[Myrislignan Induces Apoptosis in Gastric Cancer Cell Line Through PI3K/AKT Signaling Pathway].

Objective: To investigate the effect of myrislignan (MYR) on the apoptosis of gastric cancer cell line and its relationship with phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. Methods: The gastric cells (SGC-7901) were treated with MYR at different concentrations, i.e., 0, 25, 50, 100, and 200 µmol/L, for 48 h and 72 h and the effect of MYR on the proliferation of SGC-7901 cells was measured by CCK-8 assay. Then, SGC-7901 cells were treated with different concentrations of MYR at 50, 100, and 200 µmol/L for 48 h. Meanwhile, a normal control group and a dimethyl sulfoxide (DMSO) solvent control group (0.1% DMSO) were established. Flow cytometry was used to determine the apoptosis rate of SGC-7901 cells. The protein expression levels of PI3K, AKT, Bcl-2-associated X protein (BAX), cysteine-dependent aspartate-specifc protease-3 (Caspase-3), and Caspase-9 were determined by Western blot. Then, PI3K activator (20 µmol/mL) was used to treat SGC-7901 cells for 48 h in 4 groups, the control group, 0.1% DMSO group, MYR group, and MYR+PI3K activator group, and the effect on MYR's induction of apoptosis and regulation of the protein expression levels of PI3K, AKT, BAX, Caspase-3, and Caspase-9 in SGC-7901 cells. Results: Compared with the control group, MYR at 50, 100 and 200 µmol/L inhibited the proliferation of gastric cancer cells, increased the apoptosis rate, down-regulated the protein expression levels of PI3K and AKT, and up-regulated the protein expression levels of BAX, Caspase-3, and Caspase-9 in a dose-dependent manner ( P<0.05). However, PI3K activator attenuated MYR-induced apoptosis in gastric cancer cells and MYR's regulation of PI3K, AKT, BAX, Caspase-3, and Caspase-9 protein expression ( P<0.05). Conclusion: MYR induces the expression of BAX, Caspase-3, and Caspase-9 proteins by inhibiting the PI3K/AKT signaling pathway, thereby promoting the apoptosis of gastric cancer cells.

[Therapeutic Effect of Artesunate on Influenza A Viral Pneumonia].

Objective: To investigate the therapeutic effect of artesunate (ART) on influenza A viral pneumonia. Methods: A total of 36 mice were evenly and randomly assigned to six groups, a normal control group (C group), a solvent control group (M group, 10% DMSO), a positive drug group (P group, oseltamivir, 1.25 mg/kg/day), ART high-dose group (ART-G group, 120 mg/kg/day), ART medium-dose group (ART-Z group, 60 mg/kg/day), and ART low-dose group (ART-D group, 30 mg/kg/day). Except for group C, which did not receive any influenza A virus intervention or intraperitoneal injection, mice in the five other groups were infected with influenza A virus through intranasal drip. Then, after 12 hours, mice in the five other groups received intraperitoneal injection of the assigned drugs and dosage once a day. The signs, body weight, and survival of the mice were observed over the course of treatment. After 7 days of treatment, the lung tissue of the mice was collected and weighed, and the lung index was calculated accordingly. HE staining was performed to observe the pathological changes in the lung tissue. The mRNA and protein expression levels of Toll-like receptor 4 (TLR4), nuclear factor kappa-B (NF-κB [p65]), tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and IL-1ß were examined with RT-qPCR and Western blot, respectively. Results: Compared with those in C group, mice in the M group had worse physical signs and lower body mass and survival, increased lung index, severe pathological changes in lung tissue, and increased levels of TLR4, NF-κB (p65), TNF-α, IL-6 and IL-1ß mRNA and protein expression in their lung tissue ( P<0.05). Compared with those in M group, the mice in the ART groups had better physical signs, higher body mass and survival rate, decreased lung index, improvement of pathological changes in the lung tissue, and decreased levels of level of TLR4, NF-κB (p65), TNF-α, IL-6 and IL-1ß mRNA and protein expression in the lung tissue ( P<0.05). Furthermore, the most prominent changes in these indexes were observed in the ART-G group. Conclusion: ART has therapeutic effects on influenza A viral pneumonia, and the mechanisms are related to the inhibition of TLR4/p65 signaling pathway activation and anti-inflammation.

[Determination of Aromatic Compounds Metabolites in Human Urine by Solid Phase Extraction-liquid Chromatography-Tandem Mass Spectrometry].

OBJECTIVE: To establish the method based on high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) with solid phase extraction (SPE) for simultaneous determination of the biological metabolites of aromatic compounds, including N-acetyl-S-phenyl-L-cysteine (SPMA), N-acetyl-S-benzyl-cysteine (SBMA), p-nitrophenol (PNP), methylhippuric acids (MHA), p-Aminophenol (PAP), mandelic acid (MA), phenylglyoxylic acid (PGA) and 1-hydroxypyrene (1-OHP) in urine. METHODS: After adding 20 µL of ß-glucuronidase and 1 mL ammonium acetate buffer solution in 1 mL of urine, the sample was digested in a 37 ℃ incubator for 20 h. After digestion, the enzymatic hydrolysate was purified by PRIME HLB solid phase extraction column. The target compounds were eluted with 4 mL of acetonitrile and blown to dryness with nitrogen, reconstituted with 0.20 mL of methanol. Injected the sample solution into LC-MS/MS system for analysis after filtering with 0.22 µm filter membrane. LC separation was carried out on a reversed-phase C18 column (2.1 mm×150 mm, 3.5 µm); gradient eluting was performed at a flow rate of 0.2 mL/min. The water containing 0.1% formic acid was used as mobile phase A and methanol was used as mobile phase B. The mass spectrometry was performed with multiple reaction monitoring (MRM) mode, using alternating positive and negative ions, and internal standard curves were used for quantification. RESULTS: The eight metabolites showed good linearity within the range of 1-100 ng/mL, with a correlation coefficients greater than 0.995, and the relative precision deviation (RSDs) was 0.050%-9.95%. The method detection limits (MDLs) of the eight target metabolites were 0.041-0.12 ng/mL. The proposed method was used for urine sample analysis and the spiked recoveries were 80.1%-114.0%. CONCLUSION: The established method is quick, sensitive and accurate; it meets the requirementof the biological monitoring of aromatic compounds for the general population and occupational population.

[Monoclonal Antibody Against N Terminal 439-451 Epitopes of Anti-Mullerian Hormone and Its Properties].

OBJECTIVE: To prepare the specific monoclonal antibody against the N-terminal specific epitope peptide of anti-mullerian hormone (AMH) and to identify its specificity. METHODS: Using bioinformatics analysis software to predict the specific peptide fragment of AMH. Then synthesized four antigenic epitope peptide segments of mature N-terminal region of AMH as the screening target antigen. Synthesized AMH wholegene.Using the prokaryotic expression system to abtain recombinant AMH protein. Immunized BALB/c mice with the recombinant AMH, and prepared mouse spleen cells for fusing with SP/20 cells. Preparation of AMH monoclonal antibody by hybridoma technology. The monoclonal antibodies against AMH were screened by using four N-terminal epitope peptides (1: 439-451 RGRDPRGPGRAQ, 2: 273-285 PPRPSAELEESPP, 3: 42-54 DLDWPPGSPQEPL, 4: 494-506 WPQSDRNPRYGNH) as antigens, and indirect ELISA and Western blot were used to identify the antigen binding characteristics of the selected monoclonal antibodies. RESULTS: Two hybridoma cell lines with stable anti-AMH-1 and anti-AMH-2 antibody activities were screened. The two antibodies were named anti-AMH-1 and anti-AMH-2 respectively. The antibody titers were 1∶12 000 and 1∶1 600 after purification. Western blot confirmed that the two McAbs recognized different antigens. Anti-AMH-1 could not only recognize the N-terminal 439-451 epitope peptide of AMH, but also recognize the amino acid sequence of recombinant AMH, as well as the ovarian tissue. Anti-AMH-2 could recognize recombinant AMH and ovarian tissue. CONCLUSION: Two monoclonal antibodies against N-terminal specific epitopes of human AMH were successfully constructed.

[The Effects and Mechanisms of Dihydroartemisinin on Influenza A Virus H1N1 Induces TNF-α and IL-6 Expression in Bronchial Epithelial Cells].

OBJECTIVE: To investigate the effects of dihydroartemis (DHA) on influenza A virus (IAV) A/PR/8/34 (H1N1) induces the pro-inflammatory factor and protein of extracellular signal regulated kinase (ERK) signaling pathway expression in bronchial epithelial cells. METHODS: The BEAS-2B cells were treated with different concentrations of DHA (i.e.,0， 12.5, 25，50 and 100 µmol/L) for 24 h and the effect of DHA on the viability of BEAS-2B cells were measure by CCK8 method. The BEAS-2B cells were absorbed with IAV for 1 h, and then were treated with different concentrations of DHA (i.e., 12.5, 25 and 50 µmol/L) for 24 h, meanwhile, the normal control group and IAV group were established. The mRNA and protein expression levels of tumor necrosis factor-α (TNF-α) and interleukin (IL-6) were measured by real time quantitative PCR (RT-qPCR) and enzyme linked immunosorbent assay (ELISA), the expression levels of phospho-ERK (p-ERK) proteins were tested by Western blot (WB). Then, an ERK agonist (20 ng/mL) was used to treat BEAS-2B cells (the groups were divided into normal control group, DHA group, DHA+IAV group, ERK agonist group and DHA+IAV+ERK agonist group) for 24 h, and to observe the effect of DHA on inhibiting IAV induce the TNF-α, IL-6 and p-ERK expression in the BEAS-2B cells. RESULTS: The BEAS-2B cells viability was not significantly different from that of the normal control group after treatment with DHA (i.e., 12.5, 25, and 50 µmol/L). The expression levels of TNF-α, IL-6 mRNA and TNF-α, IL-6, p-ERK protein in IAV group were significantly up-regulated compared with that in the normal control group ( P<0.05), meanwhile, compared with the IAV group, the expression levels of TNF-α, IL-6 mRNA and TNF-α, IL-6, p-ERK protein showed dose-dependent decrease in IAV+DHA group ( P<0.05). However, ERK agonists attenuated the DHA inhibit IAV induced the proinflammatory factors TNF-α, IL-6 secretion and the p-ERK protein expression of ERK signaling pathway in BEAS-2B cells. CONCLUSION: These data suggest that DHA can inhibit IAV induces the TNF-α and IL-6 expression in BEAS-2B cells through ERK signaling pathway.

[Expression and clinical significance of bone morphogenetic protein 6 in human breast cancer].

OBJECTIVE: To investigate the expression pattern and clinical significance of bone morphogenetic protein 6 (BMP6) in breast tissues. METHODS: The tumor and adjacent noncancerous tissues were harvested from 36 cases of breast cancer, the expression level of BMP6 mRNA of each sample was measured by quantitative RT-PCR. Immunohistochemistry study was used to examine BMP6 protein expression in 80 cases of breast cancer, then the relationship between the expression of BMP6 and relevant clinical and pathological parameters was analyzed. RESULTS: BMP6 mRNA expression in breast cancer was significantly reduced when compared with normal breast tissues (P< 0.01), BMP6 mRNA level in estrogen receptor-positive (ER) breast cancer was distinctly higher than that in ER breast cancer. The expression of BMP6 mRNA was correlated to tumor grade (P < 0.01). The expression level of BMP6 protein in breast cancer was associated to ER and PR status, histological grade and Ki-67 status (P < 0.05), but not correlated to age, tumor size, human epidermal factor receptor 2 (Her2) status and molecular subtypes of breast cancer (P > 0.05). CONCLUSION: The ectopic expression of BMP6 may play an important role in the development and progression of breast cancer.

[Establishment of in vitro co-cultue system for tumor vascular endothelial cells].

OBJECTIVE: To establish a in vitro culture model of tumor vascular endothelial cells (TECs). METHODS: We established in vitro culture model of TECs, named A549-HUVECs, through the co-culutre system culturing primary human umbilical vein endothelial cells (HUVECs) and human lung adenocarcinoma epithelial cell line A549 cells in indirect co-cultures, six-well Transwell-clear dishes (0.4 microm porous filter, Costar). We then observed TECs' cell morphology, growth characteristics, proliferation and karyotype, telomerase activity, etc. RESULTS: In the indirect co-culture system, A549-HUVECs showed migration cell-type appearance, more active cellular metabolism, increasing cell permeability, augmenting cell proliferation, which indicated A549-HUVECs being in a state of the regulation of homeostasis imbalance for cell proliferation and apoptosis. Besides, the analysis of A549-HUVECs genetic characteristics showed that the chromosome karyotype of A549-HUVECs was sub-triploid, and its telomerase activity increased, phenotypic change occurred, which possessed the similar pathological features of precancerous lesion. CONCLUSION: A549-HUVECs co-culture system could be feasible for tumor vascular research.

[Effects of oxidative stress on MuRF1 expression in skeletal muscle of diabetic rats].

OBJECTIVE: To determine whether muscle-specific RING finger protein 1 (MuRF1) expression induced by oxidative stress lead to muscle wasting in diabetes rats. METHODS: The diabetes rat model was established by high-carbohydrate, high-fat diet and injection of streptozotocin. The expression of MuRF1 in gastrocnemius was detected by immunohistochemistry and real time PCR. The level of lipid peroxidation, SOD and fiber size of gastrocnemius was also detected. Further more, C2C12 myotubes were cultured with different concentration of H2O2 (0, 0.01, 0.05, 0.10 and 0.20 mmol/L), the level of MuRF1 protein was detected by western blot. RESULTS: Compared with the control group, the diabetes rats showed higher level of thiobarbituric acid reactive substances (TBARS) and MuRF1 mRNA and lower fiber size in gastrocnemius (P < 0.01). The oxidative stress induced by H2O2 (0.05, 0.10 and 0.20 mmol/L) upregulated the expression of MuRF1 (P < 0.01) in C2C12 myotube cells. CONCLUSION: Our results indicated that diabetes modulated the expression of MuRF1 leading to muscle wasting, and the mechanism might be involved with oxidative stress.

[Simultaneous determination of five water-soluble vitamins in human serum by high performance liquid chromatography].

OBJECTIVE: To establish a method for the simultaneous determination of 5 water-soluble vitamins including VB1, VB6, Nicotinic acid, Nicotinamide and Folic acid by high performance liquid chromatography. METHODS: Methanol (200 microL) was added to 50 microL human blood sample to precipitate proteins. The sample was frozen (-18 degrees C) for 30 min, and then centrifuged at 8000 r/min for 8 min. The supernatant was subsequently applied to the solid-phase cartridge for further purification. The elute was collected and evaporated to 0.20 mL under nitrogen in a water bath at the temperature of 50 degrees C and finally 50 microL of the sample was injected onto HPLC column for analysis. The five water-soluble vitamins were determined under optimized condition of solid phase extraction and chromatograph. RESULTS: The optimized condition for solid phase extraction (SPE) was identified as: AGT cleanert ODS C18 column as SPE column and CH3OH (2.4 mL)-H2O (0.6 mL) mixed liquor as eluent, with a flow rate of elution of 0.5 mL/min. The optimized chromatographic condition was identified as, Phenomenex C18 Luna column (250 x 4.6 mm, 5 microm) as analytical column under a temperature of 20 degrees C and 0.05 mol/L potassium dehydrogenates phosphate buffer (pH 6.0) and methanol as gradient elution with mobile phase with a flow rate of 0.9 mL/min. The detection wavelength was 266 nm. The correlation coefficients for the standard curves were greater than 0.999 in the range from 0 to 100 ng (RSD < 5%). The detection limits (S/N 2) ranged from 0.076 to 0.170 microg/mL. The recovery rate ranged from 80.2% to 115.0%. Except for Nicotinamide, the other four vitamins were detected successfully in the blood sample. CONCLUSION: This quick and convenient method can be applied to the simultaneous determination of 4 water-soluble vitamins in human serum samples. But the sensitivity for detecting Nicotinamide needs to be improved.