丙泊酚对脂多糖诱导的MHCC97H细胞黏附、迁移、侵袭和炎症因子的影响及与核转录因子-κB信号通路的关系

目的探究丙泊酚与脂多糖(LPS)刺激下MHCC97H细胞功能、炎症水平及核转录因子-κB(NF-κB)表达的关联。方法该研究起止时间为2021年12月至2022年12月。体外培养人MHCC97H细胞系,分为对照组(等量的溶剂),LPS组(1 mg/L LPS),实验组(LPS+6.25组、LPS+12.5组、LPS+25组,LPS组基础上分别加入6.25、12.5和25μmol/L丙泊酚),用酶联免疫吸附试验、细胞计数试剂盒检测炎症因子白细胞介素6(IL-6)的表达水平和细胞活力筛选出丙泊酚的最适浓度进行后续实验,后将细胞分为对照组、LPS组、LPS+25组和LPS+25+抑制剂组(LPS+25组基础上联合10μmol/L BAY 11-7082),干预24 h。细胞黏附实验测定黏附细胞数;Transwell小室法检测细胞迁移与侵袭水平;蛋白质印迹法测定上皮间质转化(EMT)及NF-κB通路相关蛋白表达水平。结果根据细胞活力和炎症因子IL-6表达选择LPS+25组进行后续实验,与对照组相比,LPS组细胞黏附数、迁移数、侵袭数、N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)、纤连蛋白(FN)、IκB激酶α(IKKα)和p-NF-κB p65蛋白水平分别为(152.00±9.01)个、(84.01±10.44)个、(65.67±3.06)个、0.46±0.02、0.47±0.03、0.99±0.02、1.03±0.02、0.95±0.05上调,E-钙黏蛋白(E-cadherin)表达0.42±0.02下调(P<0.05);与LPS组相比,LPS+25组显著扭转了上述指标的变化(P<0.05);与LPS+25组相比,LPS+25+抑制剂组加入NF-κB通路抑制剂后,上述指标变化扭转得更为显著(P<0.05)。结论丙泊酚对LPS刺激下MHCC97H细胞炎症因子表达、黏附、迁移、侵袭能力及EMT进程具有抑制作用,可能与NF-κB通路活性受到抑制有关。

Hsa-miR-214-3p inhibits breast cancer cell growth and improves the tumor immune microenvironment by downregulating B7H3

Background:Immune checkpoint inhibitors play an important role in the treatment of solid tumors,but the currently used immune checkpoint inhibitors targeting programmed cell death-1(PD-1),programmed cell death ligand-1(PD-L1),and cytotoxic T-lymphocyte antigen-4(CTLA-4)show limited clinical efficacy in many breast cancers.B7H3 has been widely reported as an immunosuppressive molecule,but its immunological function in breast cancer patients remains unclear.Methods:We analyzed the expression of B7H3 in breast cancer samples using data from the Cancer Genome Atlas Program(TCGA)and the Gene Expression Omnibus(GEO)databases.MicroRNAs were selected using the TarBase,miRTarBase,and miRBase databases.The regulatory role of the microRNA hsa-miR-214-3p on B7H3 was investigated through dual-luciferase reporter assays,which identified the specific action sites of interaction.The expression levels of B7H3 and hsa-miR-214-3p in human breast cancer tissues and adjacent normal tissues were quantified using Western blotting and quantitative PCR(qPCR).In vitro experiments were performed to observe the effects of modulating the expression of B7H3 or hsa-miR-214-3p on breast cancer cell proliferation and apoptosis.Additionally,the regulatory impact of hsa-miR-214-3p on B7H3 was examined.Enzyme-linked immunosorbent assays(ELISA)and flow cytometry were employed to assess the effects of co-cultured breast cancer cells and normal human peripheral blood mononuclear cells(PBMCs)on immune cells and associated cytokines.Results:In breast cancer tissues,the expression level of B7H3 is inversely correlated with that of hsa-miR-214-3p,as well as with the regulatory effects on breast cancercell behavior.Hsa-miR-214-3p was found to inhibit breast cancer cell growth by downregulating B7H3.Importantly,our research identified,for the first time,two binding sites for hsa-miR-214-3p on the 3’UTR of B7H3,both of which exert similar effects independently.Co-culture experiments revealed that hsamiR-214-3p obstructs the suppressive function of B7H3 on CD8^(+)T cells and natural killer cells.Conclusions:This study confirms the existence of two hsa-miR-214-3p binding sites on the 3’UTR of B7H3,reinforcing the role of hsamiR-214-3p as a regulatory factor for B7H3.In breast cancer,hsa-miR-214-3p reduces tumor cell proliferation and enhances the tumor immune microenvironment by downregulating B7H3.These findings suggest new potential targets for the clinical treatment of breast cancer.

Prevalence of cardiometabolic co-morbidities in patients with vs persons without chronic hepatitis B: The FitLiver cohort study

BACKGROUND Chronic hepatitis B(CHB)affects>300 million people worldwide.The combi-nation of CHB and cardiometabolic co-morbidities increases the risk of liver-related morbidity and mortality.However,international guidelines for CHB treatment do not provide recommendations for follow-up examinations or treatment of patients with CHB and cardiometabolic comorbidities.In studies investigating cardiometabolic co-morbidity in patients with CHB,inconsistent findings have been observed,and both lower and higher prevalence of car-diometabolic co-morbidities compared to the general population have been re-ported.It is unclear whether patients with CHB living in Denmark have an increased prevalence of cardiometabolic co-morbidities.We examined patients with CHB and age-,sex-,body mass index(BMI)-,and country-of-birth matched comparison group.Defining cardiometabolic co-morbidity:Obesity(BMI>25 kg/m2/abnormal waist-to-hip ratio),metabolic dysfunction-associated steatotic liver disease(MASLD),hypercholesterolemia(total-cholesterol>5 mmol/L/statin use),hypertension(systolic≥135 mmHg/diastolic≥85 mmHg/antihypertensive medication)and type 2 diabetes(T2D)(2-hour oral glucose tolerance test glucose>11.1 mmol/L/HbA1c>48 mmol/mol/antidiabetic medication).Physical activity was evaluated using maximal oxygen consumption(VO2max),activity monitors,and a questionnaire.RESULTS We included 98 patients with CHB and 49 persons in the comparison group.The two groups were well-matched,showing no significant differences in age,sex,BMI,country-of-birth,education,or employment.Among patients with CHB,the following prevalence of cardiometabolic co-morbidity was found:77%were obese,45%had MASLD,38%had hypercholesterolemia,26%had hypertension,and 7%had T2D,which did not differ significantly from the comparison group,apart from lower prevalence of hemoglobin A1c(HbA1c)≥48 mmol/L or known T2D.Both groups had low VO2max of 27 mL/kg/minute in the patients with CHB and 30 mL/kg/minute in the comparison group,and the patients with CHB had a shorter self-assessed sitting time.CONCLUSION The patients with CHB and the comparison group were well-matched and had a similar prevalence of car-diometabolic comorbidities.Furthermore,both groups had low levels of physical fitness.

Mesalazine alleviated the symptoms of spontaneous colitis in interleukin-10 knockout mice by regulating the STAT3/NF-κB signaling pathway

BACKGROUND Excessive endoplasmic reticulum(ER)stress in intestinal epithelial cells can lead to damage to the intestinal mucosal barrier,activate the signal transducer and activator of transcription 3(STAT3)/nuclear factor kappa B(NF-κB)signaling pathway,and exacerbate the inflammatory response,thus participating in the pathogenesis of ulcerative colitis(UC).Mesalazine is a commonly used drug in the clinical treatment of UC.However,further studies are needed to determine whether mesalazine regulates the ER stress of intestinal epithelial cells,downregulates the STAT3/NF-κB pathway to play a role in the treatment of UC.AIM To study the therapeutic effects of mesalazine on spontaneous colitis in interleukin-10(IL-10)-/-mice.METHODS The 24-week-old IL-10-/-mice with spontaneous colitis were divided into the model group and the 5-amino salicylic acid group.Littermates of wild-type mice of the same age group served as the control.There were eight mice in each group,four males and four females.The severity of symptoms of spontaneous colitis in IL-10-/-mice was assessed using disease activity index scores.On day 15,the mice were sacrificed.The colon length was measured,and the histopathological changes and ultrastructure of colonic epithelial cells were detected.The protein expressions of STAT3,p-STAT3,NF-κB,IκB,p-IκB,and glucoseregulated protein 78 were identified using Western blotting.The STAT3 and NF-κB mRNA expressions were identified using real-time polymerase chain reaction.The glucose-regulated protein 78 and C/EBP homologous protein expressions in colon sections were detected using immunofluorescence.RESULTS Mesalazine reduced the symptoms of spontaneous colitis in IL-10 knockout mice and the histopathological damage of colonic tissues,and alleviated the ER stress in epithelial cells of colitis mice.Western blotting and quantitative real-time polymerase chain reaction results showed that the STAT3/NF-κB pathway in the colon tissue of model mice was activated,suggesting that this pathway was involved in the pathogenesis of UC and might become a potential therapeutic target.Mesalazine could down-regulate the protein expressions of p-STAT3,NF-κB and p-IκB,and down-regulate the mRNA expression of STAT3 and NF-κB.CONCLUSION Mesalazine may play a protective role in UC by reducing ER stress by regulating the STAT3/NF-κB signaling pathway.

Bioactive chemical constituents from the marine-derived fungus Cladosporium sp.DLT-5

A new isochromanone,cladosporinisochromanone(1),accompanied by 15 known compounds(2–16)were obtained from secondary metabolites produced by marine-derived fungus Cladosporium sp.DLT-5.NMR and HRESIMS spectra elucidation determined the planar structure of 1.Subsequent electronic circular dichroism(ECD)experiment assigned the absolute configuration of 1.Compounds 1,2,4–6,and 10 displayed different degrees of neuroprotective activities on human neuroblastoma cells SH-SY5Y.Five compounds(1,3–5,and 13)emerged resistance to protein tyrosine phosphatase 1B(PTP1B),further kinetic analysis and molecular docking study indicated that the most potent compound 13(IC50value of 10.74±0.61μmol/L)was found as a noncompetitive inhibitor for PTP1B.Surface plasmon resonance(SPR)and molecular docking studies also demonstrated the interaction between compound 12 and Niemann-Pick C1 Like 1(NPC1L1),which has been identified as significant therapeutic target for hypercholesteremia.In addition,compounds 3,6,and 14 showed attractive inhibitory activity against the phytopathogenic fungi:Colletotrichum capsici.Therefore,library of Cladosporium metabolites is enriched and new active uses of known compounds are explored.

Hepatitis B virus reactivation associated with antirheumatic therapy: risk and prophylaxis recommendations

Accompanying the increased use of biological and non-biological antirheumatic drugs,a greater number of cases of hepatitis B virus(HBV) reactivation have been reported in inactive hepatitis B surface antigen(HBs Ag) carriers and also in HBs Ag-negative patients who have resolved HBV infection. The prevalence of resolved infection varies in rheumatic disease patients,ranging from 7.3% to 66%. Through an electronic search of the Pub Med database,we found that among 712 patients with resolved infection in 17 observational cohort studies,12 experienced HBV reactivation(1.7%) during biological antirheumatic therapy. Reactivation rates were 2.4% for etanercept therapy,0.6% for adalimumab,0% for infliximab,8.6% for tocilizumab,and 3.3% for rituximab. Regarding non-biological antirheumatic drugs,HBV reactivation was observed in 10 out of 327 patients with resolved infection from five cohort studies(3.2%). Most of these patients received steroids concomitantly. Outcomes were favorable in rheumatic disease patients. A number of recommendations have been established,but most of the supporting evidence was derived from the oncology and transplantation fields. Compared with patients in these fields,rheumatic disease patients continue treatment with multiple immunosuppressants for longer periods. Optimal frequency and duration of HBV-DNA monitoring and reliable markers for discontinuation of nucleoside analogues should be clarified for rheumatic disease patients with resolved HBV infection.

Hepatitis B virus reactivation in patients with hepatocellular carcinoma undergoing anti-cancer therapy

Patients with hepatocellular carcinoma (HCC) often experience hepatic morbidity. Hepatitis B virus (HBV) reactivation is well documented as a serious hepatic morbidity during anti-cancer therapy. Reported rates of HBV reactivation in chronic carriers with HCC undergoing chemotherapy range from 4%-67%. Apart from chemotherapy, HBV reactivation has been increasingly identified in settings of hepatectomy and local ablation therapies. The rates of HBV reactivation vary with different levels of immunosuppression and depend on treatment, viral factors, and patient characteristics. The principal concern relating to reactivation is that a substantial proportion of patients with reactivation suffer from liver dysfunction during therapy, which often leads to disruption of planned, potentially life-prolonging treatments, adversely affecting the patients&#x02019; final outcome. The first step in the management of HBV reactivation is identification of patients at risk of reactivation by testing for HBV serology prior to commencing anti-cancer therapy. Although it is a serious complication, HBV reactivation is preventable with prophylactic anti-HBV drugs. Multiple publications have shown the benefit of prophylactic or preemptive antiviral therapy in this setting and justified such an approach before the start of therapy. Given the tumors and underlying cirrhosis, long-term use of antivirals with high potency and low risk of resistance is recommended in patients with HCC. This topic review will summarize the epidemiology, pathogenesis, and clinical issues related to HBV reactivation in HCC patients, and will discuss proper management against HBV reactivation during anti-cancer therapy for HCC.

Modulation of gastric mucosal inflammatory responses to <i>Helicobacter pylori</i>by ghrelin: Role of cNOS-dependent IKK-<i>β</i>S-nitrosylation in the regulation of COX-2 activation

Disturbances in nitric oxide synthase (NOS) and cyclooxygenase (COX) isozyme systems, manifested by the excessive NO and prostaglandin (PGE2) generation, are well-recognized features of gastric mucosal inflammatory responses to H. pylori infection. In this study, we report that H. pylori LPS-induced enhancement in gastric mucosal inducible (i) iNOS expression and COX-2 activation was accompanied by the impairment in constitutive (c) cNOS phosphorylation, up-regulation in the inhibitory κB kinase-β (IKKβ) activation and the increase in the transcriptional factor, NF-κB, nuclear translocation. Further, we show that abrogation of cNOS control over NF-κB activation has lead to induction of iNOS expression and COX-2 activation through S-nitrosylation. Moreover, we demonstrate that the modulatory effect of peptide hormone, ghrelin, on the LPS-induced changes was reflected in the increase in Src/Akt-dependent cNOS activation through phosphorylation and the suppression of IKK-β activity through cNOS-mediated IKK-β protein S-nitrosylation. As a result, ghrelin exerted the inhibitory effect on NF-κB nuclear translocation, thus causing the repression of iNOS gene induction and the inhibition in COX-2 activation through iNOS-dependent S-nitrosylation. Our findings point to cNOS activation as a pivotal element in the signaling cascade by which ghrelin exerts modulatory control over proinflammatory events triggered in gastric mucosa by H. pylori infection.

Solar energy‐driven C−H activation of methanol for direct C−C coupling to ethylene glycol with high stability by nitrogen doped tantalum oxide

Direct photocatalytic coupling of methanol to ethylene glycol(EG)is highly attractive.The reported photocatalysts for this reaction are all metal sulfide semiconductors,which may suffer from photocorrosion and have low stability.Thus,the development of non‐sulfide photocatalysts for efficient photocatalytic coupling of methanol to EG and H2 with high stability is urgent but extremely challenging.Herein,the first metal oxide photocatalyst,tantalum‐based semiconductor,is reported for preferential activation of C−H bond within methanol to form hydroxymethyl radical(•CH_(2)OH)and subsequent C−C coupling to EG.Compared with other metal oxide photocatalysts,such as TiO2,ZnO,WO_(3),Nb_(2)O_(5),tantalum oxide(Ta_(2)O_(5))is unique in that it can realize the selective photocatalytic coupling of methanol to EG.The co‐catalyst free nitrogen doped tantalum oxide(2%N‐Ta_(2)O_(5))shows an EG formation rate as high as 4.0 mmol gcat−1 h−1,about 9 times higher than that of Ta_(2)O_(5),with a selectivity higher than 70%.The high charge separation ability of nitrogen doped tantalum oxide plays a key role in its high activity for EG production.This catalyst also shows excellent stability longer than 160 h,which has not been achieved over the reported metal sulfide photocatalysts.Tantalum‐based photocatalyst is an environmentally friendly and highly stable candidate for photocatalytic coupling of methanol to EG.

Theoretical perspective on mononuclear copper‐oxygen mediated C–H and O–H activations:A comparison between biological and synthetic systems

Dioxygen activations constitute one of core issues in copper-dependent metalloenzymes. Upon O_(2) activation, copper-dependent metalloenzymes such as particulate methane monooxygenases(pM MOs), lytic polysaccharide monooxygenases(LPMOs) and binuclear copper enzymes PHM and DβM, are able to perform various challenging C–H bond activations. Meanwhile, various copper-oxygen core containing complexes have been synthetized to mimic the active species of metalloenzymes. Dioxygen activation by mononuclear copper active site may generate various copper-oxygen intermediates, including Cu(Ⅱ)-superoxo, Cu(Ⅱ)-hydroperoxo, Cu(Ⅱ)-oxyl as well as the Cu(Ⅲ)-hydroxide species. Intriguingly, all these species have been invoked as the potential active intermediates for C–H/O–H activations in either biological or synthetic systems. Due to the poor understanding on reactivities of copper-oxygen complex, the nature of active species in both biological and synthetic systems are highly controversial. In this account, we will compare the reactivities of various mononuclear copper-oxygen species between biological systems and the synthetic systems. The present study is expected to provide the consistent understanding on reactivities of various copper-oxygen active species in both biological and synthetic systems.

Disrupted NF-κB activation after partial hepatectomy does not impair hepatocyte proliferation in rats

AIM： To analyze the effects of NF-kB inhibition by antioxidant pyrrolidine dithiocarbamate （PDTC） or TNF inhibitor pentoxifylline （PTX） on liver regeneration after partial hepatectomy （PH）. METHODS： Saline, PDTC or PTX were injected 1 h before PH and rats were killed at 0.5 and 24 h after PH. Several control groups were used for comparison （injection control groups）. RESULTS： Compared to saline injected controls, NF-kB activation was absent 0.5 h after PH in rats treated with PDTC or PTX. At 24 h after PH, DNA synthesis and PCNA expression were identical in treated and control rats and thus occurred irrespectively of the status of NF-kB activation at 0.5 h. Signal transducer and activator of transcription 3 （Stat3） acUvatJon was observed already 0.5 h after PH in saline, PDTC or PTX group and was similar to Stat3 activation in response to injection without PH. CONCLUSION： These data strongly suggest that （1） NF-kB p65/p50 DNA binding produced in response to PH is not a signal necessary to initiate the liver regeneration, （2） star3 activation is a stress response unrelated to the activation of NF-kB. In conclusion, NF-kB activation is not critically required for the process of liver regeneration after PH.

Activation of AMPK/MnSOD signaling mediates anti-apoptotic effect of hepatitis B virus in hepatoma cells

AIM: To investigate the anti-apoptotic capability of the hepatitis B virus(HBV) in the HepG2 hepatoma cell line and the underlying mechanisms.METHODS: Cell viability and apoptosis were measured by MTT assay and flow cytometry, respectively. Targeted knockdown of manganese superoxide dismutase(Mn SOD), AMP-activated protein kinase(AMPK) and hepatitis B virus X protein(HBx) genes as well as AMPK agonist AICAR and antagonist compound C were employed to determine the correlations of expression of these genes.RESULTS: HBV markedly protected the hepatoma cells from growth suppression and cell death in the condition of serum deprivation. A decrease of superoxide anion production accompanied with an increase of Mn SOD expression and activity was found in Hep G2.215 cells. Moreover, AMPK activation contributed to the up-regulation of Mn SOD. HBx protein was identified to induce the expression of AMPK and Mn SOD. CONCLUSION: Our results suggest that HBV suppresses mitochondrial superoxide level and exerts an antiapoptotic effect by activating AMPK/Mn SOD signaling pathway, which may provide a novel pharmacological strategy to prevent HCC.

Hippocampal activation of c-Jun N-terminal kinase,protein kinase B,and p38 mitogen-activated protein kinase in a chronic stress rat model of depression

Recent studies have shown that varied stress stimuli activate c-Jun N-terminal kinase （JNK）, protein kinase B （Akt）, and p38 mitogen-activated protein kinase （p38） signal transduction pathway, and also regulate various apoptotic cascades. JNK and p38 promote apoptosis, but Akt protects against apoptosis, in hippocampal neurons. However, changes in the transduction pathway in different regions of brain tissues in a chronic stress rat model of depression remain poorly understood. Results from this study showed that JNK phosphorylation levels were significantly greater in the stress group hippocampus compared with the control group （P 〈 0.05）. No significant difference in JNK phosphorylation levels was detected in the rat cerebral cortex between stress and control groups, and no significant difference in Akt and p38 phosphorylation levels was detected in the rat hippocampus and cerebral cortex between stress and control groups （P 〉 0.05）. These results suggested that the JNK signal pathway is activated by JNK phosphorylation and participates in pathophysiological changes in rat models of depression.

Relationship between Carbachol Hyperstimulation-induced Pancre-atic Intracelluar Trypsinogen and NF-κB Activation in Rats in vitro

The relationship between intracelluar trypsinogen activation and NF-κB activation in rat pancreatic acinar cells induced by M3 cholinergic receptor agonist （carbachol） hyperstimulation was studied. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, the active protease inhibitor （pefabloc） and NF-κB inhibitor （PDTC） in vitro. Intracelluar trypsin activity was measured by using a fluorogenic substrate. The activity of NF-κB was monitored by using electrophoretic mobility shift assay. The results showed that after pretreatment with 2 mmol/L pefabloc, the activities of trypsin and NF-κB in pancreatic acinar cells treated with high concertrations of carbachol （10^-3 mol/L） in vitro was significantly decreased as compared with control group （P〈0.01 ）. The addition of 10^-2 mol/L PDTC resulted in a significant decrease of NF-κB activities in pancreatic acinar cells after treated with high concertrations of carbachol （10^-3 mol/L） in vitro, but the intracelluar trypsinogen activity was not obviously inhibited （P〉0.05）. It was concluded that intracelluar trypsinogen activation is likely involved in the regulation of high concertrations of carbachol-induced NF-κB activation in pancreatic acinar cells in vitro. NF-κB activation is likely not necessary for high concertrations of carbachol-induced trypsinogen activation in pancreatic acinar cells in vitro.

Magnesium lithospermate B inhibits lipopolysaccharide-induced endothelial activation through NF-KB pathway

Aim Magnesium lithospermate B （MLB） is the most abundant hydrophilic active component of Salvia rniltiorrhiza Radix, a traditional Chinese herbal medicine mainly used to treat cardiovascular diseases. Studies have shown that endothelial activation contributes to the pathophysiology of cardiovascular diseases such as atherosclero- sis, diabetic vasculopathy, heart failure and hypertension. In the present study, the effects of MLB on endothelial activation were investigated. Lipopolysaccharide （LPS） 1 mg L^-1 was employed to induce endothelial activation, which was determined by relative gene expression and endothelial adhesion assay. Results showed that pretreatment with MLB attenuated LPS-induced ICAM1, VCAM1 and TNF-α upregulation in human dermal microvascular endo- thelial cells （HMEC-1） in dose-dependent manner, which contributed to the reduction of THP-1 adhesion to HMEC-1. Furthermore, it was revealed that 100 μmol · L^-1 MLB significantly decreased the nuclear translocation of NF-KB p65, a critical transcription factor in LPS-indueed inflammatory response, through the inhibition of IKBμ degradation. Besides, the transcriptional activity of NF-KB p65 was also inhibited by the pretreatment of MLB. Mo- reover, MLB pretreatment considerably inhibited LPS-induced p38 phosphorylation, which at least partly contribu- ted to the reduction of ICAM1 expression. In conclusion, these findings suggest that MLB inhibits LPS-induced nu- clear translocation and transcripitional activity of NF-KB, thus attenuates the increased expression of adhesion mole- cules and inflammatory factors, protects endothelial cells from LPS-induced activation.

Relationship between Carbachol Hyperstimulation-Induced Pancreatic Acinar Cellular Injury and Trypsinogen or NF-κB Activation in Rats in vitro

The relationship between M3 cholinergic receptor agonist （carbachol） hyperstimulationinduced pancreatic acinar cellular injury and trypsinogen activation or NF-κB activation in rats was studied in vitro. Rat pancreatic acinar ceils were isolated, cultured and treated with carbachol, the active protease inhibitor （pefabloc）, and NF-κB inhibitor （PDTC） in vitro. Intracellular trypsin activity was measured by using a fluorogenic substrate. The cellular injury was evaluated by measuring the leakage of LDH from pancreatic acinar ceils. The results showed that as compared with control group, 10-3 mol/L carbachol induced a significant increase of the intracellular trypsin activity and the leakage of LDH from pancreatic acinar cells. Pretreatment with 2 mmol/L pefabloc could significantly decrease the activity of trypsin and the leakage of LDH from pancreatic acinar cells （P〈0. 01） following the treatment with a high concentration of carbachol （10^-3 mol/L） in vitro. The addition of 10^-2mol/L PDTC didn＇t result in a significant decrease in the activity of trypsin and the leakage of LDH from pancreatic acinar cells treated with a high concentration of carbachol （10^-3 mol/L） in vitro （P〉0. 05）. It was concluded that intracellular trypsinogen activation is likely involved in pancreatic acinar cellular injury induced by carbachol hyperstimulation in vitro. NF-κB activation may not be involved in pancreatic acinar cellular injury induced by carbachol hyperstimulation in vitro.

PI3K/Akt pathway is involved in the activation of RAW 264.7 cells induced by hydroxypropyltrimethyl ammonium chloride chitosan

We previously demonstrated that 2-hydroxypropyltrimethyl ammonium chloride chitosan(HACC)promoted the production of nitric oxide(NO)and proinflammatory cytokines by activating the mitogen-activated protein kinases(MAPK)and Janus kinase(JAK)/STAT pathways in RAW 264.7 cells,indicating good immunomodulatory activity of HACC.In this study,to further investigate the immunomodulatory mechanisms of HACC,we determined the roles of phosphatidylinositol 3-kinase(PI3K)/Akt,activating protein(AP-1)and nuclear factor kappa B(NF-κB)in HACC-induced activation of RAW 264.7 cells by the western blotting.The results suggest that HACC promoted the phosphorylation of p85 and Akt.Furthermore,c-Jun and p65 were also increased after the treatment of RAW 264.7 cells with HACC,indicating the translocation of NF-κB and AP-1 from cytoplasm to nucleus.In addition,as scanning electron microscopy(SEM)analysis shows,the cell morphology changed after HACC treatment.These findings indicate that HACC activated MAPK,JAK/STAT,and PI3K/Akt signaling pathways dependent on AP-1 and NF-κB activation in RAW 264.7 cells,ultimately leading to the increase of NO and cytokines.

Influences of V5-epitope tag on the metabolic activation of AFB1 by human cytochrome P450 2A13

Objective： To explore the impact of V5-epitope tag inserted in the commercial pcDNA5/FRT/V5-His TOPO expression vector on the metabolic activation of AFB1 by human CYP2A13. Methods ： A C-terminal 6 × Histag was first introduced into CYP2A13 cDNA by PCR and subsequently transferred into the expressing vector pcDNA5/FRT. Another commercial pcDNA5/FRT/V5-His TOPO expression vector was used to develop the construct directly via PCR. Both of the constructs were then transfected into Flp-In CHO and allowed for the stable expression of CYP2A13. The mouse CYP2A5 and the vector alone were used as positive and negative control, respectively. The presence of CYP2A5 and CYP2A13 cDNA and their protein expression in the stable transfectant cells were deterrrfined by immunoblotting assay using a monoclonal antibody against 6 × Histag. The AFBl-induced cytotoxicity in these tranfected CHO cells were conducted by MTS assay and the IC50 of cell viability was used to compare the CYP enzyme metabolic activity in AFB1 metabolism among these cells. Results： In accordance with the Flp-In system working mechanism, all the transfectant cells presented same protein expression level. The CHO cells expressing CYP2A5 was more sensitive to AFB1 treatment than those cells expressing CYP2A13, there was about 30-fold ICs0 difference between the two cells （2.1 nmol/L vs 58 nmol/L）. Interestingly, CYP2A13 fused with V5-Histag had the lost of metabolic activity to AFB1 than that fused with Histag alone, the ICa, of the viability in CHO-2A13-His-V5 cells was about 20-fold less than CHO-2A13- His （〉 1 000 nmol/L vs 58 nmol/L）. However, there was no change between CYP2A5 fused with V5-Histag and Histag alone （2.4 nmol/L vs 2.1 nmol/L）. Conclusion： The results demonstrate that CYP2A13 fused with V5-epitope has a significant impact on its metabolic activation to AFB1, which indicated that it should be careful to select a new expressing vector for evaluating the enzyme activity in carcinogen metabolism.

Inhibitory Actions of Tetrandrine on Tumor Necrosis Factor α-Induced NF-κB Activation in Neovascularization of Cultured Choroidal Explants

Tetrandrine (1 μM), a bis-benzylisoquinoline alkaloid isolated from Stephania tetrandra S Moore, signifi-cantly decreased tumor necrosis factor alpha (TNFα;10 ng/ml)-induced increase in the number of micro vessels that budded from cultured rat choroidal explants. Tetrandrine also decreased the TNFα-induced in-crease in the number of cells composing the microvessels. Ammonium pyrrolidine dithiocarbamate (APDC;0.1-0.3 μM), an inhibitor of nuclear factor-κB (NF-κB), decreased the TNFα-induced increase in the number of microvessels in a concentration-dependent manner. TNFα increased the phosphorylation and degradation of inhibitor of NF-κB (IκBα), as well as increasing the DNA-binding activity of NF-κB in choroidal explants. TNF? induced an increase of vascular endothelial growth factor (VEGF)-A mRNA, but not VEGF-C mRNA or VEGF-D mRNA. TNFα-induced angiogenic action was inhibited by treatment of VEGF-A antibody in cultured choroidal capillaries. Tetrandrine inhibited the TNFα-induced increases of phosphorylation and degradation of IκBα, and reduced the TNFα-induced increase of DNA-binding activity of NF-κB in chor-oidal explants. In conclusion, tetrandrine inhibits TNFα-induced activation of NF-κB in the choroidal capil-laries via inhibition of TNFα-induced phosphorylation of IκBα.

Competition between C-C and C-H Activation in Reactions of Neutral Nickel Atom with Cycloalkanes （n = 3-7）

A theoretical investigation of the reaction mechanisms for C-H and C-C bond activation processes in the reaction of Ni with cycloalkanes C,,H2. （n = 3-7） is carried out. For the Ni ＋ CnH2, （n = 3, 4） reactions, the major and minor reaction channels involve C-C and C-H bond activations, respectively, whereas Ni atom prefers the attacking of C-H bond over the C-C bond in CnH2n （n = 5=7）. The results are in good agreement with the experimental study. In all cases, intermediates and transition states along the reaction paths of interest are characterized, It is found that both the C-H and C-C bond activation processes are proposed to proceed in a one-step manner via one transition state. The overall C-H and C-C bond activation processes are exothermic and involve low energy barriers, thus transition metal atom Ni is a good mediator for the activity of cycloalkanes CnH2n （n = 3 -7）.