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Objective To study the effect of [Gly14]-Humanin on oxidative stress and neuron apoptosis after focal cerebral ischemia reperfusion injury in rats. Methods A rat model of acute middle cerebral artery occlusion(MCAO)and reperfusion was established by suture embolism. Ninety-six healthy male Sprague-Dawley rats were randomly divided into sham-operation group, model group, normal saline group and HNG group. And 5 μL HNG(100 nmol/L)in the rats of the HNG group, normal saline in the rats of the sham-operation group and normal saline group were given to rats 3 days before operation respectively(iv, three times, qd). After cerebral ischemia 3 h and 24 h of reperfusion, neurological deficit score(NDS) were performed for each group, and the activity of sod, levels of GSH and MDA in cerebral tissue were detected. TUNEL staining were used to detect neuronal apoptosis. Results Median(Interquartile range)of NDS was 0(0)in sham-operation group, 1.5(1)in model group, 3(1)in normal saline group and 3(1)in HNG group, respectively, and the NDS of sham-operation group was significantly lower than the other three groups. The NDS of HNG group was significantly lower than the model group and normal saline group(all P<0.05). Compared with sham-operation group, the activity of SOD of model group, normal saline group and HNG group rats[(11.65±1.66),(12.15±1.56)and (19.43±1.47)U/mL]and levels of GSH[(8.84±1.23),(7.51±1.16)and (11.17±1.67)mg/L]were decreased, and levels of MDA[(42.61±1.79), (40.33±1.24)and (35.39±1.29)nmol/mL]were increased(P<0.05). The activity of SOD and levels of GSH of HNG group were significantly higher than model group and normal saline group, but the levels of MDA was significantly lower than model group and normal saline group(all P<0.05). Compared with sham-operation group[(0.13±0.01)%], percentage of neuronal apoptosis of model group, normal saline group and HNG group rats[(0.59 ±0.07)%,(0.51 ±0.08)% and (0.22 ±0.03)%, respectively]were increased(P<0.05), and percentage of neuronal apoptosis of HNG group was significantly lower than model group and normal saline group(all P<0.05). Conclusion By increasing the activity of SOD and levels of GSH,[Gly14]-Humanin can resist focal cerebral ischemia reperfusion injury, and can decrease the neuro apoptosis of ischemic area, thus mitigate the neurologic deficit.
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Objective To investigate the effects of [Gly14]-Humanin(HNG) on SOD, MDA, GSH and cell apopto?sis in a rat model of secondary brain injury. Methods One hundred thirty-five adult and healthy male rats were random?ly divided into 3 groups: sham model group (n=45), vehicle control group (n=45) and HNG group (n=45). Secondary brain injury was induced in the vehicle control and HNG groups using improved Feeney method. Vehicle control received abdominal injections of Sodium Chloride Injection (2 ml/kg) whereas the HNG group received abdominal injections of HNG (2 μL/kg) immediately and 24 h after injury. Each group was divided into three subgroups (n=15 rats per each group) by sacrificed time including 1 h, 3 d, and 7 d after injury. The expression levels of SOD, MDA and GSH of the brain tissue were analyzed and the cell apoptosis was examined using TUNEL method after brain contusion. Results MDA and cell apoptosis around the lesion started to increased at 1h, reached a peak at 3d and then gradually subsided but still remained a higher level at 7 d than 1 h. HNG significantly attenuated brain injury-induced increase in MDA and apopto?sis at all time points (P<0.05). By contrast, SOD started to decrease at 1h, reached the lowest point at 3 d and then gradu? ally recovered but still remained a lower level at 7 d than 1 h. HNG significantly mitigated brain injury-induced increase in MDA and apoptosis at all time points (P<0.05). The time course of GSH expression followed a pattern similar to that of MDA. MDA expression was strongly positive correlated with the number of cell apoptosis (r=0.720, P<0.05), strongly neg?ative correlated with the level of SOD and GSH(r=-0.702, P<0.05;r=-0.674, P<0.05). Conclusions After brain injury, HNG inhibits oxidative stress levels and reduces apoptosis, thereby mitigating secondary brain injury.
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Objective To investigate the expression of neuroprotective peptide [Gly14]-Humanin (HNG) in eukaryotic cells by gene engineering technique and analyze its biological activity. Methods By means of asymmetrical primer/template, double stranded cDNA of HNG with FLAG in its C-terminal was obtained, which was cloned into the plasmid pcDNA3.1(-), and the resultant recombinant vector pcDNA3.1(-)/HNG-FLAG was transfected into PC12 cells. At the same time, the recombinant vector pcDNA3.1(-)/EGFP was transfected to control the efficiency of transfection. The expression of HNG in the cells was determined by immunocytochemistry. In order to analyze the biological activity of the expressed HNG, 25μM Aβ25-35 peptide was added to the culture medium of the transfected cells for 24h, then cell morphology, MTT assay and Hoechst 33258 staining were observed. Results The eukaryotic expression vector of pcDNA3.1(-)/HNG-FLAG was identified by enzyme digestion and sequencing. HNG was highly expressed in PC12 cells. After exposure of PC12 cells to 25μM Aβ25-35 for 24h, cell viability decreased to (65.8±5.3)%, and the dystrophic changes of neuritis and nuclei condensation were obvious. When cells were pre-transfected with pcDNA3.1(-)/HNG-FLAG, Aβ25-35-induced cell death and morphological changes of cells and nuclei were suppressed. In contrast, pre-transfected with empty vector did not protect cells from Aβ25-35-induced toxicity. Conclusion The eukaryotic expression vector for FLAG-tagged HNG was successfully constructed and expressed in PC12 cells. Expressed HNG has biological activity.
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Objective: To investigate the expression of neuroprotective peptide [Gly14]-Humanin (HNG) in eukaryotic cells by gene engineering technique and analyze its biological activity. Methods: By means of asymmetrical primer/template, double stranded cDNA of HNG with FLAG in its C-terminal was obtained, which was cloned into the plasmid pcDNA3.1(-), and the resultant recombinant vector pcDNA3.1(-)/HNG-FLAG was transfected into PC12 cells. At the same time, the recombinant vector pcDNA3.1 (-) /EGFP was transfected to control the efficiency of transfection. The expression of HNG in the cells was determined by immunocytochemistry. In order to analyze the biological activity of the expressed HNG, 25 μM Aβ25-35 peptide was added to the culture medium of the transfected cells for 24 h, then cell morphology, MTT assay and Hoechst 33 258 staining were observed. Results: The eukaryotic expression vector of pcDNA3.1(-)/HNG-FLAG was identified by enzyme digestion and sequencing. HNG was highly expressed in PC12 cells. After exposure of PC12 cells to 25 μM Aβ25-35 for 24 h, cell viability decreased to (65.8±5.3)% , and the dystrophic changes of neuritis and nuclei condensation were obvious. When cells were pre-transfected with pcDNA3.1(-)/HNG-FLAG, Aβ25-35-induced cell death and morphological changes of cells and nuclei were suppressed. In contrast, pre-transfected with empty vector did not protect cells from Aβ25-35-induced toxicity. Conclusion: The eukaryotic expression vector for FLAG-tagged HNG was successfully constructed and expressed in PC12 cells. Expressed HNG has biological activity.
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Humanin (HN, its analogue [Gly14]-Humanin, HNG) was originally identified as an endogenous peptide that protects neuronal cells from apoptosis induced by various types of Alzheimer's disease-related insults. But the relative low content of this peptide in its natural sources limits its further characterization. An expression vector pET32a/HNG was corstructed and transformed it into E. coli BL21 trxB (DE3). HNG was expressed as a fusion protein in the soluble fraction and was purified by nickel affinity chromatography. Subsequently, the purified fusion protein was cleaved by enterokinase and was further purified by reverse-phase HPLC. A 23 mg recombinant HNG (rHNG) from 1 L bacterial culture was purified. The molecular weight of rHNG determined by ESI-MS was 2876.5 Da which was the expected size for correctly processed peptide. The N-terminal amino acid sequence of rHNG determined by Edman degradation method is identical to the theoretical sequence. Neuroprotective bioassay studies of rHNG exhibited its potential neuroprotective effect comparable to that of the natural HNG peptide.
RESUMEN
Humanin (HN, its analogue [Gly14]-Humanin, HNG) was originally identified as an endogenous peptide that protects neuronal cells from apoptosis induced by various types of Alzheimer's disease-related insults. But the relative low content of this peptide in its natural sources limits its further characterization. An expression vector pET32a/HNG was corstructed and transformed it into E. coli BL21 trxB (DE3). HNG was expressed as a fusion protein in the soluble fraction and was purified by nickel affinity chromatography. Subsequently, the purified fusion protein was cleaved by enterokinase and was further purified by reverse-phase HPLC. A 23 mg recombinant HNG (rHNG) from 1 L bacterial culture was purified. The molecular weight of rHNG determined by ESI-MS was 2876.5 Da which was the expected size for correctly processed peptide. The N-terminal amino acid sequence of rHNG determined by Edman degradation method is identical to the theoretical sequence. Neuroprotective bioassay studies of rHNG exhibited its potential neuroprotective effect comparable to that of the natural HNG peptide.
RESUMEN
Humanin (HN, its analogue [Gly14]-Humanin, HNG) was originally identified as an endogenous peptide that protects neuronal cells from apoptosis induced by various types of Alzheimer's disease-related insults. But the relative low content of this peptide in its natural sources limits its further characterization. An expression vector pET32a/HNG was corstructed and transformed it into E. coli BL21 trxB (DE3). HNG was expressed as a fusion protein in the soluble fraction and was purified by nickel affinity chromatography. Subsequently, the purified fusion protein was cleaved by enterokinase and was further purified by reverse-phase HPLC. A 23 mg recombinant HNG (rHNG) from 1 L bacterial culture was purified. The molecular weight of rHNG determined by ESI-MS was 2876.5 Da which was the expected size for correctly processed peptide. The N-terminal amino acid sequence of rHNG determined by Edman degradation method is identical to the theoretical sequence. Neuroprotective bioassay studies of rHNG exhibited its potential neuroprotective effect comparable to that of the natural HNG peptide.
RESUMEN
Humanin(HN,its analogue [Gly14]-Humanin,HNG)was originally identified as an endogenous peptide that protects neuronal cells from apoptosis induced by various types of Alzheimer's disease-related insults.But the relative low content of this peptide in its natural sources limits its further characterization.An expression vector pET32a/HNG was corstructed and transformed it into E.coli BL21 trxB(DE3).HNG was expressed as a fusion protein in the soluble fraction and was purified by nickel affinity chromatography.Subsequently,the purified fusion protein was cleaved by enterokinase and was further purified by reverse-phase HPLC.A 23 mg recombinant HNG(rHNG)from 1 L bacterial culture was purified.The molecular weight of rHNG determined by ESI-MS was 2876.5 Da which was the expected size for correctly processed peptide.The N-terminal amino acid sequence of rHNG determined by Edman degradation method is identical to the theoretical sequence.Neuroprotective bioassay studies of rHNG exhibited its potential neuroprotective effect comparable to that of the natural HNG peptide.