Effects of taurine on expression of glucose transporters in rat brain with diffused brain injury

AIM: To investigate the effect of taurine on the expression of glucose transporter 1 (GLUT1) and transporter 3 (GLUT3) in rat brain with diffused brain injury (DBI).METHODS: Sixty-four male Sprague-Dawley rats were randomly divided into 4 groups: sham-operated group, DBI group, low-dose taurine group (200 mg/kg, ig) and high-dose taurine group (300 mg/kg, ig).After fed with the corresponding drugs for 7 days, the animal model of DBI was made, and the rats were executed 24 h after DBI.The expression of GLUT1 and GLUT3 in the brain was detected by the methods of immunohistochemistry and Western blotting.The pathomorphological changes of the cerebral cortex were observed under electron microscope.RESULTS: The expression of GLUT1 was detected in capillary vascular endothelial cells in each group, and cytoplasm-positive cells or the cells with buffy membrane were observed.No significant difference of the GLUT1 expression in brain tissues between DBI group and sham-operated group was detected.Compared with DBI group, the expression of GLUT1 in the brain tissues were significantly increased in low-and high-dose taurine groups (P<0.01).The expression of GLUT1 in the brain tissues in low-dose taurine group were significantly higher than that in high-dose taurine group (P<0.05).The positive staining of GLUT3 only appeared in the periphery of the third ventricle in each group in the cells with buffy membrane or positive cytoplasm.The expression of GLUT3 in the brain tissues in DBI group was significantly higher than that in sham-operated group (P<0.01).The expression of GLUT3 in the brain tissues in low-and high-dose taurine groups was significantly higher than that in DBI group (P<0.01).Compared with low dose taurine group, the expression of GLUT3 in the brain tissues were significantly increased in high-dose taurine group (P<0.01).The pathological damage of cerebral cortex in low-dose taurine group was obviously alleviated.CONCLUSION: Taurine may take part in the neuroprotective mechanisms in DBI by increasing the expression of GLUT1 and GLUT3 at protein level to maintain the energy supply in brain tissues.     

Protective effect of ischemic preconditioning on rat brain with ischemia/reperfusion injury

AIM:To study whether ischemic preconditioning(IPC) has a protective effect against ischemia/reperfusion(I/R) injury in brain, and the possible relationship between IPC and the regulating function of microcirculation.METHODS:The I/R models were established both in I/R and IPC groups of Sprague-Dawley rats. Additional procedure was performed of short term cerebral ischemic preconditioning in IPC group 24 hours before I/R. Skull windows were performed through which microcirculation features were measured before ischemia, during ischemia, and reperfusion. Finally, brains were cut into slices and stained with red tetrazoline(TTC).RESULTS:Most TTC stained brains in I/R group presented irregular palely red areas which were few in IPC group. Compared with I/R group, IPC group presented relatively increase in accumulated length of capillaries, mean cerebral microcirculatory perfusion, and microcirculatory velocity in ischemic and reperfusion phase. There was no-reflow phenomenon in I/R group in reperfusion phase, which was substituted by the course of increasing reperfusion in IPC group.CONCLUSIONS:IPC could relieve the reduction of tissue perfusion during ischemia and the no-reflow phenomenon during reperfusion by improving the regulating function of microcirculation, which relatively promote the opening of capillaries and accelerating of microvascular flow, therefore protect brain from I/R injury.     

Effect of ginsenoside Rg1 on activity and protein expression of NOS in rats with cerebral ischemic reperfusion injury

AIM: To observe the neuroprotective effects of ginsenoside Rg1 on focal cerebral ischemia reperfusion (I/R) injury in rats. METHODS: SD rats were applied to right middle cerebral artery occlusion (MCAO) for 2 h followed by 24 h of reperfusion. The rats were randomly divided into sham-operation group, I/R group and ginsenoside Rg1 pretreatment groups. The rats in ginsenoside Rg1 pretreatment groups were pretreated with ginsenoside Rg1 at doses of 10, 20 or 40 mg/kg once a day for 7 days and then subject to MCAO. The neurological deficit score was measured by Longa's method. The neurons were observed with Nissel staining. The nitric oxide (NO) content, the activity of nitric oxide synthase (NOS) and inducible NOS (iNOS) in the brain tissues were determined. The expression of neuronal NOS(nNOS) and iNOS was detected by Western blotting. RESULTS: Compared with sham-operation group, ginsenoside Rg1 significantly reduced the neurological deficit score and increased the neuron number in the hippocampus. The activity of NOS and iNOS, and NO content were decreased. Ginsenoside Rg1 also down-regulated the expression of nNOS and iNOS. CONCLUSION: Ginsenoside Rg1 has protective effect on the brain during cerebral I/R injury in rats. The mechanism may be related to reducing the content of NO and the activiy of NOS dose-dependently.     

Effects of ischemic postconditioning on hippocampus CA1 area rCBF and astrocyte activation after cerebral ischemia in tree shrews

AIM: To study the effects of ischemic postconditioning (PC) on regional cerebral blood flow (rCBF) and astrocyte (AS) activation in hippocampus CA1 area and to explore the possible mechanism of ischemic PC affecting glial fibrillary acidic protein (GFAP) expression during focal cerebral thrombosis. METHODS: The thrombotic focal cerebral ischemia was induced by photochemical reaction in tree shrews, and ischemic postconditioning was established by cliped ipsilateral carotid of the animal at 4 h after cerebral ischemia. The rCBF and GFAP expressions in hippocampus CA1 area were detected, respectively, by laser-Doppler (LD) fowmeter and immunohistochemistry. RESULTS: The numbers of GFAP positive cells were increased markedly and GFAP expression enhanced (P<0.01). AS oncosis was apparent 24 h after cerebral ischemia. Postconditioning increased hippocampus rCBF from (2.55±0.28) PU to (10.42±3.75) PU (P<0.05) at 24 h and from (9.84±1.22) PU to (18.74±1.60) PU (P<0.05) at 72 h after the cerebral ischemia, and AS oncosis was inhibited markedly. CONCLUSION: Multiple, short, regional carotid occlusions may prolong “time window” of therapeutic cerebral ischemia. The protection mechanism of the ischemic postconditioning may be associated with the increase in rCBF and improvement of hippocampus microenvironment by regulating AS activation.     

Effects of Astragalus polysaccharide on expression of HSP70, PKB and P53 in rat cerebral cortex with cerebral ischemia and reperfusion

AIM:To explore the molecular effects of Astragalus polysaccharide(AP) on improving nervous functions and preventing neuronal apoptosis in rat cerebral cortex with cerebral ischemia and reperfusion. METHODS:One hundred and twenty male Wister rats were randomly divided into sham operation group(SOG), model groups(MG-1 d, 3 d and 7 d), low-dose AP treatment groups(L-APTG-1 d, 3 d and 7 d), and high-dose AP treatment groups(H-APTG-1 d, 3 d and 7 d). The right middle cerebral artery of the rats in MG and AGTG was intercepted by operation to induce ischemic brain injury. The rats in L-APTG and H-APTG were treated with AP at the doses of 5 mg/kg and 15 mg/kg by intraperitoneal injection, respectively. On the 1st day, 3rd day and 7th day after operation, those animals were sacrificed to collect the brain specimens for the study after cerebral blood flow reperfusion and determination of neurological deficit scores. The structural changes of the neurons were observed under electron microscope. Apoptosis was analyzed by flow cytometry. The protein levels of heat-shock protein 70(HSP70), protein kinase B(PKB) and P53 in cerebral corical neurons were determined by immunohistochemical staining and Western blotting. RESULTS:The neurological deficit scores and the apoptotic rate of cerebral cortical neurons in H-APTG were significantly lower than those in MG and L-APTG(P<0.05). The structures of the neurons in H-APTG, such as ribosome endoplasmic reticulum, nucleolus, Golgi complex, mitochondria, etc, were better than those in MG and L-APTG. On the 1st day, 3rd day and 7th day, the protein levels of HSP70 and PKB in cerebral cortical neurons in H-APTG were significantly higher than those in L-APTG, which were significantly higher than those in MG(P<0.05). However, the P53 protein level in H-APTG was significantly lower than that in L-APTG, which was significantly lower than that in MG(P<0.05). CONCLUSION:AP improves nervous functions and inhibits neuronal apoptosis during ischemia and reperfusion. The molecular mechanisms are associated with variations of protein expression in cerebral cortical neurons, such as promotion of HSP70 and PKB and inhibition of P53.     

Effect of erigeron on intercellular adhesion molecule-1 and its mRNA expression during cerebral ischemia and reperfusion in rats

AIM: To investigate the effect of erigeron on intercellular adhesion molecule-1 (ICAM-1) and mRNA expression during cerebral ischemia/reperfusion. METHODS: The rat models of middle cerebral artery (MCA) focal cerebral ischemic reperfusion were established with the suture method in the study. The ICAM-1 mRNA and protein expression were measured by RT-PCR and immunohistochemistry techniques, respectively. RESULTS: By down-regulating the expression of ICAM-1 protein and mRNA and alleviating inflammation in cerebral ischemic region, erigeron exerted a protective effect in cerebral ischemia and reperfusion. CONCLUSION: The results suggest that erigeron protects the brain against cerebral ischemia and reperfusion injury via inhibiting ICAM-1 expressino.     

Effect of aminoguanidine on TNF-α,IL-1β and neuronal apoptosis after focal cerebral ischemic injury in rats

AIM: To evaluate the effect of aminoguanidine (AG) on inflammatory factors and neuronal apoptosis after focal cerebral ischemic injury in rats and the possible mechanism of protective effect of AG against cerebral ischemic injury.METHODS: Thirty male SD rats (weighing 250 g-280 g) were randomly divided into three groups: (1) sham operated group (SH group,n=10),(2) ischemic groups (IS group,n=10),(3) AG group (n=10).In AG group,AG at dose of 100 mg·kg-1 was given intraperitoneally twice a day for 3 consecutive days.In IS group,normal saline was given instead of AG.Focal cerebral ischemia was produced by middle cerebral artery occlusion (MCAO) for 12 h.A nylon thread with rounded tip was inserted into left internal carotid artery cranially until resistance was felt.The distance from bifurcation of common carotid artery to the tip of the thread was about 18-19 mm.Focal cerebral ischemia was confirmed by left Horners syndrome and right side hemiplegia.In SH group,the carotid artery was exposed but no thread was inserted.The expression of tumor necrosis factor-α(TNF-α) was determined by immunochemistry and the content of interleulin-1β(IL-1β) was measured by radioimmunoassay.The expressions of Bcl-2 and Bax protein were detected by flow cytometry.RESULTS: The expression of TNF-α and the content of IL-1β were markedly increased after MCAO.Significantly increased DNA fragmentation,the indication of apoptosis,was detected after MCAO.The expression of TNF-α and the content of IL-1β were significantly lower in AG group than those in IS group.The percentage of apoptosis cells and expression of Bax protein were markedly lower in AG group than those in IS group but still significantly higher than those in SH group.The expression of Bcl-2 protein was markedly higher in AG group than that in IS group.No significant difference in the expression of Bcl-2 protein between IS and SH group was observed.CONCLUSION: AG inhibits the increase in the expression of TNF-α and the content of IL-1β,and protects neurons from apoptosis induced by focal cerebral ischemia through increasing the Bcl-2 protein expression and inhibiting the Bax protein expression.     

Effects of salvianolic acid B on the energy metabolism and hydrocephalus in mice with cerebral ischemia

AIM: To explore the effects of salvianolic acid B (SalB) on the energy metabolism and hydrocephalus in mice with cerebral ischemia.METHODS: NIH mice were randomly divided into four groups: sham-operated group,cerebral ischemia group,SalB-treated group and nimodipine-treated group.The brain tissue energy charge (EC),phosphocreatine (PCr),the activity of ATPase,excitability amino acid (EAA) content and water content of brain were measured when cerebral ischemia for 30 min.RESULTS: EC (0.520±0.034),PCr content ［(98.344±13.249) μmol/g］,the activity of Na⁺-K⁺-ATPase ［(0.593±0.013)×10³ U/g］ and Ca²⁺-ATPase ［(0.484±0.053)×10³ U/g］ in SalB-treated group were significantly higher than those in cerebral ischemia group {EC (0.465±0.037),PCr content ［(81.614±9.919) μmol/g］ ,the activity of Na⁺-K⁺-ATPase ［(0.244±0.065)×10³ U/g］,the activity of Ca²⁺-ATPase ［(0.321±0.086)×10³ U/g］} (P<0.01).The glutamate (Glu) content ［(0.405±0.110) μmol/g］,aspartate (Asp) content ［(0.141±0.020) μmol/g］ and water content of brain ［(38.1±0.1)%］ in SalB-treated group were markedly lower than those in cerebral ischemia group ［ Glu content (0.550±0.140) μmol/g,Asp content (0.287±0.050) μmol/g,water content of brain (44.1±0.1)%］ (P<0.05,P<0.01).CONCLUSION: The increase in cerebral energy metabolism and the activity of ATPase,and decrease in EAA content in brain tissue are the mechanism of SalB alleviating hydrocephalus at the early stage of cerebral ischemia in mice.     

Effect of cimicifugoside H-1 on amino acid neurotransmitters in striatum of rats with cerebral ischemia

AIM: To study the neuroprotective effect of cimicifugoside H-1 and to explore the mechanism involved by determining the variation of amino acid neurotransmitters in extracellular fluid in the striatum of rats with cerebral ischemia. METHODS: The rats were randomly divided into sham-operated, cerebral ischemia, high-, middle- and low-dose cimicifugoside H-1, and ginkgo groups. Focal cerebral ischemia model was established by middle cerebral artery occlusion (MCAO) with sutures. Normal saline was intraperitoneally injected into the rats in sham-operated group and cerebral ischemia group, while ginkgo and different doses of cimicifugoside H-1 were injected into the rats in ginkgo group and high-, middle- and low-dose cimicifugoside H-1 groups, respectively, once a day for 7 d. The striatal fluids were gained in vivo by brain microdialysis. The contents of aspartic acid, glutamic acid, glycine and γ-aminobutyric acid were tested by high-performance liquid chromatography electrochemical detector system. RESULTS: Compared with sham-operated group, the contents of excitatory amino acids (aspartic acid and glutamic acid) were significantly increased 2 h after cerebral ischemia (P<0.05). Compared with cerebral ischemia group, the contents of aspartic acid and glutamic acid were significantly decreased 2 h after cerebral ischemia in high-dose cimicifugoside H-1 and ginkgo groups (P<0.05). Compared with cerebral ischemia group, the contents of aspartic acid and glutamic acid did not show significant decrease 2 h after cerebral ischemia in middle- and low-dose cimicifugoside H-1 groups. Compared with sham-operated group, the contents of inhibitory amino acid (γ-aminobutyric acid and glycine) were significantly decreased 3 h after cerebral ischemia in cerebral ischemia group (P<0.05). Compared with cerebral ischemia group, the contents of γ-aminobutyric acid and glycine were significantly increased 3 h after cerebral ischemia in high-dose cimicifugoside H-1 and ginkgo groups (P<0.05). Compared with cerebral ischemia group, the contents of γ-aminobutyric acid and glycine did not show significant decrease 3 h after cerebral ischemia in middle- and low-dose cimicifugoside H-1 groups. CONCLUSION: Cimicifugoside H-1 restrains the excessive releases of excitatory amino acids and increases the contents of inhibitory amino acids during cerebral ischemia. It doesn't only penetrate through the blood brain barrier, but also regulates the disorder of excitatory amino acid during cerebral ischemia, thus showing the protective function to cerebral neuron during cerebral ischemia.     

Effect of intermittent hypobaric hypoxia preconditioning on expression of Ngb and Bcl-2 in hippocampal CA1 region in rats with global cerebral ischemia-reperfusion

AIM:To study the effect of intermittent hypobaric hypoxia preconditioning (IHHP) on the expression of neuroglobin (Ngb) and Bcl-2 in hippocampal CA1 region in the rats with global cerebral ischemia-reperfusion. METHODS:The Wistar rats were randomly divided into sham group, IHHP control group, global cerebral ischemia-reperfusion group (I/R group), and IHHP+I/R group. The 4-vessel occlusion rat model of Pulsinelli was performed in the rats in I/R group and IHHP+I/R group, in which the common carotid artery was occluded for 8 min before reperfusion. Thionine staining and immunohistochemical staining were used to observe the histological changes of the hippcampus and the expression of Ngb and Bcl-2 in the hippocampal CA1 region. RESULTS:A significant increase in the quantity of surviving cells in the hippocampal CA1 region was observed in IHHP+I/R group as compared with I/R group. There was a significant increase in the expression of Ngb and Bcl-2 in the hippocampal CA1 region in IHHP+I/R group as compared with I/R group. CONCLUSION: Through the up-regulation of hippocampal Ngb and Bcl-2 expression, intermittent hypobaric hypoxia preconditioning may play a role in neuroprotection by reducing hippocampal neuronal apoptosis from ischemia-reperfusion.     

Astragalus injection inhibits expression of Apaf-1 in rat hippocampus after cerebral ischemia and reperfusion

AIM: To investigate the effect of Astragalus injection on the expression of apoptotic protease-activating factor 1 (Apaf-1) in the hippocampus of global cerebral ische-mia-reperfusion rats. METHODS: Male SD rats were randomly divided into 4 groups with 30 each: sham operation group, cerebral ischemia-reperfusion group, cerebral ischemia-reperfusion+Astragalus injection group, and cerebral ischemia-reperfusion+vehicle group. The global cerebral ischemia-reperfusion model of the rats was established by 4-vessel occlusion. The rats in cerebral ischemia-reperfusion group, cerebral ischemia-reperfusion+Astragalus injection group and cerebral ischemia-reperfusion+vehicle group were further divided into 7 subsets, according to the reperfusion time of 0 h, 0.5 h, 2 h, 6 h, 24 h, 72 h and 120 h. After reperfusion, the brains were removed at the corresponding time points. The protein expression of Apaf-1 in hippocampal neurons was detected by immunohistochemistry and Western blotting. The mRNA expression of Apaf-1 was observed by RT-PCR. RESULTS: Compared with sham operation group, the expression of Apaf-1 at mRNA and protein levels at all time points except 0 h and 120 h increased obviously in cerebral ischemia-reperfusion group (P<0.05). Compared with cerebral ischemia-reperfusion group, the expression of Apaf-1 at mRNA and protein levels at all time points except 0 h and 120 h decreased obviously in cerebral ischemia-reperfusion+Astragalus injection group (P<0.05). However, those in cerebral ischemia-reperfusion+vehicle group had no obvious change (P>0.05). CONCLUSION: Astragalus injection inhibits the expression of Apaf-1 at mRNA and protein levels in hippocampus of global cerebral ischemia-reperfusion rats, thus inhibiting the apoptosis of hippocampal neurons.     

Upregulative effect of CGRP on expression of CREB and phospho-CREB protein during focal cerebral ischemia/reperfusion in rat parietal cortex

AIM: To investigate the effect of calcitonin gene related peptide (CGRP) on the expression of cyclic AMP response element binding protein (CREB) and phosphorylated-CREB in rat parietal cortex during focal cerebral ischemia/reperfusion (I/R).METHODS: Focal cerebral ischemia/reperfusion model was induced by occlusion of the right middle cerebral artery using the intraluminal suture method. The expressions of CREB and phospho-CREB in the parietal cortex in different groups (sham group, focal cerebral ischemia/reperfusion group and CGRP group) were detected with immunohistochemistry and Western blotting, and the positive products were analyzed by image analysis system.RESULTS: There was definite expression of CREB in right parietal cortex in sham group, while it was lesser in I/R group than that in sham group, but it became more in CGRP group than that in I/R group (P<0.05). Phospho-CREB was barely detected in right parietal cortex in sham group and it became more in I/R group than that in sham group. The expression of phospho-CREB increased in CGRP group than that in I/R group of the right parietal cortex (P<0.05).CONCLUSION: CGRP upregulates the expression of CREB and phospho-CREB in the ischemic neurons of the parietal cortex during focal cerebral ischemia/reperfusion and CREB probably involves in the mechanism of protective role of CGRP to ischemic neurons.     

Fluorocitrate inhibits regulatory effect of ischemic postconditioning on HIF-1α/iNOS signaling pathway after cerebral ischemia in tree shrews with brain injury

AIM: To investigate the regulatory effect of HIF-1α/iNOS signaling pathway on the neuroprotection of ischemic postconditioning (PC) in tree shrews, and to explore the mechanisms of deteriorated cerebral injury after inhibiting astrocyte (AS) metabolism. METHODS: Thrombotic cerebral ischemia was induced by photochemical reaction in tree shrews. Fluorocitrate (FC) was used to inhibit AS metabolism and the ischemic PC was established at 4 h after ischemia followed by clipped ipsilateral common carotid artery on the ischemia side for 3 times, 5 min/time. A total of 67 male tree shrews were randomly divided into 7 groups:control (n=9), ischemia (4 h and 24 h, n=9 for each group), ischemia with PC (4 h and 24 h, n=9 for each group), and FC pretreatment (4 h and 24 h, n=11 for each group). The cerebral infarction size was detected by TTC staining, and the histological changes of hippocampal neurons were observed under light microscope. The regional cerebral blood flow (rCBF) in ischemic cortex was monitored by laser Doppler brain flowmetry. The protein expression of iNOS in hippocampus was detected both by immunohistochemistry and Western blot. The production of NO detected by spectrophotometer. The level of HIF-1α in hippocampus analyzed by ELISA. RESULTS: The cerebral infarct volume was increased with prolonged duration of ischemia, and the changes of ischemia at 24 h were significant (P<0.05). The cortical rCBF was progressively decreased, and it was decreased at 4 h and 24 h after ischemia (P<0.05). The expression of HIF-1α and iNOS in hippocampus was enhanced, and the production of NO was increased significantly (P<0.05). Ischemic PC restored the cortical rCBF (P<0.05), reduced cerebral infarction volume (P<0.05), down-regulated iNOS expression and reduced NO production in the hippocampus (P<0.05). However, the cortical rCBF in FC pretreatment group was significantly lower than that in ischemic group (P<0.05), the neuronal damage was aggravated, and the infarction volume was increased after pretreatment with FC (P<0.05). CONCLUSION: Ischemic PC may reduce cerebral injury by regulating the expression of HIF-1α and iNOS. Inhibition of AS function may attenuate the protective effect mediated by ischemic PC and aggravate brain injury.     

Effects of uric acid on mitochondrial damage and Pgam5/Drp1 expression in rat kidney cells

AIM To investigate the effects of uric acid on mitochondrial damage and the expression of phosphoglycerate mutase family member 5 (Pgam5) and dynamin-related protein 1 (Drp1) in rat kidney cells. METHODS Normal rat kidney NRK-52E cells were treated with uric acid at 0.6 mmol/L. The cell viability was measured by MTT assay. The cell morphological change was observed by Hoechst 33258 staining. The apoptosis of the cells was analyzed by flow cytometry. The morphological structure of mitochondria was observed under transmission electron microscope. The expression of Pgam5 and Drp1 was examined by immunofluorescence staining. The mRNA expression of Pgam5 in mitochondria was detected by RT-qPCR. The protein expression of Pgam5 and Drp1 in the cytoplasmic matrix was determined by Western blot. RESULTS Uric acid significantly decreased the viability of NRK-52E cells, and significantly increased the apoptotic rate of the cells (P<0.01). Mitochondrial swelling, vacuolation and cristal rupture were observed after the NRK-52E cells were treated with uric acid, and the mRNA expression of Pgam5 in mitochondria was decreased significantly, while the protein expression of Pgam5 and Drp1 in the cytoplasmic matrix was increased significantly (P<0.01). CONCLUSION Uric acid intervention destroys the mitochondrial structure of rat renal cells, up-regulates the expression of Pgam5 and Drp1 in the cytoplasmic matrix, and induces apoptosis of the cells.     

Effects of Astragalus injection on neuronal apoptosis and expression of JNK3 in rat hippocampus after cerebral ischemia reperfusion

AIM:To investigate the effects of Astragalus injection on neuronal apoptosis and expression of c-Jun N-terminal kinase 3(JNK3) in the rat hippocampus after cerebral ischemia reperfusion. METHODS:The rat model of cerebral ischemia reperfusion was set up by a four-vessel occlusion method. The SD rats were randomly divided into 4 groups:sham operation group, cerebral ischemia reperfusion group(model group), cerebral ischemia reperfusion+Astragalus injection group(Astragalus injection group) and cerebral ischemia reperfusion+vehicle group(vehicle group). The rats in model group, Astragalus injection group and vehicle group after transient global cerebral ischemia(30 min) were then divided into 7 subgroups according to the reperfusion time of 0 h, 0.5 h, 2 h, 6 h, 24 h, 72 h and 120 h. The apoptosis of the neuron in the hippocampus was measured by the method of TUNEL staining. The expression of JNK3 at mRNA and protein levels was determined by real-time PCR and Western blotting,respectively. RESULTS:Compared with sham operation group, the number of apoptotic neurons increased in model group(P<0.05). Compared with model group, the number of apoptotic neurons decreased obviously in Astragalus injection group(P<0.05). Compared with sham operation group, the expression of JNK3 at mRNA and protein levels in the hippocampus increased obviously in model group at all time points except 120 h(P<0.05). Compared with model group, the expression of JNK3 at mRNA and protein levels in the hippocampus decreased obviously in Astragalus injection group at all time points except 120 h(P<0.05). CONCLUSION:Astragalus injection decreases neuronal apoptosis in rat hippocampus after cerebral ischemia reperfusion by inhibiting the expression of JNK3 at mRNA and protein levels.     

Effect of ginsenoside Rb1 on proliferation and serotonin transporter and 5-HT1B R expression in hypoxia-induced rat pulmonary artery smooth muscle cells via Rho/Rho-kinase pathway

AIM: To observe the effect of ginsenoside Rb1 on the proliferation and the expression of serotonin transporter (SERT), 5-hydroxytryptamine 1B receptor (5HT_1BR) in rat pulmonary artery smooth muscle cells (PASMCs) under hypoxia condition and the relationship with Rho/Rho-kinase signal pathway.METHODS: PASMCs were isolated from the adult male SD rats and primarily cultured. The subcultured cells from the 4th generation to the 6th generation were harvested and divided into normal group, and hypoxia group, different concentrations of Rb1 incubation groups treated with 50, 100 and 200 mg/L ginsenoside Rb1 under hypoxia (HR50, HR100 and HR200 groups, respectively). The viability of the PASMCs was measured by CCK-8 assay. BrdU positive cells were determined using flow cytometry. The expression of serotonin transporter and 5HT_1BR at mRNA and protein levels was detected by RT-PCR and Western blot, respectively. The PASMCs were randomly divided into normal group, hypoxia group, HR200 group and hypoxia+Y-27632 incubation group (HY group). The mRNA expression of Rho-kinase and phosphorylated myosin phosphatase target subunit 1 (p-MYPT1) protein level were investigated by RT-PCR and Western blot, respectively.RESULTS: Compared with normal group, the proliferation of PASMCs in hypoxia group was significantly increased (P<0.01). The cell viability and the expression of SERT and 5HT_1BR at mRNA and protein levels in all different concentrations of Rb1 groups were obviously decreased compared with hypoxia group (P<0.05). The mRNA expression of Rho-kinase and protein level of p-MYPT1 were markedly decreased in HR200 group, and no significant difference compared with HY group was observed (P<0.01).CONCLUSION: Treatment with ginsenoside Rb1 might prevent hypoxia-induced proliferation of PASMCs and over-expression of SERT and 5HT_1BR through inhibiting the Rho/Rho-kinase pathway.     

Nodosin induces apoptosis of HepG2 cells by increasing Apaf-1 expression and activating caspase-3

AIM:To investigate the effects of nodosin on apoptosis of human hepatocellular carcinoma HepG2 cells. METHODS:HepG2 cells were treated with nodosin at different concentrations (1.25 μmol/L, 2.5 μmol/L, 5 μmol/L, 10 μmol/L and 20 μmol/L) for 24 h. The morphological changes of the HepG2 cells were observed by Hoechst 33258 staining and electron microscopy. The apoptotic rates were analyzed by flow cytometry. The mRNA expression of apoptotic protease-activating factor-1 (Apaf-1) was detected by RT-qPCR. The protein levels of pro-caspase-3, caspase-3 and cleaved caspase-3 were determined by Western blot. RESULTS:HepG2 cells showed obvious cell shrinkage and nucleus drift when treated with nodosin as the concentration was increased. Many apoptotic bodies were observed in 5 μmol/L, 10 μmol/L and 20 μmol/L nodosin groups. The mRNA expression of Apaf-1 was increased in 5 μmol/L, 10 μmol/L and 20 μmol/L nodosin groups as compared with control group (P<0.05). The protein levels of pro-caspase-3, caspase-3 and cleaved caspase-3 were increased with the increasing dose of nodosin (P<0.05). CONCLUSION:Nodosin induces the apoptosis of HepG2 cells. This effect was related to increasing Apaf-1 mRNA expression and subsequently promoting the activation of caspase-3.     

GLP-1 down-regulates mRNA expression of SOCS-3 and SREBP-1c in rats with nonalcoholic fatty liver disease

AIM: To investigate the effects of glucagon-like peptide-1(GLP-1) on mRNA expression of SOCS-3 and SREBP-1c in the rats with nonalcoholic fatty liver disease. METHODS: Male SD rats were randomly divided into normal control(NC) group, high fat(HF) group and HF+liraglutide(Lira) group. The rats in HF group and HF+Lira group were given high-fat diet for 16 weeks. After 12 weeks of high-fat diet feeding in HF+Lira group, Lira(600 μg·kg^-1·d^-1) was intraperitoneally injected for 4 weeks. At the end of the 16th week, the rats were killed. The pathological changes of the liver were observed under optical microscope. The serum levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), triglyceride(TG) and total cholesterol(TC) were detected by automatic biochemical analyzer. TG contents of liver were measured by GPO-PAP method. The fasting insulin(FINS) was determined by ELISA, and insulin resistance index was assessed by homeostasis mode assessment(HOMA-IR). The mRNA expression of SOCS-3 and SREBP-1c in the liver tissues was detected by RT-qPCR. RESULTS: Compared with NC group, HOMA-IR, TG of liver, and the serum levels of ALT, AST, TG, TC and FINS in HF group were obviously increased(P<0.01). Compared with HF group, HOMA-IR, TG of liver, and the serum levels of ALT, AST, TG, TC and FINS in HF+Lira group were all obviously decreased(P<0.05 or P<0.01). The mRNA expression of SOCS-3 and SREBP-1c in HF group was significantly higher than that in NC group(P<0.01). The mRNA expression of SOCSV3 and SREBP-1c in HF+Lira group was significantly decreased as compared with HF group(P<0.05). CONCLUSION: Liraglutide may improve the IR and reduce TG of liver through decreasing the mRNA expression of SOCS-3 and SREBP-1c, so as to play a therapeutic role in nonalcoholic fatty liver disease.     

Effects of astragaloside IV combined with ginsenoside Rg1 on autophagy and PI3K signaling pathways of PC12 cells induced by oxygen glucose deprivation/reoxygenation

ATM: To probe the effect and the mechanism of astragaloside IV and ginsenoside Rg1 on autophagy of PC12 cells induced by oxygen glucose deprivation/reoxygenation (OGD/R). METHODS: The autophagy injury model of PC12 cells induced by OGD/R was established(PC12 cells were exposed to 2 h of OGD followed by 24 h of reoxygenation). The effects of astragaloside IV combined with ginsenoside Rg1 on autophagy of PC12 cells were observed, and the mechanism was studied through PI3K Ⅰ/Akt/mTOR and PI3K Ⅲ/becline-1/Bcl-2 signaling pathways. RESULTS: After OGD/R, LC3-Ⅱ/LC3-Ⅰin PC12 cells was increased. Astragaloside IV, ginsenoside Rg1 and astragaloside IV combined with ginsenoside Rg1 restrained the increase in LC3-Ⅱ/LC3-Ⅰ, the effect of the combination was greater than using the drug alone. Ginsenoside Rg1, astragaloside IV combined with ginsenoside Rg1 up-regulated the phosphorylation level of PI3K Ⅰ, Akt and mTOR. The effects of the combination were stronger than those of using the drug alone. Astragaloside IV, astragaloside IV combined with ginsenoside Rg1 inhibited the protein expression of PI3K Ⅲ and becline-1, the effects of the combination were better than those of single astragaloside IV and single ginsenoside Rg1. Meanwhile, the combination treatment increased Bcl-2 protein expression. CONCLUSION: The autophagy of PC12 cells induced by OGD/R is inhibited by astragaloside IV and ginsenoside Rg1. Furthermore, astragaloside IV combined with ginsenoside Rg1 plays synergitic inhibition on autophagy, the mechanism may be related to PI3K Ⅰ/Akt/mTOR and PI3K Ⅲ/becline-1/Bcl-2 signaling pathways.