Therapeutic effect of MAG gene silencing by siRNA on remyelination and motor recovery after intracerebral hemorrhage in rats

AIM:To study the functional rehabilitative effects of lentiviral vector-mediated siRNA silencing myelin-associated glycoprotein (MAG) on neurological improvement and remyelination after intracerebral hemorrhage (ICH) in rats. METHODS:ICH was induced in 45 adult male Sprague-Dawley rats by a stereotactically guided injection of bacterial type IV collagenase into the right internal capsule. Lentiviral vector-mediated siRNA targeting MAG (siRNA), or PBS was administered left intracerebroventricularly 1 d after right internal capsule ICH. The Rotarod motor test scores were determined 1 d before and 1 d, 7 d, 14 d and 21 d after treatment. Luxol fast blue staining, immunohistological staining and Western blotting were used to detect the changes of MAG. RESULTS:Compared with PBS group, the changes of Rotarod motor test scores indicated that the ICH rats receiving siRNA had significant functional recoveries. As indicated by Luxol fast blue staining, the area of demyelination in percentage was obviously reduced in siRNA treatment group as compared with PBS group after ICH (P<0.05). The expression of MAG detected by immunohistological staining and Western blotting was significantly lower in siRNA-treated brain than that in PBS group (P<0.05). CONCLUSION:siRNA targeting MAG contributes to the remarkable functional recovery after ICH. The treatment promotes nerve fiber remyelination by down-regulation of the MAG expression after ICH in rats.     

Role of autophagy in intervertebral disc degeneration in diabetic rats

AIM:To explore the levels of apoptosis and autophagy in the nucleus pulposus tissues of intervertebral discs in diabetic rats. METHODS:Sixteen weeks after injection of streptozocin (STZ), the lumbar intervertebral discs were obtained from the rats. The histological changes were observed by hematoxylin-eosin (HE) staining and alcian blue staining. The apoptosis of the nucleus pulposus cells was measured by the methods of terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL), immunohistochemistry, and Western blotting. The level of autophagy in the nucleus pulposus cells was detected by Western blotting and immunohistochemistry. RESULTS:Compared with normal group, HE and alcian blue staining suggested that the intervertebral discs of the diabetic rats became degenerate. The expression of caspase-3 and the apoptotic rate were increased in intervertebral disc nucleus pulposus of the diabetic rats. The results of immunohistochemistry and Western blotting showed that the expression levels of LC3Ⅱ/LC3Ⅰand beclin-1 in the diabetic rats were higher than those in normal group. CONCLUSION: The STZ-induced diabetes accelerates degeneration of the intervertebral discs. In addition, the apoptosis and autophagy are increased in the intervertebral discs of diabetic rats.     

Effects of microglia-derived IL-1β on differentiation of OPCs in corpus callosum of septic neonatal rats

AIM: To explore whether IL-1β inhibits the oligodendrocyte precursor cell (OPCs) differentiation and affects axonal myelination. METHODS: One-day-old SD rats were randomly divided into control group and LPS group (48 rats in each group). The rats in LPS group were intraperitoneally injected with 1 mg/kg LPS. The rats in control group were injected with an equal volume of PBS. The rats in each group were further divided into 3 h, 24 h, 3 d, 7 d, 14 d and 28 d subgroups after injection. The expression of IL-1β and IL-1R₁ in the rat corpus callosum at 3 h, 24 h, 3 d, 7 d was determined by double immunofluorescence and Western blotting. The myelin basic protein(MBP) expression in the rat corpus callosum at 14 d, 28 d after injection was also measured. In vitro, primary OPCs culture was performed and divided into control group, 30 μg/L IL-1β group, 30 μg/L IL-1β+IL-1Ra group and 30 μg/L IL-1Ra group. The expression of MBP in the OPCs induced differentiation for 3 d was observed by double immunofluorescence and Western blotting. RESULTS: The expression of IL-1β and IL-1R₁ in the rat corpus callosum at 3 h, 24 h, 3 d, 7 d after LPS injection was obviously increased and the expression of MBP in the rat corpus callosum at 14 d, 28 d in LPS group was obviously decreased compared with control group in vivo. The level of MBP was significantly decreased after IL-1β treatment for 3 d in vitro. However, IL-1Ra (IL-1R inhibitor) reversed the down-regulation of MBP expression. IL-1β inhibited the expression of p-ERK, ERK over-expression reversed the down-regulation of MBP expression compared with IL-1β group. CONCLUSION: IL-1β inhibits the differentiation of OPCs, which may be involved in ERK pathways, thus leading to axonal hypomyelination in the corpus callosum of septic neonatal rats.     

Effects of vinpocetine on inflammation of brain in rats with intracerebral hemorrhage

AIM: To investigate the effects of vinpocetine on inflammation of brain in intracerebral hemorrhage (ICH) rats and to explore the underlying mechanisms. METHODS: All rats were randomly divided into sham group, ICH group, ICH with low dose of vinpocetine treatment group, ICH with medium dose of vinpocetine treatment group, and ICH with high dose of vinpocetine treatment group. Except sham group, the rats in other groups were injected with type VⅡ collagenase to establish ICH model, and then the rats in vinpocetine treatment groups were received vinpocetine at 0.5, 1.0 or 1.5 mg/kg by intraperitoneal injection once a day for 7 days. After corresponding treatment, the impairment of neurological function in the rats was scored and the water content of the brain tissues was measured. Moreover, the activity of myeloperoxidase (MPO) was determined by ELISA, and the protein expression of Toll-like receptors 4 (TLR4), nuclear factor-κB(NF-κB), intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molcule-1 (VCAM-1) in the brain tissues was determined by Western blot. RESULTS: Compared with ICH group, vinpocetine treatment significantly decreased the scores of the impairment of neurological function and the water content of the brain tissues. Moreover, the activity of MPO and the protein expression of TLR4, NF-κB, ICAM-1 and VCAM-1 were also reduced after vinpocetine treatment (P<0.05). CONCLUSION: Vinpocetine improves neurological function in ICH rats via suppression of inflammation by inhibiting NF-κB signaling and expression of ICAM-1 and VCAM-1.     

Effects of ghrelin on brain edema and aquaporin 4 expression after cerebral ischemia/reperfusion in rats

AIM: To explore the effects of ghrelin on the brain edema, the permeability of blood-brain barrier (BBB) and the expression of aquaporin 4 (AQP4) after cerebral ischemia/reperfusion in rats. METHODS: Adult male Sprague-Dawley rats were randomly divided into sham operation group, middle cerebral artery occlusion (MCAO) group and ghrelin treatment group. The MCAO model was made with nylon thread for 2 h of occlusion following 22 h of reperfusion. Ghrelin at a dose of 10 nmol/kg was injected via femoral vein at the beginning of reperfusion. The cerebral infarct volume was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Brain functional deficits were evaluated by determining the neurological scores. The changes of brain swelling and water content were analyzed through volume calculation and dry/wet weight measurement. The permeability of BBB was detected by collecting extravascular Evans blue (EB) in the brain cortex. The changes of AQP4 expression were assessed by the methods of immunohistochemistry and Western blotting. RESULTS: Compared with MCAO group, the rats in ghrelin treatment group had smaller brain infarct volume, lower EB exudation content and neurological scores. The percentage of brain swelling, water content and AQP4 expression were lower in ghrelin treatment group than those in MCAO group. CONCLUSION: Ghrelin reduces the injury of cerebral ischemia/reperfusion, and lightens the brain edema and BBB damage in rats. Ghrelin also inhibits the expression of AQP4 in brain tissue.     

Ifenprodil reduces brain edema and cortical apoptosis after subarachnoid hemorrhage in rats

AIM:To explore the effect of ifenprodil, a negative allosteric modulator of GluN2B N-methyl-Daspartate (NMDA) receptor, on neurological deficit, permeability of blood-brain barrier, brain edema and cortical cell apoptosis after subarachnoid hemorrhage (SAH) in rats. METHODS:Adult healthy male Sprague-Dawley rats (n=90) were divided into 3 groups:sham group (n=18), SAH+vehicle group (n=36), and SAH+ifenprodil group (n=36). The rat model of SAH was established by intracranial endovascular puncture. The rats received vehicle or ifenprodil at 2 h, 24 h and 48 h after surgery by intraperitoneal injection. The modified Garcia neurological scoring, dry and wet weight method, Evans blue dye extravasation method, TUNEL staining and Western blot were used to evaluate neurological deficit, brain water content, blood-brain barrier permeability and cortical cell apoptosis at 72 h after SAH. RESULTS:The neurological score and Bcl-2 expression were decreased, while the brain water content, the Evans blue dye extravasation, TUNEL-staining positive cells, and Bax and activated caspase-3/9 protein levels in the basal cortex were increased significantly in SAH+vehicle group as compared with sham group (P<0.05). However, ifenprodil treatment significantly inhibited these SAH-induced changes (P<0.05). CONCLUSION:Ifenprodil attenuates neurological deficits and brain edema, and reduces blood-brain barrier permeability and cortical apoptosis after SAH.     

let-7a inhibits MAP4K3/MKK4/JNK signaling pathway to decrease neuronal apoptosis in rats with intracerebral hemorrhage

AIM To investigate the effect of microRNA let-7a on neuronal apoptosis in a rat model of intracerebral hemorrhage (ICH). METHODS Forty-eight healthy SD rats were selected, 8 of which served as sham group, and 2 μL of type VII collagenase was injected into globus pallidus of the remaining rats to construct ICH model. These ICH rats were randomly divided into model (2 μL normal saline) group, let-7a agomir group, NC agomir group, let-7a antagomir group and NC antagomir group, with 8 rats in each group, and the corresponding drugs were injected into the lateral ventricle. After 7 d, the neurological function scoring was performed, and HE staining was used to observe the pathological damage. RT-qPCR and Western blot were used to detect the expression of related proteins. TUNEL staining was used to detect neuronal apoptosis. The targeting relationship between let-7a and mitogen-activated protein kinase kinase kinase kinase 3 （MAP4K3） was predicted by bioinfomatic software, and further verified by dual-luciferase reporter assay. RESULTS In animal experiments, when let-7a was over-expressed, neurological function scores were reduced,and the pathological damage was alleviated. Glial fibrillary acidic protein (GFAP) expression was down-regulated, neuronal apoptosis was reduced, and the protein levels of cleaved caspase-3 and cleaved PARP were decreased (P<0.05). In cell assays, MAP4K3 was one of the target genes of let-7a, and MAP4K3 was negatively regulated by let-7a. let-7a over-expression inhibited the expression of MAP4K3/MKK4/JNK. CONCLUSION let-7a reduces neuronal apoptosis in ICH rats by inhibiting the MAP4K3/MKK4/JNK signaling pathway.     

Therapeutic efficacy of Ang-1 gene-modified MSCs in cerebral infarction

AIM: To observe the therapeutic efficacy of Ang-1 gene-modified mesenchymal stem cells (MSCs) in cerebral infarction. METHODS: The constructed lentiviral vector carrying the Ang-1 gene was used to infect the rat mesenchymal stem cells (rMSCs) to establish the Ang-1 gene-modified rMSCs (Ang-rMSCs). Adult male F344 rats were subjected to transient (2 h) middle cerebral artery occlusion (MCAO) with modified Zea Longa method. Phosphate buffered saline (PBS, 1 mL 0.1 mol/L, for control group), or Ang-rMSCs suspension (1 mL, for Ang-rMSCs group), or rMSCs suspension (1 mL, for pNL-rMSCs group), were infused into tail vein of rat respectively at 24 h after MCAO (n=8 in each group). Functional recovery measurements using the modified neurological severity scores (mNSS) were performed at 24 h post-MCAO and 1 week, 1 month and 3 months post-transplantation, respectively. The quantitative evaluation of blood-brain barrier permeability was performed at 1 week post-transplantation. The distribution, differentiation and malignant sign of grafted rMSCs were observed with immunofluorescence staining and histological method. RESULTS: Significant neurological function improvement was observed in groups treated with Ang-rMSCs or pNL-rMSCs at 1 week, 1 month post-transplantation compared with that in control group, as evidenced by mNSS (P<0.01). Better neurological function improvement was also found in Ang-rMSCs group than that in pNL-rMSCs group (P<0.01). The results of quantitative evaluation of blood-brain barrier permeability showed that the permeability in Ang-rMSCs group was the lowest compared to those in other two groups (P<0.01), and in the pNL-rMSCs group was the lower than that in control group (P<0.01). The grafted rMSCs survived in Ang-rMSCs and pNL-rMSCs groups, most were localized around the ischemic focus, and a few of them expressed NSE, NF and GFAP. The grafted rMSCs expressed BDNF abundantly in Ang-rMSCs group. These grafted rMSCs survived up to 3 months at least. No malignant sign was observed in these grafted cells. CONCLUSION: Ang-1 gene-modified MSCs transplantation has better therapeutic efficacy in cerebral infarction than that of MSC transplantation. The transplantation of cells with gene engineering is an effective therapeutic method for stroke patients.     

Tetramethylpyrazine combined with bone marrow mesenchymal stem cell transplantation inhibits neuronal apoptosis in rats with cerebral ischemia

ATM: To investigate the effects of tetramethylpyrazine (TMP) combined with bone marrow mesenchymal stem cells (BMSCs) on neuronal apoptosis, and Bcl-2 and Bax expression in rats with cerebral ischemia. METHODS: The BMSCs were isolated by the whole bone marrow adherent method and cultured, and those in the 3rd passage were used for tail-vein transplantation. The rats were subjected to right middle cerebral artery occlusion (MCAO) using suture method, and the rats except sham group were randomly divided into model group, BMSCs (1×10⁹ cells/L) group, TMP (40 mg/kg) group and combination (TMP+BMSCs) group with 12 rats in each group. Neurological function was evaluated by modified neurological severity scoring (mNSS) on 1 d, 7 d and 14 d after cerebral ischemia. Toluidine blue staining was performed to detect cerebral infarct volume, HE staining was used to observe brain histopathological change, neuronal apoptosis was observed by TUNEL staining, and the mRNA and protein expression of Bcl-2 and Bax was detected by real-time fluorescence quantitative PCR and Western blot at 14 d after cerebral ischemia. RESULTS: Compared with BMSCs group and TMP group, TMP combined with BMSCs significantly reduced the score of mNSS (P<0.01) and the infarct volume (P<0.01), alleviated the pathological damage in the peripheral area of cerebral ischemia, decreased the number of TUNEL positive cells (P<0.01), increased the expression of Bcl-2 and decreased the expression of Bax at mRNA and protein levels (P<0.01).CONCLUSION: Tetramethylpyrazine combined with transplantation of BMSCs improves the functional recovery, reduces the infarct volume, relieves the ischemic injury of the brain tissue, and attenuates neuronal apoptosis in the rats with cerebral ischemia. The mechanism may be related to regulating the expression of Bcl-2 and Bax.     

Effects of RNA interference of FABP5 on subcutaneous tumor of human hepatocellular carcinoma cell line HepG 2 transplanted in nude mice

AIM: To investigate the effect of recombinant lentiviral vector for RNA interference (RNAi) on the expression of fatty acid-binding protein 5 (FABP5) gene in hepatocellular carcinoma HepG2 cells and tumor formation in nude mice.METHODS: RNAi lentiviral vector was used in the experiment. Human hepatocellular carcinoma HepG2 cells were divided into 3 groups:the HepG2 cells in experimental group were transfected with the recombinant lentivirirus vector LV-shRNA-FABP5, the cells in negative control group were transfected with a control lentiviral vector LV-shRNA-NC, and the cells in normal control group were without any treatment. The nude mice were randomly divided into 3 groups. The growth of the transplanted tumor cells in the nude mice was observed. The tumor growth curve, volume and weight were determined 4 weeks after the cell inoculation. The expression of FABP5 was detected by real-time PCR, Western blot and immunohistochemical staining.RESULTS: Transfection of the lentiviral vector FABP5-shRNA obviously reduced FABP5 expression in the HepG2 cells. Tumor formation was all positive in the 3 groups of the nude mice inoculated with the tumor cells. Compared with normal control group and negative control group, the tumor growth slowed significantly in experimental group with smaller volume and weight. FABP5 expression in the transplanted tumor tissues was significantly down-regulated at mRNA and protein levels in experimental group as compared with normal control group and negative control group.CONCLUSION: RNAi-induced down-regulation of FABP5 effectively inhibits the growth of transplanted hepatocellular carcinoma, suggesting that FABP5 gene may be an effective target for gene therapy in treating liver cancer.     

Construction of a lentiviral RNA interference vector targeting rat myocardin and its effect on vascular smooth muscle cell differentiation

AIM: To construct a lentiviral RNA interference(RNAi)vector targeting rat myocardin mRNA and to investigate its effect on the differentiation of vascular smooth muscle cells(VSMCs).METHODS: Three pairs of dsDNA targeting rat myocardin mRNA were designed, synthesized and cloned into lentiviral vector pGCSIL-GFP to generate pGCSIL-GFP-shMyocd lentvirus. A Flag-tagged myocardin-overexpression vector pEGFP-N1-Myocd was constructed with pEGFP-N1/X124G. After these two vectors were cotransfected into 293T cells, the flag protein was assessed by Western blotting to analyze the knockdown effect of pGCSIL-GFP-shMyocd. The expression of myocardin and SM22α was also detected by RT-PCR and Western blotting after the pGCSIL-GFP-shMyocd viruses were transfected into primary cultured rat aortal VSMCs.RESULTS: The rat myocardin lentviral RNAi vector pGCSIL-GFP-shMyocd and myocardin-overexpression vector pEGFP-N1-Myocd were successfully constructed. After these two kinds of vectors were cotransfected into 293T cells,the No.1 interfering vector displayed the highest inhibitory effect on flag expression.After the No.1 lentvirus at the titer of 1×10¹² TU/L was transfected into VSMCs, the myocardin and SM22α expression was significantly attenuated. CONCLUSION: The lentiviral pGCSIL-GFP-shMyocd RNAi vector is successfully constructed, which is useful for further study regarding the molecular mechanism of the phenotypic switching in VSMCs under special pathological conditions such as atherosclerosis. Inhibition of myocardin expression in VSMCs leads to the decrease in the expression of differentiation marker, and implies a crucial role of myocardin in VSMCs differentiation.     

Effects of basic fibroblast growth factor on proliferation and collagen production in human umbilical cord mesenchymal stem cells

AIM：To observe the growth pattern and surface markers of human umbilical cord mesenchymal stem cells(hUCMSCs) and to explore the influence of basic fibroblast growth factor (bFGF) on the proliferation and collagen production in hUCMSCs cultured in vitro. METHODS：hUCMSCs were isolated by enzyme digestion method and adherent culture. The surface markers CD45, CD34, CD105, CD29 and HLA-DR of the cells were analyzed by flow cytometry. The osteogenic ability and adipogenic differentiation were confirmed with oil red O and alizarin red staining. The optimal concentration of bFGF to promote the proliferation of hUCMSCs was 20 μg/L. The cells in control group were cultured in the growth medium consisting of DMEM/F12 and 10% volume fraction of fetal bovine serum. The cells in experiment group were cultured under the same condition of control group but plus 20 μg/L bFGF. The proliferation of hUCMSCs was analyzed by MTT assay. The expression of type I and III collagens at mRNA and protein levels was determined by RT-PCR and Western blotting. RESULTS：The growth curves indicated that bFGF promoted the proliferation of hUCMSCs. The hUCMSCs expressed CD29, but did not express CD34, CD45 or HLA-DR in the presence or absence of bFGF unanimously. The cells were alizarin red staining-positive and oil red O staining-positive. Compared with control group, the expression of type I and III collagens significantly decreased at mRNA and protein levels in experiment group. CONCLUSION：bFGF promotes the proliferation of hUCMSCs and does not change the expression of the surface markers. bFGF inhibits the expression of type I and III collagens at mRNA and protein levels, indicating that bFGF enhances the healing of wound without inducing scar hyperplasia.     

Hepatocyte growth factor protects hepatocytes from apoptosis induced by actinomycin D

AIM: To investigate the anti-apoptotic effect of hepatocyte growth factor (HGF) on actinomycin D (ActD)-induced apoptosis in hepatocytes and the possible pathway. METHODS: Hepatocytes were exposed to ActD and HGF. The cytotoxic effects of ActD were tested by MTT. Apoptotic cells were identified by Hoechst 33342 staining, flow cytometry and detection of DNA fragmentation with agarose gel. Akt and phospho-Akt were detected by Western blotting analysis. RESULTS: The results showed that ActD induced apoptosis in hepatocytes 5 h after treatment. Phosphatidylinositol-3 kinase (PI-3K) specific inhibitor wortmannin enhanced the apoptotic effect of ActD. Furthermore, HGF significantly reduced the apoptosis in hepatocytes induced by ActD in a concentration-dependent manner. In the presence of wortmannin, HGF did not overcome apoptosis. CONCLUSION: Wortmannin enhances the apoptotic effect of ActD. HGF protects hepatocytes from apoptosis induced by ActD through a PI3K/Akt pathway.     

Dynamic expression of PTEN in liver tissue of rats with hepatic fibrosis

AIM: To investigate the dynamic expression of phosphatase and tensin homolog deleted on chromosome ten (PTEN) in liver tissue in the process of hepatic fibrosis in rats. METHODS: The rat model of hepatic fibrosis used in this study was induced by common bile duct ligation (BDL). Hematoxylin and eosin (HE) and Masson’s trichrome staining were used for observing the histological changes in hepatic fibrosis tissue. At 4 time points, the expressions of PTEN protein and mRNA in hepatic tissues of rats were detected by immunohistochemical staining, Western blotting and real-time fluorescent quantitative PCR assay, respectively. RESULTS: The rat model of hepatic fibrosis was established successfully. With each consecutive week after BDL, increased fibrosis, degeneration and necrosis were found in rat liver cells. Not surprisingly, a disruption of normal architecture and a decrease in normal hepatic cells was concomitantly observed. The immunohistochemical staining indicated that there was extensive expression of PTEN in liver tissues of normal rats, it expressed mainly in the cytoplasm, and with the aggravation of hepatic fibrosis, the expression of PTEN in liver tissues decreased gradually (P<0.01). Western blotting and real-time fluorescent quantitative PCR at weekly time points (1, 2, 3, and 4 weeks) after BDL showed that, the expression of PTEN protein and mRNA in fibrotic rat liver tissue decreased gradually with increasing severity of hepatic fibrosis (P<0.01). Furthermore, all values from BDL rats were significantly lower than those from the sham operation group (P<0.01). CONCLUSION: The expressions of PTEN protein and mRNA in fibrotic liver tissue of rat decrease gradually with the progression of hepatic fibrosis, and increasing severity of fibrosis correlated well with decreasing PTEN expression.     

Effects of NF-κB inhibitor on nerve injury in rats after intracerebral hemorrhage

AIM: To investigate the regulatory effect of nuclear factor-κB (NF-κB) inhibitor, pyrrolidine dithiocarbamate (PDTC), on nerve function and neural cell apoptosis in rats after intracerebral hemorrhage (ICH). METHODS: SPF Sprague-Dawley rats were randomly divided into 4 groups with 6 rats in each group:sham group, ICH group, PDTC at low concentration (P_low) group and PDTC at high concentration (P_high) group. Autologous blood injection was used to establish ICH model. After 2 h of surgery, the rats in P_low group and P_high group were intraperitoneal injected with PDTC at 100 mg/kg and 200 mg/kg, respectively, while rats in sham group and ICH group were injected with the same volume of saline. The neurological function score was classified with modified Longa grading method. TUNEL assay was used to detected the neural cell apoptosis, and the content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) were measured. Furthermore, the protein levels of p-P65 and cleaved caspase-3 in brain tissues were determined by Western blot. RESULTS: Compared with sham group, the rats in ICH group had higher neurological function score (P<0.05). After treatment with PDTC, the neurological function score was decreased (P<0.05), but no significant difference between P_low group and P_high group was observed. Compared with sham group, the number of apoptotic cells in ICH group was increased (P<0.05). After treatment with PDTC, the neural cell apoptosis was restrained, and the number of apoptotic cells in P_high group was lower than that in P_low group (P<0.05). Compared with sham group, the content of MDA was increased and the activity of SOD was decreased in ICH group (P<0.05). After treatment with PDTC, the content of MDA was decreased while the activity of SOD was increased, and the variation trend was more obvious in P_high group (P<0.05). Compared with sham group, the protein levels of p-P65 and cleaved caspase-3 in ICH group were increased (P<0.05). After treatment with PDTC, the protein levels of p-P65 and cleaved caspase-3 were decreased, and those in P_high group were lower than those in P_low group. CONCLUSION: NF-κB inhibitor PDTC plays a role in the se-condary brain injury after ICH, and the protective effect increases at the higher dose. The mechanism may be related to reducing MDA content and increasing SOD activity, and further inhibiting neural cell apoptosis.     

Effect of olfactory ensheathing cell transplantation on rat experimental autoimmune encephalomyelitis

AIM: To explore the way and the feasible time of olfactory ensheathing cells (OECs) transplanting to experimental autoimmune encephalomyelitis (EAE) rats, and to investigate the migration of OECs after transplanted to EAE and the possible mechanism of inducing the protective effect. METHODS: The Lewis rats, which were divided into MOG group and GPSCH group according to the induction by MOGIgd and GPSCH separately, were used to made EAE model. The animals in MOG group were divided into 3 subsets: OECs blank group (MOG0 group, 4 rats ); OECs transplantation by vena caudalis (MOG1 group, 7 rats); OECs transplantation by lateral cerebral ventricle (MOG2 group, 4 rats). The animals in GPSCH group were also divided into 2 subsets: OECs blank group (GPSCH0 group, 4 rats); OECs transplanted by vena caudalis (GPSCH1 group, 4 rats). OECs transplanted through different ways in peak incidence, then the rats were measured to determine whether the symptom was ameliorated. Two weeks after transplantion, the rats were killed and the methods of histofluorescence and histopathology (HE staining and Luxol fast blue staining) were used to examine the distribution of the labeled OECs in EAE rats’ bodies and to explore whether there was some amelioration in histology. RESULTS: After OECs transplantation by vena caudalis or lateral cerebral ventricle, the rats’ symptom improved. Compared to OECs blank groups, there was significant difference in clinical scores (F=18.470, P<0.01; t=-7.147, P<0.01). No significant difference between MOG1 group and MOG2 group was observed (P>0.05). The labeled OECs entered into the brain through the broken blood-brain-barrier after transplantation by vena caud-alis, the labeled OECs near the subpial area and around the lesions were found. OECs transplantation by the way of lateral cerebral ventricle showed great migration potential, the cells moved towards to the lesions extensively. No significant difference between OECs transplantation group and OECs blank group in histopathology (HE staining and LFB staining) was observed (P>0.05), and the same thing also happened between MOG1 group and MOG2 group (P>0.05). CONCLUSION: The transplantation of adult rats’ OECs improves the EAE rats’ symptom at some degree by the ways of vena caudalis or lateral cerebral ventricle.     

Lentivirus-mediated RNA interference targeting vascular endothelial growth factor gene enhances the sensitivity of K562 cells to STI571

AIM: To investigate the effects of RNA interference (RNAi) inhibiting the expression of vascular endothelial growth factor (VEGF) gene mediated by lentiviral vector on the proliferation and apoptosis of K562 leukemic cell line. METHODS: A lentiviral vector containing short hairpin RNA (shRNA) targeting VEGF was constructed and cotransfected with the packaging plasmids mixture into 293T cells by Lipofectamine 2000. K562 cells were infected with the packaged lentivirus. The levels of VEGF mRNA and protein were detected by real-time quantitative RT- PCR, Western blotting and ELISA. Cellular proliferation was determined by trypan blue dye exclusion and MTT assay. STI571 (imatinib mesylate)-induced apoptosis was analyzed by flow cytometry. RESULTS: The lentiviral shRNA vector targeting VEGF was successfully constructed and transfected into K562 cells. The expressions of VEGF mRNA and protein in K562-shVEGF cells transfected with pRNAT-shRNA were significantly inhibited when compared with those of K562 and K562-con cells (mock transduction). The proliferation rate of K562-shVEGF cells slowed down. After STI571 treatment, the percentages of apoptotic cells in K562-shVEGF cells increased more significantly than those of K562 and K562-con cells (P<0.05). CONCLUSION: Inhibition of VEGF by lentivirus-mediated RNAi effectively inhibits proliferation and increases the sensitivity of K562 cells to STI571.     

Tristetraprolin attenuates subarachnoid hemorrhage-induced early brain injury in rats

AIM: To explore the expression level of tristetraprolin (TTP) in rats after subarachnoid hemorrhage (SAH) as well as the potential role of TTP in the early brain injury (EBI) after SAH in rats.METHODS: In the first experiment setting, total 56 adult male SD rats were randomly divided into sham group and SAH group. The SAH model was performed by endovascular perforation. The brain tissues were taken out after SAH at 5 different time points (0, 12, 24, 48, 72 h and 1 week). The expression of TTP in the brain tissues was detected by Western blot. In the second experiment, a total of 60 SD rats were divided into 4 groups:sham group, SAH group, SAH+vector group and SAH+TPP group. Neurological score, brain water content and blood-brain barrier were evaluated at 48 h after SAH. TUNEL staining was performed to detect cell apoptosis in the rat brain tissue. ELISA method was used for quantitative detection of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). The protein levels of TTP, Bax, Bcl-2 and cleaved caspase-3 in the rat brain tissue were detected by Western blot.RESULTS: The protein expression of TTP in the brain was downregulated markedly from 12 h after SAH, reached the lowest level at 48 h, and then had an upward trend. After modeling for 48 h, Garcia neurological score was significantly reduced, and brain water content and Evans blue (EB) content of the brain tissue of the rats in SAH group were significantly higher than those in sham group (P<0.05). SAH induced significant increases in IL-6 and TNF-α levels in the brain tissue (P<0.05). The number of TUNEL-stained cells was increased in the subcortical brain region after SAH compared with sham group. In addition, a lower level of Bcl-2 and higher levels of Bax and cleaved caspase-3 in the rat brains were observed at 48 h after SAH. However, the neurological deficit score was significantly increased, and the brain water content and EB content in the rat brains were significantly reduced in SAH+TTP group in comparison with SAH+vector group (P<0.05). Over-expression of TTP dramatically suppressed the levels of IL-6 and TNF-α in the rat brains, and reduced the number of TUNEL positive cells. Furthermore, upregulation of TTP significantly decreased the levels of cleaved caspase-3 and Bax, and evidently enhanced the expression of Bcl-2 (P<0.01).CONCLUSION: The expression of TTP is significantly decreased in early period after SAH, and enhancing the level of TTP effectively inhibits EBI following SAH in rats.     

Effects of gastrin on expression of cyclooxygenase and growth factors in rat gastric mucosa

AIM: To clarify the effects of gastrin on the expression of cyclooxygenase (COX) and several growth factors in rat gastric mucosa. METHODS: Male Sprague Dawley rats were fasted for 24 hours and subcutaneously injected with saline or gastrin 17 at doses of 1 μg/kg, 10 μg/kg and 100 μg/kg, respectively. The expression of COX-1, COX-2, heparin-binding epidermal growth factor-like growth factor (HB-EGF) and hepatocyte growth factor (HGF) in the gastric mucosa were examined using Western blotting and immunohistochemical staining. Effects of a potent gastrin receptor antagonist YM022 on the expression of COX-1, COX-2, HB-EGF and HGF in gastric mucosa were also evaluated. RESULTS: Gastrin dose-dependently increased the expression of COX-2 and HB-EGF in rat gastric mucosa while the expression of COX-1 and HGF did not change significantly after treatment with gastrin. However, pretreatment with YM022 dose-dependently abolished the up-regulation of COX-2 and HB-EGF expression induced by gastrin. CONCLUSIONS: This study demonstrates that gastrin up-regulates COX-2 and HB-EGF expression in rat gastric mucosa, indicating that COX-2 and HB-EGF are involved in pathogenesis of the gastrin-related gastric mucosal hyperplasia and carcinoma of stomach.