AIM:To investigate the protective effect of lycopene on primary mouse cerebrocortical neurons ex -posed to tert-butyl hydroperoxide ( t-BHP) and its mechanisms of in vitro.METHODS:Primary cerebrocortical neurons of newborn C57 mice were extracted and divided into normal group , t-BHP group, lycopene +t-BHP group and lycopene group.The neuronal damage was induced by t-BHP exposure for 24 h, and the cell viability was examined by MTT assay . ROS content was measured by flow cytometry , and the protein levels of Bax , Bcl-2, caspase-3, cleaved caspase-3 and cyto-chrome C were examined by Western blot .RESULTS:The primary mouse cortical neurons expressed MAP-2 protein.Ly-copene at concentration of 4μmol/L reversed the decrease in cell viability .Flow cytometry revealed that lycopene treatment attenuated ROS content under the condition of t-BHP exposure.In addition, the protein level of Bcl-2 was increased, and the expression of Bax , cleaved caspase-3 and cytochrome-C was suppressed in lycopene +t-BHP group.CONCLUSION:The protective effect of lycopene on cortical neurons with t-BHP-induced injury may be involved in the mechanism of neuro-nal antioxidative response by down-regulating caspase-3 and Bax/Bcl-2 through the mitochondrial apoptotic pathway .

AIM:To screen and verify the effective ingredient of traditional Chines medicine (TCM) Q0409 in improving learning and memory ability of mice.METHODS:The mouse learning and memory impairment model was induced by intraperitoneal injection of scopolamine.The mice in each group were given the corresponding drug by gavage at the same time for 14 d in succession.Morris water maze test was used to measure the learning and memory ability of the mice, and then the hippocampal tissue homogenate was taken to determine the activity of acetylcholinesterase (AChE).The animals were divided into 8 groups according to L8(27) orthogonal table.The variance analysis and factorial analysis were used to analyze the pharmacological effects of seven kinds of single herbal in TCM Q0409 and determine the screening ingredients.The animals were divided into 6 groups according to the results of the preliminary screening results, and further testing and validation of TCM Q0409 screening ingredients were performed to get the final simplified ingredients.RESULTS:Three medicinal herbs of Polygalae, Panax ginseng and Acori graminei rhizome were screened by the orthogonal results of Morris water maze test and the activity of AChE in mouse hippocampal tissues.The simplified ingredients of TCM Q0409 were obtained through the variance results of Morris water maze test and the activity of AChE in mouse hippocampal tissues.CONCLUSION:Polygala and ginseng were eventually determined as simplified ingredients of TCM Q0409 and it was verified that they improve the learning and memory ability of the mice with learning and memory impairment.

AIM:To establish a mouse model of immuno-inflammation in central nervous system (CNS) associated with cognitive dysfunction.METHODS:C57BL/6J male mice were divided into 3 groups.Lipopolysaccharide (LPS) was intraperitoneally injected into the mice to induce cognitive impairment.Morris water maze test, passive avoidance test and pole test were used to observe the behavioral changes of mice.The histomorphology was analyzed by the method of immunofluorescence.The detailed molecular mechanism was determined by Western blot.RESULTS:Compared with saline group, LPS induced mouse sickness behavior and memory loss.Microglia activation and neuronal loss in the hippocampus were observed.The expression of neuroinflammatory proteins COX-2 and iNOS in the brain of LPS-induced mice was increased.CONCLUSION:Intraperitoneal injection of LPS induces cognitive dysfunction in mice.

AIM: To observe the effects of berberine (Ber) on enterocyte apoptosis in septic mice and its pos-sible mechanism.METHODS: Male C57BL/6 mice (8 ~10 weeks old) were randomly divided into sham group, cecal ligation and puncture (CLP) group, CLP +Ber group and sham +Ber group.The mice in CLP group underwent CLP ope-ration, and the mice in sham groups suffered a similar operation except the ligation and puncture.After the sham or CLP operation, the mice were administered intragastrically with distilled water or berberine (50 mg/kg) within 2 h.After 20 h, the mice were killed with excess pentobarbital sodium and the ileum tissues were removed.The histological changes of the intestine were observed and the enterocyte apoptosis was examined by determining the protein level of cleaved caspase-3. Furthermore, mitochondrial Bax, cytoplasm cytochrome C (Cyt C) and the total proteins of Bcl-2, Fas, FasL and Fas-as-sociated protein with death domain (FADD) were examined by Western blot.The mRNA expression of tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) was measured by real-time PCR.RESULTS: The extensive ileum injuries, including remarkably increased leukocytes and necrosis of intestinal villus were observed 20 h after CLP.In CLP group, the protein levels of cleaved caspase-3, cytoplasm Cyt C, as well as Fas, FasL were significantly increased, but the Bcl-2 level was decreased.Bax translocation into mitochondria was promoted.However, FADD was not changed significantly.The mRNA expression of TH and DBH was also increased sharply in CLP group.On the contrary, treatment with berberine made a considerable alleviating alteration in the ileum of the septic mice.CONCLUSION: Treatment with berberine pro-vides protective effects on intestinal injury in septic mice by reducing enterocyte apoptosis, and its possible mechanism may be involved in the inhibition of the endogenous and exogenous apoptosis pathways.

AIM:To observe the effect of B-HT933, a selective α2-adrenoceptor agonist, on lipopolysaccha-ride ( LPS )-induced TNF-αproduction in neonatal rat cardiomyocytes and to explore the underlying mechanisms . METHODS:The neonatal rat cardiomyocytes were cultured .The localization of α2A-adrenoceptor in the cardiomyocytes was examined by immunofluorescence staining .The cardiomyocytes were exposed to LPS or/and B-HT933 for different time.The level of TNF-αin the supernatants and the mRNA expression of TNF-αwere detected by ELISA and real-time PCR, respectively.In addition, LPS-associated signal molecules in the cardiomyocytes were also examined by Western blotting.RESULTS: Immunofluorescence staining showed that α2A-adrenoceptors were localized in the cardiomyocytes . LPS stimulated TNF-αproduction in the cardiomyocytes in a dose and time-dependent manner .B-HT933 pretreatment sig-nificantly inhibited the expression of TNF-αat mRNA and protein levels in LPS-treated cardiomyocytes .Furthermore, LPS exposure induced IκBαand p38 phosphorylation in cardiomyocytes and only IκBαphosphorylation was prevented by B-HT933 treatment.CONCLUSION:α2A-adrenoceptors are present in neonatal rat cardiomyocytes and its agonist B -HT933 inhibits LPS-induced TNF-αproduction in cardiomyocytes via suppressing IκBαphosphorylation .

[ ABSTRACT] AIM:To explore the role of Huoxue Jiangzhi Recipe in preventing and treating fatty liver in mice and its underlying mechanisms.METHODS:Healthy Kunming mice were fed with high-fat diet and treated intragastrically with different doses of Huoxue Jiangzhi Recipe ( compound of ginseng, panax notoginseng and rhizoma gastrodiae, named as GST) for 2 weeks.The levels of blood lipids and triglyceride ( TG) in hepatic tissues were measured.Meanwhile, liver in-dex and hepatic pathology were observed.The optimized dosage of Huoxue Jiangzhi Recipe was determined by the experi-ments.The mice were divided into normal control group ( NC group, fed with normal diet) and model group ( fed with high-fat diet) .The model mice were subdivided into 3 subgroups 12 weeks later:HF group ( fed continuously with high-fat di-et) , ND group ( fed with normal diet) , GSL group ( fed with normal diet and treated intragastrically with GSL) .The mice in NC, HF and ND groups were given distilled water by gastric perfusion.Two weeks later, all mice were killed, and blood was collected for measuring serum total cholesterol (TC),TG,high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol ( LDL-C) contents, hepatic TC, TG, malondialdehyde ( MDA ) levels and superoxide dismutase ( SOD) activity were detected.Moreover, liver index and hepatic pathology were also observed.The mRNA expression of peroxisome proliferator-activated receptor alpha (PPARα) and cytochrome-P450 2E1 (CYP2E1) in the liver was examined by RT-PCR.RESULTS:GST significantly decreased serum lipid, hepatic lipid and MDA levels and elevated SOD activi-
ty.Furthermore, GST markedly reduced liver index, improved hepatic adipose infiltration, increased PPARαmRNA ex-pression and inhibited CYP2E1 mRNA expression.CONCLUSION:GST is effective in the treatment of fatty liver in mice by up-regulating PPARα, thus reducing serum and hepatic TG levels, down-regulating CYP2E1 and inhibiting lipid peroxi-dation.

AIM:To study the expression of glycine receptorα1 subunit in neonatal rat myocardial cells and to investigate the effect of lipopolysaccharide (LPS), hypoxia/reoxygenation, isoproterenol (ISO) and high concentration of glucose (HG) on the expression of glycine receptorα1 subunit in the neonatal rat myocardial cells.METHODS:Neonatal rat myocardial cells were cultured in vitro.The expression of glycine receptorα1 subunit was detected by Western blotting. The neonatal rat myocardial cells were treated with LPS (20 mg/L), ISO (100 μmol/L) or high concentration of glucose (25 mmol/L) for 24 h, or were exposed to hypoxia for 3 h followed by reoxygenation for 3 h.Subsequently, the cell viabil-ity was measured by CCK-8 assay, and the expression of glycine receptorα1 subunit was determined by Western blotting. RESULTS:The expression of glycine receptor α1 subunit in the neonatal rat myocardial cells was positively detectable by Western blotting.Compared with control group, no significant difference of the cell viability ( P>0.05) in LPS group, ISO group, hypoxia/reoxygenation group and HG group was observed.The expression of glycine receptor α1 subunit was in-creased (P<0.01) in LPS group, ISO group and hypoxia/reoxygenatio group, but decreased (P<0.01) in HG group. CONCLUSION:Glycine receptorα1 subunit exists in the neonatal rat myocardial cells.A certain concentration of LPS or ISO, or hypoxia/reoxygenation for a certain period upregulate the expression of glycine receptorα1 subunit, but HG down-regulates the expression of glycine receptor α1 subunit in cultured neonatal rat myocardial cells.

AIM:To investigate the effects of thalidomide ( THD) on the activation of connective tissue growth factor ( CTGF) gene promoter induced by transforming growth factor β1 ( TGF-β1 ) in human embryonic lung fibroblasts ( HELF) .METHODS:DNA sequence of CTGF gene promoter was cloned into luciferase reporter gene vector to construct the recombinant eukaryotic expression vector pGL 3-CTGFP, and the recombinant vector was transfected into HELF cell line.The effects of TGF-β1 and THD on the activation of CTGF gene promoter were detected by dual-luciferase analysis . RESULTS:TGF-β1 increased the reporter gene activity dose-dependently (P<0.05), with a plateau at 5 μg/L being 2.16 folds as high as the control .TGF-β1-induced increase in the reporter gene activity was also time-dependent ( P<0.05).After exposure to TGF-β1(5 μg/L), the level of luciferase activity reached its peak at 12 h and was 2.52 folds as high as the control .THD significantly inhibited TGF-β1-induced increase in the reporter gene activity in a dose-dependent manner , but its basal activity was not changed .CONCLUSION: TGF-β1 stimulates the transcriptional activity of CTGF gene promoter in HELF cells in a dose-and time-dependent manner , while THD may inhibit the effects dose-dependently .

AIM:To explore the preliminary mechanism of senegenin ( Sen) on inhibiting hypoxia/reoxygenation ( H/R)-induced apoptosis of primary cortical neurons .METHODS:The cultured cortical neurons were randomly divided in-to normal group (control group), model group (H/R group), Sen+H/R group and Sen group.Flow cytometry was used to evaluate the effect of Sen on H/R-induced cell apoptosis .The protein levels of JNK , p-JNK, c-Jun, p-c-Jun, Bcl-2 and Bax were assessed by Western blotting .RESULTS:The apoptotic rate in H/R group was obviously higher than that in control group (P<0.05), while the apoptotic rate in Sen +H/R group was obviously lower than that in H/R group (P<0.05), suggesting that the model of apoptosis was established successfully .The results of Western blotting showed that Sen increased the expression of JNK and c-Jun, inhibited the phosphorylation of JNK and c-Jun (P<0.05), increased the protein level of Bcl-2 and inhibited the protein level of Bax in H/R treated primary cortical neurons (P<0.05).CONCLUSION:Sen has a protective effect against H/R-induced neuronal apoptosis by increasing the expression of JNK and c-Jun, inhibiting the phosphorylation of JNK and c-Jun, increasing the protein level of Bcl-2 and decreasing the protein level of Bax .

Objective To establish the CTRP4 transgenic mouse model and investigate the function of the novel adipocytokine CTRP4.Methods CTRP4 overexpressing vector in pCAGGS was firstly constructed and then microinjected into zygote to establish the founder transgenic mice .F1 heterozygotes were generated by founder mice mating with wildtype mice, and the CTRP4 transgenic homozygotes were generated by F 1 littermates.The genotype was confirmed by PCR and test cross method .The expression level of CTRP 4 in transgenic mice was detected by western blot .Result The human CTRP4 transgenic homozygote mice line was established , and the expression level of CTRP 4 was confirmed raletively high in detected tissues including heart , liver, brain and kidney . Conclusion The human CTRP4 transgenic mice was successfully established .