The purpose of this study is to explore depression metabolic markers in rat hippocampus and to investigate the anti-depressant effect of genipin and its mechanisms using nuclear magnetic resonance (NMR) metabonomics. Chronic unpredictable mild stress (CUMS) procedure was conducted to establish the depressive rat model. At the beginning of the third week, genipin low dose (25 mg x kg(-1)), middle dose (50 mg x kg(-1)), high dose (100 mg x kg(-1)), and venlafaxine (50 mg x kg(-1)) were given to the CUMS rats separately once daily for two weeks except control and model groups. Rat hippocampus was analyzed by 1H NMR based metabonomics after drug administration for 2 weeks. Significant differences in the metabolic profile of rat hippocampus of the CUMS treated group and the control group were observed with metabolic effects of CUMS including decreasing in glycine and N-acetylaspartate, increasing in inositol, glutamate, lactate, glutamine, taurine and alanine. Genipin showed ideal antidepressive effects at a dose of 50 mg x kg(-1) in rats, decrease of inositol, glutamate, lactate, alanine were observed, while glycine and N-acetylaspartate were increased. Important influence has been found on normal nervous system function of these significant changed metabolites, which suggests that the antidepressant effect of genipin may be played by enhancing the activity of neurons in hippocampus, repairing and improving the function of the neuron. The metabonomics approach is an effective tool for the investigation of the anti-depressant effect and pharmacologic mechanisms of genipin.
Alanine ; metabolism ; Animals ; Antidepressive Agents ; isolation & purification ; pharmacology ; Aspartic Acid ; analogs & derivatives ; metabolism ; Behavior, Animal ; drug effects ; Chronic Disease ; Depression ; drug therapy ; metabolism ; physiopathology ; Gardenia ; chemistry ; Glutamic Acid ; metabolism ; Glycine ; metabolism ; Hippocampus ; drug effects ; metabolism ; Inositol ; metabolism ; Iridoids ; isolation & purification ; pharmacology ; Lactic Acid ; metabolism ; Magnetic Resonance Spectroscopy ; Male ; Metabolomics ; Plants, Medicinal ; chemistry ; Rats ; Rats, Sprague-Dawley

Abstract: To report the diagnosis, treatment and outcome of 4 patients with hematological diseases complicated with Aeromonas hydrophila bloodstream infection in the Second Affiliated Hospital of Kunming Medical University, further clarify the importance of blood culture and deepen the clinical understanding of the disease. Four patients with hematological diseases complicated with Aeromonas hydrophila bloodstream infection treated in the Second Affiliated Hospital of Kunming Medical University from 2017 to 2021 were recruited as the study objects. The clinical manifestations, blood culture collection, detection time of Aeromonas hydrophila, laboratory examination, treatment and prognosis of the patients were retrospectively analyzed. In this study, 4 cases were male patients with hematological diseases, who were in myelosuppression after chemotherapy. After fever, blood culture was collected and Aeromonas hydrophila was detected. The positive time of blood culture in 4 cases ranged from 4 to 11 hours. The results of antibiotic sensitivity showed that it was highly sensitive to the second, third and fourth generation cephalosporins, quinolones and carbapenems. Four patients were treated with imipenem cilastatin sodium in the early stage, and one patient recovered after active anti infection and leukocyte raising treatment. One patient did not complete chemotherapy due to a request for discharged, and the follow-up was unknown. Two patients developed rapidly into necrotizing fasciitis and died later. Hematological diseases complicated with Aeromonas hydrophila bloodstream infection are rare, but the mortality rate is high. For patients with repeated fever and considering infection, blood culture should be carried out as soon as possible to confirm the pathogen and drug sensitivity test. During clinical treatment, the treatment should be adjusted in time in combination with the patient's situation. In addition to anti-infection treatment, the patient's immunity should be improved and the development of necrotizing fasciitis should be vigilant. Keywords: Aeromonas hydrophila; hematologic diseases; leukemia; bloodstream infection; blood culture; necrotizing fasciitis

BACKGROUNDTo determine the antigenicity of recombinant hepatitis E virus ORF2 (rHEV ORF2) protein expressed in Pichia pastoris (P. pastoris).

METHODSBy using the rHEV ORF2 protein from E.coli as control, an indirect ELISA was adopted to identify the sensitivity, specificity and stability of rHEV ORF2 protein from P. pastoris in detection of HEV IgM and IgG antibody in sera from patients with hepatitis E. The reactivity of the rHEV ORF2 against 5 HEV ORF2 monoclonal antibodies (McAbs) was also tested.

RESULTSThe minimum concentration of coated antigen with which HEV IgG could be detected was 12.5 ng/ml, while the highest serum dilution to detect both IgM and IgG antibodies against HEV was 1:5 120. No cross-reaction was found with sera from patients with any other types of hepatitis. The 37 degree C acceleration test showed that the rORF2 was highly stable within 12 months at 4 degrees C. The 5 HEV ORF2 McAbs showed better reaction with the rORF2 from P. pastoris, especially that 4B2, 2E2, whose reaction against the rORF2 were 125 and 25 times respectively higher than that of rORF2 from E.Coli.

CONCLUSIONThere may be more extensive conformational epitopes in the rHEV ORF2 from P. pastoris. The excellent antigenicity, sensitivity and stability suggest that it can be served as a new candidate antigen for the development of diagnostic reagents of hepatitis E.

Gene Expression ; Hepatitis Antibodies ; blood ; Hepatitis Antigens ; genetics ; immunology ; Hepatitis E ; immunology ; Hepatitis E virus ; genetics ; immunology ; Humans ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; genetics ; immunology ; Viral Proteins ; genetics ; immunology

OBJECTIVE: To investigate the expression and cellular localization of nucleolin C23 during human umbilical vein endothelial cell (HUVEC) apoptosis induced by hydrogen peroxide (H(2)O(2)). METHODS: Apoptosis of HUVEC was induced by exposure to 0.5 mmol/L H(2)O(2) for different periods and detected by flow cytometry and activity of caspase-3. The mRNA and protein expression of nucleolin were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively. The intracellular distribution of nucleolin was observed by indirect immunofluorescence. RESULTS: The percentage of apoptotic cells was increased significantly after treatment with H(2)O(2) for 12, 24 and 36 hours. The activity of caspase-3 reached the peak after treatment with H(2)O(2) for 4 h. RT-PCR showed that nucleolin C23 mRNA was decreased after 2, 4, and 8 hours treatment with H(2)O(2). Western blot showed that C23 protein level was decreased after 12 hours with an additional cleft band of 80 kD appeared after 8 hours. Density analysis showed that the 80 kD cleft band increased in a time-dependent manner. Immunofluorescence analysis demonstrated that H(2)O(2)-induced C23 redistribution from the nucleus to the cytoplasm. CONCLUSION H(2)O(2) could induce apoptosis accompanying with C23-cleavage and C23-translocation from the nucleus to the cytoplasm.
Apoptosis ; drug effects ; Cell Nucleus ; metabolism ; Cells, Cultured ; Cytoplasm ; metabolism ; Endothelial Cells ; cytology ; metabolism ; Humans ; Hydrogen Peroxide ; pharmacology ; Phosphoproteins ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; RNA-Binding Proteins ; biosynthesis ; genetics ; Umbilical Veins ; cytology

OBJECTIVE: To observe whether HSP70 could protect against H2O2-induced apoptosis in C2C12 myogenic cells by inhibiting Smac release from the mitochondria. METHODS: HSP70 gene and full length Smac gene was transiently transfected in C2C12 myogenic cells by lipofectamine and the protein levels of HSP70 and Smac were analysed by Western blotting. Hoechst 33 258 staining was used to examine cell morphological changes and to calculate percentage of apoptotic nuclei. DNA ladder pattern on agarose gel electrophoresis was used to observe the DNA fragmentation. Activities of caspase-3 and caspase-9 were assayed with Western blotting. The release of Smac from the mitochondria to the cytoplasm was observed by immunofluorescence. RESULTS: H2O2 ( 0.5 mmol/L ) activated caspase-3, caspase-9 8 h after the treatment and specific morphological changes of apoptosis 12 h after the treatment, and overexpression of Smac significantly promoted H2O2-induced activation of caspase-3, caspase-9 and apoptosis in C2C12 myogenic cells. HSP70 overexpression significantly inhibited H2O2-induced and Smac-promoted apoptosis, as shown by no specific DNA ladder pattern in agarose gel electrophoresis, decrease of percentage of apoptotic nuclei, and marked inactivation of caspase-3 and caspase-9. HSP70 could inhibit the release of Smac from the mitochondria to the cytoplasm 2 h after the treatment by H2O2. CONCLUSION HSP70 inhibits Smac release from the mitochondria and protects against H2O2-induced apoptosis in C2C12 myogenic cells.
Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; Cells, Cultured ; HSP70 Heat-Shock Proteins ; pharmacology ; Humans ; Hydrogen Peroxide ; Intracellular Signaling Peptides and Proteins ; metabolism ; Mitochondria, Heart ; drug effects ; metabolism ; Mitochondrial Proteins ; antagonists & inhibitors ; metabolism ; Myoblasts ; metabolism ; Myocytes, Cardiac ; drug effects ; metabolism

OBJECTIVETo explore the role of EGF in regulating HaCaT apoptosis through peroxisome proliferator-activated receptor beta (PPARbeta).

METHODSCultured HaCaT cells were divided into different groups with different additives in culture medium as control (normal culture), TNF-alpha (with addition of 10 ng/mL TNF-alpha), EGF (with addition of 20 ng/mL EGF), EGF + TNF-alpha (cells were treated with 10 ng/mL TNF-alpha for 60 mins after the exposure to 20 ng/mL EGF for 4 hs) groups. Conjugation activity and transcription activity of PPARbeta of HaCaT cells in each group were detected by electrophoretic mobility shift assay (EMSA) and luciferase gene analysis (LGA). Protein expression of PPARbeta of HaCaT cells after transfected by missense oligonucleotide (scrODN) and antisense oligonucleotide (asODN) was determined by Western blot. Caspase-3 activity and apoptosis rate were detected by flow cytometry.

RESULTSConjugation and transcription activity of PPARbeta DNA were enhanced as shown in EMSA and LGA. Compared with that of cells in groups transfected by scrODN, protein expression of PPARbeta in cells of groups transfected by asODN was obviously inhibited as shown in Western blot. Caspase-3 activity of cells in TNF-alpha and EGF + TNF-alpha groups transfected by asODN was stronger than that of cells in TNF-alpha and EGF + TNF-alpha groups transfected by scrODN (P < 0.01). Apoptosis rate of cells in control, EGF, TNF-alpha, and EGF + TNF-alpha groups which were transfected by scrODN was (7.31 +/- 0.45)%, (7.43 +/- 0.21)%, (39.78 +/- 0.65)%, (28.34 +/- 0.54)% respectively, and that in those groups transfected by asODN was (8.22 +/- 0.51)%, (7.83 +/- 0.67)%, (46.78 +/- 0.48)%, (44.69 +/- 0.83)%. Apoptosis rate of cells in TNF-alpha and EGF + TNF-alpha groups transfected by asODN was respectively higher than that in TNF-alpha and EGF + TNF-alpha groups transfected by scrODN (P < 0.01).

CONCLUSIONSEGF inhibits HaCaT KC apoptosis caused by TNF-alpha in a PPARbeta-dependent manner.

Apoptosis ; drug effects ; Cell Culture Techniques ; Cell Line ; Epidermal Growth Factor ; pharmacology ; Humans ; PPAR-beta ; genetics ; metabolism ; Transcription, Genetic ; Tumor Necrosis Factor-alpha ; antagonists & inhibitors

OBJECTIVETo investigate the influence of antisense phosphorothioate oligonucleotides on peroxisome proliferator-activated receptors (PPARbeta) in the TNF-alpha mediated apoptosis of HaCat cells.

METHODSHaCat cells were resuscitated and randomly divided into normal control (without transfection), sham (merely with liposome transfection), scrODN (with transfection of 4 micromol/L PPARbeta scrODN), asODN (with transfection of 4 micromol/L PPARbeta asODN), TNF-alpha with transfection of 10 micromol/L TNF-alpha), scrODN + TNF-alpha with 10 micromol/L TNF-alpha stimulation after transfection of 4 micromol/L PPARbeta scrODN), asODN + TNF-alpha with 10 micromol/L TNF-alpha stimulation after transfection of 4 micromol/L PPARbeta asODN) groups. The mRNA and protein levels of PPARbeta were determined with RT-PCR and Western blotting, respectively. The changes in cell morphology were observed with Hoechst 33258 fluorescent staining to quantitate apoptotic rate of nuclei. The effect of PPARbeta asODN on HaCat cell viability was assayed with MTT method. Activation of caspase-3 was evaluated with caspase colorimetric analysis kit.

RESULTSThe mRNA and protein expression of PPARbeta in normal control, sham, scrODN groups were similar, but it decreased obviously in asODN group. The nuclear apoptotic rate in normal control, scrODN and asODN groups were rather low, and the caspase-3 activity in these groups was also low. After 24 hours of culture, the nuclear apoptotic rate in TNF-alpha and scrODN + TNF-alpha groups were (33.1 +/- 2.7)% and (32.9 +/- 3.0)%, respectively, while that in asODN + TNF-alpha group was obviously increased (58.8 +/- 4.6)%, with the caspase-3 activity significantly higher, but the number of live cells markedly lower than that in the former 2 groups (P < 0.05).

CONCLUSIONPPARbeta expression can promote the apoptosis of HaCat cells mediated by TNF-alpha.

Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Cycle ; Cell Line ; Cell Proliferation ; Humans ; Oligonucleotides, Antisense ; genetics ; pharmacology ; PPAR-beta ; genetics ; pharmacology ; RNA, Messenger ; metabolism ; Transfection ; Tumor Necrosis Factor-alpha ; pharmacology

A comprehensive HPLC-DAD-MS method was developed to study the chemical components of semi-bionic extract of Coptis-Evodia herb couple. The extract was isolated on a Hypersil BDS C18 column (4.6 mm x 200 mm, 5 microm) using acetonitrile-ammonium formic buffer as mobile phase by gradient elution. Detection was performed on DAD and MS equipped with an electrospray ionization (ESI) source by full scan and product full scan on positive mode. The chromatogram of Coptis-Evodia showed seventeen main peaks, eight of which were from Evodia while the others were from Coptis. By comparison of the retention time, the on-line UV spectra and MS spectra, four peaks were identified as jatrorrhizine, hydroxevodiamine, palmatine and berberine, and three peaks were deduced as epiberberine, columbamine and coptisine. In addition, berberine and palmatine were quantitatively determined. No new component was created in the semi-bionic extract of the herb couple, yet the solubilities of berberine and palmatine decreased.
Berberine ; analogs & derivatives ; chemistry ; isolation & purification ; Berberine Alkaloids ; chemistry ; isolation & purification ; Chromatography, High Pressure Liquid ; methods ; Coptis ; chemistry ; Drugs, Chinese Herbal ; Evodia ; chemistry ; Plant Extracts ; chemistry ; Spectrometry, Mass, Electrospray Ionization

OBJECTIVETo investigate the effects of Weile Powder (WLP) on bicarbonate transporters in rats with gastric ulcers, and to probe its functional mechanisms.

METHODSThe 48 SD rats were randomly divided into the normal control group, the model group, the low dose WLP group (at the daily dose of 0.075 g/mL), the middle dose WLP group (at the daily dose of 0.150 g/mL), the high dose WLP group (at the daily dose of 0.030 g/mL), and the ranitidine group (at the daily dose of 0.030 g/mL), 8 in each group. The gastric ulcer rat model was prepared by the glacial acetic acid cauterization method. Rats in each medication group were administered from the 2nd day of modeling. Rats were sacrificed after 14-day successive medication. The protein was extracted from the ulcer tissue. The protein expressions of solute carrier26A3 (SLC26A3)and solute carrier26A6 (SLC26A6) were detected using Western blot. The gastric ulcer and its peripheral tissue were sectioned. The changes of cystic fibrosis transmembrane conductance regulator (CFTR) were measured by immunofluorescence.

RESULTSCompared with the model control group, the expression levels of SLC26A3 increased in the high dose WLP group and the ranitidine group with statistical difference (P < 0.05). The expression levels of SLC26A6 increased in the high and middle dose WLP groups and the ranitidine group with statistical difference (P < 0.05). The expression level of CFTR also obviously increased in the high and middle dose WLP groups (P < 0.01).

CONCLUSIONWLP could elevate the expression levels of SLC26A6, SLC26A3, and CFTR, increase the secretion of bicarbonate, thus protecting the gastric mucosa.

Animals ; Antiporters ; metabolism ; Bicarbonates ; metabolism ; Cystic Fibrosis Transmembrane Conductance Regulator ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Female ; Gastric Mucosa ; drug effects ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Stomach Ulcer ; metabolism

OBJECTIVE: To explore the role of cell-surface nucleolin in lipopolysaccharide (LPS)-stimulated expression and secretion of TNF-alpha and IL-1beta in human THP-1 monocytes. METHODS: Immuno-fluorescence assay and Western blot were used to identify the expression of nucleolin on the surface of THP-1 monocytes. Inactivation of nucleolin was induced by anti-nucleolin monoclonal antibody blockage, and the expression and secretion of TNF-alpha and IL-1beta were observed by using reverse transcription polymerase chain reaction (RT-PCR) and enzyme linked immuno-sorbent assay (ELISA)respectively in LPS-mediated human THP-1 monocyte inflammatory model. RESULTS: Immuno-fluorescence showed that nucleolin was localized on the cell surface of THP-1 monocytes as indicated by dotted red fluorescence. Western blot assay indicated that nucleolin existed in the cell membrane fractions. RT-PCR assay showed that the expressions of TNF-alpha and IL-1beta mRNA significantly increased at 2 h and 3 h after the treatment with 1000 microg/L LPS. After 1 h pretreatment with anti-nucleolin antibody, the levels of TNF-alpha and IL-1beta mRNA decreased compared with an anti-nucleolin antibody untreated group and an irrelevant IgG+LPS group (P<0.05). ELISA assay showed that the pretreatment with anti-nucleolin antibody inhibited significantly the secretion of LPS-induced levels of TNF-alpha and IL-1beta after 4, 12 and 24 h treatment with 1000 microg/L LPS. CONCLUSION Nucleolin expresses on the cell surface of THP-1 monocytes and involves in the LPS-mediated expression and secretion of TNF-alpha and IL-1beta.
Cell Line ; Cell Membrane ; metabolism ; Humans ; Interleukin-1beta ; biosynthesis ; metabolism ; Lipopolysaccharides ; pharmacology ; Monocytes ; cytology ; metabolism ; Phosphoproteins ; metabolism ; physiology ; RNA-Binding Proteins ; metabolism ; physiology ; Tumor Necrosis Factor-alpha ; biosynthesis ; metabolism