Effects of hyperglycemia on ionic homeostasis in hippocampal microenvironment and secondary neuronal injury after focal ischemia in cerebral cortex of tree shrews

AIM：To observe the effects of hyperglycemia on the ionic homeostasis in hippocampal microenvironment after thrombotic cortical ischemia in tree shrews, and to explore the action and mechanisms of hyperglycemia in secondary neuronal injury after ischemia. METHODS：High blood glucose in tree shrews was induced by intraperitoneal injection of streptozocin. Focal thrombotic cortical ischemia was induced by photochemical method in tree shrews. At 4, 24 and 72 h after ischemia, the changes of pH, K+, Na+, Ca2+ and Cl- in the ipsilateral ischemic hippocampal microenvironment were tested by a single-pumped push-pull microdialysis system and an ion analyzer. The histopathological changes and hippocampal neuronal density were also examined. RESULTS：After cortical ischemia in tree shrews, the pH and the concentrations of Na+, Ca2+ and Cl- in the hippocampal microenvironment decreased, while the concentration of K+ increased. These differences were the most significant at 4 h, the second at 24 h and insignificant at 72 h. Combination of hyperglycemia and cerebral ischemia worsened the turbulence of ionic homeostasis. Compared with the normoglycemic ischemic animals, the changes of pH, K+ and Ca2+ concentrations at 4 h as well as pH and Na+ at 24 h in the hyperglycemic ischemic animals were more significant (P<0.05). The results of histopathological examination showed that there was ischemic neuronal damage in the exposed cerebral cortex and the ipsilateral hippocampal CA1 region at 4 h after photochemical reaction, and the damage was the most severe at 24 h, subsequently accompanied with glial proliferation at 72 h. The hyperglycemic ischemic animals suffered from greater neuronal injury in the cortex and hippocampus than the normoglycemic ischemic animals, especially at 24 h (P<0.01) and 72 h (P<0.05). CONCLUSION: The disturbance of acid-base equilibrium and ionic homeostasis in hippocampal microenvironment, following the spreading of the microenvironment in ischemic core, may be an important reason for secondary neuronal injury in the hippocampus after thrombotic cortical ischemia in tree shrews. Hyperglycemia aggravates the turbulence of ischemic ionic microenvironment.     

Relationship between HBV genotype,PreS/S gene mutation and immunoprophylaxis failure to prevent HBV mother-to-child transmission

AIM：To investigate the relationship between hepatitis B virus (HBV) genotype, PreS/S gene mutation and immunoprophylaxis failure to prevent HBV mother-to-child transmission.
METHODS：Pregnant women with positive HBV surface antigen (HBsAg) and HBV DNA≥1×1010 IU/L were divided into case group (15 cases) and control group (45 cases) according to their neonates with immunoprophylaxis failure or not. The genotypes of HBV and the mutation rate and mutational hot spots in PreS/S gene were detected by PCR amplification technique in the two groups.
RESULTS：(1) Genotypes B and C of HBV were detected in both case and control groups, and the majority of HBV genotype was B in the two groups. Genotype distribution difference between case and control groups was not statistically significant (P>0.05). (2) There was no significant difference in the mutation rate of PreS/S gene between case and control groups (P>0.05). The mutation rates of PreS/S gene between genotypes B and C were significantly different (P<0.05), but when the HBV genotype was the same, the mutation rate of PreS/S gene had no significant difference between case and control groups. Homology tree model based on PreS2/S gene formed genotype B and genotype C clusters, and in each cluster, the sequences of case and control groups did not formed smaller different clusters further. (3) 529G-A, 530A-G, 826A-G1 and 166het-dupC were hot spots of mutation in PreS2/S gene and were found in 4 cases in case group, respectively. A530T (1 case), A530G (2 cases), T531C (3 cases) were found in control group.
CONCLUSION：(1) The mutation rates of PreS/S gene are different in various genotypes. (2) The mutation in PreS/S gene of HBV is prevalent, but not all of the mutations are related to immunoprophylaxis failure to prevent HBV mother-to-child transmission. To find mutational hot spots which are related to immunoprophylaxis failure is more important.     

Expression of Cdc25a in cross-species hepatocellular carcinoma tissues and its significance

AIM：To study the expression of cell division cycle 25a protein (Cdc25a) in hepatocellular carcinoma (HCC) tissues of different species, including human, rat and tree shrew, and to verify the feasibility of the strategy of cross-species oncogenomics screening. METHODS：Real-time fluorescence quantitative PCR and Western blotting were applied to detect the expression of Cdc25a at mRNA and protein levels in the HCC tissues, corresponding HCC-adjacent liver tissues and normal liver tissues collected from humans, rats and tree shrews. RESULTS：The mRNA expression of Cdc25a in the HCC tissues of humans, rats and tree shrews was higher than that in normal liver tissues (P<0.05). The mRNA levels of Cdc25a in the HCC tissues of humans and rats were higher than those in the corresponding HCC-adjacent liver tissues (P<0.05). The mRNA expression of Cdc25a in the HCC tissues was significantly correlated with the portal vein tumor thrombus, the extrahepatic metastasis and the clinical stage, but was not correlated with the recurrence of tumor, the diameter of tumor, the number of tumor, the level of serum alpha-fetoprotein and the differentiation of tumor. The protein levels of Cdc25a in the HCC tissues of humans, rats and tree shrews were higher than those in the corresponding HCC-adjacent liver tissues and the normal liver tissues. CONCLUSION：Cdc25a may be a particularly crucial molecule for hepatocarcinogenesis. The cross-species oncogenomics screening may represent a feasible and convenient way for identifying key molecules of human HCC.     

Ischemia postconditioning induces tight junction protein expression and inhibits brain edema after thrombotic cerebral ischemia in tree shrews

AIM: To assess whether the expression of tight junction(TJ) proteins, occludin/zonula occludins(ZO)-1, and regional cerebral blood flow(rCBF) link to brain edema in tree shrews during thrombotic cerebral ischemia and ischemic postconditioning(PC), and to explore how TJ affects brain edema and cerebral infarction. METHODS: Tree shrews were randomly grouped into control, ischemia and cerebral ischemia+PC(n=23), and the remaining 3 animals were used for magnetic resonance imaging(MRI). The local cerebral thrombosis were induced by photochemical reaction in the tree shrews, and ischemic PC was established at 4 h after induction of cerebral ischemia followed by clipped ipsilateral common carotid artery(5 min×3). The changes of the neural ultrastructure were observed under electron microscope. The neuronal apoptosis was analyzed by the method of TUNEL. Laser Doppler brain flowmetry was used to monitor the rCBF. The protein levels of occludin/ZO-1 were determined by immunochemistry and Western blot. The cerebral infarction volume was detected by MRI. The brain water content was measured by dry-wet weight method. RESULTS: Induction of cerebral ischemia led to a significant reduction of the normal neuron numbers in the hippocampal CA1 area, and conversely, the number of neurons with abnormal ultrastructure was increased. The TUNEL positive cells were increased significantly(P<0.01) in ischemia group. Moreover, the rCBF decreased significantly(P<0.01), and occludin/ZO-1 protein expression decreased(P<0.01). The brain water content and cerebral infarction volume were significantly increased(P<0.01). Ischemic PC increased the rCBF and the occludin/ZO-1 expression, but reduced the brain water content, the TUNEL positive cells, and the infarction volume(P<0.01). CONCLUSION: Ischemic PC increases the rCBF but not the local water content, suggesting that reduced cerebral infarction volume after ischemia PC is associated with the attenuation of cerebral edema by the enhancement of occludin/ZO-1 protein expression.     

Effects of hyperglycemia and cerebral ischemia on VEGF expression in different subfield of cerebral cortex in tree shrews

AIM: To observe the changes of VEGF expression in different subfield of brain in tree shrews during hyperglycemia and focal cerebral ischemia, in order to explore the relationship between cerebral ischemia, hyperglycemia and VEGF. METHODS: High blood glucose in tree shrews was induced by intraperitoneal injection of streptozotoctin. Focal cortical thrombotic cerebral ischemia was induced by photochemical method in tree shrews. At 4 h, 24 h and 72 h after cerebral ischemia, the histopathological changes and hippocampal neuronal density were examined. VEGF expressions in the ischemic core, penumbra and contralateral cerebral cortex were detected by immunohistochemistry technique at different times after cerebral ischemia. RESULTS: The results of histopathological study showed that there was infarction zone in the exposured cerebral cortex at 4 h after photochemical reaction, and the damage was most severe at 24 h, subsequently accompanied with the glia multiplication and rehab reaction at 72 h. The animals in hyperglycemic ischemic group suffered from greater neurological lesion than the normoglycemic stroke animals, especially at 24 h (P<0.01) and 72 h (P<0.05) after cerebral ischemia. Immunohistochemical analyses of VEGF expression revealed that it started to increase at 4 h after brain ischemia in the penumbra, reached a peak at 24 h, and weakened at 72 h. The stimulated VEGF production was also observed in hyperglycemic only group. When hyperglycemia and brain ischemia were combined, the VEGF expression was higher than that in hyperglycemic only group (P<0.05). Compared to normoglycemic ischemic group, no additivity of the effects of hyperglycemia combined with brain ischemia was observed. CONCLUSION: (1) The model of experimental hyperglycemia and cerebral ischemia is replicated successfully by applying the method combined in vivo injection of streptozotocin in the lower primate tree shrew with thrombotic focal cerebral ischemia. (2) This study shows that hyperglycemia aggravates the focal cerebral ischemia damage. (3) Cerebral ischemia and hyperglycemia both can independently up-regulate VEGF expression, but there is no additional increase in VEGF expression when hyperglycemia combined with brain ischemia is applied.     

Changes of hippocampal neuron microenvironment and contents of amino acid after focal cortical brain ischemia in tree shrews

AIM: To study the changes of hippocampal neuronal microenvironment and alterations of excitatory amino (EAA) and inhibitory amino acid (IAA) in extracellular fluid (ECF) in different time after thrombotic cerebral ischemia in tree shrews. METHODS: The model of focal thrombotic cerebral ischemia was induced by photochemistry-technology in tree shrews. Hippocampal ECF was collected by microperfusion. pH, PCO2, PO2 and HCO3- were analyzed by blood gas analyzer, and Asp, Glu, Gly and GABA were measured by high-performance liquid chromatography (HPLC)-PITC technology after occlusion. RESULTS: The contents of Asp, Glu, Gly and GABA in hippocampal ECF increased, and pH, PO2 and HCO3- decreased after photochemical induced cerebral ischemia in tree shrews. There were significant differences between ishemic group and sham group (P<0.01, P<0.05). CONCLUSIONS: Equilibration disturbance of amino acids is either the result of ischemia, or the reason of secondary neurons damage after photochemistry-induced focal cerebral ischemia in tree shrews. Changes of amino acid metabolism are related to alteration of hippocampal cellular microenvironment.     

Effects of ethanol on HBV gene mutations in patients with chronic hepatitis B by gene chip

AIM: To study ethanol influence on gene mutations of HBV DNA and to offer testimony for clinical diagnosis and treatment of chronic hepatitis B. METHODS: 85 patients with chronic hepatitis B were divided into alcoholic group and non-alcoholic group. Gene chip technique was used to detect gene mutations located in Pre-C nt G1896A and nt A1814C, basal core promoter (BCP) nt A1762T and nt G1764A, P nt C528A and nt T552C. RESULTS: The mutation frequency on BCP nt A1762T and nt G1764A in alcoholic group was significantly higher than that in non-alcoholic group (P<0.05). No difference of mutation frequency on pre-c nt G1896A nt A1814C and P nt C528A nt T552C between alcoholic and non-alcoholic group was observed (P>0.05). CONCLUSION: Ethanol stimulates HBV gene mutations on BCP nt A1762T and nt G1764A, enhances HBV DNA replication and gene expression, deteriorates the state of the illness.     

Effects of low-frequency electric stimulation on serum hormone levels in Chinese tree shrews

AIM：To study the low-frequency electric stimulation on the serum hormone levels in Chinese tree shrews. METHODS：Chinese tree shrews were given low-frequency electric stimulation, or intramuscularly injected with reserpine and then given low-frequency electric stimulation. The serum levels of norepinephrine (NE), endothelin (ET), thromboxane B 2 (TXB 2) and testosterone (T) were detected by radioimmunoassay. RESULTS：In electric stimulation group, the serum levels of NE, ET, TXB 2 and T were significantly increased as compared with control group. After low-frequency electric stimulation, the serum level of T at 24 h was higher than those at 1 h, 72 h and 7 d by 12.49%, 7.27% and 15.79%, respectively. Reserpine treatment significantly reduced the T level as compared with control group, and the T level at 24 h, 72 h and 7 d after treated with reserpine was lower than that at 1st h by 5.42%, 11.80% and 5.42%, respectively. The levels of TXB 2 in electric stimulation group and reserpine group were significantly higher than that in control group, and no difference in electric stimulation groups or in reserpine groups between 1 h, 24 h, 72 h and 7 d after electric stimulation was observed. The level of ET in electric stimulation group at 24 h after electric stimulation was higher than those at 1 h, 72 h and 7 d by 9.12%, 11.41% and 10.85%, respectively. The level of ET in reserpine group was significantly higher than that in control group. However, no difference of the ET level in reserpine groups between 1 h, 24 h, 72 h and 7 d was observed. The level of NE after electric stimulation at 1 h was higher than those at 24 h, 72 h and 7 d by 10.65%, 5.83% and 19.29%, respectively. The levels of NE in reserpine groups at 24 h, 72 h and 7 d after electric stimulation were lower than that at 1st h by 32.85%, 43.38% and 32.85%, respectively. Compared with control group, the level of NE in reserpine group was significantly decreased. CONCLUSION：Low-frequency electric stimulation promotes the releases of NE, ET, TXB 2 and T in Chinese tree shrews. Reserpine attenuates the stress effect of low-frequency electric stimulation on the animals.     

NRF2 attenuates oxidative stress and lysosomal dysfunction in doxorubicin-induced H9C2 cells

AIM:To study the effect of nuclear factor E2-related factor 2 (NRF2) on oxidative stress injury and lysosomal dysfunction in doxorubicin (DOX)-induced rat myocardial H9C2 cells. METHODS:The H9C2 cells were treated with DOX. The expression of NRF2 at mRNA and protein levels was determined by real-time PCR and Western blot. The H9C2 cells stably over-expressing NRF2 were established by lentiviral infection. Real-time PCR and Western blot were used to identify the efficiency of over-expression. After DOX treatment, the cell viability was measured by CCK-8 assay, the activity of lactate dehydrogenase (LDH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), and the content of malondialdehyde (MDA) in the cell supernatant were detected. FITC-dextran was used to analyze lysosomal pH, and the protein expression of lysosomal-associated membrane protein 1 (LAMP1) and cathepsin B was determined by Western blot.RESULTS:The expression of NRF2 at mRNA and protein levels in DOX-treated H9C2 cells was significantly decreased (P<0.05). Over-expression of NRF2 significantly up-regulated the mRNA and protein expression of NRF2 in DOX-treated H9C2 cells (P<0.05). After DOX treatment, the cell viability was decreased, and LDH activity was increased. The activity of SOD, GSH-Px and CAT was decreased, and the content of MDA was increased (P<0.05). The lysosomal pH was increased, and the protein expression of LAMP1 and cathepsin B decreased (P<0.05). Over-expression of NRF2 increased the cell viability, decreased LDH activity, increased the activity of SOD, GSH-Px and CAT, and decreased the content of MDA in cell supernatant (P<0.05). Over-expression of NRF2 also decreased the lysosomal pH, and increased the protein expression of LAMP1 and cathepsin B (P<0.05). CONCLUSION:DOX inhibits the expression of NRF2 in the myocardial H9C2 cells. Over-expression of NRF2 attenuates oxidative stress and lysosomal dysfunction in the H9C2 cells induced by DOX.     

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.     

Significant anti-proliferation of human acute lymphoblastic leukemia cells by combined treatment with chloroquine and dexamethasone

AIM：To explore the promoting action of chloroquine on the anti-proliferation effect of dexamethasone on acute lymphoblastic leukemia cells. METHODS：CCK-8 assay was used to assess the viability of the dexamethasone-resistant human acute lymphoblastic leukemia CEM-C1 cell line treated with the combination of chloroquine and dexamethasone. Western blotting, quantitative real-time PCR and LysoTracker Red staining were utilized to examine the mechanism. RESULTS：Combination of chloroquine and dexamethasone significantly inhibited the proliferation of CEM-C1 cells compared with control group (P<0.01). The combination of chloroquine and dexamethasone increased the abundance of glucocorticoid receptor and inhibited lysosomal function, while lysosomal inhibitor bafilomycin A1 also increased glucocorticoid signaling. CONCLUSION：Dexamethasone combined with chloroquine triggers an anti-proliferation effect on CEM-C1 cells via a lysosome-mediated pathway.     

Paired study on hepatitis B virus S gene mutation in immunoprophylaxis failure to prevent HBV vertical transmission

AIM：To explore the characteristics of hepatitis B virus S gene mutation in the vertical transmission after active and passive vaccination. METHODS：Fifteen cases of immunoprophylaxis failure were enrolled in the study. HBV S gene (including pres-S and S) from the mothers, newborns before active and passive vaccination and 7-month-old infants with immunoprophylaxis failure were detected by PCR amplification. The characteristics of HBV S gene mutation were compared among the 3 groups. RESULTS：The genotype of HBV in the newborns and the infants was the same as that in the mothers. The frequencies of mutation in the 2 fragments of the HBV S gene had no significant difference between the 3 groups. The homology tree model based on HBV S gene was analyzed in the 3 groups, in which every group had their own cluster. There were 15 different mutation sites between 7 pairs of mothers and newborns. There were 3 different mutation sites between 3 pairs of newborns and infants (nt273A→A/G, nt512C→C/T and nt1139C→A), among which the first 2 were located in the S gene region but not in the “a” determinant, and the latter was located in the overlap region of S and X genes. There were 25 different mutation sites between 9 pairs of mothers and infants, but only 1 case had a different mutation site between the mother, newborn and infant. CONCLUSION：The HBV species in newborns and infants with immunoprophylaxis failure were transmitted from the mothers. The mutations in the HBV S gene with immunoprophylaxis failure happened before and after active and passive vaccination, mainly before vaccination. The relationship between HBV S gene mutations and immunoprophylaxis failure should be further explored.     

Association between IL-6-572C/G as well as IFNAR1-168G/C and hepatitis B virus infection in populations of Dai and Han ethnicities in Yunnan Province

ATM: To explore the association between IL-6-572C/G (rs1800796) as well as interferon alpha receptor 1 (IFNAR1)-168G/C (rs2257167) and prognosis after hepatitis B virus (HBV) infection in populations of Dai and Han ethnicities in Yunnan Province. METHODS: The blood samples were collected from Dai people and Han people, each nation including 100 healthy controls and 200 infected individuals (100 spontaneous recovery individuals and 100 chronic patients). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing were used to identify the gene type. RESULTS: In Dai people, no significant difference was found between genetic polymorphism of -572C/G and prognosis after HBV infection. The differences of C and G alleles between spontaneous recovery group and chronic hepatitis B group, and healthy controls and HBV infection group were not statistically significant. Meanwhile, GG and CG genotypes were a vital protective factor for the person who developed into a chronic heptatitis B patient under the G allele dominance mode (GG+CG/CC) (P<0.05). In Han people, no statistically significance for IL-6-572C/G genotype and allele distribution in each group comparisons had been found, as well as the C allele recessive mode and C allele dominance mode. For the above 4 indicators, no statistically significant difference of IFNAR1-168C/G in Dai and Han people had been found.CONCLUSION: The GG+CG genotype of IL-6-572C/G may be a protective factor for the HBV-infected Dai people to develop into chronic hepatitis B patients. However, there is no significant association between the IFNAR1-168G/C polymorphism and prognosis after HBV infection in the 2 ethnicities.     

Role of renal preS1/S2-Ag and other HBV antigen determination in diagnosis of HBV-associated glomerulonephritis

AIM：To detect the expression of preS1/S2 antigen (preS1/S2-Ag) and other antigens of hepatitis B virus (HBV) in renal tissues of patients with HBV-associated glomerulonephritis (HBV-GN), and to analyze their roles in the diagnosis of HBV-GN.
METHODS：Patients hospitalized in our department from January in 2003 to January in 2013 were retrospectively studied. A total of 49 patients with positive HBV surface antigen (HBsAg) serology, clinical manifestations of hematuria and/or proteinuria, and pathological diagnosis of glomerulonephritis, without systemic lupus erythematosus, anaphylactic purpura, diabetes or hepatitis C, were selected. PreS1/S2-Ag, HBV e antigen (HBeAg), HBsAg and HBV core antigen (HBcAg) in the renal tissues were examined. Five cases of glomerular minimal-change disease (MCD) with negative HBsAg and 5 cases of non-glomerulonephritis with positive HBsAg served as controls. RESULTS：The positive rates of preS1/S2-Ag, HBeAg, HBsAg and HBcAg in the renal tissues from the 49 patients of glomerulonephritis with HBV infection were 32.7% (16 cases), 38.8% (19 cases), 14.3% (7 cases) and 46.9% (23 cases), respectively. Total antigen positive rate was 70.2% (36 cases). The expression of preS1/S2-Ag was located in the cytoplasm of renal tubular epithelial cells, glomerular epithelial cells, endothelial cells and mesangial cells, and positively correlated with the expression of HBcAg (r=0.459, P<001). The 4 antigens were not detected in the 5 cases of HBsAg-negative patients with glomerular MCD. In the 5 cases of HBsAg-positive patients with non-glomerulonephritis, there were 2 cases expressing HBeAg and 1 case expressing HBcAg, but no cases expressing preS1/S2-Ag or HBsAg. CONCLUSION：The expression of preS1/S2-Ag in renal tissues suggests that HBV may invade the cells of renal tissue. Combined detection of the 4 antigens could elevate the rate of diagnosis of HBV-GN.     

Effects of inhibition of Kupffer cell and splenectomy on thioacetamide-induced hepatic injury

AIM: To examine the effects of inhibition of Kupffer cell and splenectomy on intestinal endotoxemia and hepatic injury. METHODS: The hepatic injury model was established by treatment with thioacetamide (TAA). At the same time, inhibition of Kupffer cells by intravenous GdCl₃ and splenectomy were performed. Serum alanine aminotransferase (ALT), TNF-α, endotoxin content and phagocytic index were observed. RESULTS: In the TAA+GdCl₃ group, and TAA+splenectomy group, the endotoxin content was significently higher than that in normal and TAA group (P<0.05). The plasma TNF-α, ALT and total bilirubin were significantly lower than those in TAA group (P<0.05). CONCLUSION: Inhibition of Kupffer cells and splenectomy increase plasma endotoxin level, but decreases the plasma TNF-α levels and alleviates the hepatic injury induced by TAA.     

Effects of ginkgolide B on glial fibrillary acidic protein expression after thrombotic cerebral ischemia in tree shrews

AIM: The present study was designed to examine the changes in glial fibrillary acidic protein (GFAP) expression during cerebral ischemia and the effects of ginkgolide B on GFAP expression. METHODS: The focal thrombotic cerebral ischemia was formed by photochemistry-induced in tree shrews. GFAP stained by ABC immunohistochemistry and absorbance were measured with image analyze system. RESULTS: GFAP expression in astrocytes increased significantly (P<0.01) at 24 h and kept in higher level at 72 h (P<0.01) within penumbra after focal cerebral ischemia. GFAP expression declined when the animals were given GB at 6 h after thrombotic cerebral ischemia. CONCLUSIONS: Neuronal necrosis resulted in GFAP expression in astrocytes after local cerebral ischemia and GB protected neurons by antagonizing PAF receptor and inhibiting GFAP expression.     

Endothelin-1 and eNOS expression in pulmonary aterioles of patients with congenital heart diseases with pulmonary hypertension

AIM: To clarify the role of eNOS and ET-1 in development of pulmonary hypertension (PH) associated with congenital heart diseases. METHODS: 40 patients were randomly divided into three groups: severe or moderate PH group (group A, 12 cases), slight PH group (group B, 14 cases) and normal group (group C, 14 cases). ET-1 and eNOS were examined by using the technique of immunohistochemistry. RESULTS: ① Plasma ET-1 concentration was significantly higher in group A and B than that in group C (P<0.01). Serum NO concentration in group A and B was significantly lower than that in group C (P<0.01). ② The proliferation of medial smooth muscle cells and collagenous fibers of pulmonary arteriole in patients with PH were observed by electron microscopy. ③ ET-1 in pulmonary arteriole was significantly higher in group A and group B than that in group C (P<0.01). eNOS in pulmonary arteriole was significantly lower in group A and group B than that in group C (P<0.01). CONCLUSION: ET-1 and eNOS may play an important role in the pathogenesis of PH.     

Relationship between expression of heme oxygenase-1 mRNA and pulmonary hypertention induced by hypoxic hypercapnia

AIM: To study the effect of chronic hypoxic hypercapnia on expression of heme oxygenase-1 (HO-1). METHODS: Sprague-Dawley rats were randomly divided into three groups: control group(A),hypoxic hypercapnic group(B), hypoxic hypercapnia+hemin group(C). HO-1 and HO-1 mRNA were observed in pulmonary arterioles by the technique of immunohistochemistry and in situ hybridization. RESULTS: ① mPAP and weight ratio of right ventricle (RV) to left ventricle plus septum (LV+S) were significantly higher in rats of B group than those of A and C group (P<0.01). Differences of mCAP were not significant in three groups(P>0.05). ② Blood CO concentration was significantly higher in rats of B group than that of A group (P<0.01), it was much higher in C group than that of B group(P<0.01). ③ Light microscopy showed that vessel well area/total area (WA/TA), density of medial smooth muscle cell (SMC) and media thickness of pulmonary arterioles were much higher in rats of B group than those of A and C group (P<0.01). ④ The observation by electron microscopy showed proliferation of medial smooth muscle cells and collageous fibers of pulmonary arterioles in rats of B group, hemin could reverse the changes mentioned above. ⑤ HO-1 and HO-1 mRNA in pulmonary arterioles was significantly higher in rats of B group than those of A group(P<0.01), and they were significantly higher in rats of C group than those of B group (P<0.01). CONCLUSION: Expression of HO-1 mRNA and HO-1 in pulmonary arterioles was enhanced by hypoxic hypercapnia. Hemin partly inhibited pulmonary hypertension and pulmonary vessel remodeling by enhancing the expression of HO-1 mRNA and HO-1.     

Direct differentiation of embryonic stem cells into neural cells without embryonic body culture period in vitro

AIM:To investigate the feasibility and effect of directly differentiation of embryonic stem cells (ESC) into neural cells induced by retinoid acid (RA) without embryonic body (EB) culture period in vitro. METHODS:ESC were digested and divided into 4 groups:group A and B were undergone EB culturing. After that, cells in group A were induced by RA, cells in group B were differentiated spontaneously, cells in group C were committedly induced by RA directly without EB culturing, and cells in group D were differentiated spontaneously without EB period. Morphologic changes were observed under inverted microscope and scanning electron microscope. MAP-2 and GFAP were detected by immunocytochemistry and flow cytometry after differentiated for 9 days. RESULTS:In groups A or C, neuron-like cells increased gradually, forming neural network. At the 9th day, a large part of cells in these groups were MAP-2 positive cells, and the positive rate was higher than that in groups B or D (P<0.01). Groups B and D were almost epithelial-like cells. At the 9th day, GFAP positive cells were predominant. The rate of GFAP staining cells in groups A or C was significantly lower than that in groups B or D (P<0.01). There was no significant difference of MAP-2 or GFAP positive rates between group A and C, nor between group B and D (P>0.05). CONCLUSION:ESC was directly induced into neural cells by RA without EB culture period in vitro. This modified method has the same effect as the traditional RA 4－/4＋ assay and can replace the traditional method.      

Effect of diltiazem on pulmonary arterial pressure and ceNOS mRNA expression in pulmonary arteries in chronic hypoxic hypercapnic rats

AIM: To investigate the effect of diltiazem on mean pulmonary arterial pressure (mPAP) and nitric oxide synthase (NOS) in arterioles in chronic hypoxic hypercapnic rats. METHODS: Twenty-four rats were randomly divided into three groups: control group (A), hypoxic hypercapnic group (B), hypoxic hypercapnia+ diltiazem group (C), constitutive endothelial NOS (ceNOS) were observed in arterioles of rats using the technique of immunohistochemistry, ceNOS mRNA were observed by the technique of in situ hybridization. RESULTS: (1) mPAP was significantly higher in rats of B group than that of A and C group(P<0.01). Differences of mCAP were not significant between A group and B groups (P>0.05), but mCAP was lower in rats of C group than that in B group. (2) Light microscopy showed WA/TA (vessel wall area/total area) was significantly lower in rats of C group than that of B group (P<0.01), electron microscopy showed that diltiazem inhibited the proliferation of smooth muscle cells and collageous fibers of pulmonary arterioles in chronic hypoxic hypercapnic rats. (3) Immunohistochemistry showed the average value of integral light density (LD) of ceNOS in pulmonary arterioles was significantly higher in rats of C group than that of B group (P<0.01), in situ hybridization showed LD of ceNOS mRNA in pulmonary arterioles was significantly higher in rats of C group than that of B group (P<0.01). CONCLUSION: Diltiazem inhibited pulmonary hypertension, the proliferation of smooth muscle cells and collagenous fibers of pulmonary arterioles in chronic hypoxic hypercapnic rats by incresing the expression of ceNOS in pulmonary arterioles.