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Objective The objective of this study was to investigate the effect of salinomycin on proliferation and autophagic flux of human melanoma M21 cells. Methods The cell survival rate was determined by MTS assay and IC50values( half inhibitory concentration)were calculated. The morphological changes of cells after salinomycin administration were observed under optical micro-scope. Flow cytometry was used to examine the apoptosis rate of M21 cells. Western blot was used to detect the expression of autophag-ic-related protein LC3B and p62 in M21 cells. The presence of autophagosomes in M21 cells after salinomycin administration was ob-served under transmission electron microscope. Western blot and immunofluorescence were used to detect the level of p62 protein and localizing changes in M21 cells. Results Salinomycin significantly inhibited proliferation of M21 cells, and the IC50values were (1. 38 ± 0. 18)μM. After salinomycin administration,the proliferation rate of M21 cells was slowed down,and obvious vacuoles ap-peared in the cells. Salinomycin could not only induce cell apoptosis,but it also increased the ratio of LC3B-Ⅱ/LC3B-Ⅰ in M21 cells. The increase and accumulation of autophagosomes were directly observed under transmission electron microscope. The level of p62 protein was slightly elevated after salinomycin treatment and gradually aggregated into the cytoplasm,indicating that autophagic flux was inhibited. Conclusion Salinomycin can inhibit the proliferation of human malignant melanoma M21 cells,and its mechanism may be related to the accumulation autophagosomes granules and inhibition of autophagic flux.
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Objective To investigate the effects of 20 Hz repetitive transcranial magnetic stimulation (rTMS) with different intensities on neurobehavior and expression of glial fibrillary acidic protein (GFAP) in ischemic penumbra of rats with cerebral infarction,so as to explore the probable mechanism. Methods Forty-three rats were randomly divided into a blank control group( n =7 ),a model control group( n =7),a sham stimulation control group(n =8) and a rTMS group (n =21) ; the rTMS group was further subdivided into 3 subgroups:80％ MT subgroup,100％ MT subgroup and 120％ MT subgroup,with 7 rats in each subgroup.The cerebral infarction model was established by right middle cerebral artery occlusion (MCAO) in each group except the blank control group.The 3 rTMS subgroups were given 14 successive blocks of 20 Hz rTMS with corresponding intensity.The sham stimulation control group received sham treatment (without any output).The model control group was given no stimulation,and the blank control group did not receive any special treatment.Functional assessments were performed at 3 different time points.After 14-day treatment,the expression of GFAP proteins in ischemic penumbra were detected by immunohistochemistry technique. Results Functional outcome reflected from 3 behavioral tests in 100％ MT subgroup after 14-day stimulation was better than 1 day after operation,while in the other rTMS subgroups functional outcomes were just better in 2 behavioral tests.The expressions of GFAP in 3 rTMS subgroups were all less than that in model control group. Conclusions The 20 Hz rTMS with 80％ MT and 100％ MT might be safe and effective to improve the functional outcome in rats with acute cerebral infarction,especially 100％ MT.Decrease of expression of GFAP in ischemic penumbra might be one of the mechanisms of beneficial effects of rTMS in ischemia brain injury.
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Objective To investigate the effects of high-frequency repetitive transcranial magnetic stimulation (rTMS) at different intensities on the ultrastructure of an ischemic brain penumbra and the expression of brainderived neurotrophic factor (BDNF) using rats with permanent middle cerebral artery occlusion (MCAO).Methods Forty-two rats were randomly divided into a blank control group,an MCAO model control group,a sham stimulation control group and an rTMS group.The rTMS group was divided further into 3 subgroups:an 80％ of motor threshold (MT) subgroup,a 100％ of MT subgroup and a 120％ of MT subgroup.The cerebral infarction model was established by right MCAO.rTMS treatment was given 24 hours after the MCAO model was successfully established.The rTMS group and sham stimulation control group were given 20 Hz rTMS with the planned intensities.The MCAO model control group was not given any stimulation.After 14 days of treatment,transmission electron microscopy,immunohistochemical and Western blotting ( WB ) methods were used to investigate the ultrastructure of the ischemic penumbra and the expression of BDNF.Results Damage reflected in the ultrastructure in the 3 rTMS subgroups was less than in the model control group and the sham stimulation control group.Expression of BDNF protein increased significantly in 100％ of the MT group and blank control group rats as compared with that in the sham stimulation control group,while the blank control group and the 3 rTMS subgroups had no statistically significant difference in comparison with the MCAO model control group.The expression of BDNF protein had no statistically significant difference between any of the groups.Conclusion 20 Hz rTMS might,especially at 100％ of the MT,promote the recovery of the ultrastructure of neural tissues in the ischemic penumbra after acute cerebral infarction and enhance the expression of BDNF in the ipsilesional hemisphere.This may be one of the important mechanisms of rTMS's effectiveness in the treatment of ischemic stroke.
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Objective:To investigate the effects of U0126 of different doses on the ability of low frequency magnetic stimulation promoting astrocyte migration and to select suitable dose of U0126.Method:Twenty-four adult healthy SD rats were selected to receive Injection of 0.5ml of 1% ethidium bromide(EB) in PBS into the dorsal spinal cord funiculus on the left side at T10-11 level to make located spinal cord injury models and randomly divided into four groups.The four groups were exposed to magnetic stimulation(1Hz,1.52T.30pulses)at the following dose respectively:Omg/kg U0126(control group).0.1mg/kg U0126(low-dose group), 0.2mg/kg U0126(middle-dose group),0.4mg/kg U0126(high-dose group).On the day 14 after stimulation,the rats were sacrificed and the expressions of glial fibfillary acidic protein(GFAP),microtubule associated protein-2(MAP-2),extracellular signal-regulated kinase1/2(ERg1/2)and the volume of holes were detected with immunohistochemistry.Quantitative analysis 0f the expressions of GFAP,MAP-2 and ERK1/2 were performed with the image analysis system.Result:With the increase of U0126 dose,the volume of hole increased on day 14(p<0.05).In the lesion area,the expressions of GFAP and ERK1/2 could be found,while MAP-2 could not.Significant differences were revealed in the expressions of GFAP、ERK1/2 among the four groups,it Was significantly lower in U0126 groups than that in control greup(P<0.05).while the middle-dose group had similar effect with the high-dose group(P>0.05).Conclusion:U0126 of different doses all could resupinate astrocyte migrations which were coused by low frequency magnetic stimulation,and 0.2mg/kg was the suitable dose.
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Objective To detect the serum prohibitin protein(PHB)level in children with renal interstitial damage and analyze the correlation between PHB and renal interstitial fibrosis(RIF). Methods Serum PHB protein levels were determined by Western blot analysis in 36 children with kidney diseases,and 30 healthy children were studied as control. Levels of BUN,Scr,and urinary microprotein series(including ALBU/Cr,NAGU/Cr,IgG U/Cr,α1-MU/Cr)were studied by automatic biochemical analyzer. Renal interstitial damage was semiquantitatively graded according to Katafuchi's method. The correlation between serum levels of serum PHB protein and those of BUN,Scr as well as urine microprotein were analyzed. Results Serum PHB protein was positive in children with diverse kidney diseases however it was negative in the normal controls(P ＜ 0.05). Serum PHB levels were significantly higher in children with proliferative glomerulonephritis than those with non-proliferative glomerulonephritis(P ＜ 0.05). Statistical analysis indicated that serum PHB levels positively correlated with the degree of tubulointerstitial lesions(r ＝ 0.868,P ＜ 0.001)as well as the glomerular injuries(r ＝ 0.753,P ＜ 0.001). And,serum PHB levels were also positively correlated with urinary microprotein including NAG(r ＝ 0.586,P ＜ 0.001)and IgG(r ＝ 0.341,P ＜ 0.001). Conclusions Serum PHB levels were significantly increased in children with kidney diseases and were positively correlated with the degrees of renal interstitial damage,suggesting that PHB might be a potential clinical marker for detecting tubulointerstitial lesions.
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Objective To investigate the effects of epidural spinal cord electrical stimulation (ESCES) on spinal cord reflexes in normal adult rats, and to find out where and how the spinal cord reflexes are generated. Methods Ten adult female Sprague Dawley rats were anaesthetized and an electrode was placed at the S, spinal cord segment. Single electric pulses with 200 μs pulse width and voltages of 400 mV, 600 mV and 1200 mV were used in the ESCES. 1200 mV voltages with 50 Hz, 60 Hz, 80 Hz, 100 Hz frequency were also tested. EMG signals were re-corded with concentric needle electrodes in the rats' semitendinosus muscles to observe the characteristics of spinal cord reflexes. Results The voltage threshold for generating semitendinosus muscle response was 300 mV. The three ESCES voltages induced 2 kinds of spinal cord reflexes. The 400 mV and 600 mV stimulation induced spinal cord reflexes with short latency (5.27±0.36 ms and 5.19±0.67 ms respectively). The 1200 mV stimulation volt-age induced spinal cord reflexes with long latency (2.57±0.23 ms). Spinal cord reflexes could be generated by 50 Hz, 60 Hz, 80 Hz, and 100 Hz ESCES. At the higher frequencies, spinal cord reflexes declined late in the ex-periments and then appeared irregular. In some of the rats, spinal cord reflexes vanished entirely late in the stimula-tion experiments. The latency and duration of the spinal cord reflexes induced by 50 Hz ESCES were (4.46 ± 1.07) ms and (7.33±1.00)ms respectively. These were significantly different from the latency and duration initia-ted by 60 Hz, 80 Hz or 100 Hz ESCES. Conclusions Different ESCES voltages induce different spinal cord refle-xes generated differently. The long latency reflexes might be monosynaptic responses mediated by dorsal root excite-ment, while the short latency reflexes might be sarcous exciting electric activity mediated by direct excitement of mo-tor neurons or motor fibers. The irregular spinal cord reflexes induced by higher frequency ESCES might be one kind of monosynaptic response. Irregularly appearing spinal cord reflexes induced by higher frequency stimulation might due to the inhibitory effect of higher frequency stimulation.
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Objective To explore the potential of creating a cat model of spinal cord infarction initiated by a photochemical reaction in terms of the neuroethology, motor evoked potential, and morphological outcomes. Meth-ods Fifteen cats were divided into three groups at random. T_13 in the spinal cord was photochemically irradiated for 40 min, 60 min or 80 min in different groups. A photochemically-induced infarction was produced by intravenous in-jection of rose Bengal (35 mg/kg) combined with immediate cold light irradiation (3000 klx) of the spinal cord.Neuroethology changes were observed every day after the surgery far 21 days, and morphological changes were exam-ined at day 21, before which the motor evoked potential was examined and compared with measurements taken before injury. Results The spinal cord infarctions induced by intravenous injection of rose Bengal plus cold light irradia-tion for 40 min were stable by day 8, while the other two groups were stable by day 12. The size of the infarction area in the spinal cord was stable, while the neuroethology, electrophysiological and histopathological changes in the cats were significant. Conclusions All of the cats demonstrated decreased functional mobility after photochemically in-duced thrombosis of the spinal cord, with corresponding pathomorphological and electrophysiologic changes. The model of infarction was stable and reliable.
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Objective To investigate the effect of magnetic stimulation on astrocyte migration and its mech-anism. Methods Twenty-four adult, healthy Spragne-Dawley rats were injected with 0.5 μl of 1% ethidium bro-mide (EB) in the left side of the dorsal spinal cord funiculus at the T_(10-11) level to make a local spinal cord injury mod-el. They were then randomly divided into four groups and exposed to 30 pulses of magnetic stimulation at 1 Hz and the following intensities: O T (Group A);1.9x40% T (Group B); 1.9x80% T (Group C); 1.9x100% T (Group D). On the 14th day after stimulation, the rats were sacrificed and the expression of glial fibrillary acidic protein (GFAP), microtubule associated protein-2 (MAP-2) and extracellular signal-regulated kinase 1/2 (ERK 1/2) were detected, and the volume of holes in the injured area of the spinal cord was measured. Quantitative analysis of the GFAP, MAP-2 and ERK1/2 expression was performed using immunohistochemistry and an image anal-ysis system. Results The volume of holes in the injured area of the spinal cord decreased with increasing stimula tion intensity. In the reduced area of the holes, the expression of GFAP and ERK 1/2 could be seen, but not MAP-2. Significant differences were revealed in the expression of GFAP and ERK 1/2 among the four groups, but it was always significantly higher in the magnetic stimulation groups than in the controls. Conclusions After magnetic stimulation, astrocytes migrate into the injured spinal cord's holes. Astroeyte migration increases with increased mag-netic stimulation intensity, which may be associated with high expression of ERK 1/2.
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Objective:To investigate the effects of epidural spinal cord stimulation (ESCS) and treadmill training on the locomotion function and ultrastructure of spinal cord anterior horn after moderate spinal cord injury in rats. (IT, n=3). All rats received a moderate spinal cord injury surgery. Four weeks after surgery, rats in SE group received an electrode implantation procedure, with the electrode field covering spinal cord segments L2-S1. Four weeks after electrode implantation, rats received subthreshold ESCS for 30 min/d. Rats in TY group received 4cm/s treadmill training for 30min/d. Rats in SI group received no intervention, as a control group. All procedures in these three groups lasted four weeks.The open field Basso,Beattie and Bresnahan (BBB) scale was used before and after intervention to evaluate rats' hindlimb motor function. Result:After four weeks intervention, rats in TT group improved their open field locomotion scores to 20. In contrast, no significant improvement was observed in groups SI and SE. The morphology of synapses and neurons were similar regardless of whether rats had undergone ESCS, treadmill training or not. Conclusion:ESCS alone was not sufficient to improve the walking ability of spinal cord injured rats. ESCS or treadmill training alone might not contribute to the changes of ultrastructure in anterior horn of spinal cord that underlie the recovery of walking ability. Further research is needed to understand the contributions of combination of ESCS and treadmill training to the rehabilitation of spinal cord injured rats.
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Objective:To investigate the expression pattern of Mtsl/S100A4 in mouse spinal cord;to investigate the effects of Mtsl/S100A4 on glial cell responses.Method:The study was carried out on Mtsl/S100A4 wild type and knock-out mice.The degenerative spinal cord model was established by dorsal root or sciatic nerve injury.The de-myelinated spinal cord model was established by ethidium bromide injections.Then the expressions of S100A4,GFA P,NG2 and Mael were measured.Result:The expressions of Mtsl/S100A4 in mice spinal cord were similar to that in rats.In WT mice this protein expressed in a thin layer of fiber bundles in the tract of Lissauer,and in white matter astrocytes.There was intracellular up-regulation of Mtsl/S100A4 in white matter astrocytes of WT mice after dorsal root or sciatic nerve injury,with no difference in glial cell response between WT and KO mice.However,7 days after ethidium bromide injection,in WT mice,the astroglial reaction was restricted on operated side,where a distinct glial scar had formed.While in KO mice,no distinct glial scar formed in demyelinated area.Conclusion:Mtsl/S100A4 expression in mouse spinal cord is similar to the pattern as in rats;intracellular Mtsl/ S100A4 up-regulation does not affect glial responses in degenerative spinal cord;the presence of extracellular Mtsl/ S100A4,which entered the spinal cord after ethidium bromide induced demyelination,markedly affects the glial cell responses in demyelinative spinal cord,including glial scar formation.
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BACKGROUND: Puerarin possesses various biological efficacies, such as the protective efficacy on hypertension, cardiac disease, diabetes mellitus and blood disease, and the extracts of puerarin can inhibit the proliferation of S180 sarcoma and Lewis lung cancer to some extent.OBJECTIVE: To investigate the modulation effects of total flavone of puerarin (TFP) on the growth of pheochromocytoma PC12 cells and the protective efficacy on the H2O2-induced cellular oxidative damage.DESIGN: Complete randomization design and control experiment.SETTING: Department of Biochemistry and Institute of Geriatrics, Chinese PLA General Hospital.MATERIALS: Puerarin was bought from Tongrentang drug store, and TFP was extracted and purified routinely by ethanol and ether acetate, then was evaluated with thin-layer chromatography. The content of puerarin in extracts was 31.79% in quantitative assay. Methyl thiazolyl tetrazolium (MTT), luminal and anti-oxidative activity reagent xanthine oxidase were all from Sigma Company. PC12 cells were given by Institute of Geriatrics,Chinese PLA General Hospital as a present.METHODS: The experiment was conducted at the Institute of Geriatrics, Chinese PLA General Hospital from January to July in·2001.① The cells were cultured in 20 g/L DMEM (pH 7.1-7.2), and randomized into two groups: TFP group and H2O2-injury+TFP group, and each group was divided into 5 mass concentrations (0, 1.0, 10, 100 mg/L and 1.0 g/L). There were 8 holes for parallel culture with 100 mL culture medium in each hole (containing 1 ×l09 L-1 cells). TFP group:TFP was added for 72-hour culture at 37 ℃; H2O2 injury+TFP group:TFP was firstly cultured for 48 hours at 37 ℃, then 500 mmol/L H2O2 was added and co-cultured for other 24 hours.②The activity of cultured PC12 cells was monitored by MTF assays, the content of nitrite was measured by Griess reagents, and the antioxidant activity of superoxidedismutase (SOD) was monitored by hypoxanthine/xanthine oxidase.H2O2-initiated PC12 cellular oxidative damage had been used as experimental model to study the protective efficacy of TFP, and expressed as inhibition ratio [(blank A value-detection A value)/blank A value × 100%]. The higher inhibition ratio indicated the strong ability of clearing O2-. ③ One-factor analysis of variance was used to compare the difference between data. MAIN OUTCOME MEASURES: The influence of TFP on the nitrite content, SOD activity and cell activity in PC12 cells.RESULTS: ①Effect of TFP on PC12 cell activity: 1-10 mg/L TFP hadno obvious effects on the growth of PC12 cells, and 100 mg/L TFP in creased the cell growth (P ＜ 0.05), whereas the TFP concentration was increased to 1.0 g/L, the activity of PC12 cells was inhibited obviously (P ＜ 0.01). TFP of 1-100 mg/L could protect the cultured cells from the oxidative damages of H2O2 concentration dependently (P ＜ 0.05).② Effect of TFP on clearing O2-: The ability of clearing O2 increased withthe mass concentrations of TFP in both groups with obvious dose-effect relation, except when 1 mg/L TFP was added in the H2O2 injury+TFP group. The SOD activity in PC12 cell culture liquid was obviously en hanced after adding 100 mg/L and 1 g/L TFP, compared with that without TFP addition (P ＜ 0.05-0.01). ③Modulation of TFP on nitrite: TFP of low concentration (1-100 mg/L) reduced the production of cellnitrite, whereas increased the nitrite production at the concentration of1.0 mg/mL.CONCLUSION: ①TFP can regulate the growth of PC12 cells, which canbe enhanced by low-concentration (1-100 mg/L) TFP whereas inhibited byhigh-concentration (1 g/L) TFP. However, the anti-oxidation of TFP is themost powerful. ②TFP can protect the PC12 cells obviously from the oxidative damages induced by H2O2 at low concentration.