Lipoxygenase(LOX) is related to emergence and development of many diseases, such as acute and chronic inflammation, atherosclerosis, hypertension and tumor. Therefore, the inhibition of LOX may play an important role in the prevention and treatment of these diseases. Many kinds of flavonoids, such as chalcones, flavonols, flavones and flavanols, have inhibitory effect on 5-LOX, 12-LOX and 15-LOX probably through inhibiting expression of LOX, bonding to the enzyme or reacting with free radicals generated at the active site of the enzyme. Their inhibitory activities are related to their structures. The inhibitory effects of flavonoids on LOX maybe one of mechanisms of flavonoids' some biological functions, such as anti-inflammatory, anti-tumor and reducing the risk of cardiovascular disease and cerebrovascular disease.

AIM To study the therapeutic effect of bone marrow transplantation on mustard gas-poisoned mice. METHODS Mustard-poisoned mice model was constructed by injected mustard gas 20 mg·kg~(-1) subcutaneously. At 4 h, 1 d and 2 d after poisoned by mustard gas, the suspended bone marrow cells (106 cells per time) were injected into mice respectively. After poisoned 3, 5, 7 and 10 d, the numbers of leukocyte and marrow nucleated cells were recorded periodically. At the same time, after poisoned 4 h and 1 d by mustard gas, mice in another group were injected granulocyte colony stimulating factor (G-CSF) 74 μg·kg~(-1) combined with transplantation and changes of the cell number were observed. RESULTS Compared with poisoned group without transplantion, leukocyte number in bone marrow transplanted group presented ascending trend, but there was no statistical significance until the 10 d after poisoned. However, the number of marrow nucleated cell increased significantly from the 5th day. After mice injected bone marrow cells combined with G-CSF, leukocyte increased markedly in earlier stage compared with transplantation only. CONCLUTION The bone marrow transplantation has significant effects on improvement of marrow hematogenic system inhibited by mustard gas.

AIM To express recombinant human cytochrome P450 3A4 mutants CYP3A4.3, CYP3A4.4, CYP3A4.5 and CYP3A4.18, and to employ them for in vitro metabolism studies of CYP3A4. METHODS Use Bac-to-Bac baculovirus expression system to recombinant baculovirus carrying cDNA of CYP3A4 mutants CYP3A4.3, CYP3A4.4, CYP3A4.5 and CYP3A4.18. Spodoptera frugiperda 9 (Sf9), cells were co-infected by recombinant viruses of CYP3A4 mutants, human NADPH-P450 oxidoreductase and cytochrome b5 to obtain recombinant proteins CYP3A4.3, CYP3A4.4, CYP3A4.5 and CYP3A4.18 with metabolic activity. RESULTS The mRNA transcription of CYP3A4 mutants in Sf9 cells were validated by RT-PCR. Testosterone and 7-benzyloxy-4-(trifluoromethyl) coumarin were metabolized by the lysates of Sf9 cells infected by the recombinant viruses. CONCLUSION CYP3A4 mutants CYP3A4.3, CYP3A4.4, CYP3A4.5 and CYP3A4.18 with metabolic activity were successfully expressed by baculovirus-insect cell expression system. The results indicated that recombinant CYP3A4. 5 showed lower activity comparing to the wild type protein towards testosterone, while CYP3A4. 18 with higher activity, and for CYP3A4.3 and CYP3A4.4 showing similar activity to the wild type protein.

AIM To study protective effect of [D-Ala~2, D-Leu~5]-enkephalin (DADLE) against hydrogen peroxide (H_2O_2) induced myocardial cell injury and its possible mechanisms. METHODS Myocardial cells were isolated from neonatal rats and cultured for 48 h. Then the cells were randomly assigned into normal control, H_2O_2（200 μmol·L~(-1)）, H_2O_2+DADLE(1 μmol·L~(-1)), H_2O_2+DADLE +naltrindole(10 μmol·L~(-1)) and H_2O_2+DADLE +U0126(10 nmol·L~(-1)) groups and cultured for another 48 h.[~3H]TdR incorporation assay and flow cytometry were used to measure the cell proliferation and apoptosis rate. The lactate dehydrogenase (LDH) activities in culture supernatant measured by using LDH activity kit. The superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in cells were measured with xanthine oxidase method and color reaction of thiobarbituric acid, respectively. The expressions of extracellular signal-regulated kinase (ERK) and phosphorylated-ERK (p-ERK) were observed with Western blot. RESULTS ① Compared with normal control group, the incorporation of [~3H]TdR in myocardial cells of H_2O_2 group was significantly lower, apoptosis rate was higher, LDH activity and MDA content in cells were higher, while SOD activity in cells was lower. In addition, the ratio of IA_( p-ERK) /IA_( ERK) was decreased. ② Compared with H_2O_2 group, the incorporation of ［3H］TdR in H_2O_2+DADLE group was significantly higher, apoptosis rate was lower, LDH activity and MDA content in cells decreased, while SOD activity increased significantly. The ratio of IA_( p-ERK) /IA_( ERK) was increased. ③ δ-Opioid receptor antagonist naltrindole and ERK antagonist U0126 inhibited this effect of DADLE on the above index changes induced by H_2O_2. CONCLUSION The δ-opioid receptor has protective effect against H_2O_2-induced myocardial cell injury, and its possible mechanism may be related to its promotion of antioxide capacity and ERK phosphorylation.

AIM To investigate whether pioglitazone can protect cortical neurons from lipopolysaccharides(LPS)-induced neurotoxicity and the mechanisms responsible for this protective effect. METHODS After 7 d cultures，cultured cortical neurons were incubated with LPS 10 mg·L~(-1) for 4-24 h with or without other drugs. In co-incubation experiments, other drugs were added to the neurons 30 min or 1 h prior to incubation with LPS. The cell viability was assessed by MTT assay. The neuronal apoptosis was quantified by scoring the percentage of cells with apoptotic nuclear morphology after Hoechst 33258 staining. The cultured cells were then fixed on the 7th day and immunocytochemically stained with phosphorylated JNK1 antibody. The protein expressions of active caspase 3 and phosphorylated JNK1 were measured by Western blot. Nitric oxide (NO) generation was measured by Griess method. RESULTS The decrease of cell viability and the increase of apoptotic cells in cultured cortical neurons were observed incubated with LPS for 24 h compared with the normal controls. The cell viability of cortical neurons was decreased from (100.0±10.9)% in the normal control group to (72.3±2.1)% in the LPS-treated group and the apoptotic cell percentages were increased from (11.5±4.2)% in the normal control group to (39.5±8.2)% in the LPS group. LPS induced the increases in phospho-JNK1, active caspase 3 expression, and NO generation. Pioglitazone 0.01, 0.1 and 1 μmol·L~(-1), respectively inhibited LPS-induced decrease in cell viability and increase of apoptotic morphology, active caspase 3 expression in cultured neurons. In LPS+pioglitazone 1 μmol·L~(-1) group, cell viability was （97.8±9.7）%, the apoptotic cells percentage was (20.6±5.0)%, NO generation (6.8±1.3)μmol·L~(-1). Furthermore, pioglitazone also inhibited LPS-induced the increase in JNK1 phosphorylation and NO generation. JNK inhibitor SP600125 5 μmol·L~(-1) significantly inhibited LPS-induced neurotoxicity, cell viability was increased from (72.3±2.1)% to (109.8±11.8)%, the apoptotic cells percentage from (39.5±8.2)% decreased to (19.1±4.8)%, NO generation from （21.1±5.0）μmol·L~(-1) decreased to（4.0±1.3）μmol·L~(-1). The PPARγ antagonist GW9662 10 μmol·L~(-1) did not reverse the effects of pioglitazone. In LPS+pioglitazone 1 μmol·L~(-1)+GW9662 10 μmol·L~(-1) group, cell viability was (90.7±6.9)%, the apoptotic cells percentage was (23.4±4.1)%, and NO concentration was (5.8±0.7)μmol·L~(-1). CONCLUSION Pioglitazone protects cortical neurons against LPS insult at least via inhibiting JNK activity and NO generation, but not PPARγ activation.

AIM To investigate the effect of tocotrienol rich fraction of palm oil (TRF) on glucose metabolism in atherosclerotic mice and the possible mechanism. METHODS Apolipoprotein E gene deficient(ApoE~(-/-)) mice were divided into 3 groups as model control, TRF 0.05% and 0.2%(W/W) groups. 10% (W/W) fat and 0.2% (W/W) cholesterol were added into the diets to induce atherosclerosis formation. Oral glucose tolerance test and insulin tolerance test were conducted after mice were treated by TRF for 12 and 14 weeks respectively. Serum cholesterol, triglyceride, free fatty acid, and insulin levels were measured using corresponding kits. The mRNA expression levels for peroxisome proliferator-activated receptor γ(PPARγ), adiponectin and glucose transporter 4 (Glut4) in white adipose tissue (WAT) were determined by using quantitative real-time PCR. Activation of PPARγ by TRF was tested using luciferase reporter assays. RESULTS Compared with the model control group, TRF decreased non-fasting or fasting blood glucose levels and improved insulin sensitivity of ApoE~(-/-) mice. Both TRF groups showed decreased levels of triglyceride and free fatty acid. The mRNA level of adiponectin in WAT was up-regulated by (1.73±0.32) times in TRF 0.2% group compared with the control group. Glut4 mRNA level was increased (1.89±0.24) and (2.01±0.61) times compared with control group in TRF 0.05% group and TRF 0.2% group respectively. The fold inductions of TRF on PPARγ-ligand-binding domain, PPARγ1 and PPARγ2 activities were (2.7±0.2), (6.1±0.65) and (5.3±0.1) times compared with DMSO by using luciferase reporter assay. CONCLUSION TRF can improve glucose metabolism in atherosclerotic mice and this effect may be partly due to modulating the activity of PPARγ.

AIM To identify potential amino acid residues that contribute to different catalytic characteristics of CYP2A6 and CYP2A13 enzymes in nicotine metabolism. METHODSWild type of CYP2A6 and CYP2A13 and their mutants CYP2A6V117A, CYP2A6G164H, CYP2A6I208S, CYP2A6R372H, CYP2A6S465P and CYP2A13A117V, CYP2A13H164G, CYP2A13S208I, CYP2A13H372A and CYP2A13P465S, were subjected kinetic analysis in nicotine 5'-hydroxylation. RESULTS For CYP2A6, substitution of isoleucine 208 to serine caused dramatic kinetic property changes with K_m and V_( max) varied from 62.25 μmol·L~(-1) and 6.53 mol·min~(-1)·moL~(-1) to 345 μmol·L~(-1) and 2.19 mol·min~(-1)·moL~(-1). However, the corresponding serine 208 to isoleucine mutation did not heavily affect the enzyme activity in CYP2A13. The histidine 372 to arginine mutation resulted in a remarkable catalytic efficiency decrease with K_m and V_( max) changes from 26.01 μmol·L~(-1) and 24.51 mol·min~(-1)·moL~(-1) to 148.7 μmol·L~(-1) and 6.11 mol·min~(-1)·moL~(-1) in CYP2A13, but the switching of argenine 372 to histidine did not show expected corresponding crucial influence in CYP2A6 activity. Substitutions on the other positions changed enzyme activities in different rates. CONCLUSION The isoleucine 208 is crucial to human CYP2A6, while the 372 histidine is a key amino acid residue for CYP2A13 in nicotine 5'-hydroxylation.

AIM To evaluate the prevention and treatment of N-(2-mercaptopropionyl)-glycine sodium (MPG-Na) and tiopronin (MPG) on acute liver injury. METHODS The experimental mouse model of hepatotoxicity induced by D-galactosamine (Gal) was applied to investigate preventive and remedial effects. In the preventive experiment, the mice were ip administered with MPG-Na or MPG 37.5，75 and 150 mg·kg~(-1), respectively, for 7 d. Gal 800 mg·kg~(-1) was ip given into the mice 30 min after the last administration. In the remedial experiment, the mice were ip given Gal 800 mg·kg~(-1) and 30 min later followed by MPG-Na or MPG 37.5, 75 and 150 mg·kg~(-1) , respectively, for 2 d. The mice were euthanized and serum was prepared 24 h (pre-treatment) or 48 h (post-treatment) after Gal injection. The activities of serum glutamyl pyruvic transaminase (GPT) and glutamyl oxaloacetic transaminase (GOT), the contents of total protein (TP) and albumin (Alb), and the Alb/globulin (A/G) ratio were determined. The liver tissues were collected for histopathological assessment (HE staining) under light microscope. RESULTS Compared with normal control group, the activities of serum GPT and GOT in model group were significantly increased. The injuries such as fatty degeneration and liver cell necrosis were observed. Compared with model group, the activities of GPT and GOT in pre-treatment groups were obviously decreased in MPG-Na 150 mg·kg~(-1) group. In post-treatment groups, the activity of GPT decreased in 3 MPG-Na groups. The contents of TP, Alb and A/G ratio had little change. In addition, MPG-Na alleviated the injuries such as fatty degeneration and liver cell necrosis obviously. Compared with MPG, MPG-Na showed similar effect. CONCLUSION MPG-Na has an obvious protective effect against Gal-induced acute liver injury in mice and the efficiency is equivalent as MPG.

AIM To explore effects of policosanol on depressing cholesterol in hyperlipidemia rats and the correlated biochemistry mechanism. METHODS The rats were randomly divided into normal control, policosanol 4 mg·kg~(-1) prevention, hyperlipidemia model, policosanol 4, 6 and 8 mg·kg~(-1) and lovastatin positive control groups. The later 5 group rats were fed with high-cholesterol diets for 4 weeks in order to make hyperlipidemia model and beginning from the 5th week, in addition to the normal control and model groups, other groups were ig given policosanol or lovastatin once a day for 6 weeks, respectively, and policosanol protection group rats were ig given with policosanol 4 mg·kg~(-1) once a day for 10 weeks, together with high-cholesterol diets everyday. Total cholesterol(TC), triglycerides (TG), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) concentrations in the serum and fecal bile acid (FBA) in the exrement were determined by auto-biochemistry analyzer. The activity of 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase in hepatocellular microsomes was detected by ultraviolet spectrophotometric analysis and activity of low density lipoprotein receptor (LDL-R) in peripheral blood lymphocyte was detected by fluorescence labelled integrator method. RESULTS Compared with hyperlipemia model group, the levels of TC decreased (39.1%-46.4%), LDL-C decreased (66.6%-80.7%), and FBA increased (9.7%-19.0%), the activity of HMG-CoA reductase decreased （13.8%-23.6%）, and activity of LDL-R increased (27.5%-129.6%) in policosanol prevention, policosanol 4, 6 and 8 mg·kg~(-1) and lovastatin groups, respectively; HDL-C increased （12.2%-16.7%） in policosanol prevention and policosanol 8 mg·kg~(-1) groups; TG decreased in lovastatin group. CONCLUSION Policosanol has significant effects on decreasing cholesterol. The decreasing cholesterol mechanism should include: ① increasing FBA excretion; ② decreasing the activity of HMG-CoA reductase; ③ increasing activity of LDL-R.

AIM To investigate the effects and mechanism of cardiomyocyte hypertrophy induced by urocortin. METHODS The cardiomyocytes were divided into 8 groups: normal control, urocortin, staurosporine(Sta), verapamil(Ver), H89, urocortin+Sta, urocortin+Ver, and urocortin+H89 groups. The cardiomyocytes diameter was measured by computer photograph analysis system. The protein synthetic rate was obtained through measuring the incorporation of [~3H]-leucine into myocyte protein by liquid scintillation method. The total protein content was assayed by Lowry method. The expression of atrial natriuretic peptide (ANP) was determined by Western blot. [Ca~(2+)]_i transient was measured by Till image system by cell loading Fura-2/AM. RESULTSUrocortin group enhanced cardiomyocyte volume, protein synthesis, total protein content and expression of ANP by 30.9%, 36.3%, 35.5% and 34.7%;urocortin+Sta group decreased cardiomyocyte diameter, protein synthesis, total protein content and expression of ANP by 16.5%, 22.1%, 18.1% and 21.3%;urocortin+H89 group decreased the cardiomyocyte diameter, the protein synthesis,total protein content and expression of ANP by 16.6%, 21.5%, 19.5% and 20.6%;urocortin+Ver decreased the cardiomyocyte diameter, the protein synthesis, total protein content and the expression of ANP by 17.1%, 20.9%, 17.9% and 19.9%;Sta, H89 and Ver could decrease the [Ca~(2+)]_i transient induced by urocortin. CONCLUSION The hypertrophic effect of urocortin in rat neonatal cardiomyocytes is mediated via activation of protein kinase C and protein kinase A pathway and L-type calcium channels.