Morphological changes of optic nerve and retina after injury in the guinea pig

AIM: In order to understand the pathological changes, characteristic of degeneration in optic nerve and retina after strike of optic nerve. METHODS: According to methods of Allen's spinal injury, a 600gcm-strike power was put on the intraocular portion of the optic nerve and created a striking injury on optic nerve. After a survival interval of 48 h, 1 week, 2 weeks, 4 weeks, 3 months, the animal's optic nerves and retinas were collected and fixed for morphological examination. RESULTS: Forty-eight hours after nerve injuries, the optic nerves were slight enlargement and vacuolation. In 1 week, the optic nerve began to degenerate in injured part and the glia cell had proliferated, but the forms of retinal ganglion cells(RGCs) were normal. In 2 weeks, the vacuolation and focal necrosis were appeared between nerve fiber. The number of RGCs began to decrease. Condensed nuclei presented in the retina. In three month, the diameter of the optic nerve decreased in injury part and collo-scar was formed. The phenomenon mentioned above was more obviously. The internal nuclear neurons and outer nuclear neurons appeared rare. The thickness of retina decreased. The number of RGCs began to decrease in 48 hours and progressed thereafter. It decreased about 3.35%, 13.23%, 19.74%, 23.20%, 29.28% in 48 h, 1 week, 2 weeks, 1 month, 3 months compared with the number of normal RGCs. RGCs began to apoptosis in 48 h. CONCLUSION: The model in this experiment could make definite uncompleted optic nerve and retina injuries. The degree of neuron injuries decreased from RGC, internal nuclear neurons to outer nuclear neurons. The number of RGCs began to decrease in 48 hours, and most quickly periods from 48 hours to one week.     

Protective effect of activated macrophages on retinal ganglion cells after optic nerve crush

AIM:To investigate effects of factors derived from activated macrophages on survival of retinal ganglion cells (RGCs) after optic nerve crush. METHODS:Adult Wistar rats were randomly divided into normal control, crush control, medium treatment and zymosan treatment groups. All the rats were retrogradly by injecting fluorogold into superior colliculi of both sides. Partial optic nerve injury model was induced in the left eyes by a special designed optic nerve clip with 40 gram power for 9 seconds at optic nerve 2 mm behind the eyeball except for normal control group. The numbers of the labeled RGCs in each group were counted. RESULTS:The numbers of RGCs in crush group was 92.6%, 82.9%, 72.6% and 57.6% of normal controls on the third, 7th, 15th and 21th day, respectively. In the group with zymosan injection, the number of RGCs were much higher than that in controls on the third, 7th, 15th and 21th day (P<0.01). CONCLUSION:Intravitreal injection of zymosan activates macrophages. Macrophages-derived factors might protect RGCs after optic nerve crush and improve their survival.     

Effect of VEGF on proliferation and differentiation of neural stem cells from the hippocampal gyrus of rat in vitro

AIM: To observe the effects of vascular endothelial growth factor (VEGF) on the proliferation and differentiation of neural stem cells (NSCs) of rats in vitro.METHODS: NSCs isolated from the hippocampal gyrus of SD rats were primary cultured and subcultured,and then divided into two groups: (1) the cells in VEGF group were treated with 150 μg/L VEGF in the culture system,and VEGF was removed at the 7 th day;(2) control group (without VEGF treatment).The cellular morphology of two groups was observed by contrast phase microscope.Nestin and NF-200 expressing cells were detected via immunofluorescence method.The percentages of the immunostaining positive cells in each group at the 7 th day and at the 11 th day were determined.RESULTS: At the 7 th day,the percentage of nestin positive cells in VEGF group was 52.19%±7.95%,vs 29.26%±4.12% in control group (P<0.01).The percentage of NF positive cells in VEGF group was 22.33%±4.13%,vs 38.62%±5.31% in control group (P<0.01).At the 3 th day after VEGF was removed,the percentage of NF positive cells in VEGF group was 43.10%±3.70%,vs 30.56%±4.16% in control group (P<0.01).CONCLUSION: VEGF stimulates the proliferation of neural stem cells and inhibits their differentiation.     

Effects of recombinant basic fibroblast growth factor on retinal ganglion cells of rats

AIM:To investigate the pharmacological effects of recombined basic fibroblast growth factor (rbFGF) on retinal ganglion cells (RGCs) of rats.METHODS:Using calibrated cross-action forceps a moderate crush injury was inflicted on the nerve. After crush injury, rbFGF, saline and VB₁₂ were administered by retrobublar injection. Four weeks after injury, the apoptosis of RGCs was measured with flow cytometer.RESULTS:Four weeks after operation, it was shown that the rbFGF, but not saline or VB₁₂ injection could significantly improve the maintainance of RGCs of rats. After 800 U, 1600 U and 2400 U rbFGF injection, the injured RGCs were rescued by 24.5%, 27.3% and 28.5% respectively. Furthermore, it was also found that rbFGF injection could effectively prevent the axons from injury. The flow cytometer showed that the rate of apoptosis was reduced markedly on 7 days at rbFGF group. CONCLUSION:rbFGF can significantly promote the functional repair of injured optic nerve.     

Functional improvement of infarct heart by implantation of human adipose-derived mesenchymal stem cells delivered by matrigel

AIM: To investigate the implantation of matrigel carrying human adipose-derived mesenchymal stem cells to enhance the cell survival and the improvement of the ventricular functions in infarct heart.METHODS: Human adipose-derived mesenchymal stem cells (ADMSCs) were isolated and cultured from adult adipose tissue. SD rats with one-week-old myocardial infarction were randomly received the following 4 treatments: injection of PBS, matrigel, PBS+ADMSCs or matrigel+ADMSCs, respectively. Labeled ADMSCs either in matrigel or in saline were injected into the border area of ischemia. The controls received the injection of matrigel or saline only. Four weeks after injection, the heart functions were determined by echocardiography. The densities of the micro-vessels within the infarct area were also measured.RESULTS: Four weeks after implantation of ADMSCs, the cell graft size, the heart functions and the micro-vessel densities within the infarct area improved in matrigel+ADMSCs group as compared to other groups.CONCLUSION: The co-injection of ADMSCs with matrigel enhances the graft survival, increases the density of the micro-vessels in the myocardium and improves the cardiac functions.     

Survival and neuronal differentiation of transplanted stem cells derived from embryonic hippocampus in adult rats with stroke lesions

AIM: To study whether exogenous neural stem cells (NSCs) derived from embryonic hippocampus differentiates into the neurons after transplanted into the infarct periphery of the brain in a stroke model and to further investigate the behavioral improvement in the rats.METHODS: The NSCs were prepared after isolated from the embryonic hippocampus of green fluorescent protein (GFP)-transgenic rats and cultured. The NSCs were identified using nestin and doublecortin(DCX) as markers. The cortical infarction in rats was induced using photochemical method, named photothrombotic cortical injury (PCI). Twenty adult rats were randomly divided into NSC transplantation group (NSC group) and control group. The cultured NSCs were transplanted into the infarct periphery of the rats in NSC group, and nothing in control group at first day was applied after PCI. The locomotor behavior of animals was checked using the rotarod test at 1st, 7th, 14th and 21st d after PCI. The survival and differentiation of transplanted NSCs were evaluated by immunocytochemical method at 12th week after PCI.RESULTS: The cells derived from the embryonic hippocampus significantly expressed the markers of NSCs. The grafted cells survived at least 12 weeks in the infarct periphery of adult rats and differentiated into mature glial cells and neurons. The density of NeuN+/GFP+ and volume of grafts at 12th week were less than those at 3rd week after transplantation (P<0.05). The time standing on the rod was longer in NSC group than that in control group at 7th, 14th and 21st d after PCI (P<0.01).CONCLUSION: The NSCs derived from embryonic hippocampus survive in the infarct periphery of adult rats up to 12 weeks and differentiate into mature neurons, which might be associated with the improvement of locomotor behavior of stroke animals. The neuronal replacement of neurons differentiated from NSCs may be the underlying mechanism.     

Minocycline suppresses sodium nitroprusside-induced apoptosis in PC12 cells by regulating PI3K/Akt signaling

AIM：To explore the role of PI3K/Akt signaling in the anti-apoptotic effect of minocyline (MC) on the apoptosis of PC12 cells induced by sodium nitroprusside (SNP). METHODS：PC12 cells were divided into 4 groups: blank control group, SNP (500 μmol/L) group, MC (10 μmol/L)+SNP group and LY294002+MC+SNP group. The cell viability was observed by MTT assay. The expression of Akt and p-Akt was determined by Western blotting. RESULTS： The viability of the PC12 cells decreased after exposed to 500 μmol/L SNP for 24 h. Meanwhile, MC at concentration of 10 μmol/L significantly blocked the effect of SNP, such as decreasing the cell viability. Pretreatment with LY294002 for 60 min prior to exposure of the PC12 cells to MC and SNP down-regulated the expression of p-Akt induced by SNP. CONCLUSION：Minocycline regulates PI3K/Akt signaling pathway to restrain the apoptosis of PC12 cells induced by SNP.     

Rat mesenchymal stem cells differentiate into neuron-like cells induced by berberine in vitro

AIM: To investigate whether berberine can induce rat mesenchymal stem cells (MSCs) to differentiate into neuron -like cells in vitro. METHODS: MSCs were separated from young rat femurs marrow and expanded in culture medium. MSCs were induced to differentiate by berberine. The morphological changes of MSCs were evaluated by light microscope.Neuron-spcific enolase (NSE), neurofilament (NF), glial fibrillary acidic protein (GFAP) were detected by immunohistochemistry. RESULTS: Induced by berberine for 8 hours,MSCs exhibited neurotype . The expression of NSE and NF in the neuron-like cells was positive, but the glial astrocyte marker GFAP didn't express. CONCLUSION: Berberine may induce adult rat MSCs to differentiate into neuron-like cells in vitro.     

Influence of murine cytomegalovirus infection on the differentiation and the differentiation genes expression in neural stem cells

AIM: The influence of MCMV infection on differentiation and differentiation gene expression in neural stem cells(NSCs) in vitro were investigated for studying the mechanisms of brain abnormalities caused by congenital cytomegalovirus infection.METHODS: NSCs were separated from fetal BALB/C mouse, and cultured and identified in vitro. The differentiation potency of NSCs was observed by immunofluorescence. The NSCs infected by MCMV at dosage of MOI(multiplicity of infection) equaled to 5, 1 and 0.1,respectively, were cultured in differentiation medium. The morphological changes of infected cells were observed under inverted microscope. The ratios of NSCs and its differentiated cells were detected by flow cytometry. The expressions of nestin, GFAP and NSE, markers of NSCs and its differentiated cells, were studied by immunofluorescence(MOI=1). The expression of early antigen(EA) of MCMV was detected to observe the infection process. Real-time RT-PCR method was employed to measure the expression levels of the key genes Neurog2, Myc and Ccnd1 in Wnt signal pathway of NSCs at early stage of differentiation culture.RESULTS: NSCs isolated from embryonic mouse brains proliferated to form neurospheres, strongly expressed nestin and differentiated into NF-200 positive neurons or GFAP positive astrocytes. The infected NSCs did not adhere to the wall and appeared differentiation growths, but showed swollen gradually after differentiation culture. The nestin expression in the infected cells downregulated slowly and was higher than that in control groups(P<0.05). The GFAP and NSE expressions of the infected cells were lower than those in control groups(P<0.05). The early antigen(EA) of MCMV was always detected in the cells in infected groups. The ratios of nestin positive cells in infected groups were higher than those in control groups, but the ratios of GFAP and NSE positive cells of former were lower than that of the latter from 3rd to 9th d after differentiation culture(P<0.05). The levels of Neurog2 mRNA and Myc mRNA in infected groups were markedly lower than those in normal control groups on 1st d and from 1st to 4th d after differentiation culture, respectively(P<0.05). The levels of Ccnd1 mRNA of infected groups were obviously lower than those in normal control groups from 12th h to 1st d(P<0.05). These changes in infected groups became more obvious as MCMV MOI increased.CONCLUSION: MCMV significantly inhibits differentiation of NSCs to neurons and astrocytes, and leads to the decrease in differentiated cells. MCMV inhibits or interferes with the gene expression of Neurog2, Myc and Ccnd1 in Wnt signal pathway of NSCs. The effect that MCMV inhibits the expressions of differentiation and the differentiation genes in NSCs shows dose-dependent with MCMV MOI. The inhibitory effect of MCMV on the differentiation of NSCs might be induced by interfering with the expression of differentiation gene in NSCs, which is possibly the one of primary causes of brain development disorders induced by congenital CMV infection.     

Effects of iNKT cells from OVA-induced asthmatic mice combined with OVA on phenotype and function of bone marrow-derived dendritic cells in vitro

AIM:To investigate the effects of invariant natural killer T-cells (iNKT cells) from ovalbumin (OVA)-induced asthmatic mice combined with OVA on the phenotypic and functional characteristics of bone marrow-derived dendritic cells (BMDCs) in vitro. METHODS:The BMDCs from wild-type (WT) BLAB/c mice were co-cultured with purified iNKT cells from WT mice immunized and challenged with OVA in the presence of 100 mg/L OVA (iNKT cells plus OVA group) or PBS (iNKT cells plus PBS group) for 20 h, and were also cultured with 50 mg/L LPS (LPS group), 100 mg/L OVA (OVA group) or PBS (PBS group) for 20 h. The expression of MHC-Ⅱ, CD40, CD86, and CD80 on the BMDCs was measured by flow cytometric analysis, and the levels of interleukin-12 (IL-12) p70, IL-6, tumor necrosis factor-α (TNF-α) and IL-10 in the culture supernatant were measured by ELISA. Splenic CD4⁺ T cells from DO11.10 transgenic mice were co-cultured for 48 h with the above mentioned BMDCs, and then the concentrations of IL-4 and interferon-γ (IFN-γ) in culture supernatants were measured by ELISA. RESULTS:The expression of MHC-Ⅱ, CD80, CD86 and CD40, and releases of proinflammatory cytokines by BMDCs in iNKT cells plus OVA group were comparable to those in the LPS group (P>0.05), but significantly higher than those in iNKT cells plus PBS group, OVA group, and PBS group (P<0.05 or P<0.01). The concentration of IL-4 in culture supernatants from BMDCs in iNKT cells plus OVA group co-cultured with DO11.10 CD4⁺ T cells was similar to that in LPS group (P>0.05), but markedly higher than that in iNKT cells plus PBS group, OVA group, and PBS group (P<0.01). CONCLUSION:Bone marrow-derived dendritic cells undergo immunogenic maturation upon interaction with iNKT cells in the presence of OVA.     

Grafting neural stem cells improve the impaired cognitive deficits and spatial recognition after ischemic-hypoxic brain damage in neonatal rats

AIM: To investigate whether grafting neural stem cells (NSCs) improves the impaired cognitive deficits and spatial recognition after ischemic-hypoxic brain damage (HIBD) in neonatal rats. METHODS: Non-immunosuppressed 7-day-old SD rats were used as research subject and randomly divided into 3 groups: (1) sham group (n=10); (2) HIBD group (n=11); (3) transplant group (n=13). (2) and (3) were anesthetized and subjected to a hypoxic/ischemic injury obtained by combination of left carotid ligation and exposure to 8% oxygen for 2 h. At 3 days post injury, hypoxic-ischemic brain damaged animals were re-anesthetized and randomized to receive stereotactic injection of NSCs prelabeling with BrdU or control media into the hippocampus in the ipsilateral hemisphere. Cognitive (i.e., learning) deficits were assessed at 2 to 4 weeks after transplantation. At the end of the behavioral tests, the animals were killed and evaluated for NSC survival and histopathological analysis. RESULTS: Transplant group showed significantly improved cognitive function in selected tests as compared with HIBD group during the 4-week observation period. They took less time than HIBD group in finding the 3 arms baited with water and had a decreased number of working and reference memory errors in radial maze acquisition tests. Histological analysis showed that transplanted NSCs attenuated CA1 cell loss after HIBD, and NSCs survived for as long as 4 weeks after transplantation and were detected in the hippocampus. CONCLUSION: These data suggest that transplanted NSCs attenuate brain damage and cognitive dysfunction after hypoxic-ischemic brain damage. This approach warrants continued investigation in light of potential therapeutic uses.     

Differentiation of neurons from mouse induced pluripotent stem cells in vitro

AIM: To study the induction method of mouse induced pluripotent stem cells (iPSCs) that differentiate into neurons in vitro. METHODS: Mouse iPSCs were cultured in non-adherent culture dishes for 2 d to form embryoid bodies (EBs). The EBs were cultured for consecutive 2 d in the presence of retinoic acid (RA), and then were plated in the serum-free medium for adherent culture. Seven days later, Pasteur pipette was used to detach the differentiated cells around adherent EBs into “fragment” cell colonies with the help of dissecting microscopes, and these “fragments” were transferred to culture dishes with neural stem cell medium. Another 7 days later, the cells were plated onto the culture dishes using differentiation medium containing fetal bovine serum (FBS) and RA. The morphological changes of the cells were observed under inverted microscope. The iPSCs markers Oct4, Sox2 and SSEA1, the neural stem cell (NSC) marker nestin, the neuronal marker microtubule-associated protein 2 (MAP-2), the astrocyte marker glial fibrillary acidic protein (GFAP) and oligodendrocyte marker myelin basic protein (MBP) were detected by immunofluorescence method. The mRNA expression of GFAP, nestin, β3-tubulin, MAP-2 and MBP was detected by RT-RCR. MAP-2 gene sequence was identified. The proportions of NSCs differentiated from iPSCs and neurons from NSCs were detected by flow cytometry. RESULTS: Mouse iPSCs strongly expressed Oct4, Sox2 and SSEA1, and formed spherical EBs by suspended culture. The EBs were induced by RA and serum-free medium in adherent culture for 2 d, and rosette structure was observed under the microscope. “Fragments” separated by Pasteur pipette from the rosette structure formed neurosphere-like colonies. After the colonies were cultured in adherent condition for 5 d to 7 d in the presence of RA and FBS, the typical neurite was observed under the microscope. The neurospheres expressed nestin and their differentiated derivatives expressed MAP-2, GFAP and MBP, respectively. RT-PCR analysis and gene sequencing showed that the neurons were induced successfully. The results of flow cytometry demonstrated that 63.93%±1.47% of iPSCs differentiated into NSCs and 21.4%±1.70% of NSCs differentiated into neurons. CONCLUSION: Mouse iPSCs proliferate stably and differentiate into neurons in vitro, which provide a reliable source for the treatment of spinal cord injury.     

Adult rat and human bone marrow mesenchymal stem cells differentiate into neurons with Musk's polypeptide

AIM: To investigate the differentiation from adult rat and human bone marrow mesenchymal stem cells (BMMSCs) into neuron with musk polypeptide (Mu-P).METHODS: Adult rat and human BMMSCs were induced with Mu-P.Neuron-specific enolase (NSE),neurofilament (NF),Nestin,glial fibrillary acidic protein (GFAP) were detected by immunohistochemistry.RESULTS: Simple methods with Mu-P induced adult rat and human BMMSCs exhibiting a neuronal phenotype,expressing Nestin at 3 hours to 5 hours,and expressing NE and NF at 5 hours to 7 days.But the neuron-like cells didn't express the glial astrocyte marker GFAP.CONCLUSION: Adult rat and human BMMSCs can be induced to differentiate into neurons with Mu-P.     

Curcumin promotes proliferation and migration of neural stem cells in cerebral ischemic rats by regulating Notch signaling

AIM: To investigate the proliferation and migration of neural stem cells (NSCs) in the subventricular zone (SVZ) on focal cerebral ischemia with curcumin treatment, and to explore the relationship between these effects and Notch signaling. METHODS: The animal model was established by an intraluminal suture method to induce middle cerebral artery occlusion in rats. The rats were randomly divided into sham group, cerebral ischemia/reperfusion (I/R) group, and I/R+curcumin group. The animals were given curcumin for 7 d intraperitoneally. One hour after the model was successfully established, the rats were sacrificed. Immunofluorescence was used to label the NSCs by BrdU and BrdU/DCX, and the migration tendency was observed. The expression of Notch intracellular domain (NICD, the Notch signaling pathway intermediate product) was detected by Western blotting. RESULTS: Compared with I/R group, BrdU-positive and BrdU/DCX double positive cells in I/R+curcumin group were significantly higher than those in I/R group (P<0.05), and more positive cells were on the way of migration to the ischemic lesion zone. The expression level of NICD in I/R+curcumin group was significantly higher than that in I/R group (P<0.05). CONCLUSION: Curcumin promotes NSC proliferation and migration in SVZ after focal cerebral ischemia. The possible mechanism may be that curcumin activates Notch signaling.     

Human mesenchymal stem cells differentiate into neuron-like cells by increase in intracellular cyclic AMP

AIM: To investigate the differentiation from human mesenchymal stem cells(hMSC) into neuron-like cells by the increase in intracellular cyclic AMP. METHODS: hMSC were separated from human marrow with Ficoll-Paque reagent and expanded in culture medium. hMSC were induced to differentiate into neurons with Forskolin and 3-isobutyl-1-methyl-xanthine (IBMX). Neuron-specific enolase(NSE), neurofilament(NF), glial fibrillary acaidic protein(GFAP) were detected by immunohistochemistry. RESULTS: hMSC were expanded as undifferentiated cells in culture for more than10passages. Forskolin/IBMX can induce hMSC to exhibit a neuronal phenotype, expressing NSE and NF-M in 5 hours. But the neuron-like cells didn't express the glial astrocyte marker GFAP. CONCLUSION: hMSC can be induced to differentiate into neurons by increase in the intracellular cAMP.     

Expression of TNF-α and TNF-R1 in retinal glial cells of a rat chronic elevated intraocular pressure model

AIM: In order to study the effects of retinal glial cells on glaucomatous retinal ganglion cells damage, expression of TNF-α and TNF-R1 in retinal glial cells was observed in rat model with chronic elevated intraocular pressure glaucoma. METHODS: (1)Rats were rendered elevated intraocular pressure by ligating 2 episcleral veins with subconjunctival injection of 5-Fu. (2)Four weeks after operation, immuno-histological assays were carried out on cryostat sections. The co-expressions of TNF-α-GFAP, TNF-α-OX42, TNFR-1-GFAP, and TNFR-1-NeuN were observed via a confocal laser scanning microscope, respectively. RESULTS: (1)Elevated intraocular pressure was consistently maintained for up to 4 weeks in model group. (2)The co-expressions of TNF-α and GFAP, TNF-α and OX42 were detected in retina in treatment group,respectively. (3)The co-expressions of TNFR-1 and GFAP were also detected in retina, but the co-expressions of TNFR-1 and NeuN were not detected in retina in treatment group. CONCLUSION: TNF-α that activated retinal glial cells may play an important role in glaucomatous retinal ganglion cell damage.     

Effect of shRNA-mediated survivin gene silencing on sensitivity of lymphoma cells to adriamycin

AIM: This study was designed to use RNA interference technique to down-regulate the expression of survivin gene in human Burkitts lymphoma cell line Daudi and to explore the effect on sensitivity of Daudi cells to adriamycin. METHODS: The survivin-shRNA expression vector was constructed and transfected into Daudi cells. Expression of survivin mRNA and protein were assessed by RT-PCR and Western blotting analysis, respectively. Apoptosis index of transfected Daudi cells was quantified by flow cytometry. The sensitivity of Daudi cells to adriamycin (ADR) before and after transfection was detected by MTT test. RESULTS: The mRNA and protein levels of survivin were down-regulated by 62.32% and 61.88%, respectively, compared to those in control-shRNA treated group and PBS treated group (P<0.05). Meanwhile, the apoptosis index was significantly increased (19.10%±2.15%), compared to that in control group (4.48%±1.54%) and PBS group (4.35%±1.37%, P<0.05). The 50% inhibition concentration (IC50) of ADM to Daudi cells was significantly decreased (0.25±0.43) μmol/L, compared to that in control group (0.87±0.21) μmol/L and PBS group (0.91±0.36) μmol/L, P<0.05. CONCLUSION: Down-regulation of survivin expression in Daudi cells by shRNA effectively induces apoptosis and increases the sensitivity of Daudi cells to ADR.     

Protective effect of anti-aging Klotho protein on human umbilical vein endothelial cells treated with high glucose

AIM: To study the protective effect of anti-aging Klotho protein on human umbilical vein endothelial cells (HUVECs) treated with high glucose (HG).METHODS: HUVECs were cultured in vitro, and divided into PBS control group, 5.5 mmol/L glucose group, 33.3 mmol/L glucose group, 0.1 μmol/L Klotho+33.3 mmol/L glucose group, 1 μmol/L Klotho+33.3 mmol/L glucose group, and 10 μmol/L Klotho+33.3 mmol/L glucose group. The viability of the HUVECs was measured by MTT assay. The content of malondialdehyde (MDA), and the activities of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and glutathione (GSH) in cell culture supernatants were observed. The production of reactive oxygen species (ROS) in HUVECs was analyzed by flow cytometry. The levels of nitric oxide (NO), endothelin (ET-1), intercellular adhesion molecule-1 (ICAM-1) in HUVEC culture medium were detected by ELISA. The protein expression of nuclear factor-kappa B (NF-κB) in the HUVECs was determined by Western blot. RESULTS: Compared with PBS control group, 33.3 mmol/L glucose significantly decreased the HUVEC viability, increased ROS, LDH and MDA levels, reduced the activities of SOD and GSH, decreased the NO secretion, and induced the ET-1 and ICAM-1 secretion and the protein expression of NF-κB in HUVECs. When HUVECs were treated with Klotho protein at different concentrations combined with 33.3 mmol/L glucose, the cell viability was increased significantly, the ROS, LDH and MDA levels were decreased significantly, the antioxidant SOD and GSH activities were significantly increased, the secretion of NO was increased, but ET-1 and ICAM-1 releases and protein expression of NF-κB were significantly reduced.CONCLUSION: Anti-aging Klotho protein promotes the viability of HUVECs treated with HG, reduces the oxidative damage and ROS production, and restores the normal secretory function of HUVECs, thus playing a protective role in vascular endothelial cells through reducing the protein expression of NF-κB.     

Protective effect of senegenin on retinal ganglion cells in oxidative stress induced injury

AIM: To evaluate the effect of senegenin (Sen) on H₂O₂-treated retinal ganglion cells (RGCs) and to explore its underlying mechanisms. METHODS: RGCs were retrograde labeled by injection of fluorogold into the superior colliculi of SD rats on the postnatal day 3. On the postnatal days 6 to 8, the retinas were dissociated with papain and cultured. Primary RGCs cultured in vitro were treated with H₂O₂ and/or various doses of Sen. The viability of RGCs was evaluated by counting the fluorescence-labeled neurons under microscope. The morphological changes of the nuclei in the retinal neurons were observed by Hoechst 33258 staining. Western blotting was applied to determine the expression of cleaved caspase-3, cytochrome C and Bcl-2 in cultured retinal neurons. RESULTS: Compared with the control cells, Sen at doses of 10, 20 or 40 μmol/L had no toxicity to RGCs (P>0.05). However, Sen at doses of 80 and 160 μmol/L had significant toxicity to RGCs (P<0.01). Compared with H₂O₂-injured group, Sen at doses of 10, 20 and 40 μmol/L effectively protected against H₂O₂-induced injury in RGCs (P<0.05) with the best efficiency at 40 μmol/L. Hoechst 33258 staining showed that the neuronal apoptosis caused by H₂O₂ was reduced by Sen. The results of Western blotting showed an up-regulation of Bcl-2, and decreased cytochrome C and cleaved caspase-3 levels by Sen in H₂O₂-treated retinal neurons. CONCLUSION: Sen is able to protect RGCs from H₂O₂-induced injury by enhancing Bcl-2 expression and inhibiting cell apoptosis.     

Study on the potential to differentiate into myocytes of the CD34+ cells

AIM:To investigate the potential of differentiatng into myocytes of the granulocyte colony-stimulating factor(G-CSF)-mobilized CD34⁺ cells. METHODS:Three hours after intraperitoneal injecction of isoprenaline(ISO)to develop acute ischemic model,rats’bone marrow hematopoietic stem cells were mobilized to the site of myocardial infarction by G-CSF.The techniques of immunohistochemisty and HE stain were used to detect the infiltration of CD34⁺ cells and the regeneration of myocytes in the infarct zones. RESULTS:24 hours after administration of ISO, a large amount of infiltrative monocytes and regenerative myocytes which were CD34 positive expression could be found in the infarct zones of the G-CSF treatment group, while majority of the infiltrative inflammatory cells in control group were neutrophils and there was no infiltrative cells and myocytes which were CD34 positive expressio, 2 weeks after administration of ISO, there were a plenty of scar in control group, but not in the G-CSF treatment group. CONCLUSION:G-CSF-mobilized CD34⁺ cells possess the potential to differentiate into myocytes and it may be used in treating acute myocardial infarction.