| Objective In this study,a rat SCI model was established in order to study the protective effect of harpagide administration,and to explore its possible mechanism(s)of action.This provided an experimental basis for the application of harpagide in the prevention and treatment of SCI.We demonstrated that there was potential future therapeutic value for harpagide in the treatment of SCI.Materials and Methods 1.Animals and grouping,Healthy adult male Sprague-Dawley rats(weighing 190–230 g)were purchased from the Animal Center of Nanjing Medical University(Nanjing,Jiangsu,China).Rats were housed in an SPF laboratory animal center in a constant environment(23±0.5°C)with a 12:12 h light:dark cycle,Rats were randomly divided into three groups: control group,SCI-only group,and harpagide group.The SCI group and the harpagide group were given intraperitoneal injections of physiological saline and harpagide(10 mg/kg,dissolved in DMSO at 3 mg/m L),respectively,1 h after spinal cord injury,and then once a day for 2 days.2.SCI model.A rat model of acute SCI was established using a modification of the Allen method.3.The well-established Basso-Beattie-Bresnahan(BBB)scoring method was used to evaluate motor function.4.Primary neuron culture.Newborn SD rats were immersed in 75% ethanol within 4 days after birth.Skin and cartilage were incised on the back,spinal cord was separated,cultured and observed.The purity of neuron culture was evaluated by anti-microtubule-associated protein MAP-2 and Neu N antibodies after 7 days.5.Cell viability assay.Cell viability was assessed using the Cell Counting Kit-8(CCK-8)assay(Dojindo,Kumamoto,Japan).6.Apoptosis detection.After 24 h of treatment with 100 ?M GLU,neurons were incubated with or without 50 ?M harpagide.7.Western blot analysis.Total protein was extracted from cells,and the protein concentration was measured using a BCA assay.8 Tissue processing.Rats were anesthetized with lethal dose of chloral hydrate in abdominal cavity to separate the intact spinal cord.After treatment,the spinal cord was sliced and stored for use.9.Neuronal immunofluorescence staining.The apoptotic cells of spinal cord were identified and quantified by TUNEL on the7 th and 28 th day after injury according to the manufacturer's procedure.The images were obtained and the apoptotic nuclei were characterized by green fluorescence.10.Spinal cord tissue immunofluorescence.11.Statistical analysis Analysis was performed using SPSS 17.0 statistical software.Results are expressed as the mean±standard deviation.Differences between groups were analyzed using a one-way analysis of variance followed by Bonferroni's post-testResults 1.Harpagide treatment improves functional behavioral recovery after SCI.The BBB score of the harpagide group was consistently and significantly(p<0.05)higher compared to the SCI group 2.Neun staining showed that the number of motoneurons in the anterior horn of rats in the Harpagide group was larger than that in the SCI group,suggesting that Harpagide increased the survival rate of motoneurons and reduced the size of lesions after spinal cord injury.3.The number of TUNEL positive neurons in SCI group was more than that in control group.However,the percentage of TUNEL-positive neurons in the Harpagide group was significantly lower than that in the SCI group.These results suggest that Harpagide protects neurons from spinal cord injury in rats.4.The decrease of NF200 staining and the increase of GFAP staining in the lesion area of Harpagide group were much lower than those of SCI group,suggesting that Harpagide administration significantly promoted axon regeneration and inhibited glial scar formation after SCI.5.Results showed that the level of ?-catenin protein in spinal cord neurons was significantly higher in rats treated with harpagide,compared to the control and SCI-only groups.In addition,harpagide significantly enhanced the expression of c-myc and cyclin D1 proteins 14 days post-SCI.These results indicated that harpagide activated the Wnt/?-catenin signaling pathway in spinal cord neurons after SCI.6.With increased harpagide concentration,a decrease in cell viability was observed.A harpagide concentration of 100 ?M showed significantly higher cytotoxicity than other concentrations(p<0.05).7.Harpagide can reduce neuronal cell apoptosis in vitro.8.Inhibition of the Wnt/?-catenin signaling pathway reduces the anti-apoptotic effect of harpagide in primary spinal cord neurons.Conclusions Specifically,the administration of harpagide after SCI increased the expression levels of ?-catenin,c-myc and cyclin D1 proteins in spinal cord neurons,as well as increased the number of motor neurons and reduced the size of the SCI lesion area.In addition,the administration of harpagide after SCI also decreased the protein expression levels as well as the number of cells immuno-stained for the pro-apoptotic proteins Bax and cleaved-caspase 3.The expression level of the anti-apoptotic protein Bcl-2 was also increased.When the Wnt /?-catenin signaling pathway was inhibited,a weakened anti-apoptotic effect of harpagide was observed.Additionally,the application of harpagide led to an increase in NF200 staining and a reduction in GFAP staining in the SCI injury site.In summary,our study suggested that harpagide may be a promising drug for the treatment of SCI. |
PDF Full Text Request