Cardiac and hepatic side effects of fluoxetine in male and female adolescent rats.

BACKGROUND: Fluoxetine (FLX) is widely prescribed as an antidepressant medicine in the juvenile population. OBJECTIVES: Although some adverse effects of FLX have been reported in adults, the present study aimed to investigate the side effects of FLX treatment during adolescence on the cardiac and hepatic systems. METHODS: Male and female rats were gavaged with FLX (5 mg/kg/day) on postnatal days (PND) 21 to PND 60. Following treatment, blood samples were collected and hepatic enzymes were evaluated. The specimens of the liver and heart of animals were subjected to histopathological assessment. RESULTS: Fluoxetine significantly raised serum alanine aminotransferase (ALT) and alkaline phosphatase (ALP) in males, whereas the aspartate aminotransferase (AST) level increased in both male and female animals. In the histopathological study, hepatic plates were more seriously affected, and the sinusoids were irregular in adolescent male rats. Degenerative changes were observed especially in the first and second hepatic zones of FLX-treated male rats. Signs of inflammation and accumulation of lymphoid groups were frequently observed in the portal triad of the hepatic lobules. These alterations were more severe in male livers. Minimum or nearly normal changes were observed in female liver slides. In addition, the histological assessment indicated that treatment with FLX during adolescence also increased the heart's weight and the wall thickness of the right and left ventricles (hypertrophy) in male and especially female animals. CONCLUSION: Our findings may provide new insights into the cardiac and hepatic adverse effects of FLX.

Carvedilol attenuates inflammatory reactions of lipopolysaccharide-stimulated BV2 cells and modulates M1/M2 polarization of microglia via regulating NLRP3, Notch, and PPAR-γ signaling pathways.

Microglial cells coordinate immune responses in the central nervous system. Carvedilol (CVL) is a non-selective ß-blocker with anti-inflammatory and anti-oxidant effects. This study aims to investigate the anti-inflammatory effects and the underlying mechanisms of CVL on lipopolysaccharide (LPS)-induced inflammation in microglial BV2 cells. BV2 cells were stimulated with LPS, and the protective effects of CVL were investigated via measurement of cell viability, reactive oxygen species (ROS), and interleukin (IL)-1ß liberation. The protein levels of some inflammatory cascade, Notch, and peroxisome proliferator-activated receptor (PPAR)-γ pathways and relative markers of M1/M2 microglial phenotypes were assessed. Neuroblastoma SH-SY5Y cells were cultured with a BV2-conditioned medium (CM), and the capacity of CVL to protect cell viability was evaluated. CVL displayed a protective effect against LPS stress through reducing ROS and down-regulating of nuclear factor kappa B (NF-κB) p65, NLR family pyrin domain containing-3 (NLRP3), and IL-1ß proteins. LPS treatment significantly increased the levels of the M1 microglial marker inducible nitric oxide synthase (iNOS) and M1-associated cleaved-NOTCH1 and hairy and enhancer of split-1 (HES1) proteins. Conversely, LPS treatment reduced the levels of the M2 marker arginase-1 (Arg-1) and PPAR-γ proteins. CVL pre-treatment reduced the protein levels of iNOS, cleaved-NOTCH1, and HES1, while increased Arg-1 and PPAR-γ. CM of CVL-primed BV2 cells significantly improved SH-SY5Y cell viability as compared with the LPS-induced cells. CVL suppressed ROS production and alleviated the expression of inflammatory markers in LPS-stimulated BV2 cells. Our results demonstrated that targeting Notch and PPAR-γ pathways as well as directing BV2 cell polarization toward the M2 phenotype may provide a therapeutic strategy to suppress neuroinflammation by CVL.

Anti-angiogenic effects of aqueous extract from Agrostemma githago L. seed in human umbilical vein endothelial cells via regulating Notch/VEGF, MMP2/9, ANG2, and VEGFR2.

Abnormal angiogenesis contributes to the pathogenesis of various diseases. The medicinal usage of Agrostemma githago L. seed (A. githago herein) has been stated in traditional medicine. This study aims to investigate the anti-angiogenic potential of aqueous extract of A. githago. In order to test the effect of A. githago extract, its impact on HUVECs, T98G, and HGF2PI2 cells was assessed by looking at cellular viability, changes in the distribution of cells in different phases of the cell cycle, induction of oxidative stress, and apoptosis. In addition, the release of VEGF, ANG2, and MMP2/9 factors, along with the expressions of the critical Notch signaling pathway players and VEGF receptors (VEGFR), was measured. Furthermore, a γ-secretase inhibitor (LY411575) was applied to determine whether Notch inhibition restores A. githago effects. As a further characterization, total phenolic and flavonoid contents of A. githago were estimated, and five triterpene saponin compounds were identified using LC-ESI-MS. In response to A. githago extract, a reduction in total cell viability, along with the induction of ROS and apoptosis, was detected. Exposure to the A. githago extract could modulate the release of VEGF and ANG2 from T98G and HUVECs, respectively. In addition, A. githago reduced the release of MMP2/9. Furthermore, Notch1, DLL4, and HEY2 transcripts and protein expressions were up-regulated, while VEGFR2 was down-regulated in treated HUVEC cells. Treatment with the A. githago extract resulted in a dose-dependent inhibition of AKT phosphorylation. Inhibition of Notch signaling retrieved the viability loss, reduced intracellular ROS, and alleviated the impaired tube formation in A. githago-treated HUVECs. Overall, these data underscore the anti-angiogenic potential of A. githago via inducing apoptosis, modifying the expression levels of VEGF/VEGFR2, and impacting the release of MMP2/9 and ANG2, effects that are most probably modulated through the Notch/VEGF signaling axis.

Carvedilol attenuates brain damage in mice with hepatic encephalopathy.

Brain injury is the most common and serious consequence of hepatic encephalopathy (HE), and its pathophysiology is poorly understood. Excessive inflammatory, oxidative and apoptotic responses are the major mechanisms involved in the progression of brain injury induced by HE. Carvedilol is an adrenergic receptor antagonist with pronouncedantioxidant and anti-inflammatory activity. The present study aimed to investigatethe effects and underlying mechanisms of carvedilol on HE-induced brain damage in mice. Experimental model of HE was induced by the injection of thioacetamide (200 mg/kg) for two consecutive days and then mice were treated with carvedilol (10 or 20 mg/kg/day, orally) for 3 days in treatment groups. After the behavioral test, animals were sacrificed and the brain tissues were collected for biochemical, real time PCR and immunohistochemical analysis. The results showed that carvedilol improved locomotor impairment and reduced mortality rate in mice with HE. Carvedilol treatment decreased the brain levels of oxidative stress markers and induced Nrf2/HO-1 pathway. Carvedilol inhibited the activity of nuclear factor kappa B (NF-κB) and the expression of pro-inflammatory cytokines TNF-α, IL1ß and IL-6 in the brain tissues. Treatment of mice with carvedilol caused a significant reduction in the brain levels of iNOS/NO, myeloperoxidase (MPO), cyclooxygenase (COX)-2 and chemokine MCP-1 as proinflammatory mediators in HE. Moreover, the ratio of Bcl2/Bax was increased and apoptotic cell death was decreased in the brain of mice treated with carvedilol. In conclusion, carvedilol exerted protective effect against HE-induced brain injury through increasing antioxidant defense mechanisms and inhibitionof inflammatory and apoptotic pathways.

Methylsulfonylmethane protects against ethanol-induced brain injury in mice through the inhibition of oxidative stress, proinflammatory mediators and apoptotic cell death.

Excessive ethanol consumption causes brain injury through oxidative stress, inflammation and apoptotic cell death. Methylsulfonylmethane (MSM) is a natural compound that has therapeutic effects on oxidative and inflammatory disorders. The aim of this study was to investigate the protective effect and underlying mechanisms of MSM on ethanol-induced brain injury in an experimental model. Male C57BL/6 mice were exposed to binge ethanol (5 g/kg/day, orally) and treated with MSM (200 and 400 mg/kg/day) concomitantly for 12 days. At the end of the experiment brain tissues were removed for histological and biochemical analysis. The results showed that MSM reduced ethanol-mediated oxidative stress by decreasing the levels of malondialdehyde (MDA) and carbonyl protein. The Nrf2/HO-1 pathway and the levels of cytoprotective antioxidants superoxide dismutase (SOD), catalase and glutathione (GSH) were increased by MSM in the brain tissue. MSM treatment reduced the ethanol-induced inflammatory factors including myeloperoxidase (MPO), iNOS/NO, cyclooxygenase (COX)-2, nuclear factor kappa B (NF-κB), NLRP3 inflammasome and proinflammatory cytokines including TNF-α, IL-1ß, IL-6 and MCP-1. MSM also decreased the levels of pro-apoptotic caspase-3 and TUNEL positive cells while increased the level of anti-apoptotic Bcl-2 in the brain tissue. Our findings demonstrated that MSM protects against ethanol-induced brain injury by improving anti-oxidant defense mechanism and reducing ethanol-mediated inflammation and apoptosis. Therefore, MSM may be a potential protective approach for brain damage caused by high levels of alcohol.

Acquisition of paclitaxel resistance modulates the biological traits of gastric cancer AGS cells and facilitates epithelial to mesenchymal transition and angiogenesis.

PURPOSE: This study aims to develop a paclitaxel (PTX)-resistant gastric cancer AGS cells (AGS-R) and evaluate the mechanisms of drug resistance. METHODS: AGS cells were successively treated with increasing PTX concentrations. Cross-resistance of established AGS-R, the molecular patterns of cell survival, evasion of apoptosis, epithelial-mesenchymal transition (EMT), and the angiogenic potential were evaluated. RESULTS: AGS-R was induced within six months of PTX exposure. Extension of the treatment resulted in PTX-resistance beyond clinical levels. The established AGS-R showed resistance to vincristine and doxorubicin but not cisplatin. Upon induction of resistance, the expressions of MDR-1 (P < 0.001) and MRP-1 (P < 0.01) genes and proteins significantly increased. AGS-R cells had elevated levels of BCL-2, pro-CASP3, cleaved-NOTCH1, HES1, HEY1, NF-κB, PI3K, p-AKT, HIF-1α, Cyclin A, and B1 as compared with parental cells (at least P < 0.01). The protein levels of BAX, CASP3, P53, and P21 (at least P < 0.01) as well as intracellular ROS (P < 0.001) were reduced in AGS-R. A relative arrest at the G2/M phase (15.8 ± 0.75 vs. 26.7 ± 1.67) of the cell cycle and enrichment of AGS-R cells for CD44 marker (9 ± 0.6 vs. 1 ± 0.8) (P < 0.001) were detected by flow cytometry. While the E-cadherin expression was reduced (P < 0.001), the protein levels of Vimentin, N-cadherin, SLUG, and SNAIL were increased (at least P < 0.05). The angiogenic activity and release of VEGF and MMP2/9 were increased in AGS-R cells relative to the AGS line (P < 0.001). CONCLUSION: AGS-R cells could bypass chemotherapy stress by expressing the genes coding for efflux pumps and altering some key signaling in favor of survival, EMT, and angiogenesis.

Postnatal depletion of serotonin affects the morphology of neurons and the function of the hippocampus in male rats.

Serotonin (5-HT) is an essential neurotransmitter for the refined organization of the cerebral cortex. Studies have suggested that altered serotonin signaling contributes to cognitive impairment and psychiatric disorders. However, the exact role of this neurotransmitter on the development of hippocampal neurons is not recognized. Here we aimed to examine the effects of the para-chlorophenylalanine (PCPA; 100 mg/kg/daily, s.c. during the postnatal days 10-20), a reversible inhibitor of 5-HT synthesis, on the serotonin level of the hippocampal and prefrontal cortex. We also focused on the morphology of the neurons in the hippocampus and spatial learning and memory. Our results indicated that the administration of PCPA led to a decrease in serotonin levels in the hippocampus and prefrontal cortex. Postnatal serotonin depletion also induced subtle alterations in the neuronal populations of the hippocampus and impaired spatial memory in the adulthood period of life. We found that critical developmental periods of serotonin depletion caused degeneration and swelling of neurons as well as significant neuronal loss in the hippocampal CA1, CA3, and dentate gyrus (DG) areas. Thus, serotonin, a strikingly important neurotransmitter, can affect neuronal morphology, development, and hippocampal-dependent memory.

Antibacterial effect of TiO2 modified with poly-amidoamine dendrimer - G3 on S. aureus and E. coli in aqueous solutions.

This study investigated bacterial removal using TiO2 nanoparticles (NPs) modified with poly-amidoamine dendrimer macromolecule (PAMAM, G3). The PAMAM G3/TiO2 (nanohybrid) was used to specify antibacterial properties via broth microdilution (MBC-Minimum Bactericidal Concentration and MIC-Minimum Inhibitory Concentration-determination), paper disc diffusion, and surface plate count methods. The nanohybrid was characterized via the different techniques. The effects of different factors including initial bacteria count, run time, solution pH, and the nanohybrid concentration were studied. The nanohybrid cytotoxicity was studied on AGS and MKN45 cells line by MTT assay. It was revealed that the nanohybrid was effective in intercepting both bacterial strains growth. The MIC value for S. aureus and E. coli were determined to be 4 and 2 µg/mL, respectively. The MBC value for both strains were calculated to be 32 µg/mL. The results showed removal efficiency of 100% for S. aureus and E. coli bacteria in optimum situation. The decrease in cell viability in the dosage of 32 µg/mL after 72 h treatment for AGS and MKN45 cells line were shown to be 6.2 and 4.6%, respectively. The nanohybrid was able to decrease the S. aureus and E. coli count in solution, which meets the drinking water criterions aligned with WHO guidelines.

Transdifferentiation of Human Umbilical Cord-Derived Mesenchymal Stem Cells in Dopaminergic Neurons in a Three-Dimensional Culture.

Introduction: The induction of human umbilical cord-derived mesenchymal stem cells (HUC-MSCs) toward dopaminergic neurons is a major challenge in tissue engineering and experimental and clinical treatments of various neurodegenerative diseases, including Parkinson disease. This study aims to differentiate HUC-MSCs into dopaminergic neuron-like cells. Methods: Following the isolation and characterization of HUC-MSCs, they were transferred to Matrigel-coated plates and incubated with a cocktail of dopaminergic neuronal differentiation factors. The capacity of differentiation into dopaminergic neuron-like cells in 2-dimensional culture and on Matrigel was assessed by real-time polymerase chain reaction, immunocytochemistry, and high-performance liquid chromatography. Results: Our results showed that dopaminergic neuronal markers' transcript and protein levels were significantly increased on the Matrigel differentiated cells compared to 2D culture plates. Conclusion: Overall, the results of this study suggest that HUC-MSCs can successfully differentiate toward dopaminergic neuron-like cells on Matrigel, having great potential for the treatment of dopaminergic neuron-related diseases.

Differentiation of human dental pulp stem cells into functional motor neuron: In vitro and ex vivo study.

There are several therapeutic options for spinal cord injury (SCI), among these strategies stem cell therapy is a potential treatment. The stem cells based therapies have been investigating in acute phase of clinical trials for promoting spinal repair in humans through replacement of functional neuronal and glial cells. The aim of this study was to evaluate the differentiation of Human Dental Pulp Stem Cells (hDPSCs) into functional motor neuron like cells (MNLCs) and promote neuroregeneration by stimulating local neurogenesis in the adult spinal cord slice culture. The immunocytochemistry analysis demonstrated that hDPSCs were positive for mesenchymal stem cell markers (CD73, CD90 and CD105) and negative for the hematopoietic markers (CD34 and CD45). hDPSCs were induced to neurospheres (via implementing B27, EGF, and bFGF) and then neural stem cells (NSC). The NSC differentiated into MNLCs in two steps: first by Shh and RA and ; then with GDNF and BDNF administration. The NS and the NSC were assessed for Oct4, nestin, Nanog, Sox2 expression while the MNLCs were evaluated by ISLET1, Olig2, and HB9 genes. Our results showed that hDPSC can be differentiated into motor neuron phenotype with expression of the motor neuron genes. The functionality of MNLCs was demonstrated by FM1-43, intracellular calcium ion shift and co- culture with C2C12. We co-cultivated hDPSCs with adult rat spinal slices in vitro. Immunostaining and hoechst assay showed that hDPSCs were able to migrate, proliferate and integrate in both the anterolateral zone and the edges of the spinal slices.

Promoting motor functions in a spinal cord injury model of rats using transplantation of differentiated human olfactory stem cells: A step towards future therapy.

Human olfactory ecto-mesenchymal stem cells (hOE-MSCs) derived from the human olfactory mucosa (OM) can be easily isolated and expanded in cultures while their immense plasticity is maintained. To mitigate ethical concerns, the hOE-MSCs can be also transplanted across allogeneic barriers, making them desirable cells for clinical applications. The main purpose of this study was to evaluate the effects of administering the hOE-MSCs on a spinal cord injury (SCI) model of rats. These cells were accordingly isolated and cultured, and then treated in the neurobasal medium containing serum-free Dulbecco's Modified Essential Medium (DMEM) and Ham's F-12 Medium (DMEM/F12) with 2% B27 for two days. Afterwards, the pre-induced cells were incubated in N2B27 with basic fibroblast growth factor (bFGF), fibroblast growth factor 8b (FGF8b), sonic hedgehog (SHH), and ascorbic acid (vitamin C) for six days. The efficacy of the induced cells was additionally evaluated using immunocytochemistry (ICC) and real-time polymerase chain reaction (RT-PCR). The differentiated cells were similarly transplanted into the SC contusions. Functional recovery was further conducted on a weekly basis for eight consecutive weeks. Moreover, cell integration was assessed via conventional histology and ICC, whose results revealed the expression of choline acetyltransferase (ChAT) marker at the induction stage. According to the RT-PCR findings, the highest expression level of insulin gene-enhancer protein (islet-1), oligodendrocyte transcription factor (Olig2), and homeobox protein HB9 was observed at the induction stage. The number of engraftment cells also rose (approximately by 2.5 % ± 0.1) in the motor neuron-like cells derived from the hOE-MSCs-grafted group compared with the OE-MSCs-grafted one. The functional analysis correspondingly revealed that locomotor and sensory scores considerably improved in the rats in the treatment group. These findings suggested that motor neuron-like cells derived from the hOE-MSCs could be utilized as an alternative cell-based therapeutic strategy for SCI.

The Neuroprotective Effect of Mesna on Cisplatin-Induced Neurotoxicity: Behavioral, Electrophysiological, and Molecular Studies.

Peripheral neuropathy and cognitive impairments following cisplatin administration may interfere with the clinical usage of the drug. Mesna is a chemoprotective agent with anti-inflammatory and anti-oxidant effects. Our study aimed to investigate the protective effects of mesna against cisplatin-induced neurotoxicity. Neurotoxicity was induced by the administration of 2.5 mg/kg cisplatin twice a week for four consecutive weeks in male Wistar rats. The neuroprotective effect of mesna (150 mg/kg/day) was evaluated through behavioral, electrophysiological, and molecular studies. Cisplatin treatment caused passive avoidance memory impairment, increased anxiety-like behaviors, altered thermal sensitivity, and decreased muscle strength in a grip strength test. Our electrophysiological studies indicated that administration of cisplatin induced peripheral sensory neuropathy and decreased the amplitudes of the compound action potential of sensory nerves. Cisplatin administration increased MDA and 4-HNE levels and decreased anti-oxidant (SOD and GPx) enzymes. Proinflammatory cytokines (IL-1ß and TNF-α) and metalloproteinase-2 and 9 (MMP-2/9) were increased by cisplatin treatment. Morphological alterations were observed in the dorsal root ganglion (DRG) of cisplatin-treated rats. Cognitive impairments, anxiety, muscle strength, and thermal sensitivity changes induced by cisplatin were improved with mesna treatment. The reduced conduction velocity in sensory nerves was recovered in the cisplatin + mesna group. Mesna partially alleviated redox imbalance, reduced the proinflammatory cytokines, and MMP-2/9 levels. Mesna administration also relieved the morphological changes in DRG of cisplatin-treated rats. In conclusion, our results revealed that mesna can alleviate cisplatin-induced central and peripheral nervous system toxicity. These results support the concept that chemotherapy-induced neuropathy can be partially inhibited via mesna.

Role of cerebrospinal fluid in differentiation of human dental pulp stem cells into neuron-like cells.

Human dental pulp stem cells (hDPSCs) could be differentiated into neuron like-cells under particular microenvironments. It has been reported that a wide range of factors, presented in cerebrospinal fluid (CSF), playing part in neuronal differentiation during embryonic stages, we herein introduce a novel culture media complex to differentiate hDPSCs into neuron-like cells. The hDPSCs were initially isolated and characterized. The CSF was prepared from the Cisterna magna of 19-day-old Wistar rat embryos, embryonic cerebrospinal fluid (E-CSF). The hDPSCs were treated by 5% E-CSF for 2 days, then neurospheres were cultured in DMEM/F12 supplemented with 10-6 µm retinoic acid (RA), glial-derived neurotrophic factor and brain-derived neurotrophic factor for 6 days. The cells which were cultured in basic culture medium were considered as control group. Morphology of differentiated cells as well as process elongation were examined by an inverted microscope. In addition, the neural differentiation markers (Nestin and MAP2) were studied employing immunocytochemistry. Neuronal-like processes appeared 8 days after treatment. Neural progenitor marker (Nestin) and a mature neural marker (MAP2) were expressed in treated group. Moreover Nissl bodies were found in the cytoplasm of treated group. Taking these together, we have designed a simple protocol for generating neuron-like cells using CSF from the hDPSCs, applicable for cell therapy in several neurodegenerative disorders including Alzheimer's disease.

2,4-D causes oxidative stress induction and apoptosis in human dental pulp stem cells (hDPSCs).

2,4-Dicholorophenoxy acetic acid (2,4-D) is a worldwide used hormone herbicide. Human dental pulp stem cells (hDPSCs) as a potential source of mesenchymal stem cells provide a confident model system for the assessments of chemicals in vitro. The main objective of this study was to examine the biological effects and damages attributed to 2,4-D on hDPSCs. hDPSCs were isolated from third molar pulp tissues and their mesenchymal identity were evaluated. Then, hDPSCs were treated with increasing concentrations of 2,4-D (0.1 µM-10 mM). Cell viability assay and cumulative cell counting were carried out to address 2,4-D effects on biological parameters of hDPSCs. Cell cycle distribution, ROS level and ALP activity were measured before and after treatment. AO/EB staining and caspase 3/7 activity were investigated to detect the possible mechanisms of cell death. Flow-cytometric immunophenotyping and differentiation data confirmed the mesenchymal identity of cultivated hDPSCs. 2,4-D treatment caused a hormetic response in the viability and growth rate of hDPSCs. G0/G1 cell cycle arrest, enhanced ROS level, and reduced ALP activity were detected in hDPSCs treated with EC50 dose of 2,4-D. AO/EB staining showed a higher percentage of alive cells in lower concentrations of the herbicide. The increment in 2,4-D dose and the number of early and late apoptotic cells were increased. DAPI staining and caspase 3/7 assay validated the induction of apoptosis. 2,4-D concentrations up to 100 µM did not affect hDPSCs viability and proliferation. The intense cellular oxidative stress and apoptosis were observed at higher concentration.

Pharmacological Notch pathway inhibition leads to cell cycle arrest and stimulates ascl1 and neurogenin2 genes expression in dental pulp stem cells-derived neurospheres.

OBJECTIVE: Human dental pulp-derived stem cells (hDPSCs) are becoming an attractive source for cell-based neurorestorative therapies. As such, it is important to understand the molecular mechanisms that regulate the differentiation of hDPSCs toward the neuronal fate. Notch signaling plays key roles in neural stem/progenitor cells (NS/PCs) maintenance and prevention of their differentiation. The aim of this study was to address the effects of Notch signaling inhibition on neurosphere formation of hDPSCs and neuronal differentiation of hDPSCs-neurospheres. RESULTS: hDPSCs were isolated from third molar teeth. The cultivated hDPSCs highly expressed CD90 and CD44 and minimally presented CD34 and CD45 surface markers. The osteo/adipogenic differentiation of hDPSCs was documented. hDPSCs were cultured in neural induction medium and N-[N-(3,5-difluorophenacetyl-L-alanyl)]-Sphenylglycine t-butyl ester (DAPT) was applied to impede Notch signaling during transformation into spheres or on the formed neurospheres. Our results showed that the size and number of neurospheres decreased and the expression profile of nestin, sox1 and pax6 genes reduced provided DAPT. Treatment of the formed neurospheres with DAPT resulted in the cleaved Notch1 reduction, G0/G1 arrest and a decline in L-lactate production. DAPT significantly reduced hes1 and hey1 genes, while ascl1 and neurogenin2 expressions augmented. The number of MAP2 positive cells improved in the DAPT-treated group. CONCLUSIONS: Our findings demonstrated the Notch activity in hDPSCs-neurospheres. DAPT treatment positively regulated proneural genes expression and increased neuronal-like differentiation.

Inhibitory Effect of Gamma-Secretase on the Survival Rate of Dental Pulp Stem Cells: An in Vitro Study

Objective: To evaluate in vitro the effect of gamma-secretase inhibition on the survival of dental pulp stem cells. Material and Methods: Sound teeth have been used. Dental pulp stem cells were isolated by enzymatic digestion. An appropriate number of cells were treated with different concentrations of gamma secretase enzyme (DAPT) (1, 3, 6.25, 12.5, 25.5, 37.5, 50 and 100 µM). The metabolic activity of cells and the distribution of cells in different phages of cell cycle was evaluated by MTT assay and flow cytometry, respectively. Statistical analysis was made one-way ANOVA. Comparison was made between the groups on the level of p<0.05. Results: In low concentration of DAPT (1, 3, 6.25, 12.5) the growth rate of the cells increases, whereas in high concentration of DAPT (25.5, 37.5, 50, 100) can significantly reduce the viability of the treated cells. The results also indicate that DAPT can interrupt the cell cycle in G1 phase. Conclusion: The DAPT for dose-dependent survival rate of dental pulp stem cells and affect cell population increase in the G1 phase of the cell cycle.

Combination treatment of all-trans retinoic acid (ATRA) and γ-secretase inhibitor (DAPT) cause growth inhibition and apoptosis induction in the human gastric cancer cell line.

Current medication for gastric cancer patients has a low success rate with resistance and side effects. According to recent studies, γ-secretase inhibitors is used as therapeutic drugs in cancer. Moreover, all-trans retinoic acid (ATRA) is a natural compound proposed for the treatment/chemo-prevention of cancers. The aim of this study was to explore the effects of ATRA in combination with N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT) as γ-secretase inhibitor on viability and apoptosis of the AGS and MKN-45 derived from human gastric cancer. AGS and MKN-45 gastric cancer cell lines were treated with different concentrations of ATRA or DAPT alone or ATRA plus DAPT. The viability, death detection and apoptosis of cells was examined by MTT assay and Ethidium bromide/acridine orange staining. The distribution of cells in different phases of cell cycle was also evaluated through flow cytometry analyses. In addition, caspase 3/7 activity and the expression of caspase-3 and bcl-2 were examined. DAPT and ATRA alone decreased gastric cancer cells viability in a concentration dependent manner. The combination of DAPT and ATRA exhibited significant synergistic inhibitory effects. The greater percentage of cells were accumulated in G0/G1 phase of cell cycle in combination treatment. The combination of DAPT and ATRA effectively increased the proportion of apoptotic cells and the level of caspase 3/7 activities compared to single treatment. Moreover, augmented caspase-3 up-regulation and bcl-2 down-regulation were found following combined application of DAPT and ATRA. The combination of DAPT and ATRA led to more reduction in viability and apoptosis in respect to DAPT or ATRA alone in the investigated cell lines.

Epi/perineural and Schwann Cells as Well as Perineural Sheath Integrity are Affected Following 2,4-D Exposure.

2,4-dicholorophenoxy acetic acid (2,4-D) is a worldwide-known hormone herbicide. However, there are increasing concerns about its exposure and risks of developing pathological conditions for the peripheral nervous system. The aim of this study was to investigate the mechanism(s) involved in the toxicity of 2,4-D on peripheral nerve's cellular components. The epi/perineural and Schwann cells and a total of three cell lines were treated with 2,4-D. The viability of cells at different doses of 2,4-D was measured by MTT assay. The cell cycle analyses, cumulative cell counting, fluorescent staining, antioxidant and caspase enzymes activity were examined on epi/perineural and Schwann cells. The epi/perineural cells were assessed as having biological macromolecular changes. Some tight junction-related genes and proteins were also tested on explants of 2,4-D treated epi/perineural tissue. The viability of 2,4-D treated cells was reduced in a dose-dependent manner. Reduced growth rate and G1 cell cycle arrest were verified in 2,4-D treated epi/perineural and Schwann cells. The use of staining methods (acridine orange/ethidium bromide and DAPI) and caspase 3/7 activity assay along with malondialdehyde, glutathione peroxidase, and superoxide dismutase activity assays indicated the apoptotic and oxidant effects of 2,4-D on epi/perineural and Schwann cells. Data obtained from FTIR revealed changes in epi/perineural proteins and cell membrane lipids. Additionally, claudin-1, occludin, and ZO-1 gene/protein expression profiles were significantly reduced in 2,4-D-treated epi/perineural pieces. Our data indicated that oxidative stress, apoptosis of epi/perineural and Schwann cell and impaired blood-nerve barrier may have contributed to nerve damage following 2,4-D exposure.

Coenzyme Q10 protects skeletal muscle from ischemia-reperfusion through the NF-kappa B pathway.

OBJECTIVE: Coenzyme Q10 (CoQ10) has antioxidant and anti-inflammatory activity. The aim of this study was to investigate the effects of CoQ10 on the inhibition of nuclear factor-kappa B (NF-κB) activation during ischemia-reperfusion (I/R) of skeletal muscle. METHODS: For ischemia induction, the animals were anesthetized and the external iliac vessels blocked for three hours. CoQ10 or vehicle was given intraperitoneally during ischemia, just before reperfusion. Four groups received 3,7,14 and 28 days' reperfusion, respectively, after the intraperitoneal injection of CoQ10 and four corresponding groups received vehicle only. After reperfusion, the gastrocnemius muscles were removed, fixed and stained for the analysis of edema and mast cell infiltration. RESULTS: Immuno-histochemistry staining was performed for the detection of tumor necrosis factor alpha (TNF-α) and NF-κB. CoQ10-treated groups showed a significant decrease of mast cell infiltration in the gastrocnemius muscle and edema as compared with the corresponding non-treated groups. Also, CoQ10-treated groups showed a significant TNF-α and NF-κB expression decrease when compared to the corresponding non-treated controls. The results of this study showed CoQ10 administration with ischemia decreased interstitial edema, degeneration of muscle fibers and infiltration of mast cells. CONCLUSIONS: It seems that CoQ10 has inhibitory effects on NF-κB and TNF-α activation.

Human platelet lysate versus minoxidil stimulates hair growth by activating anagen promoting signaling pathways.

Minoxidil and human platelet lysate (HPL) are commonly used to treat patients with hair loss. However, the roles of HPL versus minoxidil in hair follicle biology largely remain unknown. Here, we hypothesized that bulge and dermal papilla (DP) cells may express specific genes, including Kras, Erk, Akt, Shh and ß-catenin after exposure to minoxidil or HPL. The mouse hair follicles were isolated on day 10 after depilation and bulge or DP regions were dissected. The bulge and DP cells were cultured for 14days in DMEM/F12 medium. Then, the cells were treated with 100µM minoxidil and 10% HPL for 10 days. Nuclear morphology was identified using DAPi staining. Reverse transcriptase and real-time polymerase chain reaction (PCR) analysis were also performed to examine the expression of Kras, Erk, Akt, Shh and ß-catenin mRNA levels in the treated bulge and DP regions after organ culture. Here, we found that minoxidil influences bulge and DP cell survival (P<0.05). Apoptosis in DP cells was also meaningfully decreased by HPL treatment (P=0.014). In addition, Kras, Akt, Erk, Shh and ß-catenin mRNA levels were changed in response to minoxidil treatment in both bulge and DP cells. HPL mediated Erk upregulation in both bulge and DP cells (P<0.05), but Kras and Akt mRNA levels were not considerably different in the HPL-treated cells. ß-catenin mRNA level was also significantly increased in the bulge region by HPL. We also found that Shh mRNA level was considerably higher in HPL-treated bulge cells than in minoxidil-treated bulge cells. In contrast, the expression of ß-cateinin and Shh in the DP cells was not meaningfully increased after treatment with HPL. Our results suggest that minoxidil and HPL can promote hair growth by activating the main anagen inducing signaling pathways.