[The effects of p38MAPK and HBxAg on cell proliferation and apoptosis in human hepatocarcinogenesis].

OBJECTIVE: To investigate the effects of host-derived p38 mitogen-activated protein kinase subunit 38 (p38MAPK) and the hepatitis B virus X antigen (HbxAg) on cell proliferation and apoptosis in human hepatocellular carcinoma (HCC), and to study the mechanism underlying hepatocarcinogenesis. METHODS: Liver tissues were biopsied from healthy individuals and patients with chronic hepatitis B (CHB), liver cirrhosis, paratumor cirrhosis, and HCC. Immunohistochemical staining was used to detect expressions of HBxAg, p38MAPK, cell cycle G2/M phase-related factors (cdc25B, p34cdc2, cyclin B1), and cell proliferation factor ki-67.The terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling method (known as TUNEL) was used to detect apoptosis. RESULTS: The highest rates of HBxAg were detected in CHB (65.0%) and HCC (44.4%) liver samples, and the antigen was mainly expressed in nuclei. Increasingly higher rates of p38MAPK, cdc25B, cyclin B1, and p34cdc2 expression were detected with increases in disease severity: normal liver (40.0%, 20.0%, 20.0%, and 30.0%, respectively), chronic hepatitis B (60.0%, 65.0%, 40.0%, and 50.0%, respectively), liver cirrhosis (65.0%, 75.0%, 70.0%, and 55.0%, respectively), paratumor cirrhosis (66.7%, 75.0%, 75.0%, and 63.9%, respectively), and HCC (77.8%, 80.6%, 80.6%, and 72.2%, respectively). In addition, the intracellular location of p38MAPK expression was different under different disease conditions, showing nuclear expression in CHB and liver cirrhosis samples and cytoplasmic expression in paratumor cirrhosis and HCC samples (x2 = 1.11, P more than 0.05). The proliferation index (PI) and the apoptosis index (AI) were both increased along with disease severity (normal more than CHB more than paratumor cirrhosis more than HCC) (PI: 0.0000+/-0.000, 0.0502+/-0.011, 0.0411+/-0.009, 0.0762+/-0.017; AI: 0.0351+/-0.024, 0.0607+/-0.022, 0.0562+/-0.013, 0.0716+/-0.011), with the notable exception for liver cirrhosis (PI: 0.1810+/-0.036 and AI: 0.1200+/-0.018). PI in poorly-differentiated HCC (0.2285+/-0.062) was significantly higher than in well-differentiated HCC (0.1216+/-0.032, t = 2.082, P = 0.044). AI in well-differentiated HCC (0.152+/-0.026) was significantly higher than in poorly-differentiated HCC (0.081+/-0.022, t = 2.129, P = 0.041). CONCLUSIONS: In the process of hepatocarcinogenesis, HBxAg may cause a series of abnormal changes in cell cycle, proliferation and apoptosis by affecting the expression of p38MAPK.

[Role of p38MAPK in apoptosis of human paratumor cirrhosis hepatocellular cell line QSG-7701 induced by cisplatin].

OBJECTIVE: To investigate the roles of p38 MAPK in apoptosis of the normal liver cell, the paratumor cirrhosis hepatocellular cell and the hepatocellular carcinoma cell. METHODS: Three cell lines were adopted (the normal liver cell line HL-7702, the paratumor cirrhosis hepatocellular cell line QSG-7701 and the hepatocellular carcinoma cell line QGY-7703) and treated with Diamminedichloroplatin (DDP, cisplatin) and p38MAPK inhibitor SB203580. The apoptosis and cell cycles were detected by flow cytometry and electromicroscopy. The expressions of p38MAPK, CDC25B, p34cdc2 and cyclinB1 were detected by immunocytochemical staining , confocal microscopy and western blot. RESULTS: The apoptotic rates in all three cell lines pretreated with DDP increased obviously and the rates in normal liver cells and HCC cells increased continuously even after SB203580 treatment, whereas in paratumor cirrhosis cells the rate decreased and the cell cycle stopped at S phase. CONCLUSION: Cisplatin induces apoptosis in the paratumor cirrhosis hepatocellular cell line QSG-7701 via activation of p38MAPK pathway and it differs in the normal liver cells from the hepatocellular carcinoma cells.

Learning large-scale spatial relationships in a maze and effects of MK-801 on retrieval in the rhesus monkey.

Monkeys have strong abilities to remember the visual properties of potential food sources for survival in the nature. The present study demonstrated the first observations of rhesus monkeys learning to solve complex spatial mazes in which routes were guided by visual cues. Three monkeys were trained in a maze (6 m x 6 m) included of four different mazes. We recorded the cue and cup errors, latencies, and pathway for each trial. The data showed that monkeys learned the target place after three days in the first maze and spent a shorter time in learning the following mazes. The maze was an efficient method to measure the ability and proceeding of spatial memory in monkeys. Moreover, working memory can also be tested by using the maze. MK-801 at 0.02 mg/kg but not at 0.005 mg/kg impaired monkeys' retrieval of spatial memory after they learned all four mazes. The present maze may provide an efficient method to help bridging the gap in cognition between nonhuman primates and humans, and in particular to gain insight into human cognitive function and dysfunction.

Effect of qi-tonifying and stasis-eliminating therapy on expression of vascular endothelial growth factor and its receptors Flt-1, Flk-1 in the brain of intracerebral hemorrhagic rats.

OBJECTIVE: To investigate the effects and mechanism of qi-tonifying and stasis-eliminating (QTSE) therapy on the expression of vascular endothelial growth factor (VEGF) and its receptors Flt-1 and Flk-1 in the brains of intracerebral hemorrhagic (model) rats. METHODS: One hundred and eighty Sprague-Dawley rats were randomly divided into six groups: the normal group (n=5), the sham-operative (SO) group (n=35), the model group (n=35), the QTSE group (n=35), the QT group (n=35) and the SE group (n=35). All the rats except those in the normal group and SO group were established into an intracerebral hemorrhage(ICH) model by intracerebral injection of collagenase type VII and the latter three were orally administered with Buyang Huanwu Decoction (a classical recipe for QTSE) or with some of its components for qi-tonification and for stasis-elimination, respectively. To the other three groups, normal saline solutions were given instead. Behavioral tests were carried out in the animals randomly chosen from each group on days 1, 2, 4, 7, 14, 21 and 28 after modeling. The expressions of VEGF, Flk-1 and Flt-1 were determined by immunohistochemistry and the number of vascular segments with positive expression in the injured brain area of the rats was calculated. RESULTS: From day 7 onwards, the asymmetric forelimb use rate in the QTSE group recovered more significantly than that in the other model groups. In the model group, the expressions of VEGF, Flk-1 and Flt-1 appeared on day 1 and reached a peak on day 21, then weakened gradually. In the QTSE group, as compared with the other model groups, a higher level of VEGF expression was shown from day 7 (P<0.01) and a higher level of Flt-1 expression was shown from the 7th day to the 21st day (P<0.01). CONCLUSION: QTSE therapy can up-regulate the expressions of VEGF and its receptors (Flk-1 and Flt-1) and improve the recovery of kinetic function in the ICH rats, which may be correlated with its action in modulating vascular regeneration to promote the reconstruction of microvascular networks in the damaged areas.

Expression and hypermethylation of p27 kip1 in hepatocarcinogenesis.

AIM: To study the expressions of p27 kip1 protein and p27mRNA, the hypermethylation of p27 kip1 and the relation between them in various stages of hepatocarcinogenesis. METHODS: p27 protein and p27mRNA were detected by immunohistochemical staining and in situ hybridization respectively in 68 cases of normal liver, liver cirrhosis, pericancerous cirrhosis and hepatocellular carcinoma (HCC). The hypermethylation of p27 kip1 was detected by methylation-specific PCR (MSP) in 44 cases of normal liver, liver cirrhosis, and HCC. RESULTS: The positive rate of p27 protein was 66.7% (4/6) in normal liver, 60.0% (6/10) in liver cirrhosis, 50.0% (12/24) in pericancerous cirrhosis and 21.4% (6/28) in HCC. There were no statistical differences in normal liver, liver cirrhosis and pericancerous cirrhosis, but the positive rate of p27 protein significantly decreased in HCC compared to that in the other groups (P = 0.006, chi2 = 7.664). The positive rate of p27 kip1 mRNA was 83.3% (5/6) in normal liver, 70.0% (7/10) in liver cirrhosis, 75.0% (18/24) in pericancerous cirrhosis and 25.0% (7/28) in HCC. There were no statistical differences in normal liver, liver cirrhosis and pericancerous cirrhosis, but the positive rate of p27 kip1 mRNA also significantly decreased in HCC compared to that in the other groups (P = 0.000, chi2 = 16.600). In addition, there was a significant correlation between the expression of p27 protein and p27mRNA in the integrated group of normal liver and liver cirrhosis. However, no significant correlation was found between pericancerous cirrhosis and HCC. Using MSP, we found that 1 HCC in 44 cases (including 6 cases of normal liver, 10 cases of liver cirrhosis and 28 cases of HCC) was methylated, whose p27 protein and p27mRNA were negative. CONCLUSION: The reduction or loss of p27 protein and p27mRNA are potentially involved in hepatocarcinogenesis. The hypermethylation of p27 might lead to the loss of p27mRNA transcription.

[Effects of naoyian serum on VEGF protein expression in cultured rat cerebral microvascular endothelial cell with hypoxia].

OBJECTIVE: To determine the effects of naoyian (NYA) serum on the expression of vascular endothelial growth factor (VEGF) protein in cultured rat cerebral microvascular endothelial cell (RCMEC) with hypoxia. METHODS: NYA serum was separated from rat heart which had been filled stomach with NYA successively for 3 days. The rat cerebral microvascular endothelial cells were taken from the Sprageu-Dawley rat brain at postborn 7 days. The rat cerebral microvascular endothelial cells were incubated at anaerobic incubator to establish the hypoxia models. The vigo of RCMEC was determined by MTT. The level of expression of VEGF protein was measured by cell immunohistochemistry and Western blot. RESULTS: The OD value of NYA serum group was higher than the control groups after hypoxia for 18 hours. VEGF protein was increased by hypoxia in cerebral microvascular endothelial cells (P < 0.05). The content of VEGF protein in NYA serum containing medium was more significantly elevated than those cultured in other control media (P < 0.01). CONCLUSION: VEGF protein was induced by hypoxia in rat cerebral microvascular endothelial cells, and NYA could upregulate the expression of VEGF protein, which may be one of the protection mechanisms for cerebral microvascular endothelial cells.

Activation of endogenous neural stem cells in experimental intracerebral hemorrhagic rat brains.

BACKGROUND: Many researchers suggest that adult mammalian central nervous system (CNS) is incapable of completing self-repair or regeneration. And there are accumulating lines of evidence which suggest that endogenous neural stem cells (NSCs) are activated in many pathological conditions, including stroke in the past decades, which might partly account for rehabilitation afterwards. In this study, we investigated whether there was endogenous neural stem cell activation in intracerebral hemorrhagic (ICH) rat brains. METHODS: After ICH induction by stereotactical injection of collagenase type VII into globus pallidus, 5-Bromo-2 Deoxyuridine (BrdU) was administered intraperitoneally to label newborn cells. Immunohistochemical method was used to detect Nestin, a marker for neural stem cells, and BrdU. RESULTS: Nestin-positive or BrdU-Labeled cells were predominantly located at 2 sites: basal ganglion around hemotoma, ependyma and nearby subventricular zone (SVZ). No positive cells for the 2 markers were found in the 2 sites of normal control group and sham group, as well as in non-leisioned parenchyma, both hippocampi and olfactory bulbs in the 4 groups. Nestin+ cells presented 4 types of morphology, and BrdU+ nucleus were polymorphologic. Positive cell counting around hemotoma showed that at day 2, Nestin+ cells were seen around hemotoma in model group, the number of which increased at day 4, day 7 (P <0.01), peaked at day 14 (P <0.05), and reduced significantly by day 28 (P <0.01). CONCLUSION: Endogenous neural stem cells were activated in experimental intracerebral hemorrhagic rat brains.

[Effect of naoyian on phosphorylated Akt in the neural stem cell injured with anoxia].

OBJECTIVE: To determine the effect of naoyian (NYA) on the expression of phosph-Akt in neural stem cells injured with anoxia. METHODS: The hippocampal neural stem cells (NSC) taken from the Sprageu-Dawley rat brain at 5 days after birth were cultured in DMEM/F12 solution containing EGF and bFGF. After being treated with anoxia for 6 hours, the neural stem cells were incubated in solution containing the NYA serum or normal serum. The neural stem cells were then detected for phosph-Akt and TUNEL stain. RESULTS: After the neural stem cells were injured with anoxia, the expression of the phosph-Akt in NYA serum group was higher than that in the normal serum group and the serum-free group. Treated with NYA serum, the amount of apoptotic NSC decreased. CONCLUSION: After the neural stem cell injured by anoxia was treated with NYA serum, the phosphorylation of Akt increased, the apoptosis of NSC was inhibited.

[Effects of naoyian granule on the expression of IL-6 mRNA and protein in the experimental intracerebral hemorrhagic brain in rats].

OBJECTIVE: To explore the protective mechanisms of the traditional Chinese medicine complex Naoyian granule (NYA) on intracerebral hemorrhagic (ICH). METHODS: ICH rat models were established by stereotaxically injecting 0.4 U type VII collagenase into the right globus pallidus (GP), the expression of IL-6mRNA and protein were investigated by in situ hybridization and Western blotting. RESULTS: In the NYA group the IL-6 mRNA level was higher than that of the model group on 4 - 7 d and the IL-6 protein level was remarkably higher than that of the model group on 6 h - 7 d after ICH injury (P <0.01). CONCLUSION: Up-regulating the expression of IL-6 protein, and stabilizing the increasing expression of IL-6 mRNA and protein may be the protective mechanisms of NYA.

[Characteristics of Bcl-2 and Bax expressions in rat brain after experimental intracerebral hemorrhage and intervention of naoyian granule].

OBJECTIVE: To investigate the characteristics of Bcl-2 and Bax expressions in rat brain after experimental intracerebral hemorrhagic (ICH), and to determine the protective mechanisms of the Traditional Chinese medicine complex, naoyian granule (NYA). METHODS: ICH models were induced according to Rosenbergs method. The rats were divided into the model group, the NYA-treatment group and the sham group. Bcl-2 and Bax expressions in the rat brain after ICH were detected by immunohistochemistry method. RESULTS: Bax expressed in the hemorrhagic core, and the immunoreactive neurons were seriously injuried and the shape varied. Bcl-2 expressed in the penumbra with the normal shape of the immunoreactive neurons. Both Bax and Bcl-2 expressed simultaneously in the marginal region of the cerebral hemorrhagic core. Bcl-2 expression in the rat brain reached the peak at the 2nd day and maintained at a relatively high level until the 7th day, and NYA could increase it. Bax expression peaked at the 1st day, and NYA could down-regulate it. CONCLUSION: The expressions of Bcl-2 and Bax after the cerebral hemorrhage are related to the degree of injury, the time, and the location of the brain tissues. NYA can up-regulate the Bcl-2 expression and down-regulate the Bax expression, which may be one of its neuroprotective mechanisms.

[Comparison and analysis of expression of c-myc and p16 in cervical carcinoma].

BACKGROUND & OBJECTIVE: Activation of proto-oncogene and inactivation of tumor suppressor genes is related to the carcinogenesis of many tumors. It is still unclear whether abnormal expression of c-myc and p16 cooperate in the occurrence and progression of cervical carcinoma, and whether there exists a connection between the expression of two genes and the chemotherapy response of cervical carcinoma. This study was designed to investigate the correlation between the expression of c-myc and p16 and their roles in the genesis and development of the uterine cervical carcinoma and chemotherapy response. METHODS: Using in situ hybridization, 37 cases of cervical carcinoma (including 11 cases after chemotherapy), 21 cases of precancerous lesion and 5 cases of normal cervix were observed for c-myc and p16 mRNA with dig-labeled probes. An image analytic system was used to detect the gray degree values of the positive signals. RESULTS: The positive expression rates of p16 in normal cervix,CIN (cervical intraepithelial neoplasia) and cervical carcinoma were 100%, 71.4%, and 21.6%, respectively (P=0.0001), whereas the expression rates of c-myc were 0%, 42.9%, and 75.7% (P=0.0011), respectively. Statistically significant difference was found among the three groups for both p16 and c-myc. The expression of positive signals of c-myc increased with the increase of malignant degree, and the positive signals in CIN III were also higher than that in CIN II and CIN I. The expression rates of c-myc were decreased in cervical carcinoma after chemotherapy. There was a tendency of negative correlation between the expression of c-myc and p16(r(s)=-0.907). Expression of p16 and c-myc showed no significant difference between effectual and ineffectual chemotherapy groups. CONCLUSION: Both over expression of c-myc and descended expression of p16 may play an important role in the genesis and development of uterine cervical carcinoma. The increased expression of c-myc in different grade CIN suggests that carcinogenesis of cervix be progressive.

Detection of HBV, PCNA and GST-pi in hepatocellular carcinoma and chronic liver diseases.

AIM: To investigate the change of HBV DNA, PCNA and GST-pi in chronic liver disease and hepatocellular carcinoma (HCC). METHODS: Hepatitis B surface antigen (HBsAg), proliferating cell nuclear antigen (PCNA) and glutathione S-transferases (GST-pi) were detected by immunohistochemical staining and HBV DNA was detected by in situ hybridization (ISH) in formalin-fixed and paraffin-embedded sections with a total of 111 specimens of chronic hepatitis, liver cirrhosis, paratumorous tissue, HCC and normal liver tissue. RESULTS: The positive rates of HBsAg and HBVDNA were 62.5 %(15/24) and 75.0 %(12/16) in chronic hepatitis, 64.0 %(16/25) and 83.3 %(15/18) in liver cirrhosis, 72.7 %(16/22) and 85.7 %(12/14) in the paratumorous tissu and 45.0 %(14/31) and 64.3 %(9/14) in HCC. The positive HBVDNA granules in chronic hepatitis, liver cirrhosis and the paratumorous tissue were more intense than that in HCC. The positive rates of PCNA and GST-pi were 34.8 %(8/23) and 25.0 %(4/16) in chronic hepatitis, 73.7 %(14/19) and 17.6 %(3/17) in liver cirrhosis, 86.7 %(13/15) and 53.3 %(8/15) in the paratumorous tissue, 100 %(15/15) and 60.0 %(9/15) in HCC, respectively, and the positive rate of GST-pi in the paratumorous tissue was significantly higher than that in the liver cirrhosis without tumor (P<0.05), but same as that in HCC (P>0.05). CONCLUSION: The HBV infection may increase expression of PCNA and GST-pi. The paratumor cirrhosis may be a sequential lesion of precancerous cirrhosis around HCC.

[Expression of GST-pi and HBV infection in hepatocellular carcinoma].

BACKGROUND & OBJECTIVE: The expression of glutathione S-transferases(GST-pi) might abnormally increase in many carcinogenesis, and the alteration of GST-pi preceded than that alteration of cell morphology. This study was designed to investigate the expression of glutathione S-transferases (GST-pi) and its relationship with hepatitis B virus (HBV) infection in hepatocellular carcinoma (HCC). METHODS: Hepatitis B surface antigen (HBsAg), Hepatitis B core antigen (HBcAg), and GST-pi were detected by immunohistochemical staining and HBV DNA was detected by in situ hybridization (ISH) in total 86 samples of chronic hepatitis, liver cirrhosis, paratumor cirrhosis, HCC, and normal liver tissue. RESULTS: The positive expression rates of HBsAg, HBcAg, and HBV DNA were 61.9% (13/21), 42.9% (9/21), and 75.0% (12/16) in chronic hepatitis; 64.0% (16/25), 36.0% (9/25), and 83.3% (15/18) in liver cirrhosis; 72.7% (16/22), 61.1% (11/18), and 85.7% (12/14) in the paratumor cirrhosis, as well as 45.0% (14/31), 50.0% (14/28) and 64.3% (9/14) in HCC. HBV DNA positive granules in chronic hepatitis, liver cirrhosis, and the paratumor cirrhosis were more and stronger than that in HCC, respectively. The positive expression rates of GST-pi were 25.0% (4/16), 17.6% (3/17), 53.3% (8/15), and 60.0% (9/15) in chronic hepatitis, liver cirrhosis, the Paratumor cirrhosis, and HCC, respectively. The positive rate of GST-pi in the paratumor cirrhosis was significantly higher than that in the liver cirrhosis without tumor (P < 0.05), but the same as in HCC (P > 0.05). CONCLUSIONS: Most of the HCC cases were closely associated with chronic hepatitis and liver cirrhosis of HBV infection. The HBV infection may increase expression of GST-pi. The paratumor cirrhosis may be a sequential lesion of precancerous cirrhosis around HCC.

[Effects of nao-yi-an granule on bcl-2 expression in the rat brain after experimental cerebral hemorrhage].

OBJECTIVE: To investigate the protective effects and mechanisms of the traditional Chinese medicine complex, nao-yi-an granule (NYA) on experimental intracerebral hemorrhage (ICH) in rats. METHODS: ICH models anesthesized were induced by stereotaxical injection of 2 microliters normal saline containing 0.4 U bacterial collagenase(type VII) into the right globas pallidus(GP), and the protein expression of bcl-2 in the rat brain after ICH was detected with the immunohistochemistry method, with which the effects of NYA were observed. RESULTS: Bcl-2 positive cells were found in the cerebral cortex and basal ganglia area after ICH. Immunoreactivity was localized in the cytoplasm as a fine granular precipitate. The levels of bcl-2 expression in the model group varied at different time-points, and reached the peak at 2 d. In the NYA group, the protein expression of bcl-2 was increased significantly both in the cortex and in the basal ganglia compared with those in the model group. CONCLUSION: NYA can significantly upregulate the protein expression of bcl-2.

[Effects of nao-yi-an granule on the intercellular expression of IL-6 in the experimental intracerebral hemorrhagic brain of rats].

OBJECTIVE: To define the cellular localization of IL-6 and the effects of the traditional Chinese medicine complex nao-yi-an granule (NYA) on the intercellular expression of IL-6 in the experimental intracerebral hemorrhagic (ICH) brain of rats, so as to investigate the efficacy mechanisms of NYA on ICH. METHODS: ICH rat models were established by stereotaxically injecting 0.4 U type VII collagenase into the right globus pallidus (GP). The distribution and content of IL-6 protein expression together with the reaction of glial cells were investigated with the immunohistochemistry method. RESULTS: Microglial activation was detected from 6 h to 4 d after ICH in the hematoma region, and reactive astrocytes were found from 6 h to 7 d after ICH around the hematoma region; there was an up-regulation of IL-6 in the hematoma at 6 h after ICH; it reached its peak at 12 h, and disappeared on 7 d. CONCLUSION: The major sources of IL-6-positive protein were neurons and the activated microglia in the brain of ICH rats. Stabilizing the increasing expression of IL-6 may be one of the neuroprotective mechanisms of NYA.