ABSTRACT
Resumen La humanina es un péptido derivado de la mitocondria con efectos protectores robustos contra una gran variedad de estímulos citotóxicos en diversos tipos celulares. Esto la convierte en un blanco terapéutico interesante para muchas enfermedades, como el cáncer y enfermedades neurodegenerativas, entre otras. Además, este péptido podría utilizarse como un biomarcador en estas enfermedades. Durante la última década, han sido desarrollados análogos y péptido-miméticos de la humanina que muestran resultados prometedores en modelos preclínicos. A su vez, también se está explorando el potencial terapéutico de vectores de terapia génica que puedan sobreexpresar o silenciar la humanina endógena. Varios puntos importantes a considerar antes de trasladar estas estrategias terapéuticas a la clínica son su posible papel en la progresión del cáncer y la eventual generación de quimiorresistencia. Todos estos temas serán abordados en este artículo de revisión.
Abstract Humanin is a mitochondrial-derived peptide which shows robust protective effects against large series of cytotoxic stimuli in many cell types. This makes it an interesting therapeutic target for many diseases, including cancer and neurodegenerative diseases, among others. Furthermore, this peptide could be used as a biomarker for such diseases. Over the last decade, humanin analogs and peptide mimetics have been developed, which exert highly promising results in preclinical models. Besides, the therapeutic potential of gene therapy vectors that overexpress or silence endogenous humanin is under evaluation. Nonetheless, its possible role in cancer progression and chemoresistance are critical issues to be addressed before translating these therapeutic approaches to the clinic. All these matters will be covered in this review.
Subject(s)
Neurodegenerative Diseases , Volition , Disease , NeoplasmsABSTRACT
Humanin (HN) is a mitochondrial peptide that exhibits cytoprotective actions against various stresses and diseases. HN has been shown to induce the phosphorylation of AMP-activated protein kinase (AMPK), which is a negative regulator of receptor activator of nuclear factor-κB ligand (RANKL). However, the role of HN in osteoclastogenesis or other skeletal disorders remains unknown. Here, we examined whether HN regulates osteoclastogenesis via AMPK activation using bone marrow-derived macrophage (BMM) cultures. Our results show that HN inhibited RANKL-induced osteoclast formation and reduced the expression of genes involved in osteoclastogenesis, including nuclear factor of activated T-cells cytoplasmic 1, osteoclast-associated receptor, cathepsin K, and tartrate-resistant acid phosphatase. Moreover, HN increased the levels of phosphorylated AMPK protein; compound C, an AMPK inhibitor, recovered HN-induced osteoclast differentiation. In addition, we found that HN significantly decreased the levels of RANKL-induced reactive oxygen species in BMMs. Therefore, these results indicate that HN plays an important role in osteoclastogenesis and may function as an inhibitor of bone disorders via AMPK activation.
Subject(s)
Acid Phosphatase , AMP-Activated Protein Kinases , Cathepsin K , Cytoplasm , Macrophages , Osteoclasts , Phosphorylation , Reactive Oxygen Species , T-LymphocytesABSTRACT
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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GH/IGF-1 axis is involved in the whole process of human's growth,and its trait of activity would change in varied ages that might affect human longevity.Humanin is a novel mitochondriai-associated polypeptide which has wide-spectrum neural and cellular protection.Hormone and its derivatives play a positive role on a variety of age-related diseases,such as stroke,myocardial ischemia reperfusion,atherosclerosis,diabetes,etc.Recently,relevant studies show a negative correlation between the plasma level of Hormone and the activity of GH/IGF-1 axis,while a positive correlation between the plasma level of Hormone and human longevity.Additionally,evidences have demonstrated that Hormone and its derivatives can protect the beta cells as well as improve insulin sensitivity,which is greatly expected to become an effective means of prevention and treatment to diabetes in the future.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
AIM: To study the effects of [Gly~(14)]-humanin,one of the strongest derivate of humanin,on proliferation and differentiation of neural stem cells(NSCs) and the protective action of cell death or apoptosis induced by ?-amyloid protein 1-42.METHODS: NSCs were treated with different concentrations of [Gly~(14)]-humanin and ?-amyloid protein 1-42.RESULTS: 10 nmol/L [Gly~(14)]-humanin made NSCs resistant to the apoptotic action induced by A?P1-42 and prevented NSCs from death induced by 25 ?mol/L ?-amyloid protein 1-42.The differentiated neural stem cells yield more neuronal cells than that in control groups when 10 nmol/L [Gly~(14)]-humanin was added to the culture media.The number of cells increased and the cultures grown with a manner of floating cell clones likes that cultured in the presence of mitogen when 100 nmol/L [Gly~(14)]-humanin was added to the differentiation culture media.CONCLUSION: The [Gly~(14)]-humanin significantly promoted the proliferation and neuronal differentiation of neural stem/progenitor cells and also inhibited the toxic action of ?-amyloid protein 1-42 on cultured neural stem/progenitor cells.