Epstein-Barr病毒胸苷激酶(TK)基因表达质粒的构建

目的 :构建高效表达 EBVTK的重组克隆体系。方法 :以 p UC8X为模板 ,5’- CTGAATTCATG-GCTGGATTTCC- 3’及 5’CAACTGCAGCCTAGTCCCGATT- 3’为引物 ,用 PCR技术扩增出 EBV tk基因的 DNA片段。Eco R I/ Pst I双酶切 PCR产物和载体 p BV2 2 0 ,T4连接酶使目的基因 tk定向克隆至选定质粒 p BV2 2 0中。结果 :重组质粒 p BV2 2 0 - tk经 Eco R I/ Pst I双酶切后得两条电泳带分别位于 1.8kb、3.6 kb处 ;以 p BV2 2 0 - tk为模板 ,两引物同上进行 PCR扩增 ,产物电泳带于 1.8kb处。结论 :目的基因 tk已定向克隆至质粒 p BV2 2 0中。

Galangin induces apoptosis of hepatocellular carcinoma cells via the mitochondrial pathway

AIM:To investigate the mechanism by which galangin,a polyphenolic compound derived from medicinal herbs,induces apoptosis of hepatocellular carcinoma(HCC) cells.METHODS:The 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay was used to measure cell viability.Apoptosis was evaluated by in situ uptake of propidium iodide and Hoechst 33258 and was then detected by fluorescence microscopy.Protein expressions were detected by Western blotting.To confirm the apoptotic pathway mediated by galangin,cells were transfected by bcl-2 gene to overexpress Bcl-2 or siRNA to down-regulate Bcl-2 expression.RESULTS:Galangin(46.25-370.0 μmol/L) exerted an anti-proliferative effect,induced apoptosis,and decreased mitochondrial membrane potential in a dose and time-dependent manner.Treatment with galangin induced apoptosis by translocating the pro-apoptotic protein Bax to the mitochondria,which released apoptosis-inducing factor and cytochrome c into the cytosol.Overexpression of Bcl-2 attenuated galangin-induced HepG2 cell apoptosis,while decreasing Bcl-2 expression enhanced galangin-induced cell apoptosis.CONCLUSION:Our data suggests that galangin mediates apoptosis through a mitochondrial pathway,and may be a potential chemotherapeutic drug for the treatment of HCC.

Effects of baicalin in CD4 + CD29 + T cell subsets of ulcerative colitis patients

AIM:To evaluate the role of baicalin in ulcerative colitis(UC) with regard to the CD4+CD29+ T helper cell,its surface markers and serum inflammatory cytokines.METHODS:Flow cytometry was used to detect the percentage of CD4+CD29+ cells in patients with UC.Real time polymerase chain reaction was used to detect expression of GATA-3,forkhead box P3,T-box expressed in T cells(T-bet),and retinoic acid-related orphan nuclear hormone receptor C(RORC).Western blotting was used to analyze expression of nuclear factor-κB(NF-κB) p65,phosphorylation of NF-κB(p-NF-κB) p65,STAT4,p-STAT4,STAT6 and p-STAT6.The concentrations of interferon-γ(IFN-γ),interleukin(IL)-4,IL-5,IL-6,IL-10 and TGF-β in serum were determined by ELISA assay.RESULTS:The percentages of CD4+CD29+ T cells were lower in treatment with 40 and 20 μmol/L baicalin than in the treatment of no baicalin.Treatment with 40 or 20 μmol/L baicalin significantly upregulated expression of IL-4,TGF-β1 and IL-10,increased p-STAT6/STAT6 ratio,but downregulated expression of IFN-γ,IL-5,IL-6,RORC,Foxp3 and T-bet,and decreased ratios of T-bet/GATA-3,p-STAT4/STAT4 and p-NF-κB/NF-κB compared to the treatment of no baicalin.CONCLUSION:The results indicate that baicalin regulates immune balance and relieves the ulcerative colitis-induced inflammation reaction by promoting proliferation of CD4+CD29+ cells and modulating immunosuppressive pathways.

Bioinformatics analysis of the structure and linear B-cell epitopes of aquaporin-3 from Schistosoma japonicum

Objective:To analyze the structure of aquaporins-3(AQP-3) from Schistosoma japonicum(SJAQP-3) using bioinformalical methods,and to provid of references for vaccine targets research.Methods:Protparam,BepiPred,TMHMM Server,MLRC,Geno3d,DNA star software packages were used to predict the physical and chemical properties,hydrophilicity plot, flexibility regions,antigenic index,surface probability plot,secondary structure,and tertiary structure of amino acid sequence of SJAQP-3.Results:SJAQP-3 had six transmembrane regions and two half-spanning helices that form a central channel.The half-spanning helices fold into the centre of the channel.Either of the half-spanning helix had a conserved motif of NPA common to all aquaporins.Predicted linear B-Cell epitopes were most likely at the N-terminal amino acid residues of Saa-7aa,59aa- 62aa,225aa-230aa,282aa -288aa,294aa -29Saa and 305aa -307aa area.59aa- 62aa,22Saa-230aa located outside the membrane,the others located inside the cell.Conclusions:SJAQP-3 is a integral membrane protein in Schistosoma japonicum tegument.There are six potential epitopes in SJ AQP-3.It might be a potential molecular target for the development of vaccines.

Dihydromyricetin inhibits migration and invasion of hepatoma cells through regulation of MMP-9 expression

AIM:To investigate the effects of dihydromyricetin(DHM)on the migration and invasion of human hepatic cancer cells.METHODS:The hepatoma cell lines SK-Hep-1 and MHCC97L were used in this study.The cells were cultured in RPIM-1640 medium supplemented with 10%fetal bovine serum at 37℃in a humidified 5%CO2incubator.DHM was dissolved in dimethyl sulfoxide and diluted to various concentrations in medium before applying to cells.MTT assays were performed to measure the viability of the cells after DHM treatment.Wound healing and Boyden transwell assays were used to assess cancer cell motility.The invasive capacity of cancer cells was measured using Matrigel-coated transwell chambers.Matrix metalloproteinase(MMP)-2/9 activity was examined by fluorescence analysis.Western blot was carried out to analyze the expression of MMP-2,MMP-9,p-38,JNK,ERK1/2 and PKC-δproteins.All data were analyzed by Student’s t tests in GraphPad prism 5.0software and are presented as mean±SD.RESULTS:DHM was found to strongly inhibit the migration of the hepatoma cell lines SK-Hep-1(without DHM,24 h:120±8μmol/L vs 100μmol/L DHM,24h:65±10μmol/L,P<0.001)and MHCC97L(without DHM,24 h:126±7μmol/L vs 100μmol/L DHM,24h:74±6μmol/L,P<0.001).The invasive capacity of the cells was reduced by DHM treatment(SK-Hep-1cells without DHM,24 h:67±4μmol/L vs 100μmol/L DHM,24 h:9±3μmol/L,P<0.001;MHCC97L cells without DHM,24 h:117±8μmol/L vs 100μmol/L DHM,24 h:45±2μmol/L,P<0.001).MMP2/9 activity was also inhibited by DHM exposure(SK-Hep-1 cells without DHM,24 h:600±26μmol/L vs 100μmol/L DHM,24 h:100±6μmol/L,P<0.001;MHCC97L cells without DHM,24 h:504±32μmol/L vs 100μmol/L DHM 24 h:156±10μmol/L,P<0.001).Western blot analysis showed that DHM decreased the expression level of MMP-9 but had little effect on MMP-2.Further investigation indicated that DHM markedly reduced the phosphorylation levels of p38,ERK1/2 and JNK in a concentration-dependent manner but had no impact on the total protein levels.In addition,PKC-δprotein,a key protein in the regulation of MMP family protein expression,was up-regulated with DHM treatment.CONCLUSION:These findings demonstrate that DHM inhibits the migration and invasion of hepatoma cells and may serve as a potential candidate agent for the prevention of HCC metastasis.

Dihydromyricetin alleviates carbon tetrachloride-induced acute liver injury via JNK-dependent mechanism in mice

AIM: To assess the effects of dihydromyricetin(DHM) as a hepatoprotective candidate in reducing hepatic injury and accelerating hepatocyte proliferation after carbon tetrachloride(CCl4) treatment.METHODS: C57 BL/6 mice were used in this study. Mice were orally administered with DHM(150 mg/kg) for 4 d after CCl4 treatment. Serum and liver tissue samples were collected on days 1, 2, 3, 5 and 7 after CCl4 treatment. The anti-inflammatory effect of DHM was assessed directly by hepatic histology detection and indirectly by serum levels of aspartate aminotransferase(AST), alanine aminotransferase(ALT), albumin, and superoxide dismutase(SOD). Inflammatory cytokines, such as interleukin(IL)-1β, IL-6 and tumor necrosis factor-α(TNF-α), were detected using ELISA kits. Proliferating cell nuclear antigen(PCNA) staining was used to evaluate the role of DHM in promoting hepatocyte proliferation. Hepatocyte apoptosis wasmeasured by TUNEL assay.Furthermore,apoptosis proteins Caspases-3,6,8,and 9 were detected by Western blot.SP600125 were used to confirm whether DHM regulated liver regeneration through JNK/TNF-αpathways.RESULTS:DHM showed a strong anti-inflammatory effect on CCl4-induced liver injury in mice.DHM could significantly decrease serum ALT,AST,IL-1β,IL-6 and TNF-αand increase serum albumin,SOD and liver SOD compared to the control group after CCl4 treatment(P<0.05).PCNA results indicated that DHM could significantly increase the number of PCNA positive cells compared to the control(348.9±56.0 vs 107.1±31.4,P<0.01).TUNEL assay showed that DHM dramatically reduced the number of apoptotic cells after CCl4 treatment compared to the control(365.4±99.4 vs 90.5±13.8,P<0.01).Caspase activity detection showed that DHM could reduce the activities of Caspases-8,3,6 and 9 compared to the control(P<0.05).The results of Western blot showed that DHM increased the expression of JNK and decreased TNF-αexpression.However,DHM could not affect TNF-αexpression after SP600125 treatment.Furthermore,DHM could significantly improve the survival rate of acute liver failure(ALF)mice(73.3%vs 20.0%,P<0.0001),and SP600125 could inhibit the effect of DHM.CONCLUSION:These findings demonstrate that DHM alleviates CCl4-induced liver injury,suggesting that DHM is a promising candidate for reversing liver injury and ALF.

Comparison of 5-hydroxytryptophan signaling pathway characteristics in diarrhea-predominant irritable bowel syndrome and ulcerative colitis

AIM: To study differences in the visceral sensitivity of the colonic mucosa between patients with diarrheapredominant irritable bowel syndrome(IBS-D) and those with ulcerative colitis(UC) in remission and to relate these differences with changes in the 5-hydroxytryptophan(5-HT) signaling pathway. METHODS: Gastrointestinal symptoms were used to determine the clinical symptom scores and rectal visceral sensitivity of patients with IBS-D and patients with UC in remission. Blood levels of 5-HT and5-hydroxyindoleacetic acid(5-HIAA) were measured using an HPLC-electrochemical detection system. The levels of 5-HT 3 receptor(3R), 4R, and 7R m RNAs in colonic biopsy samples were detected using reverse transcription-polymerase chain reaction. The protein expression of TPH1 was analyzed by Western blot and immunohistochemistry.RESULTS: Abdominal pain or discomfort, stool frequency, and the scores of these symptoms in combination with gastrointestinal symptoms were higher in the IBS-D and UC groups than in the control groups. However, no significant differences were observed between the IBS-D and UC remission groups. With respect to rectal visceral sensitivity, the UC remission and IBS-D groups showed a decrease in the initial perception threshold, defecating threshold and pain threshold. However, these groups exhibited significantly increased anorectal relaxation pressure. Tests examining the main indicators of the 5-HT signaling pathway showed that the plasma 5-HT levels, 5-HIAA concentrations, TPH1 expression in the colonic mucosa, and 5-HT3 R and 5-HT5 R expression were increased in both the IBS-D and the UC remission groups; no increases were observed with respect to 5-HT7 R expression.CONCLUSION: The IBS-D and UC groups showed similar clinical symptom scores, visceral sensitivity, and levels of serotonin signaling pathway indicators in the plasma and colonic mucosa. However, the pain threshold and 5-HT7 R expression in the colonic mucosa were significantly different between these groups. The results reveal that(1) IBS-D and UC are related to visceral sensitivity pathogenesis and the clinical manifestations of these conditions and(2) the observed differences in visceral hypersensitivity are possibly due to differences in levels of the 5-HT7 receptor, a component of the 5-HT signaling pathway.

Hepatoprotective effects of baicalein against CCl 4-induced acute liver injury in mice

AIM:To investigate the hepatoprotective effect of baicalein against carbon tetrachloride(CCl 4)-induced liver damage in mice.METHODS:Mice were orally administered with baicalein after CCl 4 injection,and therapeutic baicalein was given twice a day for 4 d.The anti-inflammation effects of baicalein were assessed directly by hepatic histology and serum alanine aminotranferease and aspartate aminotransferase measurement.Proliferating cell nuclear antigen was used to evaluate the effect of baicalein in promoting hepatocyte proliferation.Serum interleukin(IL)-6,IL-1β and tumor necrosis factor-α(TNF-α) levels were measured by enzyme-linked immunosorbent assay and liver IL-6,TNF-α,transforming growth factor-α(TGF-α),hepatocyte growth factor(HGF) and epidermal growth factor(EGF) genes expression were determined by quantitative real-time polymerase chain reaction.RESULTS:CCl4-induced acute liver failure model offers a survival benefit in baicalein-treated mice.The data indicated that the mRNA levels of IL-6 and TNF-α significantly increased within 12 h after CCl 4 treatment in baicalein administration groups,but at 24,48 and 72 h,the expression of IL-6 and TNF-α was kept at lower levels compared with the control.The expression of TGF-α,HGF and EGF was enhanced dramatically in baicalein administration group at 12,24,48 and 72 h.Furthermore,we found that baicalein significantly elevated the serum level of TNF-α and IL-6 at the early phase,which indicated that baicalein could facilitate the initiating events in liver regeneration.CONCLUSION:Baicalein may be a therapeutic candidate for acute liver injury.Baicalein accelerates liver regeneration by regulating TNF-α and IL-6 mediated pathways.

Heme oxygenase-1 and gut ischemia/reperfusion injury: A short review

Ischemia/reperfusion (I/R) injury of the gut is a significant problem in a variety of clinical settings and is associated with a high morbidity and mortality. Although the mechanisms involved in the pathogenesis of gut I/R injury have not been fully elucidated, it is generally believed that oxidative stress with subsequent inflammatory injury plays an important role. Heme oxygenase (HO) is the rate-limiting enzyme in the catabolism of heme, followed by production of CO, biliverdin, and free iron. The HO system is believed to confer cytoprotection by inhibiting inflammation, oxidation, and apoptosis, and maintaining microcirculation. HO-1, an inducible form of HO, serves a vital metabolic function as the rate-limiting step in the heme degradation pathway, and affords protection in models of intestinal I/R injury. HO-1 system is an important player in intestinal I/R injury condition, and may offer new targets for the management of this condition.

Antioxidative effects of berberine pre-treatment on hydrogen peroxide-induced PC12 cell toxicity

Oxidative stress has been implicated in the pathogenesis of Alzheimer＇s disease. Oxidative damage could be prevented by augmenting the endogenous defense capacity against oxidative stress by antioxidant intake. As an effective alkaloid component of Chinese herbal medicine Rhizoma coptidis extract, berberine exhibits antioxidative properties and ameliorates memory impairment in a rat model of Alzheimer＇s disease. The present study investigated the protective effects of berberine on H2O2-induced PC12 cell toxicity. Results demonstrated that berberine protects PC12 cells from H2O2-induced apoptosis and increases PC12 cell viability. Lactate dehydrogenase release, reactive oxygen content, and malonyl dialdehyde levels were significantly decreased （P 〈 0.01）. The protective effects of berberine on H2O2-induced PC12 cell toxicity were achieved via the antioxidative effects of berberine.

Critical protein GAPDH and its regulatory mechanisms in cancer cells

Glyceraldehyde-3-phosphate dehydrogenase(GAPDH), initially identified as a glycolytic enzyme and considered as a housekeeping gene, is widely used as an internal control in experiments on proteins, m RNA, and DNA. However, emerging evidence indicates that GAPDH is implicated in diverse functions independent of its role in energy metabolism; the expression status of GAPDH is also deregulated in various cancer cells. One of the most common effects of GAPDH is its inconsistent role in the determination of cancer cell fate. Furthermore, studies have described GAPDH as a regulator of cell death; other studies have suggested that GAPDH participates in tumor progression and serves as a new therapeutic target. However, related regulatory mechanisms of its numerous cellular functions and deregulated expression levels remain unclear. GAPDH is tightly regulated at transcriptional and posttranscriptional levels, which are involved in the regulation of diverse GAPDH functions. Several cancer-related factors, such as insulin, hypoxia inducible factor-1(HIF-1), p53, nitric oxide(NO), and acetylated histone, not only modulate GAPDH gene expression but also affect protein functions via common pathways. Moreover, posttranslational modifications(PTMs) occurring in GAPDH in cancer cells result in new activities unrelated to the original glycolytic function of GAPDH. In this review, recent findings related to GAPDH transcriptional regulation and PTMs are summarized. Mechanisms and pathways involved in GAPDH regulation and its different roles in cancer cells are also described.

Inhibition of inflammatory mediator release from microglia can treat ischemic/hypoxic brain injury

Interleukin-1α and interleukin-1β aggravate neuronal injury by mediating the inf1αmmatory reaction following ischemic/hypoxic brain injury. It remains unclear whether interleukin-1α and interleukin-1β are released by microglia or astrocytes. This study prepared hippocampal slices that were subsequently subjected to oxygen and glucose deprivation. Hematoxylin-eosin staining verified that neurons exhibited hypoxic changes. Results of enzyme-linked immunosorbent assay found that interleukin-1α and interleukin-1β participated in this hypoxic process. Moreover, when hypoxic injury occurred in the hippocampus, the release of interleukin-1α and interleukin-1β was mediated by the P2X4 receptor and P2X7 receptor. Immunofluorescence staining revealed that during ischemia/hypoxia, the P2X4 receptor, P2X7 receptor, interleukin-1α and interleukin-1β expression was detectable in rat hippocampal microglia, but only P2X4 receptor and P2X7 receptor expression was detected in astrocytes. Results suggested that the P2X4 receptor and P2X7 receptor, respectively, mediated interleukin-1α and interleukin-1β released by microglia, resulting in hippocampal ischemic/hypoxic injury. Astrocytes were activated, but did not synthesize or release interleukin-1α and interleukin-1β.

Ent-11α-Hydroxy-15-oxo-kaur-16-en-19-oic-acid Inhibits Growth of Human Lung Cancer A549 Cells by Arresting Cell Cycle and Triggering Apoptosis

Objective： To examine the apoptotic effect of ent-llα-hydroxy-15-oxo-kaur-16-en-19-oic-acid （5F）, a compound isolated from Pteris semipinnata L （PsL）, in human lung cancer A549 cells. Methods： A549 cells were treated with 5F （0-80 lag/ml） for different time periods. Cytotoxicity was examined using a Ml-I- method. Cell cycle was examined using propidium iodide staining. Apoptosis was examined using Hoechst 33258 staining, enzyme-linked immunosorbent assay （ELISA） and caspase-3 activity analysis. Expression of representative apoptosis-related proteins was evaluated by Western blot analysis. Reactive oxygen species （ROS） level was measured using standard protocols. Potential interaction of 5F with cisplatin was also examined. Results： 5F inhibited the proliferation of A549 cells in a concentration- and time-dependent manner. 5F increased the accumulation of cells in sub-G1 phase and arrested the cells in the G2 phase. Exposure to 5F induced morphological changes and DNA fragmentation that are characteristic of apoptosis. The expression of p21 was increased. 5F exposure also increased Bax expression, release of cytochrome c and apoptosis inducing factor （AIF）, and activation of caspase-3. 5F significantly sensitized the cells to cisplatin toxicity. Interestingly, treatment with 5F did not increase ROS, but reduced ROS production induced by cisplatin. Conclusion： 5F could inhibit the proliferation of A549 cells by arresting the cells in G2 phase and by inducing mitochondrial-mediated apoptosis.

Clinical evaluation of serum concentrationsof intercellular adhesion molecule-1 in patients with colorectal cancer

AIM: To investigate the correlation between the serum soluble intercellular adhesion molecule-1 (sICAM-1) and the clinicopathologic features and to evaluate the possible prognostic significance of sICAM-1 concentration in colorectal cancer.METHODS: A total of 56 patients (mean age 57.3 years)having transitional cell carcinoma of the colorectal and 25 control patients (mean age 42.6 years) were enrolled in the study. The serum samples of the patients were obtained on the day before surgery. Sera were obtained by centrifugation, and stored at -80 ℃ until assay. Serumconcentrations of ICAM-1 were measured with enzymelinked immunoassay. Differences between the two groups were analyzed by Student's t-test.RESULTS: No significant increase of serum sICAM-1 could be demonstrated in the Dukes A1 patients (352.63±61.82μg/L) compared to the control group (345.72±49.81 μg/L,P＞0.05), Dukes A1 patients (352.63±61.82 μg/L)compared to Dukes A2,3 patients (491.17±86.36 μg/L,P＜0.05). Furthermore, the patients with Dukes B had significantly higher serum concentrations of sICAM-1than those of the control group (496.82±93.04 μg/L vs 345.72±49.81 μg/L, P＜0.01). Compared with Dukes A2,3,B colorectal cancer patients, patients with more advanced clinical stage (Dukes C and D) had higher levels of sICAM-1 (743.68±113.74 μg/L vs491.17±86.36 μg/L and 496.82±93.04 μg/L, P＜0.001). The difference was statistically significant in sICAM-1 levels between patients with positive lymph node status and those without lymph node involvement (756.25±125.57 μg/L vs445.62±69.18 μg/L, P＜0.001).Patients with poorly differentiated colorectal cancer had a higher level of sICAM-1 than those with differentiated and highly differentiated cancer (736.49±121.97 μg/Lvs 410.23±67.47 μg/L, P＜0.001).CONCLUSION: In this study, serum ICAM-1 levels were found to be related to tumor presence, clinical stages,and grade. Increased ICAM-1 in patients with colorectal cancer which should be considered when the diagnostic and/or prognostic usefulness of soluble ICAM-1 is to be evaluated, sICAM-1 should prove useful for monitoring malignant disease stage and for evaluating the effectiveness of various therapeutic approaches for colorectal carcinomas.

Theoretical insight into the enhanced CH_4 desorption via H_2O adsorption on different rank coal surfaces

The density functional theory was used to investigate the adsorption of CH4and H2O on different rank coal surfaces. The coal rank is the dominant factor in affecting the adsorption capacity of coal. In order to better understand gas and water interaction with coal of different maturity, we developed fourteen coal models to represent the different rank coal. The interactions of CH4and H2O with coal surfaces were studied and characterized by their adsorption energies, Mulliken charges and electrostatic potential surfaces. The results revealed that the interaction between coal and CH4was weak physical adsorption, and that the interaction between coal and H2O consisted of physical and chemical adsorption. Adsorption energy of coal–H2O system was larger than that of coal–CH4on all rank coals, suggesting that the adsorption priority in the coal models is H2O > CH4. Consequently, the injection of H2O into the different rank coal could effectively enhance the coal bed methane (CBM) recovery. © 2016 Science Press

TET1 knockdown inhibits the odontogenic differentiation potential of human dental pulp cells

Human dental pulp cells （hDPCs） possess the capacity to differentiate into odontoblast-like cells and generate reparative dentin in response to exogenous stimuli or injury. Ten-eleven translocation 1 （TET1） is a novel DNA methyldioxygenase that plays an important role in the promotion of DNA demethylation and transcriptional regulation in several cell lines. However, the role of TET1 in the biological functions of hDPCs is unknown. To investigate the effect of TET1 on the proliferation and odontogenic differentiation potential of hDPCs, a recombinant shRNA lentiviral vector was used to knock down TET1 expression in hDPCs. Following TET1 knockdown, TET1 was significantly downregulated at both the mRNA and protein levels. Proliferation of the hDPCs was suppressed in the TET1 knockdown groups. Alkaline phosphatase activity, the formation of mineralized nodules, and the expression levels of DSPP and DMP1 were all reduced in the TETl-knockdown hDPCs undergoing odontogenic differentiation. Based on these results, we concluded that TET1 knockdown can prevent the proliferation and odontogenic differentiation of hDPCs, which suggests that TET1 may play an important role in dental pulp repair and regeneration.

Phycocyanobilin accelerates liver regeneration and reduces mortality rate in carbon tetrachloride-induced liver injury mice

AIM: To investigate the hepatoprotective effects of phycocyanobilin(PCB) in reducing hepatic injury and accelerating hepatocyte proliferation following carbon tetrachloride(CCl4) treatment.METHODS: C57BL/6 mice were orally administered PCB 100 mg/kg for 4 d after CCl4 injection, and then the serum and liver tissue of the mice were collected at days 1, 2, 3, 5 and 7 after CCl4 treatment. A series of evaluations were performed to identify the curative effects on liver injury and recovery. Aspartate aminotransferase(AST), alanine aminotransferase(ALT), albumin and superoxide dismutase(SOD) were detected to indirectly assess the anti-inflammatory effects of PCB. Meanwhile, we detected the expressions of hepatocyte growth factor, transforming growth factor alpha(TGF-α), TGF-β, tumor necrosis factor-alpha(TNF-α) and interleukin-6(IL-6), the factors which are associated with inflammation and liver regeneration. The protein expressions of proliferating cell nuclear antigen(PCNA), TNF-α and cytochrome C were detected by western blot. Furthermore, the survivalrates were analyzed of mice which were administered a lethal dose of CCl4(2.6 mg/kg)with or without PCB.RESULTS:In our research,PCB showed a strongly anti-inflammatory effect on CCl4-induced liver injury in mice.The ALT was significantly decreased after CCl4 treatment from day 1(P<0.01)and the AST was significantly decreased from day 2(P<0.001).Both albumin and liver SOD were increased from day2(P<0.001 and P<0.01),but serum SOD levels did not show a significant increase(P>0.05).PCB protected the structure of liver from the injury by CCl4.TUNEL assay showed that PCB dramatically reduced the number of apoptotic cells after CCl4 treatment compared to the control(101.0±25.4 vs 25.7±6.4,P<0.01).The result of western blotting showed that PCB could increase PCNA expression,decrease TNF-αand cytochrome C expression.Furthermore,data shows that PCB could improve the survival rate of acute liver failure(ALF)mice which were injected with a lethal dose of CCl4(60.0%vs 20.0%).CONCLUSION:Our study indicated that PCB could be an ideal candidate for reversing acute liver injury or ALF.

LC-MS/MS determination and pharmacokinetic study of bergenin, the main bioactive component of Bergenia purpurascens after oral administration in rats

Bergenin, a C-glucoside of 4-O-methyl gallic acid from Bergenia purpurascens, is a naturally antitussive and expectorant agent. A rapid and sensitive liquid chromatography tandem mass spectrometry （LC-MS/MS） method has been developed and validated for the determination of the active component--bergenin, in rat plasma after oral administration of aqueous B. purpurascens extract. The plasma samples were pretreated by protein precipitation with acetonitrile and chromatographic separation was achieved on a Diamonsil C18 column （150mim×4.6mm, 5μm） with isocratic elution using a mobile phase consisting of water- methanol （30：70, v/v） at a flow rate of 0.6 mL/min. The detection was accomplished by a triple- quadrupole tandem mass spectrometer in multiple-reaction monitoring （MRM） scanning via an electrospray ionization （ESI） source operating in the negative mode. The optimized mass transition ion-pairs （m/z） for quantitation were 327.3/192.0 for bergenin, and 431.1/311.1 for IS. The time for each analysis run was only 3.5 min between injections. The calibration curve exhibited good linearity （r2〉 0.99） over a range of 1.00-2000 ng/mL for bergenin. The lower limit of quantitation （LLOQ） was 1.00ng/mL. The intra- and inter-day precisions were no more than 11.8%, and relative errors （RE） were within the range of 0.0-4.4%. The validated method was successfully applied to investigate the pharmacokinetics of bergenin after oral administration of B. purpurascens extract in rats.

Using cholecystokinin to facilitate endoscopic clearance of large common bile duct stones

AIM:To evaluate the effect of cholecystokinin(CCK)during extracorporeal shockwave lithotripsy(ESWL)in the clearance of common bile duct(CBD)stones in endoscopic retrograde cholangiopancreatography(ERCP).METHODS:Between January 2007 and September2012,patients with large CBD stones who were treated with ESWL and ERCP were identified retrospectively.Patients were randomized in equal numbers to cholecystokinin(CCK)and no CCK groups.For each CCK case,a dose(3 ng/kg per min for 10 min)of sulfated octapeptide of CCK-8 was administered intravenously near the beginning of ESWL.ERCP was performed 4 h after a session of ESWL.The clearance rate of the CBD was assessed between the two groups.RESULTS:A total of 148 consecutive cases(CCK group:74,no CCK group:74)were tallied.Overall there were 234 ESWLs and 228 ERCPs in the 148 cases.The use of CCK showed a significantly higher rate of successful stone removal in the first ESWL/ERCP procedure(71.6%vs 55.4%,P=0.035),but resulted in similar outcomes in the second(42.8%vs 39.4%)and third(41.7%vs 40.0%)sessions,as well as total stone clearance(90.5%vs 83.8%).The use of mechanical lithotripsy was reduced in the CCK group(6.8%vs17.6%,P=0.023),and extremely large stone(≥30mm)removal was higher in the CCK group(72.7%vs41.7%,P=0.038).CONCLUSION:CCK during ESWL can aid with the clearance of CBD stones in the first ESWL/ERCP session.Mechanical lithotripsy usage was reduced and the extremely large stone(≥30 mm)clearance rate can be raised.

Effect of minocycline on cerebral ischemia-reperfusion injury

Minocylcine, a tetracycline derivate, has been shown to cross the blood-brain barrier and enter the central nervous system. In this study, cerebral ischemia-reperfusion injury models were established using the suture method, and minocycline was immediately injected intraperitoneally after cerebral ischemia-repeffusion （22.5 mg/kg, initially 45 mg/kg） at a 12-hour interval. Results showed that after minocycline treatment, the volume of cerebral infarction was significantly reduced, the number of surviving cell in the hippocampal CA1 region increased, the number of apoptotic cells decreased, the expression of caspase-3 and poly（adenosine diphosphate-ribose） polymerase-1 protein was down-regulated, and the escape latency in the water maze test was significantly shortened compared with the ischemia-reperfusion group. Our experimental findings indicate that minocycline can protect against neuronal injury induced by focal ischemia-reperfusion, which may be mediated by the inhibition of caspase-3 and poly（adenosine diphosphate-ribose） polymerase-1 protein expression.