Effect of dexmedetomidine on astrocytes in rats with focal cerebral ischemia-reperfusion

AIM: To investigate the effects of dexmedetomidine on astrocytes in rats with focal cerebral ischemia-reperfusion. METHODS: Sixty female SD rats, weighing 230~250 g, were randomly divided into sham operation group, ischemia-reperfusion group, dexmedetomidine preconditioning group 1 and dexmedetomidine preconditioning group 2. The model of middle cerebral artery occlusion (MCAO) was established by thread embolism of middle cerebral artery. In sham operation group, the carotid arteries were exposed without performing MCAO. In ischemia-reperfusion group, NS was injected intraperitoneally 30 min before focal cerebral ischemia-reperfusion. The rats in dexmedetomidine preconditioning group 1 and dexmedetomidine preconditioning group 2 received intraperitoneal injection of dexmedetomidine at doses of 20 μg/kg and 40 μg/kg, respectively. The neurological scores were studied, and the pathological changes were observed under microscope with HE staining. The expression of glial fibrillary acidic protein (GFAP) and tumor necrosis factor α (TNF-α) in astrocytes was detected by the methods of immunohistochemistry and immunoblotting 24 h after cerebral ischemia-reperfusion. RESULTS: No neurological change was observed in sham operation group. The neurological deficiency scores in ischemia-reperfusion group were markedly higher than those in dexmedetomidine preconditioning group 1 and group 2 (P<0.05). Compared with sham operation group, the expression of GFAP and TNF-α in astrocytes and the level of GFAP increased significantly 24 h after focal cerebral ischemia-reperfusion. Pretreatment with dexmedetomidine significantly attenuated the expression of GFAP and reduced the infarct size and inflammation. CONCLUSION: Dexmedetomidine has a neuroprotective effect on focal cerebral ischemia-reperfusion injury by inhibiting the astrocytes.     

Astrocyte proliferation in the rat hippocampus after middle cerebral artery occlusion

AIM: To study rat astrocyte proliferation in ipsilateral hippocampus following focal cerebral ischemia. METHODS: Ischemia was induced by temporary middle cerebral artery occlusion (MCAO). In hippocampus of rats at 3, 7 and 30 days after MCAO, the numbers and anatomic distribution of glial fibrillary acidic protein (GFAP) were detected by immunohistochemistry. The protein expression of GFAP and proliferating cell nuclear antigen (PCNA) in the ipsilateral hippocampus were analyzed by Western blot analysis. RESULTS: Astrocytes appeared hypertrophic, with increased process thickness and numbers at 7 days after MCAO, and the highest density of astrocytes were seen at 30 days in the CA1, CA2 regions of the ipsilateral hippocampus. Western blot analysis revealed that GFAP levels were normal at 3 days, but increased by 7 days and remained elevation at 30 days. Western blot analysis of PCNA protein also revealed identified upregulation PCNA at 3 days after MCAO and the expression peaked at 7 days. CONCLUSION: This study demonstrates that focal cerebral ischemia in the rat results in a rapid response, a process often referred to as reactive astrogliosis or glial scarring, from resident astrocytes of the ipsilateral hippocampus to the side of ischemia.     

Effects of β-ME and RA on expression of GFAP in mesenchymal cells derived from mouse fetal liver

AIM: To investigate the effects of β-mercaptoethanol (β-ME) and all-trans rentinal acid (RA) on glial fibrillary acidic protein (GFAP) expression in mesenchymal cells derived from mouse fetal liver in vitro. METHODS: Cells suspension from 14.5-days-old mouse fetal liver were cultured in DMEM/HEPES/F12 supplemented with 20％ FCS and mesenchymal cells were acquired after discarding nonadherent cells. The 5th passage cells were induced by β-ME and RA. The characteristics of treated cells were assayed by immunocytochemistry staining at 5 hours and 5 days after induction. β-actin as an internal control, GFAP gene expression of mesenchyal cells was detected with semi-quantitative RT-PCR. RESULTS: After being inducted by β-ME and RA, 80％ approximately of the cells exhibited typical neural morphology and about 85％ expressed GFAP phenotype. Semi-quantitative RT-PCR showed that mRNA expression of GFAP increased in treated cells versus untreated cells (P<0.01). CONCLUSION: GFAP expression in mesenchymal cells derived from mouse fetal liver in vitro increases after being treated with β-ME and RA.     

Expression of glial fibrillary acidic protein in developing rat brain after intrauterine infection

AIM: To investigate the alteration of glial fibrillary acidic protein (GFAP) immunoreactivity in developing rat brain after intrauterine infection. METHODS: Escherichia coli (E.coli) was inoculated into uterine horn of pregnant rats when gestation was 15 days and the control group was inoculated with normal saline. Immunohistochemistry was used for evaluation of GFAP expression in pup brains at postnatal day 1 (P1), P3, P7, P14, P21. RESULTS: GFAP-immunopositive cells was scarce in the periventricular white matter at P1 and P3 in two groups (P>0.05), but not in other brain regions. The number of GFAP-immunopositive cells of the E.coli-treated pups was markedly increased in periventricular white matter and hippocampus at P7 compared with the control group (P<0.05). The E.coli-treated pups at P14 showed a marked increase in GFAP expression in periventricular white matter, corpus callosum and cortex (P<0.01). However, no significant different levels of GFAP expression in any brain regions were found at P21 between two groups (P>0.05). CONCLUSION: Intrauterine infection induces an increased expression of GFAP in the neonatal brain.     

Losartan inhibits LPS-induced GFAP expression via AMPK activation in mouse hippocampus

AIM: To investigate the effects of losartan on lipopolysaccharide (LPS)-induced glial fibrillary acidic protein (GFAP) expression, and to determine whether adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) activation is involved in the mechanism.METHODS: Adult male KM mice were divided into control group, LPS model group, losartan treatment group, and losartan and Compound C co-treatment group. To establish a model of central nervous system inflammation, the mice received daily intracerebroventricular injection of LPS (24 μg/d) for 2 d. Daily losartan administration (0.5, 1 or 5 mg·kg^-1·d^-1, ip) initiated at 14 d prior to LPS injection. Compound C (10 mg/kg, ip), a selective AMPK inhibitor, started to be injected daily at 2 d prior to LPS injection. The hippocampal tissues in each group were isolated at 3 d after the last LPS injection, and then the protein levels of GFAP, AMPK, p-AMPK, mammalian target of rapamycin (mTOR) and p-mTOR were determined by Western blot.RESULTS: Twice LPS injections significantly increased the expression of GFAP in the hippocampus (P<0.01). Losartan inhibited LPS-induced GFAP expression in a concentration-dependent way, and losartan at 5 mg·kg^-1·d^-1 significantly inhibited GFAP expression and AMPK activation (P<0.05), but it had no obvious effect on mTOR activation. Furthermore, Compound C significantly reversed the effect of losartan treatment on LPS-induced GFAP expression and AMPK phosphorylation (P<0.05).CONCLUSION: Losartan inhibits LPS-induced GFAP expression in the mouse hippocampus, and AMPK activation but not mTOR, is involved in the mechanism.     

Effects of baicalin on glial fibrillary acidic protein and nuclear factor-κB expression in juvenile rat hippocampus after status convulsion

AIM: To study the effects of baicalin (BC) on glial fibrillary acidic protein (GFAP) and nuclear factor-κB (NF-κB) expression and neuronal apoptosis in juvenile rat hippocampus after status convulsion (SC). METHODS: One hundred and ninety five juvenile male Sprague-Dawley rats were randomly divided into 3 groups: normal saline pretreatment group (NS group), SC group and SC with BC pretreatment group (BC group). Each of these 3 groups would be subdivided into 5 subgroups sacrificed at 4 h, 12 h, 24 h, 48 h and 72 h after SC. The rat SC model was prepared by lithium-pilocarpine chemical method. The protein expression of GFAP and NF-κB was detected by the method of immunohistochemistry. The mRNA expression of GFAP was detected by RT-PCR. The neuronal apoptosis was observed by TdT-mediated dUTP nick end labeling (TUNEL). RESULTS: Compared with NS group, the GFAP positive cells was increased in SC group (P<0.05). Compared with SC group, the expression of GFAP was significantly reduced in BC group (P<0.05). Compared with NS group, the NF-κB positive cells was increased in SC group (P<0.05). Compared with SC group, the expression of NF-κB was significantly reduced in BC group. RT-PCR showed that the expression trend of GFAP mRNA was similar to that of the protein. Compared with NS group, the TUNEL positive cells in the hippocampus CA1 area in SC group increased significantly 12 h after SC (P<0.01), and reached a peak at 48 h. After the intervention with BC, the TUNEL positive cells decreased significantly between 12~48 h after SC (P<0.05 or P<0.01), but the number of TUNEL positive cells remained significantly greater than that in NS group (P<0.05). CONCLUSION: The expression of GFAP and NF-κB in the hippocampus increased after SC in rats. Baicalin decreases the expression of GFAP and NF-κB in hippocampus of rats with pilocarpine-induced seizures, and reduces the number of neuronal apoptosis, suggesting that baicalin may protect against the brain damage caused by status convulsion.     

Effects of betaine on expression of GFAP,glycine and glycine receptor in hippocampus of pentylenetetrazole-induced epileptic rats

AIM：To study the effects of betaine on glial fibrillary acidic protein (GFAP), glycine (Gly) and glycine receptor (GlyR) expression in the hippocampus of rats with epilepsy induced by pentylenetetrazole (PTZ). METHODS：Forty-eight healthy male Wistar rats were randomly divided into control group, PTZ (35 mg·kg-1·d-1, intraperitoneal injection) group, PTZ+betaine (450 mg·kg-1·d-1, intragastric administration) group, PTZ+betaine (225 mg·kg-1·d-1, intragastric administration) group, PTZ+betaine (112.5 mg·kg-1·d-1, intragastric administration) group and PTZ+sodium valproate (200 mg·kg-1·d-1, intragastric administration) group. The rats in control group were intraperitoneally injected with saline at the same volume as PTZ injection, and those in control group and PTZ group received intragastric administration of saline at 1.0 mL·d-1. Rat behavior was recorded. Serum homocysteine (Hcy) level was measured. The expression of GFAP in the hippocampus was measured by immunofluorescence. Hippocampal Gly content was measured by an amino acid analysis system. The expression of GlyR was detected by immunofluorescence and Western blotting. RESULTS：There was no difference in the latency of grand mal seizures among groups (P>0.05). However, betaine treatment significantly decreased the duration of the first grand mal seizure compared with PTZ group (P<0.01). Serum Hcy level in PTZ group was significantly lowered compared with control group (P<0.01), and further decreased after betaine treatment (P<0.05). GFAP in PTZ group was significantly higher than that in control group (P<0.01), and decreased after betaine treatment (P<0.05). Gly in PTZ group was significantly lowered compared with control group (P<0.01), and increased after betaine treatment (P<0.05). The content of GlyR among groups showed the same trend as Gly. CONCLUSION：Betaine treatment shows antiepileptic effect, which may be related to its effects on the metabolites of Hcy and Gly.     

miR-301a-3p regulates connexin 43 expression in rat astrocytes

AIM: To explore the mechanism of microRNA-301a-3p (miR-301a-3p) regulating connexin 43 (Cx43) expression in the rat astrocytes. METHODS: miR-301a-3p agomir, miR-301a-3p antagomir and microRNA negative control (miR-NC) were synthesized and transfected into the astrocytes. The protein expression of Cx43 was determined by Western blot. The recombinant vectors wt-pEZX-MT05-Cx43 and mut-pEZX-MT05-Cx43 were constructed. The target gene of miR-301a-3p was validated by dual-luciferase reporter assay. The expressional vector pcDNA3.1-Cx43 was constructed and the biological activity regulation of miR-301a-3p on astrocytes was analyzed by restore experiment. RESULTS: The results of Western blot showed that the Cx43 expression was inhibited by miR-301a-3p agomir transfection (P<0.05). The results of dual-luciferase reporter assay showed that miR-301a-3p bound to 3′-UTR of Cx43, thus regulating expression of Cx43 negatively. Transfection with recombinant vector pcDNA3.1-Cx43 without 3′-UTR of Cx43 into astrocytes resulted in the restoration of the negative function of miR-301a-3p on Cx43 expression, also induced the apoptosis of astrocytes. CONCLUSION: miR-301a-3p binds to 3′-UTR of Cx43 mRNA in the rat astrocytes, thus regulating expression of Cx43 negatively.     

Effects of Scutellaria barbata flavonoids on abnormal expression of NOS,HSP70 and apoE induced by Aβ 25-35 in rat astrocytes

AIM:To study the effects of Scutellaria barbata flavonoids (SBF) on abnormal expression of nitric oxide synthase (NOS), heat-shock protein 70 (HSP70) and apolipoprotein E (apoE) induced by Aβ 25-35 in rat astrocytes. METHODS:The third generation of cultured rat astrocytes was divided into 5 groups. The cells in 3 drug treatment groups were given SBF at dose of 17.5 mg/L, 35 mg/L and 70 mg/L for 24 h, and then the cells in model group and 3 doses of SBF groups were exposed to Aβ 25-35 at concentration of 100 μmol/L for 24 h. The expression of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS) in the cultured cells was assayed by immunohistochemical method. The expression of HSP70 was estimated by Western blotting and the mRNA expression of apoE was assessed by RT-PCR. RESULTS:Compared with control group, the protein level of eNOS were significantly decreased and the protein level of iNOS increased (P<0.01) in model group. The protein expression of HSP70 and mRNA expression of apoE were notably increased (P<0.01) in model group. However, these disturbances were attenuated by SBF at dose of 17.5, 35 and 70 mg/L (P<0.01). CONCLUSION:SBF has an obvious protective effect on damaged astrocytes induced by Aβ 25-35, suggesting that SBF may be helpful for the treatment of Alzheimer disease.     

Effect of GRK5 on activation of rat astrocytes

AIM: To study the effect of G-protein-coupled receptor kinase 5 (GRK5) on the activation of astrocytes in the brain cortex of newborn Wistar rats. METHODS: GRK5 gene was silenced in the model of rat brain cortex astrocytes in vitro for 24 h. N-acetylcysteine (NAC), which is a known inhibitor of NF-κB, was added into the culture medium according to gene silencing for 24 h. The expression levels of GFAP and caspase-3 were detected by the method of immunofluorescence, and the mRNA levels of NF-κB, TNF-α, IL-1β and iNOS were determined by real-time PCR. Moreover, the activity of SOD and concentrations of TNF-α and NO were measured. RESULTS: GRK5 gene silencing increased the expression of NF-κB at mRNA and protein levels obviously (P<0.01), and the mRNA levels of IL-1β and iNOS increased synchronously (P<0.01). Furthermore, caspase-3-positive cells in GRK5 siRNA group were increased compared with control siRNA group (P<0.01). Treatment with NAC obviously reduced the activity of NF-κB and weakened the effects induced by GRK5 siRNA (P<0.05). CONCLUSION: GRK5 siRNA increases NF-κB activity and induces the activation of astrocytes.     

Internal standards for mRNA quantitative expression in hypoxic astrocytes

AIM: To investigate the effect of hypoxia on glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin and endothelin-converting enzyme (ECE)-2 mRNA levels in cultured mouse brain astrocytes (AC). METHODS:AC from neonatal mouse brain was incubated for 24h in serum-free medium under hypoxic or normoxic conditions.The amount of transferred RNA was estimated using ethidium bromide stained 28SrRNA and 18S rRNA.The levels of tested mRNA were evaluated by Northern blot RNA hybridizat ion.RESULTS: The GAPDH mRNA was up-regulated to 503.0% of the normoxic controls in hypoxic AC (n=10. P<0.01), while the level of β-actin mRNA in hypoxic AC was down-regulated to 50.3% of control levels (n=6. P<0.01). In contrast, the ECE-2 mRNA level was not affected by hypoxia. CONCLUSION: The results indicate that GAPDH and β-actin are not suitable for the internal standards for quantitative mRNA expression analysis in AC after hypoxia or ischemia. ECE-2 may function as an alternative in such circumstance.     

Effects of synthetic kainic acid on microfilament expression and gap junction in astrocytes

AIM：To study the effects of synthetic kainic acid (SKA) and 1-heptanol (1-Hep), a gap junction blocker, on the cytoskeletal filament expression in the astrocytes. METHODS：The neonatal rat brain was obtained from the Wistar rats (1 day old) and primary purified astrocytes were obtained by differential attachment for removing filamentoblasts and orbital shaker for removing the oligodendrocytes. The effects of SKA and other interventions on the morphologic changes and expression levels of skeleton protein filamentous actin (F-actin) were observed in the astrocytes after 24 h of the exposures by laser scanning confocal microscopy. The effect of 1-Hep on the expression of F-actin was also explored. RESULTS：Compared with control group, the fluorescence intensities in KCl group and KCl+SKA group were increased, and highly increased in KCl+SKA group. The F-actin filaments in the above 2 groups were more intensive, thickened and concentrated than those in control group, and more obvious in KCl+SKA group. l-Hep significantly decreased the expression of F-actin in KCl group and KCl+SKA group as compared with control group, and parts of the filamentous fracture were seen in the astrocytes in all 1-Hep-treated groups, in which some of the filamentous lines were crosscut. CONCLUSION：Increase in the expression of F-actin in the astrocytes affects the structure and function of the intercellular gap junctions, which may be involved in the mechanism of SKA-induced epilepsy.     

Ubiquitin-dependent degradation of SnoN/Ski protein in glomerular mesangial cells induced by high glucose

AIM: To observe the protein expression of SnoN/Ski and ubiqutin ligase Arkadia in rat glomerular mesangial cells (GMCs) exposed to the high glucose. METHODS: Cultured rat glomerular mesangial HBZY-1 cells were divided into control group, 20 mmol/L glucose group, 30 mmol/L glucose group, 30 mmol/L glucose+MG132 group (culture medium containing 30 mmol/L glucose and 0.5 μmol/L specific proteasome inhibitor MG132), and mannitol group. The expression levels of SnoN, Ski and Arkadia were measured by Western blotting analysis, immunofluorescence and laser scanning confocal microscopy. RESULTS: In control GMCs, the expression of SnoN/Ski was rich and Arkadia was weak. After stimulated with high glucose, the expression of SnoN/Ski was decreased and Arkadia was gradually increased (P<0.05). Compared with high glucose group, the levels of SnoN/Ski and Arkadia were mostly reverted by adding the proteasome inhibitor MG132 at concentration of 0.5 μmol/L (P<0.01). The expression levels of SnoN/Ski and Arkadia were not significantly changed in mannitol group in comparison with control group (P>0.05). CONCLUSION: High glucose decreases the expression of SnoN/Ski through ubiquitin-dependent degradation of SnoN/Ski. The degradation of SnoN/Ski mediated by Arkadia may play an important role in the pathogenesis of diabetic nephropathy.     

Expression of ephrin-A3 in lipopolysaccharide-induced proliferation of astrocytes

AIM: To explore the effects of ephrin-A3 expression on the proliferation of astrocytes induced by lipopolysaccharide (LPS).METHODS: Astrocytes from cerebral cortex of newborn rats were cultured and purified.The cells were exposed to LPS at the concentration of 10 mg/L and divided into 6 groups at random: control group (without LPS) and the groups of exposure to LPS for 30 min, 6 h, 24 h, 48 h and 72 h.The morphological changes of the astrocytes were observed under inverted phase-contrast microscope.The survival rate and apoptotic rate of astrocytes were measured by MTT method and AO/EB staining,respectively.The expression of ephrin-A3 and glial fibrillary acidic protein (GFAP) was measured by fluorescein staining with CY3.The changes of the inflammatory factors TNF-α and IL-6 in the culture supernatant were detected by ELISA.RESULTS: Compared with control group, the survival rates were notably increased, the apoptotic rates were notably decreased, the expression of ephrin-A3 and GFAP was significantly up-regulated and the inflammatory cytokines were significantly increased in the cells exposed to LPS at the concentration of 10 mg/L for 24 h, 48 h and 72 h.CONCLUSION: LPS promotes the proliferation of astrocytes.The expression of ephrin-A3 increases during the proliferation of astrocytes induced by LPS in rats.The mechanism may be related to inflammatory stimulation by cytokines.     

Changes of MAPKs expression in rat hippocampal neurons after sleep deprivation

AIM: To explore the change and the possible role of MAPKs in rat hippocampus neuron after sleep deprivation. METHODS: The morphology of hippocampus neuron after sleep derivation was observed by TUNEL and HE staining, the activity of ERK was assayed by β-liquid scintillation counting and the expression of JNK was detected by Western blot. RESULTS: In paradoxical sleep deprivation (PSD) group, the number of apoptotic cells in hippocampus was increased. The scores of ERK activity were 1 764.00±941.56. Compared with control groups, the ERK activity was obviously decreased (P<0.05). The JNK expression was 87.5%, which was higher than that in control group. CONCLUSION: These results provide some important evidences that the sleep deprivation could cause changes in MAPKs activity, which may be related to the mechanism of hippocampus neuron apoptosis.     

Effects of lipopolysaccharide,IL-6 and TNFα on the tissue factor expression of astrocytes

AIM:To study the effects of lipopolysaccharide(LPS), interleukin-6(IL-6)and tumor necrosis factor α (TNFα) on tissue factor(TF) expression of astrocytes. METHODS:Astrocytes were identified with anti-glial fibrillary acidic protein antibody. The TF activity of cell lysate was measured with one stage clotting assay. RESULTS:TF activity of astrocytes of LPS,IL-6,TNFα groups were obviously higher than that of the control group(P <0.05); While LPS,IL-6 and TNFα were combined with trifluoperazine or H₇, their inductive effects were inhibited. CONCLUSION:LPS,IL-6 and TNFα promoted the TF expression of astrocytes and its mechanisms may connected with Calcium/Camodulin and protein kinase C pathway.     

Saikosaponin a inhibits PTZ-induced activation of hippocampal astrocytes in mice

AIM: To investigate the inhibitory effect of saikosaponin a (SSa) on pentylenetetrazole (PTZ)-induced activation of hippocampal astrocytes in mice. METHODS: Hippocampal astrocytes were isolated and cultured. The cells were randomly divided into control group, PTZ group, PTZ+0.625 mg/L SSa group and PTZ+1.25 mg/L SSa group. The cells were identified by detection of glial fibrillary acidic protein (GFAP). The cell viability was measured by MTT assay. The cell cycle was analyzed by flow cytometry. The expression of GFAP and connexin 43 (Cx43) was mea-sured by ELISA. The level of apoptosis was determined by flow cytometry and Hoechst 33258 staining. RESULTS: The primary hippocampal astrocytes grew by adherent culture, and the processes of the astrocytes were obvious. Immunofluorescence showed positive GFAP expression in the astrocytes. Compared with control group, the viability of the cells and the percentage of the cells in G₂/M phase in PTZ group were significantly increased (P<0.05), and the expression of GFAP and Cx43 was also markedly increased (P<0.05). Compared with PTZ group, the viability of the cells and the percentage of the cells in G₂/M phase were obviously decreased in PTZ+0.625 mg/L SSa group and PTZ+1.25 mg/L SSa group, and the expression of GFAP and Cx43 was also reduced, whereas the percentage of apoptotic cells was significantly increased (P<0.05).CONCLUSION: SSa significantly suppresses PTZ-induced activation of hippocampal astrocytes, inhibits the cell proliferation and induced apoptosis.     

Effect of genistein on ammonia-induced NF-κB activation in astrocytes

AIM: To explore the effect of genistein on ammonia-induced nuclear factor-κB (NF-κB) activation and the underlying mechanism.METHODS: Primary astrocyte cultures were prepared and challenged with NH₄Cl to establish a hyperammonemic model. The activation of ERK, Akt and NF-κB was examined by Western blot.RESULTS: AG1478 and genistein significantly inhibited ammonia-induced activation of ERK and Akt. Ammonia-induced NF-κB nuclear translocation was significantly inhibited by the pretreatment of LY294002, genistein and AG1478.CONCLUSION: Genistein significantly inhibited ammonia-induced ERK activation and Akt-mediated NF-κB activation, which might represent the important mechanism by which this naturally occurring substance exerts its swelling-inhibiting effect.