PDK activity preserved chief cell numbers and attenuated neck lineage cell expansion. CONCLUSIONS: In tracing studies of mice, we found that most chief cells are lost during metaplasia and therefore are unlikely to contribute to gastric carcinogenesis. Expansion of cells that coexpress neck and chief lineage markers, known as spasmolytic polypeptide-expressing metaplasia, does not occur via dedifferentiation from chief cells but, rather, through a compensatory response from neck progenitors to replace the eliminated chief cells.

Protective effects of eicosapentaenoic acid in adipocyte-breast cancer cell cross talk.

Breast cancer is the leading cause of death in women among all cancer types. Obesity is one of the factors that promote progression of breast cancer, especially in post-menopausal women. Increasingly, adipose tissue is recognized for its active role in the tumor microenvironment. We hypothesized that adipocytes conditioned medium can impact breast cancer progression by increasing inflammatory cytokines production by cancer cells, and subsequently increasing their motility. By contrast, eicosapentaenoic acid (EPA), an anti-inflammatory n-3 polyunsaturated fatty acid, reduces adipocyte-secreted inflammatory factors, leading to reduced cancer cell motility. To test these hypotheses, we investigated the direct effects of EPA on MCF-7 and MDA-MB-231 breast cancer cells and the effects of conditioned medium from 3 T3-L1 or human mesenchymal stem cells (HMSC)-derived adipocytes treated with or without EPA supplementation on breast cancer cells. We observed that conditioned medium from HMSC-derived adipocytes significantly increased mRNA transcription levels of cancer-associated genes such as FASN, STAT3 and cIAP2, while EPA-treated HMSC-derived adipocytes significantly reduced mRNA levels of these genes. However, direct EPA treatment significantly reduced mRNA content of these tumor-associated markers (FASN, STAT3, cIAP-2) only in MDA-MB-231 cells not in MCF-7 cells. Conditioned medium from EPA-treated 3 T3-L1 adipocytes further decreased inflammation, cell motility and glycolysis in cancer cells. Our data confirms that adipocytes play a significant role in promoting breast cancer progression and demonstrates that EPA-treated adipocytes reduced the negative impact of adipocyte-secreted factors on breast cancer cell inflammation and migration.

Pro-Apoptotic Effect of Grape Seed Extract on MCF-7 Involves Transient Increase of Gap Junction Intercellular Communication and Cx43 Up-Regulation: A Mechanism of Chemoprevention.

Growing evidence suggests dietary antioxidants reduce the risk of several cancers. Grape seeds extracts (GSE) are a rich source of polyphenols known to have antioxidant, chemopreventive and anticancer properties. Herein, we investigated the in vitro effects and putative action mechanisms of a grape seed extract (GSE) on human breast cancer cells (MCF-7). The effects of GSE were evaluated on cell proliferation, apoptosis and gap-junction-mediated cell-cell communications (GJIC), as basal mechanism involved in the promotion stage of carcinogenesis. GSE (0.05-100 µg/mL) caused a significant dose- and time-dependent inhibition of MCF-7 viability and induced apoptotic cell death, as detected by Annexin-V/Propidium Iodide. Concurrently, GSE induced transient but significant enhancement of GJIC in non-communicating MCF-7 cells, as demonstrated by the scrape-loading/dye-transfer (SL/DT) assay and an early and dose-dependent re-localization of the connexin-43 (Cx43) proteins on plasma membranes, as assayed by immunocytochemistry. Finally, real-time-PCR has evidenced a significant increase in cx43 mRNA expression. The results support the hypothesis that the proliferation inhibition and pro-apoptotic effect of GSE against this breast cancer cell model are mediated by the GJIC improvement via re-localization of Cx43 proteins and up-regulation of cx43 gene, and provide further insight into the action mechanisms underlying the health-promoting action of dietary components.

Calcium-induced differentiation in normal human colonoid cultures: Cell-cell / cell-matrix adhesion, barrier formation and tissue integrity.

BACKGROUND AND AIMS: The goal of the study was to assess calcium alone and Aquamin, a multi-mineral natural product that contains magnesium and detectable levels of 72 trace elements in addition to calcium, for capacity to affect growth and differentiation in colonoid cultures derived from histologically-normal human colon tissue. METHODS: Colonoid cultures were maintained in a low-calcium (0.25 mM) medium or in medium supplemented with an amount of calcium (1.5-3.0 mM), either from calcium alone or Aquamin for a period of two weeks. This was shown in a previous study to induce differentiation in colonoids derived from large adenomas. Changes in growth, morphological features and protein expression profile were assessed at the end of the incubation period using a combination of phase-contrast and scanning electron microscopy, histology and immunohistology, proteomic assessment and transmission electron microscopy. RESULTS: Unlike the previously-studied tumor-derived colonoids (which remained un-differentiated in the absence of calcium-supplementation), normal tissue colonoids underwent differentiation as indicated by gross and microscopic appearance, a low proliferative index and high-level expression of cytokeratin 20 in the absence of intervention (i.e., in control condition). Only modest additional changes were seen in these parameters with either calcium alone or Aquamin (providing up to 3.0 mM calcium). In spite of this, proteomic analysis and immunohistochemistry revealed that both interventions induced strong up-regulation of proteins that promote cell-cell and cell-matrix adhesive functions, barrier formation and tissue integrity. Transmission electron microscopy revealed an increase in desmosomes in response to intervention. CONCLUSIONS: These findings demonstrate that colonoids derived from histologically normal human tissue can undergo differentiation in the presence of a low ambient calcium concentration. However, higher calcium levels induce elaboration of proteins that promote cell-cell and cell-matrix adhesion. These changes could lead to improved barrier function and improved colon tissue health.

Abatacept for the treatment of psoriatic arthritis.

INTRODUCTION: Psoriatic arthritis (PsA) is a chronic inflammatory disease characterized by the presence of psoriasis, arthritis, and enthesitis, with the association of other musculoskeletal and extra-articular manifestations. Current treatment of PsA is based on the use of conventional, biological and targeted synthetic disease modifying anti-rheumatic drugs; however, patients may not respond or have a loss of response to these agents. Recently, a deeper understanding of the pathogenetic mechanisms has made possible the development of new drugs that actively interact with the activation of immune system, inhibiting the co-stimulation between antigen-presenting cells and lymphocytes. Areas covered: The aim of this paper is to review the role of the activation of the immune system in the pathogenesis and treatment of PsA, with a discussion on the emerging CTLA4Ig drugs (abatacept) for PsA. A search in PubMed and EMBASE was performed with the keywords: 'abatacept', 'CTLA4,' and 'Psoriatic Arthritis.' We considered preclinical studies, phase I, II and III clinical trials. Expert commentary: The inhibitors of co-stimulation may represent an effective treatment strategy by acting on the very early phase of the immunological process that brought about the development of inflammation and activation of the immune system, mainly for patients with peripheral joint involvement and mild psoriasis.

Modulation of Insulin Resistance and the Adipocyte-Skeletal Muscle Cell Cross-Talk by LCn-3PUFA.

The cross-talk between skeletal muscle and adipose tissue is involved in the development of insulin resistance (IR) in skeletal muscle, leading to the decrease in the anabolic effect of insulin. We investigated if the long chain polyunsaturated n-3 fatty acids (LCn-3PUFA), eicosapentaenoic and docosapentaenoic acids (EPA and DPA, respectively) could (1) regulate the development of IR in 3T3-L1 adipocytes and C2C12 muscle cells and (2) inhibit IR in muscle cells exposed to conditioned media (CM) from insulin-resistant adipocytes. Chronic insulin (CI) treatment of adipocytes and palmitic acid (PAL) exposure of myotubes were used to induce IR in the presence, or not, of LCn-3PUFA. EPA (50 µM) and DPA (10 µM) improved PAL-induced IR in myotubes, but had only a partial effect in adipocytes. CM from adipocytes exposed to CI induced IR in C2C12 myotubes. Although DPA increased the mRNA levels of genes involved in fatty acid (FA) beta-oxidation and insulin signaling in adipocytes, it was not sufficient to reduce the secretion of inflammatory cytokines and prevent the induction of IR in myotubes exposed to adipocyte's CM. Treatment with DPA was able to increase the release of adiponectin by adipocytes into CM. In conclusion, DPA is able to protect myotubes from PAL-induced IR, but not from IR induced by CM from adipocytes.

Glutamic Acid Signal Synchronizes Protein Synthesis Kinetics in Hepatocytes from Old Rats for the Following Several Days. Cell Metabolism Memory.

The kinetics of protein synthesis was investigated in primary cultures of hepatocytes from old rats in serum-free medium. The rats were fed mixed fodder supplemented with glutamic acid and then transferred to a regular mixed fodder. The amplitude of protein synthesis rhythm in hepatocytes isolated from these rats increased on average 2-fold in comparison with the rats not receiving glutamic acid supplement. Based on this indicator reflecting the degree of cell-cell interactions, the cells from old rats were not different from those of young rats. The effect was preserved for 3-4 days. These results are discussed in connection with our previous data on preservation of the effect of single administration of gangliosides, noradrenaline, serotonin, and other synchronizers on various cell populations. In contrast to the other investigated factors, glutamic acid is capable of penetrating the blood-brain barrier, which makes its effect possible not only in the case of hepatocytes and other non-brain cells, but also in neurons.

Roles of astrocytic connexin-43, hemichannels, and gap junctions in oxygen-glucose deprivation/reperfusion injury induced neuroinflammation and the possible regulatory mechanisms of salvianolic acid B and carbenoxolone.

BACKGROUND: Glia-mediated neuroinflammation is related to brain injury exacerbation after cerebral ischemia/reperfusion (I/R) injury. Astrocytic hemichannels or gap junctions, which were mainly formed by connexin-43, have been implicated in I/R damage. However, the exact roles of astrocytic hemichannels and gap junction in neuroinflammatory responses induced by I/R injury remain unknown. METHODS: Primary cultured astrocytes were subjected to OGD/R injury, an in vitro model of I/R injury. Salvianolic acid B (SalB) or carbenoxolone (CBX) were applied for those astrocytes. Besides, Cx43 mimetic peptides Gap19 or Gap26 were also applied during OGD/R injury; Cx43 protein levels were determined by western blot and cytoimmunofluorescene staining, hemichannel activities by Ethidium bromide uptake and ATP concentration detection, and gap junction intercellular communication (GJIC) permeability by parachute assay. Further, astrocyte-conditioned medium (ACM) was collected and incubated with microglia. Meanwhile, ATP or apyrase were applied to explore the role of ATP during OGD/R injury. Microglial activation, M1/M2 phenotypes, and M1/M2-related cytokines were detected. Also, microglia-conditioned medium (MEM) was collected and incubated with astrocytes to further investigate its influence on astrocytic hemichannel activity and GJIC permeability. Lastly, effects of ACM and MCM on neuronal viability were detected by flow cytometry. RESULTS: We found that OGD/R induced abnormally opened hemichannels with increased ATP release and EtBr uptake but reduced GJIC permeability. WB tests showed decreased astrocytic plasma membrane's Cx43, while showing an increase in cytoplasma. Treating OGD/R-injured microglia with ATP or OGD/R-ACM induced further microglial activation and secondary pro-inflammatory cytokine release, with the M1 phenotype predominating. Conversely, astrocytes incubated with OGD/R-MCM exhibited increased hemichannel opening but reduced GJIC coupling. Both SalB and CBX inhibited abnormal astrocytic hemichannel opening and ATP release and switched the activated microglial phenotype from M1 to M2, thus providing effective neuroprotection. Application of Gap19 or Gap26 showed similar results with CBX. We also found that OGD/R injury caused both plasma membrane p-Cx43(Ser265) and p-Src(Tyr416) significantly upregulated; application of SalB may be inhibiting Src kinase and attenuating Cx43 internalization. Meanwhile, CBX treatment induced obviously downregulation of p-Cx43(Ser368) and p-PKC(Ser729) protein levels in plasma membrane. CONCLUSIONS: We propose a vicious cycle exists between astrocytic hemichannel and microglial activation after OGD/R injury, which would aggravate neuroinflammatory responses and neuronal damage. Astrocytic Cx43, hemichannels, and GJIC play critical roles in OGD/R injury-induced neuroinflammatory responses; treatment differentially targeting astrocytic Cx43, hemichannels, and GJIC may provide novel avenues for therapeutics during cerebral I/R injury.

Petroleum coke exposure leads to altered secretome profiles in human lung models.

Petroleum coke (PC) is a coal-like product that is produced during the refinement of crude oil and bituminous sand. Fugitive dust from open storage of PC in urban areas is a potential human health concern. Animal inhalation studies suggest that PC leads to an adverse pulmonary histopathology, including areas of fibrosis and chronic inflammation; however, little is known about its impact on human health. In order to identify biomarkers and cellular pathways that are associated with exposure, we performed two-dimensional liquid chromatography-mass spectrometric analyses on secreted proteins from two human lung culture models. A total of 2795 proteins were identified and relatively quantified from an immortalized cell line and 2406 proteins from primary cultures that were either mock treated or exposed to particulate matter with a diameter of 2.5-10 µm PC or filtered urban air particulates for 16 h. Pathway analysis on secretomes from primary lung cultures indicated that PC exposure suppressed the secretion of proteins involved in the organization of the extracellular matrix and epithelial differentiation. Because these cellular processes could facilitate fibrosis, we performed chronic 12-day exposure studies on three-dimensional human lung cultures consisting of epithelia and stromal fibroblasts. Relative to mock-treated cells, matrix metallopeptidase 9 levels in the conditioned media were lower by 4 days postexposure and remained suppressed for the duration of the experiment. Immunocytochemical staining of collagen III, a marker associated with fibrosis, showed increased accumulation in the epithelial layer and at the air-liquid interface.

Piper sarmentosum Roxb. confers neuroprotection on beta-amyloid (Aß)-induced microglia-mediated neuroinflammation and attenuates tau hyperphosphorylation in SH-SY5Y cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Piper sarmentosum Roxb. (PS), belonging to Piperaceae family, is an edible plant with medicinal properties. It is traditionally used by the Malays to treat headache and boost memory. Pharmacological studies revealed that PS exhibits anti-inflammatory, anti-oxidant, anti-acetylcholinesterase, and anti-depressant-like effects. In view of this, the present study aimed to investigate the anti-inflammatory actions of PS and its potential neuroprotective effects against beta-amyloid (Aß)-induced microglia-mediated neurotoxicity. MATERIALS AND METHODS: The inhibitory effects of hexane (LHXN), dichloromethane (LDCM), ethyl acetate (LEA) and methanol (LMEOH) extracts from leaves of PS on Aß-induced production and mRNA expression of pro-inflammatory mediators in BV-2 microglial cells were assessed using colorimetric assay with Griess reagent, ELISA kit and real-time RT-PCR respectively. Subsequently, MTT reduction assay was used to evaluate the neuroprotective effects of PS leaf extracts against Aß-induced microglia-mediated neurotoxicity in SH-SY5Y neuroblastoma cells. The levels of tau proteins phosphorylated at threonine 231 (pT231) and total tau proteins (T-tau) were determined using ELISA kits. RESULTS: Polar extracts of PS leaves (LEA and LMEOH) reduced the Aß-induced secretion of pro-inflammatory cytokines (IL-1ß and TNF-α) in BV-2 cells by downregulating the mRNA expressions of pro-inflammatory cytokines. The inhibition of nitric oxide (NO) production could be due to the free radical scavenging activity of the extracts. In addition, conditioned media from Aß-induced BV-2 cells pre-treated with LEA and LMEOH protected SH-SY5Y cells against microglia-mediated neurotoxicity. Further mechanistic study suggested that the neuroprotective effects were associated with the downregulation of phosphorylated tau proteins. CONCLUSIONS: The present study suggests that polar extracts of PS leaves confer neuroprotection against Aß-induced microglia-mediated neurotoxicity in SH-SY5Y cells by attenuating tau hyperphosphorylation through their anti-inflammatory actions and could be a potential therapeutic agent for Alzheimer's disease.

Contact guidance diversity in rotationally aligned collagen matrices.

Cancer cell metastasis is responsible for approximately 90% of deaths related to cancer. The migration of cancer cells away from the primary tumor and into healthy tissue is driven in part by contact guidance, or directed migration in response to aligned extracellular matrix. While contact guidance has been a focus of many studies, much of this research has explored environments that present 2D contact guidance structures. Contact guidance environments in 3D more closely resemble in vivo conditions and model cell-ECM interactions better than 2D environments. While most cells engage in directed migration on potent 2D contact guidance cues, there is diversity in response to contact guidance cues based on whether the cell migrates with a mesenchymal or amoeboid migration mode. In this paper, rotational alignment of collagen gels was used to study the differences in contact guidance between MDA-MB-231 (mesenchymal) and MTLn3 (amoeboid) cells. MDA-MB-231 cells migrate with high directional fidelity in aligned collagen gels, while MTLn3 cells show no directional migration. The collagen stiffness was increased through glycation, resulting in decreased MDA-MB-231 directionality in aligned collagen gels. Interestingly, partial inhibition of cell contractility dramatically decreased directionality in MDA-MB-231 cells. The directionality of MDA-MB-231 cells was most sensitive to ROCK inhibition, but unlike in 2D contact guidance environments, cell directionality and speed are more tightly coupled. Modulation of the contractile apparatus appears to more potently affect contact guidance than modulation of extracellular mechanical properties of the contact guidance cue. STATEMENT OF SIGNIFICANCE: Collagen fiber alignment in the tumor microenvironment directs migration, a process called contact guidance, enhancing the efficiency of cancer invasion and metastasis. 3D systems that assess contact guidance by locally orienting collagen fiber alignment are lacking. Furthermore, cell type differences and the role of extracellular matrix stiffness in tuning contact guidance fidelity are not well characterized. In this paper rotational alignment of collagen fibers is used as a 3D contact guidance cue to illuminate cell type differences and the role of extracellular matrix stiffness in guiding cell migration along aligned fibers of collagen. This local alignment offers a simple approach by which to couple collagen alignment with gradients in other directional cues in devices such as microfluidic chambers.

Chinese medicine Tongxinluo capsule alleviates cerebral microcirculatory disturbances in ischemic stroke by modulating vascular endothelial function and inhibiting leukocyte-endothelial cell interactions in mice: A two-photon laser scanning microscopy study.

OBJECTIVE: The aim of this study was to examine the effect of TXL, a Chinese medicine prescription, on cerebral microcirculatory disturbances after pMCAO in mice using TPLSM and further explore the underlying mechanisms. METHODS: Adlut male C57BL/6J mice were subjected to pMCAO and orally administered with TXL (3.0, 1.5 and 0.75 g/kg/d) at 1, 3, and 21 hours after pMCAO. The following parameters were examined at 6 and 24 hours after pMCAO: neurological deficits, infarct volume, BBB permeability, cerebral microvessel structure, brain microcirculation (TPLSM imaging), vasoactive factors, and adhesion molecules. RESULTS: TXL improved neurological deficits, reduced infarct volume, attenuated BBB disruption, protected cerebral microvessel structure, increased cerebral capillary flow velocity and volume flux, and inhibited leukocyte-endothelial cell interactions at 6 or 24 hours after pMCAO. The therapeutic efficacy was exerted in a dose-dependent manner. Further study revealed that TXL (high dose) regulated the expression of PGI2, TXA2, and ET-1, and suppressed ICAM-1 and P-selectin. CONCLUSIONS: TXL alleviates cerebral microcirculatory disturbances against ischemic injury by modulating endothelial function and inhibiting leukocyte-endothelial cell interactions. These effects are associated with regulating the expression of PGI2, TXA2, and ET-1, and suppressing ICAM-1 and P-selectin expression.