Screening of blood-activating active components from Danshen-Honghua herbal pair by spectrum-effect relationship analysis.

BACKGROUND: Danshen (Salvia miltiorrhiza, DS) and Honghua (Carthamus tinctorius, HH) are commonly used traditional Chinese medicines for activating blood and removing stasis, and DS-HH (DH) herbal pair had potential synergistic effects on promoting blood circulation. Therefore, it is essential to make clear the active components of this herbal pair for better understanding their potential synergistic effects. PURPOSE: To comprehensively evaluate the activity of DH herbal pair on physiological coagulation system of rats, and seek their potential active components by spectrum-effect relationship analysis. METHODS: The water extracts of DH herbal pair with different proportions (DS: HH = 1:1, 2:1, 3:1, 5:1, 1:5 and 1:3) were prepared. Male Sprague-Dawley rats were randomly divided into eight groups: blank group, model group, model + 1:1 (DH) group, model + 2:1 group, model + 3:1 group, model + 5:1 group, model + 1:5 group and model + 1:3 group. The intragastric administration was performed for eight times with 12 h intervals. SC40 semi-automatic coagulation analyzer was employed to determine coagulation indices. Meanwhile, HPLC and LC-MS were applied for chemical analyses of DH extracts. Finally, the active ingredients were screened by spectrum-effect relationship analysis and the activities of major predicted compounds were validated in vitro. RESULTS: Different proportions of DH extracts could significantly prolong thrombin time (TT) and activated partial thromboplastin time (APTT), increase prothrombin time (PT) and decrease fibrinogen (FIB) content, reduced whole blood viscosity (WBV) and plasma viscosity (PV), decreased erythrocyte sedimentation rate blood (ESR) compared with model group. Furthermore, fifteen highly related components were screened out by the spectrum-effect relationship and LC-MS analysis, of which caffeic acid, salvianolic acid B, hydroxysafflor yellow A and lithospermate acid had significant blood-activing effect by prolong APTT and decrease FIB content at high (0.6 mM), medium (0.3 mM) and low (0.15 mM) (except lithospermate acid) concentrations in vitro. CONCLUSIONS: DH herbal pair showed strong blood-activating effect on blood stasis rat through regulating the parameters involved in haemorheology and plasma coagulation system. Four active compounds, caffeic acid, salvianolic acid B, hydroxysafflor yellow A and lithospermate acid predicted by spectrum-effect relationship analysis had good blood-activating effect. Therefore, spectrum-effect relationship analysis is an effective approach for seeking active components in herbal pairs.

pH-dependent surface electrostatic effects in retention on immobilized artificial membrane chromatography: Determination of the intrinsic phospholipid-water sorption coefficients of diverse analytes.

The passive membrane permeability of drugs may be potentially predicted by phospholipid-water sorption coefficient (KPLIPW) through immobilized artificial membrane (IAM) chromatography. However, for predominantly ionized compounds, unexpected confounding electrostatic interaction would influence the accurate determination of KPLIPW, accompanied by the mobile phase with varied pH and ionic strength. In order to measure the intrinsic phospholipid-water sorption coefficient (KIAM,intr) for diverse analytes, a mathematic model by characterizing the confounding electrostatic effects was developed in this study. Based on the developed electrostatic model, additional electrostatic repulsion/attraction interactions with the charged IAM surface were characterized for organic cations/anions. Considering that most of the IAM chromatography data was determined under physiological conditions, the KPLIPW values of all compounds were calculated with the ionic strength of 0.1 M and the pH value of 7. Generally, KPLIPW was hardly affected by the pH and ionic strength of mobile phase for those electroneutral compounds. KIAM,intr was 0.28 log units smaller than apparent phospholipid-water sorption coefficient (KIAM,app) for cations. In contrast, KIAM,intr was larger than KIAM,app by 0.28 log units for anions. Herein, KIAM,intr of diverse analytes can be easily calculated by the developed electrostatic model. For application, KIAM,intr was further compared with intrinsic Caco-2 permeability, and good sigmoidal correlations (R2 = 0.76) were established between them for organic cations and neutral compounds but not for organic anions.

Screening and Characterizing Tyrosinase Inhibitors from Salvia miltiorrhiza and Carthamus tinctorius by Spectrum-Effect Relationship Analysis and Molecular Docking.

Tyrosinase (TYR) is a rate-limiting enzyme in the synthesis of melanin, while direct TYR inhibitors are a class of important clinical antimelanoma drugs. This study established a spectrum-effect relationship analysis method and high-performance liquid chromatography-mass spectrometry (LC-MS) analysis method to screen and identify the active ingredients that inhibited TYR in Salvia miltiorrhiza-Carthamus tinctorius (Danshen-Honghua, DH) herbal pair. Seventeen potential active compounds (peaks) in the extract of DH herbal pair were predicted, and thirteen of them were tentatively identified by LC-MS analysis. Furthermore, TYR inhibitory activities of five pure compounds obtained from the DH herbal pair were validated in the test in which kojic acid served as a positive control drug. Among them, three compounds including protocatechuic aldehyde, hydroxysafflor yellow A, and tanshinone IIA were verified to have high TYR inhibitory activity (IC50 value of 455, 498, and 1214 µM, resp.) and bind to the same amino acid residues in TYR catalytic pocket according to the results of the molecular docking test. However, the other two compounds lithospermic acid and salvianolic acid A had a weak effect on TYR, as they do not combine with the active amino acid residues or act on the active center of TYR. Therefore, the developed methods (spectrum-effect relationship analysis and molecular docking) could be used to effectively screen TYR inhibitors in complex mixtures such as natural products.

Investigation of Interactions between Thrombin and Ten Phenolic Compounds by Affinity Capillary Electrophoresis and Molecular Docking.

Thrombin plays a vital role in blood coagulation, which is a key process involved in thrombosis by promoting platelet aggregation and converting fibrinogen to form the fibrin clot. In the receptor concept, drugs produce their therapeutic effects via interactions with the targets. Therefore, investigation of interaction between thrombin and small molecules is important to find out the potential thrombin inhibitor. In this study, affinity capillary electrophoresis (ACE) and in silico molecular docking methods were developed to study the interaction between thrombin and ten phenolic compounds (p-hydroxybenzoic acid, protocatechuic acid, vanillic acid, gallic acid, catechin, epicatechin, dihydroquercetin, naringenin, apigenin, and baicalein). The ACE results showed that gallic acids and six flavonoid compounds had relative strong interactions with thrombin. In addition, the docking results indicated that all of optimal conformations of the six flavonoid compounds were positioned into the thrombin activity centre and had interaction with the HIS57 or SER195 which was the key residue to bind thrombin inhibitors such as argatroban. Herein, these six flavonoid compounds might have the potential of thrombin inhibition activity. In addition, the developed method in this study can be further applied to study the interactions of other molecules with thrombin.

Helicobacter pylori infection-induced H3Ser10 phosphorylation in stepwise gastric carcinogenesis and its clinical implications.

BACKGROUND: Our previous works have demonstrated that Helicobacter pylori (Hp) infection can alter histone H3 serine 10 phosphorylation status in gastric epithelial cells. However, whether Helicobacter pylori-induced histone H3 serine 10 phosphorylation participates in gastric carcinogenesis is unknown. We investigate the expression of histone H3 serine 10 phosphorylation in various stages of gastric disease and explore its clinical implication. MATERIALS AND METHODS: Stomach biopsy samples from 129 patients were collected and stained with histone H3 serine 10 phosphorylation, Ki67, and Helicobacter pylori by immunohistochemistry staining, expressed as labeling index. They were categorized into nonatrophic gastritis, chronic atrophic gastritis, intestinal metaplasia, low-grade intraepithelial neoplasia, high-grade intraepithelial neoplasia, and intestinal-type gastric cancer groups. Helicobacter pylori infection was determined by either 13 C-urea breath test or immunohistochemistry staining. RESULTS: In Helicobacter pylori-negative patients, labeling index of histone H3 serine 10 phosphorylation was gradually increased in nonatrophic gastritis, chronic atrophic gastritis, intestinal metaplasia groups, peaked at low-grade intraepithelial neoplasia, and declined in high-grade intraepithelial neoplasia and gastric cancer groups. In Helicobacter pylori-infected patients, labeling index of histone H3 serine 10 phosphorylation followed the similar pattern as above, with increased expression over the corresponding Helicobacter pylori-negative controls except in nonatrophic gastritis patient whose labeling index was decreased when compared with Helicobacter pylori-negative control. Labeling index of Ki67 in Helicobacter pylori-negative groups was higher in gastric cancer than chronic atrophic gastritis and low-grade intraepithelial neoplasia groups, and higher in intestinal metaplasia group compared with chronic atrophic gastritis group. In Helicobacter pylori-positive groups, Ki67 labeling index was increased stepwise from nonatrophic gastritis to gastric cancer except slightly decrease in chronic atrophic gastritis group. In addition, we noted that histone H3 serine 10 phosphorylation staining is accompanied with its location changes from gastric gland bottom expanded to whole gland as disease stage progress. CONCLUSIONS: These results indicate that stepwise gastric carcinogenesis is associated with altered histone H3 serine 10 phosphorylation, Helicobacter pylori infection enhances histone H3 serine 10 phosphorylation expression in these processes; it is also accompanied with histone H3 serine 10 phosphorylation location change from gland bottom staining expand to whole gland expression. The results suggest that epigenetic dysregulation may play important roles in Helicobacter pylori-induced gastric cancer.

An ultrafiltration and high performance liquid chromatography coupled with diode array detector and mass spectrometry approach for screening and characterizing thrombin inhibitors from Rhizoma Chuanxiong.

Thrombin (THR) plays a significant role in thromboembolic diseases, direct THR inhibitors are a class of important clinical anticoagulant drugs. This study established a THR in-solution based biospecific extraction combined with ultrafiltration and high performance liquid chromatography coupled with diode array detector and mass spectrometry analysis (TUA) method to screen and identify ligands for THR in Rhizoma Chuanxiong. After evaluating the reliability of the present TUA method using positive (argatroban) and negative (adenosine, tirofiban, ticagrelor) control drugs, this method was successfully applied to detect eight potential active compounds in Rhizoma Chuanxiong. Two new THR-targeted compounds isochlorogenic acid C and senkyunolide I with high THR inhibitory activity (IC50 206.48 and 197.23µM, respectively) were identified by liquid chromatography/mass spectrometry and enzyme inhibitory activity test finally. They were reported with direct THR inhibition activity for the first time and their ligand-THR interactions were explored by in silico molecular docking research. In addition, based on the TUA screening result, four compounds gained similar structure with the two hit compounds were also investigated as promising candidates targeting THR with high binding energy (>5.0kcal/mol). These results may prove that the proposed method could effectively screen THR inhibitors in complex mixtures.

Induction of defense-related enzymes in patchouli inoculated with virulent Ralstonia solanacearum

Background: Defense-related anti-oxidative response is a vital defense mechanism of plants against pathogen invasion. Ralstonia solanacearum is an important phytopathogen. Bacterial wilt caused by R. solanacearum is the most destructive disease and causes severe losses in patchouli, an important aromatic and medicinal plant in Southeast Asia. The present study evaluated the defense response of patchouli inoculated with virulent R. solanacearum. Results: Results showed that the basic enzymatic activities differed not only between the leaves and stems but also between the upper and lower parts of the same organ of patchouli. POD, SOD, PPO, and PAL enzymatic activities were significantly elevated in leaves and stems from patchouli inoculated with R. solanacearum compared to those in control. The variation magnitude and rate of POD, PPO, and PAL activities were more obvious than those of SOD in patchouli inoculated with R. solanacearum. PAGE isoenzymatic analysis showed that there were one new POD band and two new SOD bands elicited, and at least two isoformic POD bands and two SOD bands were observably intensified compared to the corresponding control. Conclusion: Our results suggest that not only defense-related enzymatic activities were elevated but also the new isoenzymatic isoforms were induced in patchouli inoculated with R. solanacearum.

[Applications of immobilization techniques in the screening of active constituents from traditional Chinese medicines].

It has been an active approach to screen the active ingredients in traditional Chinese medicines (TCMs) according to the affinity property between small molecule compounds and biomaterials such as cells, bacteria and proteins. On the other hand, the biomaterials can be immobilized on a solid support before the screening procedure. The immobilization method not only can maintain the biological activities of biomaterials, but also have other advantages such as high efficiency, simple operation, easy to be continuous and automatic, etc. Carrier materials (solid supports) for the immobilization including silica gel, magnetic materials, hollow fiber, and the surface plasma resonance sensor chips have been used to immobilize biomaterials and successfully applied in the screening of active ingredients from TCMs. In this paper, applications of immobilization techniques in the screening of active components from TCMs were reviewed to provide a scientific reference to the future applications.

Outcomes of autologous bone marrow mononuclear cell transplantation in decompensated liver cirrhosis.

AIM: To determine the long-term efficacy of autologous bone marrow mononuclear cells (BM-MNCs) transplantation in terms of improving liver function and reducing complications in patients with decompensated cirrhosis. METHODS: A total of 47 inpatients with decompensated liver cirrhosis were enrolled in this trial, including 32 patients undergoing a single BM-MNCs transplantation plus routine medical treatment, and 15 patients receiving medical treatment only as controls. Forty-three of 47 patients were infected with hepatitis B virus. Bone marrow of 80-100 mL was obtained from each patient and the BM-MNCs suspension was transfused into the liver via the hepatic artery. The efficacy of BM-MNCs transplantation was monitored during a 24-mo follow-up period. RESULTS: Liver function parameters in the two groups were observed at 1 mo after BM-MNCs transfusion. Prealbumin level was 118.3 ± 25.3 mg/L vs 101.4 ± 28.7 mg/L (P = 0.047); albumin level was 33.5 ± 3.6 g/L vs 30.3 ± 2.2 g/L (P = 0.002); total bilirubin 36.9 ± 9.7 mmol/L vs 45.6 ± 19.9 mmol/L (P = 0.048); prothrombin time 14.4 ± 2.3 s vs 15.9 ± 2.8 s (P = 0.046); prothrombin activity 84.3% ± 14.3% vs 74.4% ± 17.8% (P = 0.046); fibrinogen 2.28 ± 0.53 g/L vs 1.89 ± 0.44 g/L (P = 0.017); and platelet count 74.5 ± 15.7 × 10(9)/L vs 63.3 ± 15.7 × 10(9)/L (P = 0.027) in the treatment group and control group, respectively. Differences were statistically significant. The efficacy of BM-MNCs transplantation lasted 3-12 mo as compared with the control group. Serious complications such as hepatic encephalopathy and spontaneous bacterial peritonitis were also significantly reduced in BM-MNCs transfused patients compared with the controls. However, these improvements disappeared 24 mo after transplantation. CONCLUSION: BM-MNCs transplantation is safe and effective in patients with decompensated cirrhosis. It also decreases the incidence of serious complications.

Anti-apoptotic proteins and catalase-dependent apoptosis resistance in nickel chloride-transformed human lung epithelial cells.

Chronic exposure to nickel compounds is associated with increased incidence of certain types of human cancer, including lung and nasal cancers. Despite intensive investigation, the oncogenic processes remain poorly understood. Apoptosis resistance is a key feature for tumor cells to escape physiological surveillance and acquire growth advantage over normal cells. Although NiCl2 exposure induces transformation of human lung epithelial cells, little information is available with regard to its molecular mechanisms, it is also not clear if the transformed cells are apoptosis resistant and tumorigenic. We explored the apoptosis resistance properties of nickel chloride­transformed human lung epithelial cells and the underlying mechanisms. The results showed that transformed BEAS-2B human lung epithelial cells are resistant to NiCl2-induced apoptosis. They have increased Bcl-2, Bcl-xL and catalase protein levels over the passage matched non­transformed counterparts. The mechanisms of apoptosis resistance are mitochondria­mediated and caspase-dependent. Forced overexpression of Bcl-2, Bcl-xL and catalase proteins reduced NiCl2-induced cell death; siRNA­mediated knockdown of their expression sensitized the cells to nickel-induced apoptosis, suggesting that Bcl-2, Bcl-xl and catalase protein expression plays a critical role in apoptosis resistance. Akt also participates in this process, as its overexpression increases Bcl-xL protein expression levels and attenuates NiCl2-induced apoptosis. Furthermore, transformed cells are tumorigenic in a xenograft model. Together, these results demonstrate that nickel-transformed cells are apoptosis­resistant and tumorigenic. Increased expression of Bcl-2, Bcl-xL and catalase proteins are important mechanisms contributing to transformed cell oncogenic properties.

Chimeric antigen receptor containing ICOS signaling domain mediates specific and efficient antitumor effect of T cells against EGFRvIII expressing glioma.

BACKGROUND: Adoptive transfer of chimeric antigen receptor (CAR)-modified T cells appears to be a promising immunotherapeutic strategy. CAR combines the specificity of antibody and cytotoxicity of cytotoxic T lymphocytes, enhancing T cells' ability to specifically target antigens and to effectively kill cancer cells. Recent efforts have been made to integrate the costimulatory signals in the CAR to improve the antitumor efficacy. Epidermal growth factor receptor variant III (EGFRvIII) is an attractive therapeutic target as it frequently expresses in glioma and many other types of cancers. Our current study aimed to investigate the specific and efficient antitumor effect of T cells modified with CAR containing inducible costimulator (ICOS) signaling domain. METHODS: A second generation of EGFRvIII/CAR was generated and it contained the EGFRvIII single chain variable fragment, ICOS signaling domain and CD3ζ chain. Lentiviral EGFRvIII/CAR was prepared and human CD3+ T cells were infected by lentivirus encoding EGFRvIII/CAR. The expression of EGFRvIII/CAR on CD3+ T cells was confirmed by flow cytometry and Western blot. The functions of EGFRvIII/CAR+ T cells were evaluated using in vitro and in vivo methods including cytotoxicity assay, cytokine release assay and xenograft tumor mouse model. RESULTS: Chimeric EGFRvIIIscFv-ICOS-CD3ζ (EGFRvIII/CAR) was constructed and lentiviral EGFRvIII/CAR were made to titer of 106 TU/ml. The transduction efficiency of lentiviral EGFRvIII/CAR on T cells reached around 70% and expression of EGFRvIII/CAR protein was verified by immunoblotting as a band of about 57 kDa. Four hour 51Cr release assays demonstrated specific and efficient cytotoxicity of EGFRvIII/CAR+ T cells against EGFRvIII expressing U87 cells. A robust increase in the IFN-γ secretion was detected in the co-culture supernatant of the EGFRvIII/CAR+ T cells and the EGFRvIII expressing U87 cells. Intravenous and intratumor injection of EGFRvIII/CAR+ T cells inhibited the in vivo growth of the EGFRvIII expressing glioma cells. CONCLUSIONS: Our study demonstrates that the EGFRvIII/CAR-modified T cells can destroy glioma cells efficiently in an EGFRvIII specific manner and release IFN-γ in an antigen dependent manner. The specific recognition and effective killing activity of the EGFRvIII-directed T cells with ICOS signaling domain lays a foundation for us to employ such approach in future cancer treatment.

Epithelial-mesenchymal transition during oncogenic transformation induced by hexavalent chromium involves reactive oxygen species-dependent mechanism in lung epithelial cells.

Hexavalent chromium [Cr(VI)] is an important human carcinogen associated with pulmonary diseases and lung cancer. Exposure to Cr(VI) induces DNA damage, cell morphological change and malignant transformation in human lung epithelial cells. Despite extensive studies, the molecular mechanisms remain elusive, it is also not known if Cr(VI)-induced transformation might accompany with invasive properties to facilitate metastasis. We aimed to study Cr(VI)-induced epithelial-mesenchymal transition (EMT) and invasion during oncogenic transformation in lung epithelial cells. The results showed that Cr(VI) at low doses represses E-cadherin mRNA and protein expression, enhances mesenchymal marker vimentin expression and transforms the epithelial cell into fibroblastoid morphology. Cr(VI) also increases cell invasion and promotes colony formation. Further studies indicated that Cr(VI) uses multiple mechanisms to repress E-cadherin expression, including activation of E-cadherin repressors such as Slug, ZEB1, KLF8 and enhancement the binding of HDAC1 in E-cadherin gene promoter, but DNA methylation is not responsible for the loss of E-cadherin. Catalase reduces Cr(VI)-induced E-cadherin and vimentin protein expression, attenuates cell invasion in matrigel and colony formation on soft agar. These results demonstrate that exposure to a common human carcinogen, Cr(VI), induces EMT and invasion during oncogenic transformation in lung epithelial cells and implicate in cancer metastasis and prevention.

[Expression of recombinant human IFNa-2b/IgG4 Fc fusion protein in a baculovirus insect cell system].

To investigate a baculovirus insect cell system for expressing an interferon alpha 2b (IFNa2b)/immunoglobulin G-4 (IgG4) Fc fusion protein, which has long-acting antiviral effects. Human IFNa2b and IgG4 Fc cDNAs were generated by molecular cloning and inserted into a baculovirus shuttle vector, which was then transposed into the DH10 Bac strain to form recombinant Bacmid-IFN/Fc. The Bacmid-IFN/Fc was transfected into High five insect cells, and expression of the IFN/Fc fusion protein was detected by Western blotting and its biological activity was assessed by the cytopathic effect inhibition method. The IFNa2b and IgG4 Fc cDNA fragments were successfully amplified by RT-PCR using human peripheral lymphocytes. After cloning into the baculovirus shuttle vector, pFastBac1, and transforming into DH10 Bac competent cells, screening identified positive clones carrying the recombinant Bacmid-IFN/Fc. A Bacmid-IFN/Fc clone was successfully transfected into the High five insect cells and packaged into the baculovirus for expression of the IFN/Fc fusion protein. Western blotting revealed that the fusion protein expression was specific, and yielded a protein of 45 kD in size. The in vitro antiviral activity of the IFN/Fc fusion protein was 580 IU/mL. A novel IFN/Fc fusion protein was successfully generated using a baculovirus insect cell system, which may prove useful for providing future experimental data for development of a new long-acting interferon to treat chronic viral hepatitis.

Derivation and validation of a prediction rule for estimating advanced colorectal neoplasm risk in average-risk Chinese.

No prediction rule is currently available for advanced colorectal neoplasms, defined as invasive cancer, an adenoma of 10 mm or more, a villous adenoma, or an adenoma with high-grade dysplasia, in average-risk Chinese. In this study between 2006 and 2008, a total of 7,541 average-risk Chinese persons aged 40 years or older who had complete colonoscopy were included. The derivation and validation cohorts consisted of 5,229 and 2,312 persons, respectively. A prediction rule was developed from a logistic regression model and then internally and externally validated. The prediction rule comprised 8 variables (age, sex, smoking, diabetes mellitus, green vegetables, pickled food, fried food, and white meat), with scores ranging from 0 to 14. Among the participants with low-risk (≤3) or high-risk (>3) scores in the validation cohort, the risks of advanced neoplasms were 2.6% and 10.0% (P < 0.001), respectively. If colonoscopy was used only for persons with high risk, 80.3% of persons with advanced neoplasms would be detected while the number of colonoscopies would be reduced by 49.2%. The prediction rule had good discrimination (area under the receiver operating characteristic curve = 0.74, 95% confidence interval: 0.70, 0.78) and calibration (P = 0.77) and, thus, provides accurate risk stratification for advanced neoplasms in average-risk Chinese.

Longitudinal increases in PCDD/F and dl-PCB concentrations in human milk in northern China.

There is a dearth of information on the temporal changes in polychlorinated dibenzodioxin/furans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (dl-PCBs) contamination, in both environmental and biological specimens, in China. We compared the concentrations of PCDD/Fs and dl-PCBs in human milk collected in Shijiazhuang, Hebei Province, in northern China in 2002 (n=30) and 2007 (n=20). The level of PCDD/Fs and dl-PCBs showed an increasing trend. The mean concentrations of PCDD/Fs plus dl-PCBs were 4.47 TEQ pg g(-1) fat and 6.24 TEQ pg g(-1) fat in human milk from Shijiazhuang in 2002 and in 2007, respectively. Based on statistical analysis of questionnaire data collected by in-person interviews with mothers, we found positive correlations between consumption of sea fish and PCDFs. The PCDDs, PCDFs, PCDD/Fs, and PCDD/Fs plus dl-PCBs levels in individuals consuming greater amounts of sea fish were higher than those consuming less sea fish, both with and without adjustments for potential confounding factors. Among 17 congeners of PCDD/Fs, the 2,3,7,8-TCDF, 1,2,3,7,8-PeCDF, 2,3,4,7,8-PeCDF, 1,2,3,4,7,8-HxCDF, 1,2,3,6,7,8-HxCDF, and 2,3,4,6,7,8-HxCDF congener concentrations in 2007 increased 134%, 55%, 53%, 57%, 65% and 130% when compared to 2002 levels, respectively. The 2007 dl-PCB congener levels were greater than those of the 2002 samples, with the exception of PCB81 and PCB77. Specifically, PCB105, PCB114, PCB118, PCB123 and PCB156 had increased greater than twofold from 2002 to 2007. Continuous surveillance of PCDD/F and dl-PCB levels in human milk is needed to accurately evaluate both environmental contamination and the human health risk to neonates in China.

[Relationship between single nucleotide polymorphisms in the promoter of COX-2 gene and hereditariness to NAFLD].

OBJECTIVE: To study the relationship between COX-2 gene and hereditariness to Nonalcoholic fatty liver disease by detecting single nucleotide polymorphisms in the promoter of COX-2 gene. METHODS: Genotypes of 200 case patients with NAFLD and 206 control subjects were examined by polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP). The DNA samples were extracted from the peripheral blood of all subjects. RESULTS: Two SNPs, -1195G more than A and -765 G more than C, were identified with frequencies of variant alleles 54% and 5% in patients with NAFLD and 48% and 2% in control, respectively. A case-control analysis revealed a 1.13-fold (95% CI = 1.01-2.46) and a 2.35-fold (95% CI = 1.17-3.65) excess risk of developing NAFLD for -1195AA or -765CG genotype carriers compared with noncarriers. Compared with G-1195-G-765 containing haplotype, a greater risk of developing NAFLD was observed for A-1195-G-765 (OR =1.42; 95% CI =1.11-1.63) and A-1195-C-765 (OR = 4.24; 95% CI =1.72-14.22) containing haplotypes. A greater risk of developing NAFLD was observed for A-1195 and C-765 containing haplotype compared with other haplotype, suggesting an interaction between the -1195A and -765C in the context of haplotype. CONCLUSIONS: These findings suggest that genetic variants in the COX-2 promoter may play an important role in mediating susceptibility to developing NAFLD in a Chinese population. -1195G more than A and -765G more than C in promoter region of Cyclooxygenase-2 gene, whose single nucleotide polymorphisms are related with development of NAFLD, are the significance factors of the susceptibility of NAFLD.