Western Blot Studies Research Articles

High fat diet-induced metabolically obese and normal weight rabbit model shows early vascular dysfunction: mechanisms involved.

Obesity contributes significantly to the development and evolution of cardiovascular disease (CVD) which is believed to be mediated by oxidative stress, inflammation and endothelial dysfunction. However, the vascular health of metabolically obese and normal weight (MONW) individuals is not completely comprehended. The purpose of our study was to evaluate vascular function on the basis of a high fat diet (HFD)-MONW rabbit model. Twenty four male rabbits were randomly assigned to receive either a regular diet (CD, n = 12) or a high-fat diet (18% extra fat on the regular diet, HFD, n = 12) for 6 weeks. Body weight, TBARS and gluthathione serum levels were similar between the groups; fasting glucose, triglycerides, C reactive protein (CRP), visceral adipose tissue (VAT), triglyceride-glucose index (TyG index) were higher in the HFD group. Compared to CD, the HFD rabbits had glucose intolerance and lower HDL-cholesterol and plasma nitrites levels. Thoracic aortic rings from HFD rabbits exhibited: (a) a reduced acetylcholine-induced vasorelaxation; (b) a greater contractile response to norepinephrine and KCl; (c) an improved angiotensin II-sensibility. The HFD-effect on acetylcholine-response was reversed by the cyclooxygenase-2 (COX-2) inhibitor (NS398) and the cyclooxygenase-1 inhibitor (SC560), and the HFD-effect on angiotensin II was reversed by NS398 and the TP receptor blocker (SQ29538). Immunohistochemistry and western blot studies showed COX-2 expression only in arteries from HFD rabbits. Our study shows a positive pro-inflammatory status of HFD-induced MONW characterized by raised COX-2 expression, increase of the CRP levels, reduction of NO release and oxidative stress-controlled conditions in an early stage of metabolic alterations characteristic of metabolic syndrome. Endothelial dysfunction and increased vascular reactivity in MONW individuals may be biomarkers of early vascular injury. Therefore, the metabolic changes induced by HFD even in normal weight individuals may be associated to functional alterations of blood vessels.

Age-Related Changes in the Expression of the Circadian Clock Protein PERIOD in Drosophila Glial Cells.

Circadian clocks consist of molecular negative feedback loops that coordinate physiological, neurological, and behavioral variables into “circa” 24-h rhythms. Rhythms in behavioral and other circadian outputs tend to weaken during aging, as evident in progressive disruptions of sleep-wake cycles in aging organisms. However, less is known about the molecular changes in the expression of clock genes and proteins that may lead to the weakening of circadian outputs. Western blot studies have demonstrated that the expression of the core clock protein PERIOD (PER) declines in the heads of aged Drosophila melanogaster flies. This age-related decline in PER does not occur in the central pacemaker neurons but has been demonstrated so far in retinal photoreceptors. Besides photoreceptors, clock proteins are also expressed in fly glia, which play important roles in neuronal homeostasis and are further categorized into subtypes based on morphology and function. While previous studies of mammalian glial cells have demonstrated the presence of functional clocks in astrocytes and microglia, it is not known which glial cell types in Drosophila express clock proteins and how their expression may change in aged individuals. Here, we conducted immunocytochemistry experiments to identify which glial subtypes express PER protein suggestive of functional circadian clocks. Glial cell subtypes that showed night-time accumulation and day-time absence in PER consistent with oscillations reported in the pacemaker neurons were selected to compare the level of PER protein between young and old flies. Our data demonstrate that some glial subtypes show rhythmic PER expression and the relative PER levels become dampened with advanced age. Identification of glial cell types that display age-related dampening of PER levels may help to understand the cellular changes that contribute to the loss of homeostasis in the aging brain.

Downregulation of neuroligin1 ameliorates postoperative pain through inhibiting neuroligin1/postsynaptic density 95-mediated synaptic targeting of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor GluA1 subunits in rat dorsal horns.

Neuroligin1 is an important synaptic cell adhesion molecule that modulates the function of synapses through protein–protein interactions. Yet, it remains unclear whether the regulation of synaptic transmission in the spinal cord by neruoligin1 contributes to the development of postoperative pain. In a rat model of postoperative pain induced by plantar incision, we conducted Western blot study to examine changes in the expression of postsynaptic membrane of neuroligin1, postsynaptic density 95 (PSD-95), and α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor GluA1 and GluA2 subunits in the spinal cord dorsal horn after injury. The interaction between neuroligin1 and PSD-95 was further determined by using coimmunoprecipitation. Protein levels of neuroligin1 and GluA1, but not GluA2 and PSD-95, were significantly increased in the postsynaptic membrane of the ipsilateral dorsal horn at 3 h and 1 day after incision, as compared to that in control group (naïve). A greater amount of PSD-95 was coimmunoprecipitated with neuroligin1 at 3 h after incision than that in the control group. Intrathecal administration of small interfering RNAs (siRNAs) targeting neuroligin1 suppressed the expression of neuroligin1 in the spinal cord. Importantly, pretreatment with intrathecal neuroligin1 siRNA2497, but not scrambled siRNA or vehicle, prevented the upregulation of GluA1 expression at 3 h after incision, inhibited the enhanced neuroligin1/PSD-95 interaction, and attenuated postoperative pain. Together, current findings suggest that downregulation of spinal neuroligin1 expression may ameliorate postoperative pain through inhibiting neuroligin1/PSD-95 interaction and synaptic targeting of GluA1 subunit. Accordingly, spinal neuroligin1 may be a potential new target for postoperative pain treatment.

Ligustrazine suppresses neuron apoptosis via the Bax/Bcl-2 and caspase-3 pathway in PC12 cells and in rats with vascular dementia.

The aim of this study was to examine the comprehensive neuroprotective mechanism of ligustrazine, which is extracted from Ligusticum Chuanxiong Hort., against vascular dementia (VD) in rats and apoptosis in oxygen and glucose deprivation (OGD) PC12 cells. Rats were subjected to bilateral common carotid artery occlusion (BCCAO) surgery and administered ligustrazine intragastrically for 6 weeks. At the end of the experiments, the hippocampal biomarkers brain-derived neurotrophic factor (BDNF), monocyte chemotactic protein 1 (MCP-1), and homocysteine (Hcy) were examined. In experiments in vitro, OGD PC12 cells were treated with ligustrazine for 0.5, 1, 3, 6, 12, or 24 h. The cell-released biomarkers BDNF, MCP-1, and Hcy were examined. Microscopy, acridine orange-ethidium bromide (AO/EB) staining, and flow cytometry assays were performed to investigate apoptosis. Cleaved caspase-3, Bcl-2 associated X protein (Bax), and B cell lymphoma 2 (Bcl-2) expression was examined using Western blot assays. The results showed that biomarkers, including MCP-1 and Hcy, were significantly increased in both the in vivo and in vitro models, while the BDNF level was significantly decreased compared with the sham or vehicle models. Microscopy, AO/EB staining, and flow cytometry analysis showed that severe cell damage occurred in OGD PC12 cells, and apoptosis played a major role in this environment. Further Western blot studies showed that the apoptosis-related Bax/Bcl-2 protein ratio and cleaved caspase-3 were significantly increased in the experiment. However, ligustrazine profoundly suppressed the imbalance of these biomarkers, reduced cell damage, decreased the Bax/Bcl-2, and downregulated cleaved caspase-3. Pro- and anti-apoptotic biomarkers of multiple pathways including BDNF, MCP-1, and Hcy played a joint role in triggering the activation of the mitochondria-related Bax/Bcl-2 and caspase-3 apoptosis pathway in VD. Ligustrazine attenuated VD by comprehensively regulating BDNF, MCP-1, and Hcy and inactivating the Bax/Bcl-2 and caspase-3 apoptosis pathway. Our data provide novel insight into ligustrazine, which is a promising neuroprotective agent for VD disease treatment strategies. © IUBMB Life, 70(1):60-70, 2018.

Putative identification of components in Zengye Decoction and their effects on glucose consumption and lipogenesis in insulin-induced insulin-resistant HepG2 cells

Zengye Decoction (ZYD) is a well-known traditional medicine in China used for treating diseases associated with “Yin deficiency” such as diabetes. However, little information is available on its components, pharmacological effects and underlying mechanisms. This study was designed to identify its active components and evaluate the effects and mechanisms of ZYD on glucose consumption and lipogenesis in insulin-induced insulin-resistant (IR)-HepG2 cells. In this study, 45 compounds of ZYD were putatively identified, in which the iridoid glycosides such as catalpol, aucubin and harpagide were identified as the main components. The insulin-resistant (IR)-HepG2 cell model was established and the effect of ZYD at three doses (0.17, 0.5 and 1.5 μg/mL) on cell growth was evaluated with an IncuCyte™ live-cell imaging system. The effects of ZYD on glucose consumption and uptake were evaluated by glucose consumption and uptake assay. Meanwhile, the effect of ZYD on lipogenesis was investigated in IR-HepG2 cells by oil red O (ORO) staining. Western blot was applied to observe the changes in some of the key factors involved in glucose metabolism and lipogenesis. It was found that the ZYD at a dose of 1.5 μg/mL exhibited an inhibitory activity on IR-HepG2 cell growth. Besides, ZYD at doses of 0.5 and 1.5 μg/mL accelerated the glucose consumption, glucose uptake and reduced the lipogenesis in the IR-HepG2 cells. Western blot studies revealed that ZYD phosphorylated AMP-activated protein kinase α subunits (AMPKα), upregulated hexokinase (HK), phosphorylated acetyl-CoA carboxylase alpha (pACC1) and carnitine palmitoyltransferase 1A (CPT1A) in the IR-HepG2 cells. These results indicate ZYD promotes glucose consumption and uptake, and attenuates lipogenesis in IR-HepG2 cells, which may be involved in activating AMPK and regulating its downstream factors including HK, pACC1 and CPT1A.

Antitumor effect of Quercetin on Y79 retinoblastoma cells via activation of JNK and p38 MAPK pathways

BackgroundQuercetin (QCT) is a flavonol present in many vegetables, it is proved to show chemo preventive effect against lung, cervical, prostate, breast and colon cancer due to its anti-inflammatory, anti-tumor and anti-oxidant property. Looking into the reported chemo-preventive effect we speculated antitumor activity in retinoblastoma (RB) Y79 cells, we also studied the molecular mechanism for antitumor activity.MethodsThe effect of QCT on Y79 cell viability count was done by cell counting kit, cell cycle distribution, apoptosis studies and mitochondrial membrane potential was evaluated by flow cytometry. Protein expression was done by western blot analysis.ResultsThe outcomes of study showed that QCT reduced Y79 cell viability and caused arrest of G1 phase in cell cycle via decreasing the expression levels of cyclin-dependent kinase (CDK)2/6 and cyclin D3 and by increasing the levels of both CDK inhibitor proteins p21 and p27. Apoptosis of Y79 cells mediated by QCT occurred via activation of both caspases-3/-9. Flow cytometry studies showed that QCT caused collapse in mitochondrial membrane potential (ΔΨm) in Y79 cells. Western blot studies confirmed that QCT brought about phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). We also established that inhibitors of JNK and p38 MAPK suppressed QCT mediated activation of both caspases-3/-9 and subdued the apoptosis of cancerous Y79 cells.ConclusionAll the results of the study suggest that QCT induced the apoptosis of Y79 cells via activation of JNK and p38 MAPK pathways, providing a novel treatment approach for human RB.

The Short-Chain Fatty Acid Propionate Inhibits Adipogenic Differentiation of Human Chorion-Derived Mesenchymal Stem Cells Through the Free Fatty Acid Receptor 2

Free fatty acid receptor 2 (FFAR2, also known as GPR43) is a G-protein-coupled receptor activated by short-chain fatty acids that are produced by gut microbiota through fermentation of nondigestible carbohydrates. FFAR2 functions as a metabolic sensor and is expressed in metabolically active tissues, such as adipose tissue. Earlier studies proved the connection between FFAR2 and adipocyte differentiation in mice. The aim of this study was to investigate the implication of FFAR2 receptor in adipogenesis in human chorion-derived mesenchymal stem cells (cMSCs). The short-chain fatty acid, propionate, and phenylacetamide a selective FFAR2 agonist resulted in a marked suppression of lipid droplet accumulation during the adipogenic differentiation of cMSCs. Western blot studies revealed that FFAR2 was detectable at any time point of the differentiation period. The direct involvement of FFAR2 in the differentiation into adipocytes was proven by the downregulation of its gene expression in cMSCs by lentiviral messenger RNA (mRNA) silencing transduction particles. Our results showed that a significant suppression in lipid accumulation upon FFAR2 agonist treatments was elicited by FFAR2-silencing. Based on these results we suggest that propionate inhibits the formation of adipocytes from MSCs and acts on adipogenesis predominantly via FFAR2.

Attenuation of the Infiltration of Angiotensin II Expressing CD3+ T-Cells and the Modulation of Nerve Growth Factor in Lumbar Dorsal Root Ganglia - A Possible Mechanism Underpinning Analgesia Produced by EMA300, An Angiotensin II Type 2 (AT2) Receptor Antagonist.

Recent preclinical and proof-of-concept clinical studies have shown promising analgesic efficacy of selective small molecule angiotensin II type 2 (AT2) receptor antagonists in the alleviation of peripheral neuropathic pain. However, their cellular and molecular mechanism of action requires further investigation. To address this issue, groups of adult male Sprague–Dawley rats with fully developed unilateral hindpaw hypersensitivity, following chronic constriction injury (CCI) of the sciatic nerve, received a single intraperitoneal bolus dose of the small molecule AT2 receptor antagonist, EMA300 (10 mg kg-1), or vehicle. At the time of peak EMA300-mediated analgesia (∼1 h post-dosing), groups of CCI-rats administered either EMA300 or vehicle were euthanized. A separate group of rats that underwent sham surgery were also included. The lumbar (L4–L6) dorsal root ganglia (DRGs) were obtained from all experimental cohorts and processed for immunohistochemistry and western blot studies. In vehicle treated CCI-rats, there was a significant increase in the expression levels of angiotensin II (Ang II), but not the AT2 receptor, in the ipsilateral lumbar DRGs. The elevated levels of Ang II in the ipsilateral lumbar DRGs of CCI-rats were at least in part contributed by CD3+ T-cells, satellite glial cells (SGCs) and subsets of neurons. Our findings suggest that the analgesic effect of EMA300 in CCI-rats involves multimodal actions that appear to be mediated at least in part by a significant reduction in the otherwise increased expression levels of Ang II as well as the number of Ang II-expressing CD3+ T-cells in the ipsilateral lumbar DRGs of CCI-rats. Additionally, the acute anti-allodynic effects of EMA300 in CCI-rats were accompanied by rescue of the otherwise decreased expression of mature nerve growth factor (NGF) in the ipsilateral lumbar DRGs of CCI-rats. In contrast, the increased expression levels of TrkA and glial fibrillary acidic protein in the ipsilateral lumbar DRGs of vehicle-treated CCI-rats were not attenuated by a single bolus dose of EMA300. Consistent with our previous findings, there was also a significant decrease in the augmented levels of the downstream mediators of Ang II/AT2 receptor signaling, i.e., phosphorylated-p38 mitogen-activated protein kinase (MAPK) and phosphorylated-p44/p42 MAPK, in the ipsilateral lumbar DRGs.

Synthesis and biological evaluation of sulfur-containing shikonin oxime derivatives as potential antineoplastic agents

As a continuation of our research on developing potent and potentially safe antineoplastic agents, a set of forty five sulfur-containing shikonin oxime derivatives were synthesized and evaluated for their in vitro cytotoxic activity against human colon cancer (HCT-15), gastric carcinoma (MGC-803), liver (Bel7402), breast (MCF-7) cancer cells and human skin fibroblast (HSF) cells. All the synthesized compounds exhibited potent cytotoxic activity selectively towards HCT-15 cells and did not display apparent toxicity to the normal HSF cells, some of which were more or comparatively effective to the parent compound against HCT-15, MGC-803 and Bel7402 cells. The most active agent 9m displayed high potency against human cancer cells with IC50 ranging from 0.27 ± 0.02 to 9.23 ± 0.12 μM. The structure-activity relationships (SARs) studies suggested that the nature of substituent group in the side chain is important for antitumor potency in vitro. Additionally, nitric oxide release studies revealed that the amount of nitric oxide generated from these oxime derivatives was relatively low. Furthermore, cellular mechanism investigations indicated that compound 9m could arrest cell cycle at G1 phase and induce a strong apoptotic response in HCT-15 cells. Moreover, western blot studies revealed that compound 9m induced apoptosis through the down-regulation of Bcl-2 and up-regulation of Bax, caspase 3 and 9. For all these reasons, compound 9m hold promising potential as antineoplastic agent.

Antioxidative and Antiapoptotic Effects of Delta-Opioid Peptide [D-Ala2, D-Leu5] Enkephalin on Spinal Cord Ischemia-Reperfusion Injury in Rabbits.

Background: In our previous study, we found that regional administration of delta-opioid peptide [D-Ala2, D-Leu5] enkephalin (DADLE) could provide dose-dependent protection on spinal cord ischemia-reperfusion (I/R) injury in rabbits. However, the relative protective molecular mechanisms underlying this neuroprotection remain unclear. The purpose of this study was to investigate whether DADLE provided the protection in spinal cord I/R injury through its antioxidant property by decreasing malondialdehyde (MDA) and nitric oxide (NO) levels and increasing glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) levels and through its antiapoptotic capacity by inhibiting caspase-3 and p53 expression.Methods: The rabbits were divided into three groups. The animals in Group NS and Group DADLE were administered with normal saline (NS) or DADLE via aorta during 30 min of ischemia respectively, while the one in Group Sham received no intervention. During the period of reperfusion, the rabbit's blood samples were collected for enzyme-linked immunoabsorbent assay (ELISA) examinations of MDA, NO, GSH-Px and SOD. At 48 h after reperfusion, the lumbar spinal cords were harvested for immunohistochemical, real-time polymerase chain reaction (PCR) and western blot studies to detect the caspase-3 and p53 expressions.Results: The activities of serum MDA and NO showed significant reductions in the DADLE group as compared with the control group. By contrast, the levels of serum GSH-Px and SOD were significantly higher in the DADLE group than those in the NS group. In addition, caspase-3 and p53 expression were significantly increased in the NS group, while DADLE mitigated these changes.Conclusions: The protective effects of DADLE at the dosage of 0.05 mg/kg may be related to its antioxidant and antiapoptosis properties in the rabbit model of spinal cord I/R injury.

Proteolytic maturation of Drosophila Neuroligin 3 by tumor necrosis factor α-converting enzyme in the nervous system

BackgroundThe functions of autism-associated Neuroligins (Nlgs) are modulated by their post-translational modifications, such as proteolytic cleavage. A previous study has shown that there are different endogenous forms of DNlg3 in Drosophila, indicating it may undergo proteolytic processing. However, the molecular mechanism underlying DNlg3 proteolytic processing is unknown. Here, we report a novel proteolytic mechanism that is essential for DNlg3 maturation and function in the nervous system. MethodsMolecular cloning, cell culture, immunohistochemistry, western blotting and genetic studies were employed to map the DNlg3 cleavage region, identify the protease and characterize the cleavage manner. Behavior analysis, immunohistochemistry and genetic manipulations were employed to study the functions of different DNlg3 forms in the nervous system and neuromuscular junction (NMJs). ResultsTumor necrosis factor α-converting enzyme (TACE) cleaved DNlg3 exclusively at its extracellular acetylcholinesterase-like domain to generate the N-terminal fragment and the short membrane-anchored fragment (sDNlg3). DNlg3 was constitutively processed in an activity-independent manner. Interestingly, DNlg3 was cleaved intracellularly in the Golgi apparatus before it arrived at the cell surface, a unique cleavage mechanism that is distinct from ‘conventional’ ectodomain shedding of membrane proteins, including rodent Nlg1. Genetic studies showed that sDNlg3 was essential for maintaining proper locomotor activity in Drosophila. ConclusionsOur results revealed a unique cleavage mechanism of DNlg3 and a neuron-specific role for DNlg3 maturation which is important in locomotor activity. General significanceOur study provides a new insight into a cleavage mechanism of Nlgs maturation in the nervous system.

L-Tetrahydropalmatine Induces Apoptosis in EU-4 Leukemia Cells by Down-Regulating X-Linked Inhibitor of Apoptosis Protein and Increases the Sensitivity Towards Doxorubicin.

L-Tetrahydropalmatine (L-THP) is a tetra-hydro protoberberine isoquinoline alkaloid. The phyto-compounds bearing isoquinoline alkaloids have been reported to show a potential effect against a number of human cancers cell lines including leukemia. We hypothesized that L-THP, being an isoquinoline alkaloid, could be a potential molecule against acute lymphoblastic leukemia (ALL), in this study, we evaluate L-THP against p53 deficient leukemia EU-4 cell lines in vitro. For the study, p53 null leukemia EU-4 cells were used and treated with LTHP. The extent of apoptosis and viability of cells were determined. Expression of apoptosis related proteins such as XIAP and MDM2 was done by western blot and PCR studies. The expression of MDM2 and XIAP was knocked down by small interfering RNA (siRNA). Outcomes of the study suggested that L-THP caused p53-indipendent apoptosis mediated by XIAP in EU-4 cells. The treatment of L-THP caused a decrease in the levels of XIAP protein with increasing dose and time. L-THP caused down-regulation of XIAP protein via inhibiting the expression of MDM2 and involving proteasomedependent pathway. Also, the outcomes of experiments suggested increased sensitivity of leukemia cells towards doxorubicin due to the inhibition of XIAP by L-THP or by siRNA. Findings of the study confirm that L-THP resulted in p53 independent apoptosis via down-regulating XIAP protein by inhibiting MDM2 associated with proteasome-dependent pathway and increased sensitivity of EU-4 cells against doxorubicin. L-THP caused activation of caspase and resulted in apoptosis, L-THP may be a novel molecule for inducing apoptosis specifically in p53 null leukemia EU-4 cells.

Blockade of LGR4 inhibits proliferation and odonto/osteogenic differentiation of stem cells from apical papillae.

During tooth root development, stem cells from apical papillae (SCAPs) are indispensable, and their abilities of proliferation, migration and odontoblast differentiation are linked to root formation. Leucine-rich repeat-containing GPCR 4 (LGR4) modulates the biological processes of proliferation and differentiation in multiple stem cells. In this study, we showed that LGR4 is expressed in all odontoblast cell lineage cells and Hertwig's epithelial root sheath (HERS) during the mouse root formation in vivo. In vitro we determined that LGR4 is involved in the Wnt/β-catenin signaling pathway regulating proliferation and odonto/osteogenic differentiation of SCAPs. Quantitative reverse-transcription PCR (qRT-PCR) confirmed that LGR4 is expressed during odontogenic differentiation of SCAPs. CCK8 assays and in vitro scratch tests, together with cell cycle flow cytometric analysis, demonstrated that downregulation of LGR4 inhibited SCAPs proliferation, delayed migration and arrested cell cycle progression at the S and G2/M phases. ALP staining revealed that blockade of LGR4 decreased ALP activity. QRT-PCR and Western blot analysis demonstrated that LGR4 silencing reduced the expression of odonto/osteogenic markers (RUNX2, OSX, OPN, OCN and DSPP). Further Western blot and immunofluorescence studies clarified that inhibition of LGR4 disrupted β-catenin stabilization. Taken together, downregulation of LGR4 gene expression inhibited SCAPs proliferation, migration and odonto/osteogenic differentiation by blocking the Wnt/β-catenin signaling pathway. These results indicate that LGR4 might play a vital role in SCAPs proliferationand odontoblastic differentiation.

The Effect of a Small Molecule EZH2 Methyltransferase Inhibitor GSK126 on p53 Negative Prostate Cancer Cell Line

To quantitatively evaluate the effect of GSK126 (an inhibitor of histone methyltransferase) in inhibiting migration of prostate cancer cell line. And to investigate whether GSK126 can regulate the radiosensitivity and reduce the survival rate of p53 negative prostate cancer cell line. Human prostatic carcinoma cells (PC-3) were separated into 5 groups and incubated with GSK126 of different concentrations (0 μM, 5 μM, 10 μM, 20 μM) for 48h. The protein expression levels of EZH2 in the cells were detected by western blotting. Cell migration was examined by both wound scratch assay and transwell assay. And then the PC-3 cells were exposure to a single dose of 4Gy delivered using a 6MV photon beam of Clinac 600 CD. Cell colony assay was used to determine cell survival rates after irradiation. One group of cells without any treatments was used as the control group. All experiments were repeated 3 times, statistical analysis was performed using independent sample T-test and p value of less than 0.05 was considered to be significant. Western blotting study showed that the expression level of EZH2 in PC-3 cells decreased with increasing concentration of GSK126. Transwell assay study showed that, when the concentration of GSK126 was increased to 10μM or higher, the EZH2 expression decreased and number of cells penetrating cell membrane decreased significantly (p<0.02). Wound scratch assay study showed that, along with decreasing EZH2 expression, cell migration speed and the ability to migrate reduced (p<0.05). This is consistent with the results of transwell experiments. Results mentioned above indicated that GSK126 can regulate migration of PC-3 cells by inhibition of EZH2. Cell colony assay study showed that with the increase of GSK126 concentration, the forming rates of colonies decreased significantly after irradiation (P<0.05). It indicated that GSK126 enhanced radiosensitivity of PC-3 cells.Abstract 3402; Table 1Forming rates of colonies after irradiation of 4GyGSK126 concentrationForming rate of coloniesReference (0μM w/o radiation treatment)10 μM0.36±0.045 μM0.33±0.0510 μM0.29±0.0820 μM0.14±0.07 Open table in a new tab Using GSK126 of 10μM or higher can inhibit migration of PC-3 cells. And GSK126 in combination with radiation can effectively reduce the survival rates of PC-3 cells.

A comparative study between platelet-rich plasma and platelet-poor plasma effects on angiogenesis.

Platelet-rich plasma (PRP) has been established as an autologous source for therapeutic angiogenesis. The purpose of this study was to evaluate PRP angiogenic effects compared to platelet-poor plasma (PPP) in vitro and in vivo. The effects of PRP on vascular endothelial growth factor receptor-2 (VEGFR2) and CD34 expression were evaluated using real-time PCR, flow cytometry, western blot, immunocytochemistry and pathological study, as were carried out in both human umbilical endothelial cell culture and rat skin. Our findings indicated significant effect of PRP and PPP on VEGFR2 and CD34 expression by human umbilical vein endothelial cells, which was greater in latter. These effects, however, were confirmed by demonstrating an earlier angiogenic effect of PPP in vivo when compared to PRP. The findings of the present study as the first comparative study of PRP versus PPP are novel. Nevertheless, further studies are needed to clarify the underlying mechanism of these findings to improve the therapeutic effects of PRP and PPP.

Validation of commercial Mas receptor antibodies for utilization in Western Blotting, immunofluorescence and immunohistochemistry studies.

Mas receptor (MasR) is a G protein-coupled receptor proposed as a candidate for mediating the angiotensin (Ang)-converting enzyme 2-Ang (1–7) protective axis of renin–angiotensin system. Because the role of this receptor is not definitively clarified, determination of MasR tissue distribution and expression levels constitutes a critical knowledge to fully understanding its function. Commercially available antibodies have been widely employed for MasR protein localization and quantification, but they have not been adequately validated. In this study, we carried on an exhaustive evaluation of four commercial MasR antibodies, following previously established criteria. Western Blotting (WB) and immunohistochemistry studies starting from hearts and kidneys from wild type (WT) mice revealed that antibodies raised against different MasR domains yielded different patterns of reactivity. Furthermore, staining patterns appeared identical in samples from MasR knockout (MasR-KO) mice. We verified by polymerase chain reaction analysis that the MasR-KO mice used were truly deficient in this receptor as MAS transcripts were undetectable in either heart or kidney from this animal model. In addition, we evaluated the ability of the antibodies to detect the human c-myc-tagged MasR overexpressed in human embryonic kidney cells. Three antibodies were capable of detecting the MasR either by WB or by immunofluorescence, reproducing the patterns obtained with an anti c-myc antibody. In conclusion, although three of the selected antibodies were able to detect MasR protein at high expression levels observed in a transfected cell line, they failed to detect this receptor in mice tissues at physiological expression levels. As a consequence, validated antibodies that can recognize and detect the MasR at physiological levels are still lacking.

Inhibitory growth evaluation and apoptosis induction in MCF-7 cancer cells by new 5-aryl-2-butylthio-1,3,4-oxadiazole derivatives.

Cancer has become one of the global health issues and it is the life-threatening disease characterized by unrestrained growth of cells. Despite various advances being adopted by chemotherapeutic management, the use of the current anticancer drugs such as Doxorubicin, Asparginase, Methotrexate, Vincristine remains limited due to high toxicity, side effects and developing drug resistance. Apoptosis is a crucial cellular process and improper regulation of apoptotic signaling pathways may lead to cancer formation. Subsequently, the synthesis of effective chemotherapeutic agents that can induce apoptosis in tumor cell has emerged as a significant approach in cancer drug discovery. The goal of this work is to develop a potential antitumor agent exerting significant inhibitory effects on cancer cell and low cytotoxicity, for which we focused on the structural features of 1,3,4-oxadiazoles as it a privileged scaffold in modern medicinal chemistry and have the ability to inhibit growth factors, enzymes and kinases potentially involved in the attainment of cellular immortality and carcinogenesis. In vitro MTT screening assay showed the compound 5-aminophenyl-2-butylthio-1,3,4-oxadiazole (5e) showing the highest inhibitory effect against MCF-7 cancer cell with IC50 value 10.05±1.08µM while it is much safer and less toxic on normal cell line (HEK-293). The dose-dependent treatment of MCF-7 cells with 5e resulted in inhibition of cell migration in the wound healing assay. The flow-cytometry analysis showed the cells arrested in G0/G1 phase of the cell cycle. Compound 5e induced apoptosis of MCF-7 cells was characterized using DAPI staining and Annexin V-PE/7-AAD dual binding assay. Reduction of NBT by compound 5e showed a reduced generation of ROS. Western blotting studies showed high activation of apoptotic protein Caspase3 and decrease in expression of anti-apoptotic protein BCL-2. Based on the results of in vitro studies, it could be concluded that compound 5e showed a significant inhibitory growth effect on MCF-7 cells and have the potential to be developed as lead molecule and further structural modifications may result in promising new anticancer agents.

Adipose stem cells enhance myoblast proliferation via acetylcholine and extracellular signal–regulated kinase 1/2 signaling

ABSTRACT Introduction: In this study we investigated the interaction between adipose tissue–derived stem cells (ASCs) and myoblasts in co‐culture experiments. Methods: Specific inductive media were used to differentiate ASCs in vitro into a Schwann cell–like phenotype (differentiated adipose tissue–derived stem cells, or dASCs) and, subsequently, the expression of acetylcholine (ACh)‐related machinery was determined. In addition, the expression of muscarinic ACh receptors was examined in denervated rat gastrocnemius muscles. Results: In contrast to undifferentiated ASCs, dASCs expressed more choline acetyltransferase and vesicular acetylcholine transporter. When co‐cultured with myoblasts, dASCs enhanced the proliferation rate, as did ACh administration alone. Western blotting and pharmacological inhibitor studies showed that phosphorylated extracellular signal–regulated kinase 1/2 signaling mediated these effects. In addition, denervated muscle showed higher expression of muscarinic ACh receptors than control muscle. Discussion: Our findings suggest that dASCs promote proliferation of myoblasts through paracrine secretion of ACh, which could explain some of their regenerative capacity in vivo. Muscle Nerve 57: 305–311, 2018

Radiation protection and mitigation potential of phenylbutyrate: delivered via oral administration

Purpose: Phenylbutyrate (PB), a histone deacetylase inhibitor (HDACi) has demonstrated radiation protection in both in vitro and in vivo models. Studies previously demonstrated that PB and other HDAC inhibitors could inhibit radiation lethality in vivo by subcutaneous (s.c) injection. The objective of this study was to test the ability of oral PB treatment to protect against or to mitigate acute gamma radiation-induced lethality in vivo.Materials and methods: Human osteoblasts cells were used to evaluate radiation survival when PB was delivered pre- or post-radiation. A 30-day radiation lethality study was used to assess the radioprotective (pre-radiation) and radiomitigative (post-radiation) capability of PB. Possible mechanisms evaluated were antioxidant activity effects, HDAC inhibition, DNA damage, and hematological recovery.Results: Treatment of HOS cells with PB 50 μM either before or after radiation increased radiation resistance as assessed by clonogenic survival. Western blot studies showed that PB treatment acetylated histones in vivo and ameliorated the radiation-induced reduction in acetylated histone-4 (H4). Pre-radiation oral administration of PB (10 mg/kg) provided radioprotection against gamma radiation (7–11.5 Gy) with a dose reduction factor of 1.25 (p = 0.001). PB oral administration post-radiation provided moderate radiation mitigation against gamma radiation (7–11.5 Gy) and demonstrated a dose reduction factor of 1.18 (p = 0.05). PB pre-radiation and post-radiation treatment was associated with significant elevations in neutrophils and platelets and attenuation of DNA damage.Conclusions: These results indicate that oral PB has potential as a radiation protector and a radiation mitigator and that potential mechanisms of action include attenuation of DNA damage, antioxidant activity, and bone marrow protection.

AIM2 Inflammasome-Mediated Pyroptosis in Enterovirus A71-Infected Neuronal Cells Restricts Viral Replication

Encephalomyelitis is a well-known complication of hand, foot, and mouth disease (HFMD) due to Enterovirus 71 (EV71) infection. Viral RNA/antigens could be detected in the central nervous system (CNS) neurons in fatal encephalomyelitis but the mechanisms of neuronal cell death is not clearly understood. We investigated the role of absent in melanoma 2 (AIM2) inflammasome in neuronal cell death, and its relationship to viral replication. Our transcriptomic analysis, RT-qPCR, Western blot, immunofluorescence and flow cytometry studies consistently showed AIM2 gene up-regulation and protein expression in EV-A71-infected SK-N-SH cells. Downstream AIM2-induced genes, CARD16, caspase-1 and IL-1β were also up-regulated and caspase-1 was activated to form cleaved caspase-1 p20 subunits. As evidenced by 7-AAD positivity, pyroptosis was confirmed in infected cells. Overall, these findings have a strong correlation with decreases in viral titers, copy numbers and proteins, and reduced proportions of infected cells. AIM2 and viral antigens were detected by immunohistochemistry in infected neurons in inflamed areas of the CNS in EV-A71 encephalomyelitis. In infected AIM2-knockdown cells, AIM2 and related downstream gene expressions, and pyroptosis were suppressed, resulting in significantly increased virus infection. These results support the notion that AIM2 inflammasome-mediated pyroptosis is an important mechanism of neuronal cell death and it could play an important role in limiting EV-A71 replication.