Neuroprotective effects of neural stem cells pretreated with neuregulin1ß on PC12 cells exposed to oxygen-glucose deprivation/reoxygenation.

Studies on ischemia/reperfusion (I/R) injury suggest that exogenous neural stem cells (NSCs) are ideal candidates for stem cell therapy reperfusion injury. However, NSCs are difficult to obtain owing to ethical limitations. In addition, the survival, differentiation, and proliferation rates of transplanted exogenous NSCs are low, which limit their clinical application. Our previous study showed that neuregulin1ß (NRG1ß) alleviated cerebral I/R injury in rats. In this study, we aimed to induce human umbilical cord mesenchymal stem cells into NSCs and investigate the improvement effect and mechanism of NSCs pretreated with 10 nM NRG1ß on PC12 cells injured by oxygen-glucose deprivation/reoxygenation (OGD/R). Our results found that 5 and 10 nM NRG1ß promoted the generation and proliferation of NSCs. Co-culture of NSCs and PC12 cells under condition of OGD/R showed that pretreatment of NSCs with NRG1ß improved the level of reactive oxygen species, malondialdehyde, glutathione, superoxide dismutase, nicotinamide adenine dinucleotide phosphate, and nuclear factor erythroid 2-related factor 2 (Nrf2) and mitochondrial damage in injured PC12 cells; these indexes are related to ferroptosis. Research has reported that p53 and solute carrier family 7 member 11 (SLC7A11) play vital roles in ferroptosis caused by cerebral I/R injury. Our data show that the expression of p53 was increased and the level of glutathione peroxidase 4 (GPX4) was decreased after RNA interference-mediated knockdown of SLC7A11 in PC12 cells, but this change was alleviated after co-culturing NSCs with damaged PC12 cells. These findings suggest that NSCs pretreated with NRG1ß exhibited neuroprotective effects on PC12 cells subjected to OGD/R through influencing the level of ferroptosis regulated by p53/SLC7A11/GPX4 pathway.

Transplantation of Human Umbilical Cord Mesenchymal Stem Cells-Derived Neural Stem Cells Pretreated with Neuregulin1ß Ameliorate Cerebral Ischemic Reperfusion Injury in Rats.

Ischemic stroke is a common cerebrovascular disease and recovering blood flow as early as possible is essential to reduce ischemic damage and maintain neuronal viability, but the reperfusion process usually causes additional damage to the brain tissue in the ischemic area, namely ischemia reperfusion injury. The accumulated studies have revealed that transplantation of exogenous neural stem cells (NSCs) is an ideal choice for the treatment of ischemia reperfusion injury. At present, the source and efficacy of exogenous NSCs after transplantation is still one of the key issues that need to be resolved. In this study, human umbilical cord mesenchymal stem cells (hUC-MSCs) were obtained and induced into NSCs byadding growth factor and neuregulin1ß (NRG1ß) was introduced during the differentiation process of NSCs. Then, the rat middle cerebral artery occlusion/reperfusion (MCAO/R) models were established, and the therapeutic effects were evaluated among groups treated by NRG1ß, NSCs and NSCs pretreated with 10 nM NRG1ß (NSCs-10 nM NRG1ß) achieved through intra-arterial injection. Our data show that the NSCs-10 nM NRG1ß group significantly improves neurobehavioral function and infarct volume after MCAO/R, as well as cerebral cortical neuron injury, ferroptosis-related indexes and mitochondrial injury. Additionally, NSCs-10 nM NRG1ß intervention may function through regulating the p53/GPX4/SLC7A11 pathway, and reducing the level of ferroptosis in cells, further enhance the neuroprotective effect on injured cells.

Lycopene-Loaded Microemulsion Regulates Neurogenesis in Rats with Aß-Induced Alzheimer's Disease Rats Based on the Wnt/ß-catenin Pathway.

Objective: To investigate the effects of lycopene-loaded microemulsion (LME) on the cognitive function and neurogenesis in the dentate gyrus (DG) of the hippocampus and subventricular (SVZ) region of rats with amyloid ß- (Aß-) induced Alzheimer's disease (AD) and its mechanism based on the Wnt/ß-catenin pathway. Methods: Healthy Wistar rats were divided into four groups: the blank control (CON), AD control, traditional lycopene (LOO), and LME groups. The CON and AD groups were fed with normal saline, while the LOO group was fed with traditional lycopene, and the LME group was fed with lycopene-loaded microemulsion. Behavioral tests were performed after three weeks of gastric administration. Immunofluorescence-labeled cells were used to observe the differentiation and maturation of new nerve cells in the DG of the hippocampus and SVZ region. qRT-PCR and Western blotting detected the expression of neurogenesis genes and Wnt/ß-catenin pathway-related proteins, respectively. Results: On the Morris water maze test, LME rats had significantly shortened movement trajectory on the searching platform, reduced escape latency time, and increased residence time on the original platform quadrant. In addition, more LME rats crossed the platform when it was removed. Thus, LME can improve the spatial learning and memory of Aß-induced AD rats. On qRT-PCR, LME significantly increased Reelin, Nestin, and Pax6 gene expressions, which regulate neurogenesis. Immunofluorescence showed that LME could significantly increase BrdU+, Dcx+, BrdU+/Neun+, BrdU+/Dcx+ cells in the DG and SVZ regions, thus promoting neurogenesis. LME also reduced the number of Iba1+ and Iba1+/BrdU+ cells, thus reducing the neuroinflammatory response. On Western blot, LME upregulated the Wnt/ß-catenin pathway by upregulating Wnt3a, ß-catenin, Disheveled (Dvl), and p-GSK3ß and downregulating p-ß-catenin and GSK3ß. Conclusion: LME attenuates cognitive impairment in Aß-induced AD rats by promoting neurogenesis in the hippocampus and SVZ region through upregulating the Wnt/ß-catenin pathway.

[Effect of Picroside II on ERK1/2 Signal Pathway in Cerebral lschemic Injury Rats].

OBJECTIVE: To explore the neuroprotective effect and mechanism of picroside II on extracellular regulated protein kinases1/2 (ERK1/2) signal transduction pathway in cerebral ischemia injuryrats. METHODS The middle cerebral artery occlusion (MCAO) model was established by inserting a monofilament into middle cerebral artery. Totally 96 successfully modeled Wistar rats were divided into the modelgroup, the treatment (picroside II) group, the Lipopolysachcaride (LPS) group, and the U0126 group according to random digit table. Each group was further divided into 3 subgroups, i.e. 6, 12, and 24 h sub-groups. Picroside II (20 mg/kg) was peritoneally injected to rats in the treatment group 2 h after ischemia.LPS (20 mg/kg) and Picroside II (20 mg/kg) were peritoneally injected to rats in the LPS group 2 h after ischemia. U0126-EtOH (20 mg/kg)and Picroside II (20 mg/kg) were peritoneally injected to rats in the U0126group 2 h after ischemia. Equal volume of normal saline was peritoneally injected to rats in the control groupand the model group. The neurobehavioral function was evaluated by modified neurological severity score(mNSS) test. The structure of neurons was observed using hematoxylin-eosinstaining (HE) staining. Theapoptotic cells were detected using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The expression of phosphorylated extracellular signal-regulated protein kinase1,2 (pERK1,2) in cortex was detected using immunohistochemistry (IHC) and Western blot. RESULTS: After cerebral ischemia injury neurological impairment score increased, the damage of neuron in the cortical area was aggravated, apoptotic cells increased in the model group as time went by. The expression of pERK1/2 increased more significantly in the model group than in the control group (P <0.05). The damage of neuron in the cortical area was milder, while apoptotic cells decreased, the expression of pERK1f2 obviously decreased more in the treatment group and the U0126 group (P < 0.05). The early damage of neuron in the cortical area was more severe, apoptotic cells and the expression of pERK12 were comparatively higher in early stage of the LPS group, but the expression of pERK1/2 was somewhat decreased in late stage. CONCLUSIONS: Activating ERK12 pathway could mediate apoptosis and inflammatory reactions of neurons after cerebral ischemia injury. Picroside II could protect the nerve system possibly through reducing activation of ERKI2 pathway, inhibiting apoptosis of neurons and inflammation reaction.

Edaravone attenuates brain damage in rats after acute CO poisoning through inhibiting apoptosis and oxidative stress.

Acute carbon monoxide (CO) poisoning is the most common cause of death from poisoning all over the world and may result in neuropathologic and neurophysiologic changes. Acute brain damage and delayed encephalopathy are the most serious complication, yet their pathogenesis is poorly understood. The present study aimed to evaluate the neuroprotective effects of Edaravone against apoptosis and oxidative stress after acute CO poisoning. The rat model of CO poisoning was established in a hyperbaric oxygen chamber by exposed to CO. Ultrastructure changes were observed by transmission electron microscopy (TEM). TUNEL stain was used to assess apoptosis. Immunohistochemistry and immunofluorescence double stain were used to evaluate the expression levels of heme oxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf-2) protein and their relationship. By dynamically monitored the carboxyhemoglobin (HbCO) level in blood, we successfully established rat model of severe CO poisoning. Ultrastructure changes, including chromatin condensation, cytoplasm dissolution, vacuoles formation, nucleus membrane and cell organelles decomposition, could be observed after CO poisoning. Edaravone could improve the ultrastructure damage. CO poisoning could induce apoptosis. Apoptotic cells were widely distributed in cortex, striatum and hippocampus. Edaravone treatment attenuated neuronal apoptosis as compared with the poisoning group (P < 0.01). Basal expressions of HO-1 and Nrf-2 proteins were found in normal brain tissue. CO poisoning could activate HO-1/Nrf-2 pathway, start oxidative stress response. After the administration of Edaravone, the expression of HO-1 and Nrf-2 significantly increased (P < 0.01). These findings suggest that Edaravone may inhibit apoptosis, activate the Keapl-Nrf/ARE pathway, and thus improve the ultrastructure damage and neurophysiologic changes following acute CO poisoning.

Combinatorial effects of miR-20a and miR-29b on neuronal apoptosis induced by spinal cord injury.

Neuronal apoptosis is one of the prominent features involved in spinal cord injury (SCI). MicroRNAs (miRNAs) are small non-coding RNAs that functions in a variety of cellular processes including apoptosis. MiRNAs have been implicated as effectors of SCI. However, role of miRNAs in SCI-associated neuronal apoptosis remains to be investigated. A number of bioinformatics approaches have suggested Mcl-1 and BH3-only family genes as potential downstream targets regulated by miR-20a and miR-29b, respectively. To determine whether miR-20a and miR-29b play a role in neuronal apoptosis of SCI by regulating those genes, we transfected Neuro-2A neuroblastoma cells with mimic and inhibitor for the two miRNAs. The miR-20a mimic decreased Mcl-1 expression and the miR-29b mimic reduced the expression of Bad, Bim, Noxa and Puma. The repressor role of miR-20a and miR-29b is confirmed by the transfection of Neuro-2A cells with their inhibitor. Moreover, miR-20a mimic or miR-29b inhibitor attenuated Neuro-2A cell viability and co-transfection of both further diminished the viability of these cells. The in vitro effects of miR-20a and miR-29b on neuronal apoptosis were corroborated by the in vivo studies. Injection of miR-20a mimic or miR-29b inhibitor into the lesion of the injured spinal cord rescued the neuronal death and co-injection of both completely abolished SCI-induced apoptosis. In conclusion, altered expression of miR-20a and miR-29b may cooperatively contribute to the neuronal cell death of SCI through down-regulating anti-apoptotic myeloid cell leukemia sequence-1 (Mcl-1) and up-regulating pro-apoptotic BH3-only proteins.

The hypoglycemic effect of the kelp on diabetes mellitus model induced by alloxan in rats.

Hypoglycemic effects and the use of kelp in diabetes mellitus (DM) model rats induced by alloxan were investigated. Sixty healthy male rats were used to establish DM models by injecting alloxan intraperitoneally. Kelp powder was added to the general forage for the rats. The levels of fasting blood glucose (FBG) were determined by an automatic blood glucose device. Electrochemiluminescence immunoassay was applied to determine the serum levels of insulin. The serum levels of malondialdehyde (MDA) were measured by thiobarbituric acid assay and nitric oxide (NO) by nitrate reductase assay. The activities of superoxide dismutase (SOD) were determined by xanthinoxidase assay and glutathione peroxidase (GSH-Px) by chemical colorimetry. The shape and structure of islet cells were observed with Hematine-Eosin staining, and the expression of superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) in islet cells were detected by immunohistochemical assay. The results showed that the serum levels of insulin after treatment with kelp powder increased significantly compared to those in the DM-model group, while the FBG in the medium-high dose treated groups decreased significantly compared to those in the DM-model group (P < 0.05). The levels of MDA and NO in the kelp powder groups were lower than those in the DM-model group, while the activities of SOD and GSH-Px were higher than those in the DM-model group, of which a significant difference existed between the medium-high dose treated groups and the DM-model group (P < 0.05). The shape and structure of islet cells improved with the up-expressing SOD and down-expressing iNOS in the medium-high dose treated groups compared to those in the DM-model group (P < 0.05). There were no significant differences between the medium and high dose treated groups, all above indexes (P > 0.05). It is suggested that kelp might aid recovery of the the islet cell secreting function and reduce the level of FBG by an antioxidant effect.

[Effect of tagalsin on p53 and Bcl-2 expression in hepatoma H(22) tumor-bearing mice].

OBJECTIVE: To explore the effect and mechanism of tagalsin on hepatoma cells. METHODS: The animal models were established by transplanting H(22) mouse hepatoma cells to mouse liver, and ten days later the mice were randomly divided into five groups: blank group, carmofur positive group and tagalsin groups, including low-dose, middle-dose and high-dose groups. Then medicine or oil was given to the mice by gastric gavage in consecutive 5 days with a 2-days interval as a course of treatment, two courses in all. All mice were killed at 24 hours after medication, and the survival period, ascites conditions, aggressive conditions intra- or extra-liver, weight changes, tumor volume and spleen index of the tumor-bearing mice were observed. Pathological changes of the tumors were examined. Apoptotic factors p53 and Bcl-2 protien and mRNA were detected by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). RESULTS: tagalsin inhibited the hepatoma growth effectively without influencing spleen index to some extent. The tumor inhibition rate of tagalsin low, middle and high dose groups were 17.9%, 63.1% and 71.8%, respectively. Immunohistochemical results showed that the p53 and Bcl-2 protein positive cell counts of the positive control and experimental groups were significantly lower than those of the blank group (P < 0.01). RT-PCR results showed that the p53 mRNA expression was significantly enhanced and Bcl-2 mRNA expression was decreased in the positive control groups and tagalsin treatment groups, especially in the high dose group, compared with those of the blank group (P < 0.05). CONCLUSIONS: tagalsin can inhibit the growth of mouse hepatoma cells significantly. The mechanism of its anti-tumor effect may work via up-regulating the wild type p53 gene expression and down-regulating Bcl-2 gene expression and thus regulating tumor cell apoptosis.

[Effect of picroside II on expressions of TLR4 and NFkappaB in rats with cerebral ischemia reperfusion injury].

OBJECTIVE: To explore the effects of picrodide II on the expressions of Toll-like receptor 4 (TLR4) and nuclear transcription factor kappaB (NFkappaB) in brain tissue of rat after cerebral ischemic reperfusion (I/R) injury. METHODS: Ten rats from 60 adult healthy female Wistar rats received sham-operation were set as the sham-operative group. Established as middle cerebral I/R model (MCAO/R) by thread tying method, the 30 successfully modeled rats were equally randomized into the negative control group, the positive control group and the treatment group. Besides, rats in the treatment group and the positive control group were respectively intervened with picrodide II (10 mg/kg) and salvianic acid A sodium (10 mg/kg) via caudal vein injection before I/R injury, while rats in the sham-operative group and the negative group were injected with equal volume of 0.1 mol/L PBS. Immunohistochemistry stain was used to determine the expressions of TLR4 and NFkappaB, and the apoptotic cells were counted by TUNEL-immunofluorescence assay. RESULTS: In the sham-operative group, the TLR4 and NFkappaB expressed weakly with few TUNEL positive cells scattering in the cortex, striatum and hippocampus. As compared with the sham-operative group, TLR4 and NFkappaB in the negative control group were significantly higher both in absorption A) value and cell number (P < 0.05). In the treatment group and the positive control group, the expressions of TLR4 and NFkappaB and the number of TUNEL positive cells were significantly lower than those in the negative control group (P < 0.05), but no significant difference was shown between the two treated groups (P > 0.05). CONCLUSIONS: Picroside II could down-regulate the expressions of TLR4 and NFkappaB, and inhibit the inflammatory response induced apoptosis in cerebral I/R injured rats.

Neuroprotective properties of picroside II in a rat model of focal cerebral ischemia.

The aim of this study was to explore the effect of picroside II on neuronal apoptosis and the expression of caspase-3 and poly ADP-ribose polymerase (PARP) following middle cerebral artery occlusion/reperfusion in male Wistar rats. Picroside II (10 mg/kg) was administered intravenously into the tail vein of the animals. The neurological function deficits were evaluated with the Bederson's test and the cerebral infarction volume was visualized with tetrazolium chloride (TTC) staining. The apoptotic cells were counted by in situ terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end labeling (TUNEL) assay. The immunohistochemistry stain and enzyme linked immunosorbent assay (ELISA) was used to determine the expressions of caspase-3 and PARP in brain tissue. The results indicated that rats in the control group showed neurological function deficit and cerebral infarction in ischemic hemisphere after two hours ischemia followed by 22 hours reperfusion. Caspase-3 and PARP expressions were also profound in the cortex, the striatum and the hippocampus, along with increased apoptotic cells in this group. Bederson's score, infarction volume, and expressions of caspase-3 and PARP, as well as apoptosis in the treatment group were, however, significantly decreased compared to those in the control group indicating that intravenous treatment with picroside II might be beneficial to inhibit neuronal apoptosis and, thus, to improve the neurological function of rats upon cerebral ischemia reperfusion injury.

[Interfering effect of neuregulin upon cerebral ischemic reperfusion injury in rhesus monkeys].

OBJECTIVE: To study the interfering effect of neuregulin-1beta (NRG-1beta) on the cerebral ischemic reperfusion injury in monkeys. METHODS: The models with middle cerebral artery occlusion reperfusion (MCAO/R) were established by inserting a micro-balloon catheter intra-arterially into MCA from femoral artery in 12 adult healthy monkeys. The NRG-1beta was injected into MCA from micro-balloon catheter in the animals of treatment group at 2 h post-ischemia while the normal saline was simultaneously injected in the animals of control group. Then the micro-balloon catheter was withdrawn from the MCA to perfuse for 22 h. The nervous behavioral function was evaluated by task-oriented score and the infarct volume measured by TTC and MRI. RESULTS: The animals of control group had an onset of nervous functional disorders at 2 h post-ischemia and had no significant difference at reperfusion 22 h (59.8 +/- 15.7); the nervous function of monkeys had no improvement after NFG-1beta treatment (61.3 +/- 16.2) (P > 0.05). The diffuse weight imaging (DWI) showed a high signal area but T(1) and T(2) imaging had no significant changes at 2 h post-ischemia in control group animals; while the T(1) and T(2) imaging showed a typical high signal areas, DWI high signal area expanded and TTC staining ischemic area appeared at reperfusion 22 h. The T(1), T(2) and DWI abnormal signal areas and TTC cerebral infarct volume did not shrink significantly in NFG-1beta treatment group (P > 0.05). CONCLUSION: The neuroprotective effects of NRG-1beta upon cerebral ischemic reperfusion injury needs to be further studied.

Effects of neuregulin on expression of MMP-9 and NSE in brain of ischemia/reperfusion rat.

The aim is to investigate the effects of neuregulin-1beta (NRG-1beta) on expression of matrix metalloproteinase-9 (MMP-9) and neuron-specific enolase (NSE) in brain tissue in rats following cerebral ischemia/reperfusion. One hundred and fifty adult healthy male Wistar rats were used in the present study. Ten of them were randomized into a sham-operation group (n = 10) and the rest suffered surgery operation of middle cerebral artery occlusion/reperfusion with intraluminal monofilament suture from the left external-internal carotid artery. As a result, 100 rats of successful models were randomly divided into a control group (n = 50) and a treatment group (n = 50). Rats in the treatment group were injected 1.5% NRG-1beta at a dosage of 0.3 microg/kg from the stump of the left external carotid artery into the internal carotid artery. The expressions of MMP-9 and NSE proteins were determined by immunohistochemical, immunofluorescent double labeling, and Western blot assay. Ischemia/reperfusion induced morphological changes of brain tissue, including neurocyte shrinkage, chromatin condensation, nuclei fragment, and gliacyte and endothelial cell swelling. NRG-1beta obviously reduced and delayed the cerebral damage. With the duration of ischemia, the expression of MMP-9 gradually increased in the control group. NRG-1beta decreased the level of MMP-9 compared with that in the control group (P < 0.01). NSE immunoreaction transiently elevated at the early stage of cerebral ischemia insult, and then gradually decreased in the control group. The administration of NRG-1beta significantly increased the level of NSE, and thus delayed the time and the degree of neuron damage. There were statistical differences in contrast to the control group (P < 0.01). There was no relationship between the expressions of the two proteins. MMP-9 might aim at various target cells at different stages and contribute to the inflammatory reaction after cerebral ischemia-reperfusion insult. NRG-1beta inhibits the activation of MMP-9 and development of inflammation, enhances the activity of NSE, improves the microenvironment of neuron survivals, and delays the phase of irreversible neuron necrosis. Therefore, NRG-1beta may play a neuroprotective role in cerebral ischemia/reperfusion.

Effect of neuregulin on apoptosis and expressions of STAT3 and GFAP in rats following cerebral ischemic reperfusion.

The aim is to investigate the effect of neuregulin-1beta (NRG-1beta) on the neuronal apoptosis and the expressions of signal transducer and activator of transcription (STAT3) and glial fibrillary acidic protein (GFAP) in rats following cerebral ischemia/reperfusion. The animal models of middle cerebral artery occlusion/reperfusion (MCAO/R) were established by an intraluminal filament method from left external-internal carotid artery in 100 cases of adult healthy male Wister rats. NRG-1beta was administered from the internal carotid artery (ICA) into MCA in the treatment group. The neuronal apoptosis was detected by terminal deoxynucleotidyl transference-mediated biotinylated deoxyuridine triphosphate nick-end labeling technique. The expression alternations of STAT3 and GFAP proteins were determined by fluorescent labeling analysis and Western blotting assay. Ischemic cerebral injury could induce neuronal apoptosis. Furthermore, with the duration of ischemia, the amount of apoptotic cells increased in the control group. These apoptotic cells distributed in various brain regions, especially the cortex, striatum, and hippocampus, while only a small amount of apoptotic cells could be observed in the treatment group, and there were significant differences compared with that in the control group (P < 0.01). The expressions of STAT3 and GFAP proteins in brain tissue gradually increased in the control group with the duration of ischemia. And NRG-1beta could elevate the expressional level of STAT3 and GFAP proteins in contrast to the control group (P < 0.05). NRG-1beta may play a neuroprotective role in cerebral ischemic insult by activating JAK/STAT signal transduction pathway, promoting the astrocyte gumnosis and regulating the anti-apoptosis mechanism in neurocytes.