Overexpression of heme oxygenase-1 protects smooth muscle cells against oxidative injury and inhibits cell proliferation

To investigate whether the expression of exogenous heme oxygenase-1 (HO-1) gene within vascular smooth muscle cells (VSMC) could protect the cells from free radical attack and inhibit cell proliferation, we established an in vitro transfection of human HO-1 gene into rat VSMC mediated by a retroviral vector. The results showed that the profound expression of HO-1 protein as well as HO activity was 1.8- and 2.0-fold increased respectively in the transfected cells compared to the non-transfected ones. The treatment of VSMC with different concentrations of H2O2 led to the remarkable cell damage as indicated by survival rate and LDH leakage. However, the resistance of the HO-1 transfected VSMC against H2O2 was significantly raised. This protective effect was dramatically diminished when the transfected VSMC were pretreated with ZnPP-IX, a specific inhibitor of HO, for 24 h. In addition, we found that the growth potential of the transfected cells was significantly inhibited directly by increased activity of HO-1, and this effect might be related to decreased phosphorylation of MAPK. These results suggest that the overexpression of introduced hHO-1 is potentially able to reduce the risk factors of atherosclerosis, partially due to its cellular protection against oxidative injury and to its inhibitory effect on cellular proliferation.

Novel miRNA, miR-sc14, promotes Schwann cell proliferation and migration

MicroRNAs refer to a class of endogenous,short non-coding RNAs that mediate numerous biological functions.MicroRNAs regulate various physiological and pathological activities of peripheral nerves,including peripheral nerve repair and regeneration.Previously,using a rat sciatic nerve injury model,we identified many functionally annotated novel microRNAs,including miR-sc14.Here,we used real-time reverse transcription-polymerase chain reaction to examine miR-sc14 expression in rat sciatic nerve stumps.Our results show that miRsc14 is noticeably altered following sciatic nerve injury,being up-regulated at 1 day and diminished at 7 days.EdU and transwell chamber assay results showed that miR-sc14 mimic promoted proliferation and migration of Schwann cells,while miR-sc14 inhiThe study was approved by the Jiangsu Provincial Laboratory Animal Management Committee,China on March 4,2015(approval No.20150304-004).bitor suppressed their proliferation and migration.Additionally,bioinformatic analysis examined potential target genes of miR-sc14,and found that fibroblast growth factor receptor 2 might be a potential target gene.Specifically,our results show changes of miR-sc14 expression in the sciatic nerve of rats at different time points after nerve injury.Appropriately,up-regulation of miR-sc14 promoted proliferation and migration of Schwann cells.Consequently,miR-sc14 may be an intervention target to promote repair of peripheral nerve injury.The study was approved by the Jiangsu Provincial Laboratory Animal Management Committee,China on March 4,2015(approval No.20150304-004).

Claudin-15 overexpression inhibits proliferation and promotes apoptosis of Schwann cells in vitro

Our previous experiments have discovered that Claudin-15 was up-regulated in Schwann cells of the distal nerve stumps of rat models of sciatic nerve injury.However,how Claudin-15 affects Schwann cell function is still unknown.This study aimed to identify the effects of Claudin-15 on proliferation and apoptosis of Schwann cells cultured in vitro and explore the underlying mechanisms.Primary Schwann cells were obtained from rats.Claudin-15 in Schwann cells was knocked down using siRNA(siRNA-1 group)compared with the negative control siRNA transfection group(negative control group).Claudin-15 in Schwann cells was overexpressed using pGV230-Claudin-15 plasmid(pGV230-Claudin-15 group).The pGV230 transfection group(pGV230 group)acted as the control of the pGV230-Claudin-15 group.Cell proliferation was analyzed with EdU assay.Cell apoptosis was analyzed with flow cytometric analysis.Cell migration was analyzed with Transwell inserts.The mRNA and protein expressions were analyzed with quantitative polymerase chain reaction assay and western blot assay.The results showed that compared with the negative control group,cell proliferation rate was up-regulated;p-AKT/AKT ratio,apoptotic rate,p-c-Jun/c-Jun ratio,mRNA expression of protein kinase C alpha,Bcl-2 and Bax were down-regulated;and mRNA expression of neurotrophins basic fibroblast growth factor and neurotrophin-3 were increased in the siRNA-1 group.No significant difference was found in cell migration between the negative control and siRNA-1 groups.Compared with the pGV230 group,the cell proliferation rate was down-regulated;apoptotic rate,p-c-Jun/c-Jun ratio and c-Fos protein expression increased;mRNA expression of protein kinase C alpha and Bax decreased;and mRNA expressions of neurotrophins basic fibroblast growth factor and neurotrophin-3 were up-regulated in the pGV230-Claudin-15 group.The above results demonstrated that overexpression of Claudin-15 inhibited Schwann cell proliferation and promoted Schwann cell apoptosis in vitro.Silencing of Claudin-15 had the reverse effect and provided neuroprotective effect.This study was approved by the Experimental Animal Ethics Committee of Jilin University of China(approval No.2016-nsfc001)on March 5,2016.

Microsatellite instability,MMR gene expression and proliferation kinetics in colorectal cancer with famillial predisposition

INTRODUCTIONGenetic instability is a conunon property of manyhuman cancers,including those of HNPCC.A novel form of genetic instability involving somaticalterations,such as deletions and insertions insimple repeated sequences,has been found.Microsatellitcs are relatively short runs of tandemlyrepeated sequences scattered throughout

Increased levels of miR-3099 induced by peripheral nerve injury promote Schwann cell proliferation and migration

MicroRNAs(miRNAs) can regulate the modulation of the phenotype of Schwann cells. Numerous novel miRNAs have been discovered and identified in rat sciatic nerve segments, including miR-3099. In the current study, miR-3099 expression levels following peripheral nerve injury were measured in the proximal stumps of rat sciatic nerves after surgical crush. Real-time reverse transcription-polymerase chain reaction was used to determine miR-3099 expression in the crushed nerve segment at 0, 1, 4, 7, and 14 days post sciatic nerve injury, which was consistent with Solexa sequencing outcomes. Expression of miR-3099 was up-regulated following peripheral nerve injury. EdU and transwell chamber assays were used to observe the effect of miR-3099 on Schwann cell proliferation and migration. The results showed that increased miR-3099 expression promoted the proliferation and migration of Schwann cells. However, reduced miR-3099 expression suppressed the proliferation and migration of Schwann cells. The potential target genes of miR-3099 were also investigated by bioinformatic tools and high-throughput outcomes. miR-3099 targets genes Aqp4, St8 sia2, Tnfsf15, and Zbtb16 and affects the proliferation and migration of Schwann cells. This study examined the levels of miR-3099 at different time points following peripheral nerve injury. Our results confirmed that increased miR-3099 level induced by peripheral nerve injury can promote the proliferation and migration of Schwann cells.

Inhibitory activity of polysaccharide extracts from three kinds of edible fungi on proliferation of human hepatoma SMMC-7721 cell and mouse implanted S180 tumor

AIM To determine the activities ofpolysaccharide extracts from Flammulina velutipes (Curt. ex Fr. ) Sing (FV), Lentinusedodes (LE) and Agaricus bisporus Sing (AB)on the proliferation of human hepatoma SMMC-7721 cells in vitro and on mouse implanted S-180tumors in vivo.METHODS The polysaccharide extracts were isolated from the fruit bodies of FV, LE and AB by the methods of hot-water extraction, Sevag’sremoval of proteins, ethanol precipitation,trypsin digestion and ethanol fractionalprecipitation. Human hepatoma SMMC-7721 cells were treated with 50 mg/L Polysaccharide extracts, and the mitosis index, mitochondria activity and cell proliferation were detected at different times in both control and experimental groups. The mice with S-180 implanted tumors were injected with the polysaccharide extracts at 24 mg/ kg body weight for 9 d and the tumorweight was measured on the 15th day.RESULTS The mitosis index of hepatoma cells in vitro could be significantly decreased by treatment with the polysaccharide extracts fromthe three kinds of edible fungi (P < 0 .005 ). Thecell numbers and mitochondria activity of SMMC7721 cells treated with polysaccharide extracts were lower than those in control groups (P <0.005). The inhibition rates of polysaccharide extracts against implanted S-180 tumors in mice were 52.8%, 56.6% and 51 .9% respectivelycompared with that in c0ntrol gr0ups.CONCLUSI0N The POIysaccharide extractsfrom the three kinds of edible fungi could inhibitnot only the Cultured malignant cells in vitfO butalso impIanted Sl80 tum0r i0 vivo.

High-level expression of human calmodulin in E.coli and its effects on cell proliferation

Calmodulin (CaM), widely distributed in almost all eukaryotic cells, is a major intracellular calcium receptor responsible for mediating the Ca2 + signal to a multitude of different enzyme systems and is thought to play a vital role in the regulation of cell proliferative cycle[1，2]. Recently, many studies showed that CaM is also present in extracellular fluid such as cell culture media and normal body fluid and has been reported to stimulate proliferation in a range of normal and neoplastic cells, apparently acting as an autocrine growth factor[3-11]. In 1988, Crocker et al reported for the first time that addition of extracellular pure pig brain CaM could promote DNA synthesis and cell [7]proliferation in K562 human leukaemic lymphocytes[7].After that, more and more research was done on extracellular CaM and evidences demonstrated that extracellular CaM could also stimulate cell proliferation in normal human umbilical vein endothelial cells[5], keratinocytes[4], suspension-cultured cells of Angelica Dahurica, etc[6]. CaM is a monomeric protein of 148 amino acids that contains four homologous Ca2 + -binding domains. CaM has been highly conserved throughout the evolution. Only 1 out of 148 amino acids of human CaM is different from that of fish CaM. Complementary DNAs encoding rat, eel, chicken, human, and trypanosome CaM have been cloned.

High-frequency repetitive transcranial magnetic stimulation promotes neural stem cell proliferation after ischemic stroke

Prolife ration of neural stem cells is crucial for promoting neuronal regeneration and repairing cerebral infarction damage.Transcranial magnetic stimulation(TMS)has recently emerged as a tool for inducing endogenous neural stem cell regeneration,but its underlying mechanisms remain unclea r In this study,we found that repetitive TMS effectively promotes the proliferation of oxygen-glucose deprived neural stem cells.Additionally,repetitive TMS reduced the volume of cerebral infa rction in a rat model of ischemic stro ke caused by middle cerebral artery occlusion,im p roved rat cognitive function,and promoted the proliferation of neural stem cells in the ischemic penumbra.RNA-sequencing found that repetitive TMS activated the Wnt signaling pathway in the ischemic penumbra of rats with cerebral ischemia.Furthermore,PCR analysis revealed that repetitive TMS promoted AKT phosphorylation,leading to an increase in mRNA levels of cell cycle-related proteins such as Cdk2 and Cdk4.This effect was also associated with activation of the glycogen synthase kinase 3β/β-catenin signaling pathway,which ultimately promotes the prolife ration of neural stem cells.Subsequently,we validated the effect of repetitive TMS on AKT phosphorylation.We found that repetitive TMS promoted Ca2+influx into neural stem cells by activating the P2 calcium channel/calmodulin pathway,thereby promoting AKT phosphorylation and activating the glycogen synthase kinase 3β/β-catenin pathway.These findings indicate that repetitive TMS can promote the proliferation of endogenous neural stem cells through a Ca2+influx-dependent phosphorylated AKT/glycogen synthase kinase 3β/β-catenin signaling pathway.This study has produced pioneering res ults on the intrinsic mechanism of repetitive TMS to promote neural function recove ry after ischemic stro ke.These results provide a stro ng scientific foundation for the clinical application of repetitive TMS.Moreover,repetitive TMS treatment may not only be an efficient and potential approach to support neurogenesis for further therapeutic applications,but also provide an effective platform for the expansion of neural stem cells.

Cerebral dopamine neurotrophic factor promotes the proliferation and differentiation of neural stem cells in hypoxic environments

Previous research found that cerebral dopamine neurotrophic factor(CDNF)has a protective effect on brain dopaminergic neurons,and CDNF is regarded as a promising therapeutic agent for neurodegenerative diseases.However,the effects of CDNF on the proliferation,differentiation,and apoptosis of neural stem cells(NSCs),which are very sensitive to hypoxic environments,remain unknown.In this study,NSCs were extracted from the hippocampi of fetal rats and cultured with different concentrations of CDNF.The results showed that 200 nM CDNF was the optimal concentration for significantly increasing the viability of NSCs under non-hypoxic environmental conditions.Then,the cells were cultured with 200 nM CDNF under the hypoxic conditions of 90%N_2,5%CO_2,and 5%air for 6 hours.The results showed that CDNF significantly improved the viability of hypoxic NSCs and reduced apoptosis among hypoxic NSCs.The detection of markers showed that CDNF increased the differentiation of hypoxic NSCs into neurons and astrocytes.CDNF also reduced the expression level of Lin28 protein and increased the expression of Let-7 mRNA in NSCs,under hypoxic conditions.In conclusion,we determined that CDNF was able to reverse the adverse proliferation,differentiation,and apoptosis effects that normally affect NSCs in a hypoxic environment.Furthermore,the Lin28/Let-7 pathway may be involved in this regulated function of CDNF.The present study was approved by the Laboratory Animal Centre of Southeast University,China(approval No.20180924006)on September 24,2018.

Growth hormone stimulates remnant small bowel epithelial cell proliferation

INTRODUCTIONCurrently the major treatment choices for shortbowel syndrome are parenteral nutrition and smallbowel transplantation.Both therapies involvegreat fiscal challenge and recurring complications.Recent years have witnessed the promisingexperimental results of pharmacologicalrehabilitation of remnant small bowel.

Extraction of Phenolic Compounds from Olive Leaf Extracts and Their Effect on Proliferation of Human Carcinoma Cell Lines

The aim of this work was to evaluate the effect of different olive leaf extracts (OLE) from different leaf growing stages on human carcinoma cell lines. OLE were tested in human carcinoma cell lines in vitro and cells were plated in 96-microtiter culture plates for each OLE concentration. Fresh (F) and freeze-dried (FD) leaves exhibited phenolic compounds in the range of 2.09 ± 0.10 to 8.44 ± 0.64 and 7.72 ± 0.56 to 24.65 ± 1.9 mg gallic acid equivalents/g leaves, respectively. OLE from several Portuguese olive tree cultivars were found to inhibit the growth of human carcinoma cell lines in a range of 2.09 - 8.44 μg phenolic compound/well (209 - 844 μg/ml) and 0.07 - 2.40 μg phenolic compounds/well (7 - 240 μg/ml) for fresh and freeze-dried leaves, respectively. Young (Y) leaves have revealed the highest cell growth inhibition ranging from about 95% for Cobran?osa, followed by 90% for Cobran?osa, 90% for Arbequina and 75% for Arbequina for cell lines A549, HeLa, A431 and OE21, respectively. The lowest cell growth inhibition (35%) was observed for Galega (Y) leaf extract on cell line A549. However, FD samples exhibited a distinctive pattern since cell growth inhibition was highest at highest extract dilution tested, for A431 (Galega Y) followed by A549 (Cobran?osa Y) with cell inhibition of 75% and 70%, respectively. The data presented in this work strongly suggest that OLEs inhibit the growth of human carcinoma cell lines.

The MORC2 p.S87L mutation reduces proliferation of pluripotent stem cells derived from a patient with the spinal muscular atrophy-like phenotype by inhibiting proliferation-related signaling pathways

Mutations in the microrchidia CW-type zinc finger protein 2(MORC2)gene are the causative agent of Charcot-Marie-Tooth disease type 2Z(CMT2Z),and the hotspot mutation p.S87L is associated with a more seve re spinal muscular atrophy-like clinical phenotype.The aims of this study were to determine the mechanism of the severe phenotype caused by the MORC2 p.S87L mutation and to explore potential treatment strategies.Epithelial cells were isolated from urine samples from a spinal muscular atrophy(SMA)-like patient[MORC2 p.S87L),a CMT2Z patient[MORC2 p.Q400R),and a healthy control and induced to generate pluripotent stem cells,which were then differentiated into motor neuron precursor cells.Next-generation RNA sequencing followed by KEGG pathway enrichment analysis revealed that differentially expressed genes involved in the PI3K/Akt and MAP K/ERK signaling pathways were enriched in the p.S87L SMA-like patient group and were significantly downregulated in induced pluripotent stem cells.Reduced proliferation was observed in the induced pluripotent stem cells and motor neuron precursor cells derived from the p.S87L SMA-like patient group compared with the CMT2Z patient group and the healthy control.G0/G1 phase cell cycle arrest was observed in induced pluripotent stem cells derived from the p.S87L SMA-like patient.MORC2 p.S87Lspecific antisense oligonucleotides(p.S87L-ASO-targeting)showed significant efficacy in improving cell prolife ration and activating the PI3K/Akt and MAP K/ERK pathways in induced pluripotent stem cells.Howeve r,p.S87L-ASO-ta rgeting did not rescue prolife ration of motor neuron precursor cells.These findings suggest that downregulation of the PI3K/Akt and MAP K/ERK signaling pathways leading to reduced cell proliferation and G0/G1 phase cell cycle arrest in induced pluripotent stem cells might be the underlying mechanism of the severe p.S87L SMA-like phenotype.p.S87L-ASO-targeting treatment can alleviate disordered cell proliferation in the early stage of pluripotent stem cell induction.

Electroacupuncture in the repair of spinal cord injury:inhibiting the Notch signaling pathway and promoting neural stem cell proliferation

Electroacupuncture for the treatment of spinal cord iniury has a good dinical curative effect, but the underlying mechanism is unclear. In our experiments, the spinal cord of adult Sprague-Daw- ley rats was clamped for 60 seconds. Dazhui （GV14） and Mingmen （GV4） acupoints of rats were subjected to electroacupuncture. Enzyme-linked immunosorbent assay revealed that the expres- sion of serum inflammatory factors was apparently downregulated in rat models of spinal cord injury after electroacupuncture. Hematoxylin-eosin staining and immunohistochemistry results demonstrated that electroacupuncture contributed to the proliferation of neural stem cells in rat injured spinal cord, and suppressed their differentiation into astrocytes. Real-time quantitative PCR and western blot assays showed that electroacupuncture inhibited activation of the Notch signaling pathway induced by spinal cord injury. These findings indicate that electroacupuncture repaired the injured spinal cord by suppressing the Notch signaling pathway and promoting the proliferation of endogenous neural stem ceils.

Hyperbaric oxygen treatment promotes neural stem cell proliferation in the subventricular zone of neonatal rats with hypoxic-ischemic brain damage

Hyperbaric oxygen therapy for the treatment of neonatal hypoxic-ischemic brain damage has been used clinically for many years, but its effectiveness remains controversial. In addition, the mechanism of this potential neuroprotective effect remains unclear. This study aimed to investigate the influence of hyperbaric oxygen on the proliferation of neural stem cells in the subventricular zone of neonatal Sprague-Dawley rats （7 days old） subjected to hypoxic-ischemic brain damage. Six hours after modeling, rats were treated with hyperbaric oxygen once daily for 7 days. Immunohistochemistry revealed that the number of 5-bromo-2＇-deoxyuridine positive and nestin positive cells in the subventricular zone of neonatal rats increased at day 3 after hypoxic-ischemic brain damage and peaked at day 5. After hyperbaric oxygen treatment, the number of 5-bromo-2＇- deoxyuddine positive and nestin positive cells began to increase at day 1, and was significantly higher than that in normal rats and model rats until day 21. Hematoxylin-eosin staining showed that hyperbaric oxygen treatment could attenuate pathological changes to brain tissue in neonatal rats, and reduce the number of degenerating and necrotic nerve cells. Our experimental findings indicate that hyperbaric oxygen treatment enhances the proliferation of neural stem cells in the subventricular zone of neonatal rats with hypoxic-ischemic brain damage, and has therapeutic potential for promoting neurological recovery following brain injury.

Infection of Schistosomiasis japanicum is likely to enhance proliferation and migration of human breast cancer cells:mechanism of action of differential expression of MMP2 and MMP9

Objective:To study whether the infection of Schistosomiasis japanicum(S.japanicum) is related to enhanced proliferation and migration of cancer cells,and the molecular mechanism pertains to cancer cell metastasis in human host.Methods:The gene of S.japanicum glutathione transferase(sjGST) cloned from 5.japanicum was expressed,purified and applied in a series of assays to explore the effect of sjGST on proliferation and migration of MDA-MB-435S,and the expression of MMP2 and MMP9.Immunofluorescence assay for the binding of sjGST to MDA-MB-435S was also carried out.Results:Results showed that sjGST enhanced proliferation and migration in human breast cancer cell MDA-MB-435S signifycantly at 50-200 nM,but did not enhance them in human lung cancer cell A549.Immunofluorescence assay for the binding of sjGST to MDA-MB-435S and A549 showed that GST was readily hound to the breast cancer cells,but showed almost no binding to human lung cancer cells.The assays for gelatinase activity showed that both MMP2 and MMP9 activities were increased significantly in the presence of sjGST(50-200 nM) in MDA-MB-435S, but they were not significant in A549.Conclusions:Our current results show strongly that S. japanicum GST binds to MDA-MB-435S probably via its i'eceptor,and enhances proliferation and migration of the cancer cells by up-regulatory expression of MMP2 and MMP9.

shRNA-interfering LSD1 inhibits proliferation and invasion of gastric cancer cells via VEGF-C/PI3K/AKT signaling pathway

BACKGROUND Histone Lysine Specific Demethylase 1(LSD1)is the first histone demethylase to be discovered,which regulates various biological functions by making lysine of histone H3K4,H3K9 and non-histone substrates demethylated.Abnormal regulation of LSD1 is closely related to the occurrence and development of gastric cancer.The change of LSD1 expression level plays an important role in the proliferation and metastasis of gastric cancer cells.The study of its function and mechanism may provide a theoretical basis for early diagnosis and targeted therapy of gastric cancer.AIM To investigate the effect of downregulation of lysine-specific demethylase 1(LSD1)expression on proliferation and invasion of gastric cancer cells and the possible regulatory mechanisms of the VEGF-C/PI3K/AKT signaling pathway.METHODS The LSD1-specific short hairpin RNA(shRNA)interference plasmid was transiently transfected,and expression of LSD1 was downregulated.The cell proliferation ability of LSD1 was observed by CCK-8 assay after downregulating expression of LSD1.Transwell invasion assay was used to observe the change of cell invasion ability after downregulating expression of LSD1.Expression of phosphorylated phosphoinositide 3-kinase(p-PI3K),PI3K,p-AKT,AKT,vascular endothelial growth factor receptor(VEGFR)-3,matrix metalloproteinase(MMP)-2 and MMP-9 in each group was detected by Western blotting.RESULTS The cell proliferation ability of transiently transfected LSD1-shRNA interference plasmid group was significantly lower than that of the control group(P<0.05).Transwell invasion assay showed that the number of cells across the membrane of the LSD1-shRNA transfection group(238.451±5.216)was significantly lower than that of the control group(49.268±6.984)(P<0.01).Western blotting showed that expression level of VEGF-C,p-PI3K,PI3K,p-AKT,AKT,VEGFR-3,MMP-2 and MMP-9 in the LSD1-shRNA group was significantly lower than that in the control group(P<0.05).CONCLUSION Downregulation of LSD1 expression inhibits metastatic potential of gastric cancer cells,and VEGF-C-mediated activation of PI3K/AKT signaling pathway,which may be an important mechanism for inhibiting lymph node metastasis in gastric cancer cells.

Dorsal root ganglion neurons promote proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells

Preliminary animal experiments have confirmed that sensory nerve fibers promote osteoblast differentiation, but motor nerve fibers have no promotion effect. Whether sensory neurons pro- mote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells remains unclear. No results at the cellular level have been reported. In this study, dorsal root ganglion neurons （sensory neurons） from Sprague-Dawley fetal rats were co-cultured with bone marrow mesenchymal stem cells transfected with green fluorescent protein 3 weeks after osteo- genic differentiation in vitro, while osteoblasts derived from bone marrow mesenchymal stem cells served as the control group. The rat dorsal root ganglion neurons promoted the prolifera- tion of bone marrow mesenchymal stem cell-derived osteoblasts at B and 5 days of co-culture, as observed by fluorescence microscopy. The levels of mRNAs for osteogenic differentiation-re- lated factors （including alkaline phosphatase, osteocalcin, osteopontin and bone morphogenetic protein 2） in the co-culture group were higher than those in the control group, as detected by real-time quantitative PCR. Our findings indicate that dorsal root ganglion neurons promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells, which pro- vides a theoretical basis for in vitro experiments aimed at constructing tissue-engineered bone.

Ceramide is involved in alcohol-induced neural proliferation

Prenatal alcohol exposure, especially during early pregnancy, can lead to fetal alcohol syndrome. The pharmacological and toxicological mechanisms of ethanol are related to the effects of ceramide In this study, we established an alcohol exposure model in wild-type mice and in knockout mice for the key enzyme involved in ceramide metabolism, sphingomyelin synthase 2. This model received daily intragastric administration of 25% ethanol, and pups were used at postnatal days 0, 7, 14, 30 for experiments. Serology and immunofluorescence staining found that ethanol exposure dose-dependently reduced blood sphingomyelin levels in two genotypes of pups, and increased neural cell proliferation and the number of new neurons in the hippocampal dentate gyrus. Western blot analysis showed that the relative expression level of protein kinase C e increased in two genotypes of pups after ethanol exposure. Compared with witd-type pups, the expression level of the important activator protein of the ceramide/ceramide-l-phosphate pathway, protein kinase C a, was reduced in the hippocampus of sphingomyelin synthase 2 knockouts. Our findings illustrate that ceramide is involved in alcohol-induced neural proliferation in the hippocampal dentate gyrus of pups after prenatal ethanol exposure, and the mechanism may be associated with increased ex- pression of protein kinase C a activating the ceramide/ceramide-l-phosphate pathway.

Electroacupuncture and moxibustion promote regeneration of injured sciatic nerve through Schwann cell proliferation and nerve growth factor secretion

Using electroacupuncture and moxibustion to treat peripheral nerve injury is highly efficient with low side effects. However, the electroacupuncture-and moxibustion-based mechanisms underlying nerve repair are still unclear. Here, in vivo and in vitro experiments uncovered one mechanism through which electroacupuncture and moxibustion affect regeneration after peripheral nerve injury. We first established rat models of sciatic nerve injury using neurotomy. Rats were treated with electroacupuncture or moxibustion at acupoints Huantiao （GB30） and Zusanli （ST36）. Each treatment lasted 15 minutes, and treatments were given six times a week for 4 consecutive weeks. Behavioral testing was used to determine the sciatic functional index. We used electrophysiological detection to measure sciatic nerve conduction velocity and performed hematoxylin-eosin staining to determine any changes in the gastrocnemius muscle. We used immunohistochemistry to observe changes in the expression of S100—a specific marker for Schwann cells—and an enzyme-linked immunosorbent assay to detect serum level of nerve growth factor. Results showed that compared with the model-only group, sciatic functional index, recovery rate of conduction velocity, diameter recovery of the gastrocnemius muscle fibers, number of S100-immunoreactive cells,and level of nerve growth factor were greater in the electroacupuncture and moxibustion groups. The efficacy did not differ between treatment groups. The serum from treated rats was collected and used to stimulate Schwann cells cultured in vitro. Results showed that the viability of Schwann cells was much higher in the treatment groups than in the model group at 3 and 5 days after treatment. These findings indicate that electroacupuncture and moxibustion promoted nerve regeneration and functional recovery; its mechanism might be associated with the enhancement of Schwann cell proliferation and upregulation of nerve growth factor.

Effect of WFDC 2 silencing on the proliferation,motility and invasion of human serous ovarian cancer cells in vitro

Objective:To investigate effect and possible mechanisms of silencing human WFDC2（HE4） gene on biological behavior changes as cell proliferation,apoplosis,movement and invasion of human serous ovarian cancer cell line SKOV3.Methods:Lentiviral WFDC2 gene sequence of small interfering siRNA was stablely transfected into SKOV3 identified by Q-PCR and western-blot. Obtained SKOV3 stable strains with silenced HE4 were measured by proliferation,apoplosis, migration,and invasion.Results:Gene sequencing showed that the oligonucleotides were successfully inserted into the expected site.After silencing HE4 in the SKOV3,proliferation was significandy inhibited（P【0.05）.G0/G1 phase was arrested by the cell cycle（P【0.01） and capacity of the migration and invasion decreased significandy（P【0.01）.Slight early apoptosis ratio and no change of late apoplosis were found without change of Caspase-3 or Bcl-2 protein.Proteins involed in ERK pathway as phosphorylated protein as p-EGFR,p- ERK decreased and protease protein involved in tissue remoding as matrix metalloproteinases MMP-9,MMP-2 and cathepsin B decreased compared with control group.Conclusions:HE4 gene plays an important role in regulating proliferation,apoptosis,migration,invasion of serous ovarian cancer cells by ERK pathway and protease system.Its role in apoptosis needs to be further explored,and it may be a potential target for serous ovarian cancer.