OBJECTIVETo investigate the effect of dimethylsulfoxide on the expression of thymic stromal lymphopoietin (TSLP) in human bronchial epithelial cell (HBE).

METHODS16HBE cells were incubated in the presence of dimethylsulfoxide at different concentrations, and the cell proliferation changes were observed. The expressions of TSLP mRNA and protein in the cells were detected by real-time quantitative PCR and ELISA, respectively.

RESULTSDimethylsulfoxide induced significantly increased TSLP mRNA expression in HBE cells (P<0.01) in a concentration-dependent manner. The level of TSLP protein in the supernatant was also increased after dimethylsulfoxide treatment, but high concentration of dimethylsulfoxide resulted in e inhibited cell proliferation.

CONCLUSIONDimethylsulfoxide may affect the immunomodulatory function of HBE cells.

Bronchi ; cytology ; metabolism ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cytokines ; genetics ; metabolism ; Dimethyl Sulfoxide ; pharmacology ; Epithelial Cells ; metabolism ; Humans ; RNA, Messenger ; genetics ; metabolism

Transition metal ions including Se2+, Cd2+, Hg2+ or Mn2+ have been thought to disturb the bone metabolism directly. However, the mechanism for the bone lesion is unknown. In this study, we demonstrated that MC3T3E1 osteoblasts, exposed to various transition metal ions; selenium, cadmium, mercury or manganese, generated massive amounts of reactive oxygen species (ROS). The released ROS were completely quenched by free radical scavengers-N-acetyl cysteine (NAC), reduced glutathione (GSH), or superoxide dismutase (SOD). First, we have observed that selenium (10 micrometer), cadmium (100 micrometer), mercury (100 micrometer) or manganese (1 mM) treatment induced apoptotic phenomena like DNA fragmentation, chromatin condensation and caspase-3-like cysteine protease activation in MC3T3E1 osteoblasts. Concomitant treatment of antioxidant; N-acetyl-L-cysteine (NAC), reduced-form glutathione (GSH), or superoxide dismutase (SOD), prevented apoptosis induced by each of the transition metal ions. Catalase or dimethylsulfoxide (DMSO) has less potent inhibitory effect on the apoptosis, compared with NAC, GSH or SOD. In line with the results, nitroblue tetrazolium (NBT) stain shows that each of the transition metals is a potent source of free radicals in MC3T3E1 osteoblast. Our data show that oxidative damage is associated with the induction of apoptosis in MC3T3E1 osteoblasts following Se2+, Cd2+, Hg2+ or Mn2+ treatment.
Acetylcysteine ; Apoptosis* ; Cadmium ; Catalase ; Chromatin ; Cysteine ; Cysteine Proteases ; Dimethyl Sulfoxide ; DNA Fragmentation ; Free Radicals ; Glutathione ; Ions ; Manganese ; Metabolism ; Metals ; Nitroblue Tetrazolium ; Osteoblasts* ; Reactive Oxygen Species ; Selenium ; Superoxide Dismutase

OBJECTIVE: To explore the effect of shikonin on the recovery of nerve function after acute spinal cord injury(SCI) in rats. METHODS: 96 male Sprague-Dawley(SD)rats were divided into 4 groups randomly:sham operation group (Group A), sham operation+shikonin group (Group B), SCI+ DMSO(Group C), SCI+shikonin group (Group D).The acute SCI model of rats was made by clamp method in groups C and D . After subdural catheterization, no drug was given in group A. rats in groups B and D were injected with 100 mg·kg^-1 of shikonin through catheter 30 min after modeling, and rats in group C were given with the same amount of DMSO, once a day until the time point of collection tissue. Basso-Beattie-Bresnahan(BBB) scores were performed on 8 rats in each group at 6, 12, and 3 d after moneling, and oblique plate tests were performed on 1, 3, 7 and 14 d after modeling, and then spinal cord tissues were collected. Eight rats were intraperitoneally injected with propidine iodide(PI) 1 h before sacrificed to detection PI positive cells at 24 h in each group. Eight rats were sacrificed in each group at 24 h after modeling, the spinal cord injury was observed by HE staining.The Nissl staining was used to observe survivor number of nerve cells. Western-blot technique was used to detect the expression levels of Bcl-2 protein and apoptosis related protein RIPK1. RESULTS: After modeling, BBB scores were normal in group A and B, but in group C and D were significantly higher than those in group A and B. And the scores in group D were higher than those in group C in each time point (P<0.05). At 12 h after modeling, the PI red stained cells in group D were significantly reduced compared with that in group C, and the disintegration of neurons was alleviated(P<0.05). HE and Nissl staining showed nerve cells with normal morphology in group A and B at 24h after operation. The degree of SCI and the number of neuronal survival in group D were better than those in group C, the difference was statistically significant at 24h (P<0.05). The expression of Bcl-2 and RIPK1 proteins was very low in group A and B;The expression of RIPK1 was significantly increased in Group C and decreased in Group D, with a statistically significant difference (P<0.05);The expression of Bcl-2 protein in group D was significantly higher than that in group C (P<0.05). CONCLUSION Shikonin can alleviate the pathological changes after acute SCI in rats, improve the behavioral score, and promote the recovery of spinal nerve function. The specific mechanism may be related to the inhibition of TNFR/RIPK1 signaling pathway mediated necrotic apoptosis.
Animals ; Male ; Rats ; Dimethyl Sulfoxide/metabolism* ; Naphthoquinones ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Rats, Sprague-Dawley ; Spinal Cord/metabolism* ; Spinal Cord Injuries/metabolism* ; Receptors, Tumor Necrosis Factor/metabolism* ; Receptor-Interacting Protein Serine-Threonine Kinases/metabolism*

OBJECTIVE: To observe the effect of electroacupuncture (EA) at "Huantiao" (GB 30) and "Weizhong" (BL 40) on the activation of glial cells, the expression of brain-derived neurotrophic factor (BDNF), excitability and the number of dendritic spines of neurons in the spinal dorsal horn in rats with spared nerve injury (SNI) of sciatic nerve, and to explore the analgesic mechanism of EA on SNI. METHODS: PartⅠ: Sixty SD rats were randomly divided into a sham operation group, a model group, an EA group and a sham EA group, 15 rats in each group. Except the sham operation group, the SNI rat model was established in the remaining groups. The rats in the sham operation group were only treated with incision without damaging the nerve. The rats in the EA group were treated with EA at "Huantiao" (GB 30) and "Weizhong" (BL 40) on the affected side, continuous wave, frequency of 2 Hz, current intensity of 1 mA, 30 minutes each time, once a day, for 14 days. The rats in the sham EA group were treated with EA at points 0.5 cm next to "Huantiao" (GB 30) and "Weizhong" (BL 40) on the affected side; the manipulation, EA parameters and treatment course were the same as the EA group. The latency of thermal foot contraction reflex and the threshold of mechanical foot contraction reflex were detected 1 day before modeling and 3, 7 and 14 days after modeling. Fourteen days after modeling, Western blot was used to detect the protein expressions of ionized binding adapter junction protein 1 (Iba-1), glial fibrillary acidic protein (GFAP), BDNF and c-Fos in the spinal dorsal horn; the expressions of Iba-1 and c-Fos proteins in the spinal dorsal horn were detected by immunofluorescence staining; immunohistochemical method was used to detect the expression of GFAP protein in the spinal dorsal horn; Golgi staining was used to detect the number of dendritic spines in spinal dorsal horn neurons. PartⅡ: Thirty SD rats were randomly divided into a control group, a BDNF group and a BDNF+anti-TrkB group, 10 rats in each group. The control group was treated with intrathecal injection of 10 μL mixture with 1︰1 of 0.9% sodium chloride solution and dimethyl sulfoxide (DMSO); the BDNF group was treated with intrathecal injection of 10 μg rat recombinant BDNF dissolved in 10 μL mixture with 1︰1 of 0.9% sodium chloride solution and DMSO; the BDNF+anti-TrkB group was treated with intrathecal injection of 10 μg rat recombinant BDNF and 30 μg tyrosine kinase receptor B (TrkB) antibody dissolved in 10 μL mixture with 1︰1 of 0.9% sodium chloride solution and DMSO. The threshold of mechanical foot retraction reflex was detected 1 day before intrathecal injection and 1, 3 and 7 days after injection. Seven days after injection, the expression of c-Fos protein in the spinal dorsal horn was detected by Western blot and immunofluorescence staining. RESULTS: PartⅠ: Compared with the sham operation group, 3, 7 and 14 days after modeling, the latency of thermal foot contraction reflex and the threshold of mechanical foot contraction reflex in the model group were decreased (P<0.05); 7 and 14 days after modeling, compared with the model group, the latency of thermal foot contraction reflex and the threshold of mechanical foot contraction reflex in the EA group were increased (P<0.05). The expressions of Iba-1, GFAP, BDNF, c-Fos proteins and the number of neuronal dendritic spines in the spinal dorsal horn in the model group were higher than those in the sham operation group (P<0.05); the expressions of Iba-1, BDNF, c-Fos proteins and the number of neuronal dendritic spines in the EA group were lower than those in the model group (P<0.05). PartⅡ: 3 and 7 days after intrathecal injection, the threshold of mechanical foot retraction reflex in the BDNF group was lower than that in the control group (P<0.05); the threshold of mechanical foot retraction reflex in the BDNF+anti-TrkB group was higher than that in the BDNF group (P<0.05). The expression of c-Fos protein in spinal dorsal horn in the BDNF group was higher than that in the control group (P<0.05); the expression of c-Fos protein in spinal dorsal horn in the BDNF+anti-TrkB group was lower than that in the BDNF group (P<0.05). CONCLUSION The analgesic effect of EA at "Huantiao" (GB 30) and "Weizhong" (BL 40) on SNI rats may be related to inhibiting the activation of microglia in the dorsal horn of the spinal cord, thereby blocking the signal of microglia-BDNF-neuron, and finally reducing the excitability of neurons.
Analgesics ; Animals ; Brain-Derived Neurotrophic Factor/metabolism* ; Dimethyl Sulfoxide/metabolism* ; Electroacupuncture ; Microglia ; Neuralgia/therapy* ; Neurons ; Proto-Oncogene Proteins c-fos/metabolism* ; Rats ; Rats, Sprague-Dawley ; Sodium Chloride/metabolism* ; Spinal Cord/metabolism*

OBJECTIVES: Long-term treatment of olanzapine, the most widely-prescribed second-generation antipsychotic, remarkably increases the risk of non-alcoholic fatty liver disease (NAFLD), whereas the mechanism for olanzapine-induced NAFLD remains unknown. Excessive hepatic fat accumulation is the basis for the pathogenesis of NAFLD, which results from the disturbance of TG metabolism in the liver. Apolipoprotein A5 (ApoA5) is a key regulator for TG metabolism in vivo that promotes TG accumulation in hepatocytes, thereby resulting in the development of NAFLD. However, there are no data indicating the role of apoA5 in olanzapine-induced NAFLD. Therefore, this study aims to investigate the role of apoA5 in olanzapine-induced NAFLD. METHODS: This study was carried out via animal studies, cell experiment, and ApoA5 gene knockdown experiment. Six-week-old male C57BL/6J mice were randomized into a control group, a low-dose group, and a high-dose group, which were treated by 10% DMSO, 3 mg/(kg·d) olanzapine, and 6 mg/(kg·d) olanzapine, respectively for 8 weeks. The lipid levels in plasma, liver function indexes, and expression levels of ApoA5 were detected. HepG2 cells were treated with 0.1% DMSO (control group), 25 μmol/L olanzapine (low-dose group), 50 μmol/L olanzapine (medium-dose group), and 100 μmol/L olanzapine (high-dose group) for 24 h. HepG2 cells pretreated with 100 μmol/L olanzapine were transfected with siRNA and scrambled siRNA (negative control), respectively. We observed the changes in lipid droplets within liver tissues and cells using oil red O staining and fat deposition in liver tissues using HE staining. The mRNA and protein levels of ApoA5 were determined by real-time PCR and Western blotting, respectively. RESULTS: After intervention with 3 and 6 mg/(kg·d) olanzapine for 8 weeks, there was no significant difference in body weight among the 3 groups (P>0.05). Olanzapine dose-dependently increased the plasma TG, ALT and AST levels, and reduced plasma ApoA5 levels (all P<0.05), whereas there was no significant difference in plasma cholesterol (HDL-C, LDL-C, and TC) levels among the 3 groups (all P>0.05). Olanzapine dose-dependently up-regulated ApoA5 protein levels in liver tissues (all P<0.05), but there was no significant change in ApoA5 mRNA expression among groups (P>0.05). In the control group, the structure of liver tissues was intact, the morphology of liver cells was regular, and only a few scattered lipid droplets were found in the cells. In the olanzapine-treated group, there was a large amount of lipid deposition in hepatocytes, and cells were balloon-like and filled with lipid droplet vacuoles. The nucleus located at the edge of cell, and the number of lipid droplets was increased significantly, especially in the high-dose group. Likewise, when HepG2 cells were treated with olanzapine for 24 h, the number and size of lipid droplets were significantly elevated in a dose-dependent manner. Moreover, olanzapine dose-dependently up-regulated ApoA5 protein levels in HepG2 cells (all P<0.05), but there was no significant difference in ApoA5 mRNA expression among groups (P>0.05). Compared with the HepG2 cells transfected with scrambled siRNA, the number and size of lipid droplets in HepG2 cells transfected with ApoA5 siRNA were significantly reduced. CONCLUSIONS The short-term intervention of olanzapine does not significantly increase body weight of mice, but it can directly induce hypertriglyceridemia and NAFLD in mice. Olanzapine inhibits hepatic apoA5 secretion but does not affect hepatic apoA5 synthesis, resulting in the pathogenesis of NAFLD. Inhibition of apoA5 secretion plays a key role in the development of olanzapine-related NAFLD, which may serve as an intervention target for this disease.
Animals ; Apolipoprotein A-V/genetics* ; Body Weight ; Dimethyl Sulfoxide/metabolism* ; Liver/metabolism* ; Male ; Mice ; Mice, Inbred C57BL ; Non-alcoholic Fatty Liver Disease/chemically induced* ; Olanzapine/metabolism* ; RNA, Messenger/metabolism* ; RNA, Small Interfering ; Triglycerides

Objective: To investigate the effect of acetyl-CoA carboxylase 1 (ACC1) knockdown on the migration of esophageal squamous cell carcinoma (ESCC) KYSE-450 cell and underlying mechanism. Methods: Lentiviral transfection was conducted to establish sh-NC control cell and ACC1 knocking down cell (sh-ACC1). Human siRNA HSP27 and control were transfected by Lipo2000 to get si-HSP27 and si-NC. The selective acetyltransferase P300/CBP inhibitor C646 was used to inhibit histone acetylation and DMSO was used as vehicle control. Transwell assay was performed to detect cell migration. The expression of HSP27 mRNA was examined by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and the expressions of ACC1, H3K9ac, HSP27 and epithelial-mesenchymal transition-related proteins E-cadherin and Vimentin were detected by western blot. Results: The expression level of ACC1 in sh-NC group was higher than that in sh-ACC1 group (P<0.01). The number of cell migration in sh-NC group was (159.00±24.38), lower than (361.80±26.81) in sh-ACC1 group (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-NC group were statistically significant compared with sh-AAC1 group (P<0.05). The migrated cell number in sh-NC+ si-NC group was (189.20±16.02), lower than (371.60±38.40) in sh-ACC1+ si-NC group (P<0.01). The migrated cell number in sh-NC+ si-NC group was higher than that in sh-NC+ si-HSP27 group (152.40±24.30, P<0.01), and the migrated cell number in sh-ACC1+ si-NC group was higher than that in sh-ACC1+ si-HSP27 group (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-NC+ si-NC group were significantly different from those in sh-ACC1+ si-NC and sh-NC+ si-HSP27 groups (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-ACC1+ si-NC group were significantly different from those in sh-ACC1+ si-HSP27 group (P<0.01). After 24 h treatment with C646 at 20 μmmo/L, the migrated cell number in sh-NC+ DMSO group was (190.80±11.95), lower than (395.80±17.10) in sh-ACC1+ DMSO group (P<0.01). The migrated cell number in sh-NC+ DMSO group was lower than that in sh-NC+ C646 group (256.20±23.32, P<0.01). The migrated cell number in sh-ACC1+ DMSO group was higher than that in sh-ACC1+ C646 group (87.80±11.23, P<0.01). The protein expressions of H3K9ac, HSP27, E-cadherin and Vimentin in sh-NC+ DMSO group were significantly different from those in sh-ACC1+ DMSO group and sh-NC+ C646 group (P<0.01). The protein expression levels of H3K9ac, HSP27, E-cadherin and Vimentin in sh-ACC1+ DMSO group were significantly different from those in sh-ACC1+ C646 group (P<0.01). Conclusion: Knockdown of ACC1 promotes the migration of KYSE-450 cell by up-regulating HSP27 and increasing histone acetylation.
Humans ; Esophageal Neoplasms/pathology* ; Esophageal Squamous Cell Carcinoma/genetics* ; Vimentin/metabolism* ; Dimethyl Sulfoxide ; HSP27 Heat-Shock Proteins/metabolism* ; Histones/metabolism* ; Cadherins/metabolism* ; Cell Movement ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Epithelial-Mesenchymal Transition/genetics* ; Gene Expression Regulation, Neoplastic

Objective To investigate the effects and mechanism of Interleukin-33 (IL-33) mediated proliferation and differentiation of pulmonary myofibroblasts (MFbs) in pulmonary fibrosis (PF). Methods C57BL/6 mice were randomly divided into four groups: a control group, a bleomycin (BLM) group, a BLM combined with IL-33 group and a BLM combined with anti-IL-33 antibody group, 12 mice in each group. The PF model was induced by intratracheal injection of BLM (5000 U/kg). The degrees of fibrosis were examined using HE and Masson staining. ELISA was used to measure the plasma levels of IL-33. Immunohistochemical staining was used to measure the expression of alpha smooth muscle actin (α-SMA) in lung tissue. Primary pulmonary fibroblasts were isolated and cultured from lung tissues of mice. The cells were divided into four groups: a control group, an IL-33 group, an IL-33 combined with dimethyl sulfoxide (DMSO) group and an IL-33 combined with pyrrolidine dithiocarbamate (PDTC) group. The cells were treated with DMSO or PDTC for 1 hour and then with IL-33 for 48 hours. Cell proliferation was measured by 5-ethynyl-2'-deoxyuridine (EdU) assay and cell cycle was measured by flow cytometry. Transwell^TM assay was used to analyze cell migration. Real-time quantitative PCR was used to measure the expression of collagen type I (Col1), Col3 and α-SMA mRNA. The protein levels of IL-33, Col1, Col3, α-SMA, eukaryotic initiation factor 3a (eIF3a), phosphorylated IκBα (p-IκBα) (total lysate), p-NF-κB p65(total lysate) and NF-κB p65 (nucleus) were measured by Western blot analysis. Results In vivo, compared with the control group, the expressions of IL-33, p-IκBα (total lysate), p-NF-κB p65 (total lysate), NF-κB p65(nucleus), eIF3a, α-SMA, Col1 and Col3 in the BLM group significantly increased. Compared with the BLM group, the expressions of p-IκBα (total lysate), p-NF-κB p65 (total lysate), NF-κB p65 (nucleus), eIF3a, α-SMA, Col1 and Col3 in the IL-33 group increased further and the PF was further aggravated. But the effect of anti-IL-33 antibody was just opposite to that of IL-33. In vitro, IL-33 markedly induced the proliferation and migration of pulmonary fibroblasts, and significantly up-regulated the expression of p-IκBα (total lysate), p-NF-κB p65(total lysate), NF-κB p65 (nucleus), eIF3a, α-SMA, Col1 and Col3. But all these effects of IL-33 were reversed by pyrrolidine dithiocarbamate. Conclusion The results suggest that IL-33 may promote the expression of eIF3a by activating NF-κB signaling pathway, thus inducing the proliferation and differentiation of MFbs and promoting the occurrence and development of PF.
Animals ; Mice ; Bleomycin/metabolism* ; Cell Differentiation ; Cell Proliferation ; Dimethyl Sulfoxide/pharmacology* ; Fibroblasts ; Interleukin-33/pharmacology* ; Mice, Inbred C57BL ; Myofibroblasts/metabolism* ; NF-kappa B/metabolism* ; NF-KappaB Inhibitor alpha/metabolism* ; Pulmonary Fibrosis ; Signal Transduction

AIMTo investigate whether NR2B-pERK1/2-pElk-1 signaling contributes to the Y-maze learning and memory of rat brain.

METHODS45 adult male SD rats were divided into 4 groups: (1) Ifenprodil peritoneal injection group (Ifenprodil ip, n = 14); (2) DMSO peritoneal injection group(DMSO ip, n = 15); (3) Ifenprodil cerebral ventricle injection group (Ifenprodil ic, n = 8); (4) DMSO cerebral ventricle injection group(DMSO ic, n = 8). Y-maze training and test were used as an learning and memory enhancing stimulus. Immunohistochemical and Western blotting methods were used for detecting pERK1/2 and pElk-1 expression intensity of different brain regions.

RESULTSCompared with the DMSO ip group, the ifenprodil ip group showed no change on the Y-maze learning score (P > 0.05), but its Y-maze memory score tested 24 after learning decreased (P < 0.05). Ifenprodil peritoneal injection made brain pERK1/2 and pElk-1 expression decreased generally. In hippocampus, marginal division of striatum(MrD), amygdala,these changes were more significant (P < 0.05). Compared with the DMSO ic group, the reconsolidation of Y-maze memory tested 6 hours after ifenprodil injection was impaired in ifenprodil ic group (P < 0.05). The OD value of pERK1/2 and pElk-1 positive bands in ifenprodil ic group attenuated generally. The pElk-1 positive bands of caudate putamen and MrD almost disappeared in ifenprodil ic group.

CONCLUSIONNR2B is essential for the formation of long-term memory, reconsolidation of Y-maze memory. The deactivation of NR2B by ifenprodil will impair these courses. Meanwhile, the deactivation of NR2B attenuates pERK1/2 and pElk-1 expression of learning and memory related regions after Y-maze learning and memory reconsolidation test. In MrD and caudate putamen, the pElk-1 expression are completely blocked by ifenprodil after memory reconsolidation test.

Animals ; Avoidance Learning ; physiology ; Dimethyl Sulfoxide ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Male ; Maze Learning ; physiology ; Memory ; physiology ; Piperidines ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Receptors, N-Methyl-D-Aspartate ; metabolism ; ets-Domain Protein Elk-1 ; metabolism

OBJECTIVETo investigate the protective effect of progesterone against high intraocular pressure-induced ischemia-reperfusion (IR) injury.

METHODSTwenty-four SD rats were randomly divided into normal control, IR model, dimethyl sulfoxide (DMSO) solvent treatment group, and progesterone treatment group. In the latter 3 groups, retinal IR injury was induced by intraocular injection of saline. In the progesterone group, intraperitoneal injections of 4 mg/kg progesterone were administered 30 min before and 2 h after ischemia, and an equivalent volume of DMSO was used in the DMSO group. The content of malondialdehyde (MDA) and superoxide dismutase (SOD) activity were measured by spectrophotometer after the treatment, and the pathological changes of the retina were observed by transmission electron microscope and light microscope.

RESULTSSix hours after reperfusion, the content of MDA in the model group was significantly higher than that in the normal control group (P<0.01), but lower than that in progesterone treatment group (P<0.01); reverse changes in SOD activity was observed. In the model group, the inner nuclear layer and nerve fiber layer became thinner with obvious cellular pathologies including nuclear condensation, mitochondria vacuolization and endocytoplasmic reticulum swelling. Progesterone treatment markedly alleviated these pathologies in the inner nuclear layer and nerve fiber layer of the retina.

CONCLUSIONProgesterone offers protection of the retina against IR injury in SD rats by increasing SOD activity and decreasing MDA content in the retina.

Animals ; Dimethyl Sulfoxide ; Female ; Ischemia ; etiology ; pathology ; Male ; Malondialdehyde ; metabolism ; Ocular Hypertension ; complications ; Progesterone ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; etiology ; prevention & control ; Retina ; metabolism ; Retinal Vessels ; physiopathology ; Superoxide Dismutase ; metabolism

OBJECTIVE: To investigate the involvement of transcription factor Foxa2 in cardiac differentiation in P19 embryonal carcinoma cells and its molecular mechanism. METHODS: P19 cells were induced to differentiate into cardiomyocytes by adding dimethyl sulfoxide (DMSO) into the culture medium of their embryoid bodies (EBs). The mRNA levels of pluripotency markers of embryonic pluripotent stem cells, cardiac differentiation related genes, and Foxa2 in the cell samples at different time points of cardiac differentiation were detected by reverse transcription PCR (RT-PCR). Differentiated and mature cardiomyocytes were identified by immunofluorescence. Eukaryotic expression plasmid pCMV-rFoxa2 (rat Foxa2) was transfected into P19 cells, and clonal populations of P19 cells that stably expressed green fluorescence protein (GFP)-rFoxa2 were isolated to enhance the expression levels of Foxa2 in P19 cells. The mRNA and protein levels of pluripotency markers and cardiac differentiation related genes in the above cell samples were detected by RT-PCR and Western blot. The mRNA levels of cardiac differentiation related genes in EBs differentiation system were also examined. RESULTS: P19 cells differentiated into cardiomyocytes in the presence of DMSO, accompanied by stimulated expression of Foxa2. Transfection of pCMV-rFoxa2 plasmids into P19 cells upregulated rFoxa2 expression transiently and activated the transcription of its downstream cardiac inducer Cerberus1 (Cer1). The expression of pluripotency marker Nanog was suppressed and the expression of cardiac inducer Sonic Hedgehog (Shh) was elevated in GFP-rFoxa2 P19 cells. The expression of Cer1 and cardiac muscle marker actin, alpha cardiac muscle 1 (Actc1) was upregulated in EBs of GFP-rFoxa2 P19 cells. CONCLUSION Foxa2 participates in cardiac differentiation in P19 embryonal carcinoma cells. Foxa2 may inhibit Nanog expression and stimulate the expression of Cer1 and Shh directly during cardiac differentiation in P19 cells in the presence of DMSO.
Animals ; Cell Differentiation ; drug effects ; Cell Line ; Cytokines ; Dimethyl Sulfoxide ; pharmacology ; Embryonal Carcinoma Stem Cells ; pathology ; Hedgehog Proteins ; metabolism ; Hepatocyte Nuclear Factor 3-beta ; physiology ; Homeodomain Proteins ; metabolism ; Mice ; Myocytes, Cardiac ; cytology ; Nanog Homeobox Protein ; Proteins ; metabolism ; Transfection