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OBJECTIVE To study the metabolites of four diterpenoids of Euphorbia fischeriana in liver microsomes of rats and to investigate its metabolic regularity. METHODS In vitro incubation system of liver microsomes of rats was built. The jolkinolide A，jolkinolide B ，17-hydroxyl jolkinolide A and 17-hydroxyl jolkinolide B were added into incubation system of liver microsomes in rats activated by reduced nicotinamide adenine dinucleotide phosphate ，incubated at 37 ℃ for 30 min，and then terminated the reaction with acetonitrile. Taking the negative group （adding acetonitrile firstly and then starting incubation for 30 min）as the reference，the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry was used ；Anaylyst®TF 1.7.1、PeakView® 2.2，MetabolitePilot 1.5 and MasterView 1.2 software were used to speculate and identify the fragmentation law of mass spectrometry and metabolites. RESULTS Four diterpenoids were easy to lose neutral fragments such as H 2O and CO in secondary mass spectrometry. Jolkinolide A and 17-hydroxyl jolkinolide A showed similar metabolism pathway ，including dihydroxylation，dehydrogenation，and monohydroxylation ；six and five metabolites were identified respectively. Jolkinolide B and 17-hydroxyl jolkinolide B showed similar metabolism pathway ，including monohydroxylation ，hydration and isomerization. Five metabolites were identified. CONCLUSIONS Both jolkinolide A and 17-hydroxyl jolkinolide A produce the metabolites of hydroxylation and dehydrogenation in liver microsomes of rats ；both jolkinolide B and 17-hydroxyl jolkinolide B produce the metabolites of hydroxylation ，hydration and isomerization in liver microsomes of rats. The metabolites of four diterpenoids are phase Ⅰ metabolites.
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Curcumin is exclusively isolated from Zingiberaceae plants with a broad spectrum of bioactivities. In the present study, we used the diketide-CoA synthase (DCS) and curcumin synthase (CURS) genes to construct a non-natural fusion gene encoding diketide-CoA synthase::curcumin synthase (DCS::CURS). This fusion protein, together with the acetyl coenzyme A carboxylase (ACC) and the 4-coumarate coenzyme A ligase (4CL), were introduced into Escherichia coli for the production of curcumin from ferulic acid. The process is divided into two stages, the growth stage using LB medium and the fermentation stage using the modified M9 medium. The yield of curcumin reached 386.8 mg/L by optimizing the induction of protein expression in the growth stage, and optimizing the inoculum volume, medium composition and fermentation time in the fermentation stage, as well as the addition of macroporous resin AB-8 into the second medium to attenuate the toxicity of the end product. The exploitation of the non-natural fusion protein DCS::CURS for the production of curcumin provides a new alternative to further promoting the production of curcumin and the related analogues.
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Curcumin/pharmacology , Escherichia coli/genetics , FermentationABSTRACT
Objective@#To observe the effect of tumor necrosis factor-α (TNF-α) monoclonal antibody on autophagy in allergic rhinitis (AR) mice.@*Methods@#Thirty six weeks old BALB/c mice were randomly divided by random number table method into five groups: control group, model group (AR group), TNF-α antibody intervention group (AR+TNF-α group), autophagy inhibitor (3-methylindole, 3-NA) intervention group (AR+3-MA group), TNF-α antibody combined with autophagy inducer rapamycin (RAP) intervention group (AR+TNF-α+RAP group), with 6 mice in each group. AR model was established by conventional method, the corresponding reagent was administered before nasal cavity stimulation sensitization and during the whole experiment. Behavioral scores of mice were obtained, blood was collected from the eye socket, and mice in each group were sacrificed to collect nasal mucosa tissue samples. Pathological changes of nasal mucosa were observed by hematoxylin-eosin staining. Expression levels of inflammatory factor and IgE in serum were detected by enzyme-linked immunosorbent assay (ELISA). Expressions of autophagy related indicators microtubule-associated protein-1 light chain-3B (LC3B), Beclin-1, sequestosome1 (p62), autophagy-related 5 (ATG5), autophagy-related 7 (ATG7) were measured by Real-time PCR and Western blot. The aggregation of LC3B protein was observed by immunofluorescence. SPSS 19.0 software was used for statistical analysis.@*Results@#Compared with the AR model group, symptoms of AR in AR+TNF-α group and AR+3-MA group were mild; the pathological changes of nasal mucosa were weak; the expression of IgE, TNF-α, interleukin 4 (IL-4), interferon-γ (IFN-γ) in serum significantly reduced (IgE: 666.19±78.35 (±s) vs. 692.38±64.29 vs. 1 059.05±146.44, TNF-α: 112.06±12.95 vs. 113.17±15.43 vs. 161.22±17.96, IL-4: 54.05±7.14 vs. 58.26±5.67 vs. 79.95±6.33, IFN-γ: 28.58±4.51 vs. 30.67±2.60 vs. 39.83±3.31, all P<0.05), and the expression of LC3B Ⅱ/Ⅰ, Beclin-1, ATG5, ATG7 in nasal mucosa significantly decreased, the expression of p62 significantly elevated. After intervention with autophagy inducer RAP, the therapeutic effect of TNF-α monoclonal antibodies on AR was antagonized.@*Conclusion@#TNF-α monoclonal antibody significantly improves nasal symptoms in AR mice by inhibiting autophagy levels.
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OBJECTIVE To investigate the cytotoxicity of cyflumetofen for SH-SY5Y cells and the mechanism. METHODS SH-SY5Y cells treated with cyflumetofen 0.03, 0.06, 0.125, 0.25, 0.5, 1, 2, 2.6, 4, 6, 8 and 16 mmol·L-1 for 48 h. Cell survival was measured with MTT assay. The reactive oxygen species (ROS) was determined with the DCFH- DA probe, and mitochondrial membrane potential (MMP) was detected by JC-1 staining. The morphological changes in cell nuclei were observed with Hoechst33258 staining. Cell cycle and apoptosis were determined by flow cytometry. The protein levels of phosphorylated Jun Kinase (p-JNK) and p-P38 were measured by Western blotting. RESULTS Compared with solvent (DMSO) control group, cyflumetofen (≥0.06 mmol · L- 1) inhibited the proliferation of SH- SY5Y cells obviously (P<0.05), and the IC50 was 2.6 mmol·L-1. MMP declined and ROS levels increased significantly in cyflumetofen 1, 2, 4 and 6 mmol·L- 1 groups (P<0.01). Cyflumetofen 2, 4 and 6 mmol·L- 1 induced nucleic accumulation, nuclear shrinkage and disintegration in SH-SY5Y cells. Apoptosis rates of cyflu? metofen 2, 4 and 6 mmol·L- 1 groups increased from (0.7±0.1)% in DMSO control group to (6.7±0.1)%, (72.4±8.6)% and (90.7±3.2)% (P<0.01). Cyflumetofen 4 and 6 mmol·L- 1 induced G1 phase cell cycle arrest (P<0.01). In addition, Western blotting showed that cyflumetofen 4 and 6 mmol·L-1 up-regulated the expression of p-JNK (P<0.01), while the level of p-P38 in SH-SY5Y cells was increased in cyflumetofen 6 mmol · L- 1 group (P<0.01). CONCLUSION Cyflumetofen induces cell damage, apoptosis and G1 phase cell cycle arrest in SH- SY5Y cells. The mechanism may be associated with oxidative damage, and activation of P38 and JNK stress-response pathways.
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Objective To compare the advantages and disadvantages of three methods of yeast-like fungi identification of vulvovaginal candidiasis (VVC).Methods The pathogenic candida strains were identified by CHROM agar chromogeinc culture medium,the chromogenic medium of Autobio and VITEK 2 system,and the result of VITEK 2 system was the gold standard.Results All candida strains were appraised.By VITEK2 system,217 candida isolates were identified as candida albicans,and 26 were detected as non-candida albicans.214 isolates were identified as candida albicans and 13 isolates were identified as non-candida albicans by Autobio.214 isolates were appraised as candida albicans,16 isolates were identified as non-candida albicans.The coincidence rate of identification of CHROMagar and the chromogenic medium of Autobio were 94.7％ and 95.06％.Compared with VITEK 2 system,there was no significant difference between these two chromogeinc culture medium identifying candida albicans and non-candida albicans.Conclusion The chromogenic medium of Autobio has higher cost-effectiveness,higher color discrimination and simpler technical operation,and is worthy of promotion in identifying candida species in clinic.
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Using bioactive compounds 7-hydroxy flavone, salicylaldehyde, cinnamic acid and 4-amino-5- mercapto-1,2,4-triazoles as starting materials, three new types of flavone derivatives containing 1,2,4-triazole structure were synthesized via different step reactions. These new compounds were characterized by 1IHNMR, ESI-MS, IR and elemental analysis. Their scavenging effects on the superoxide radical (O2·-), hydroxyl radical (·OH), DPPH · radical and their total reduction activities were tested. The results showed that all of the compounds possessed some antioxidative activity at the concentration of 0.5 mg · mL(-1), but the scavenging ability of the target compounds was lower than that of the standard compound Vc.
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OBJECTIVE@#To observe the histopathologic and morphological changes of palatopharyngeal soft tissues in patients with different degrees of obstructive sleep apnea hypopnea syndrome (OSAHS) patients.@*METHOD@#Thirty-eight male OSAHS patients were divided into 3 groups according to AHI, namely mild group (n = 10), moderate group (n = 13),and severe group (n = 15). The soft palate tissues with partial palatopharyngeal arch and palatoglossal arch tissues were obtained from surgery and processed with conventional paraffin embedding. The sections were stained by HE and observed under a light microscope. The histological quantitative changes of the specimens were measured by analyzing the constituent ratios of glandular tissue, fat tissue and interstitial elements. Statistical analysis was performed.@*RESULT@#1) Optical microscope showed that (100 times), as the aggravation of the OSAHS, the soft palate squamous epithelial cells are swollen and irregular, exhibiting hyperkeratosis, accompanied by liquefied degeneration of basal cell; The mucous membrane and submucosal connective tissue contain a certain number of lymphocytes infiltration. The mucosa and submucosal layer of loose connective tissue contain inflammatory cells and a lot of fat vacuoles can be observed; The soft palate mucous acini have inconsistent and irregular shape, among which there are a certain amount of fat cells infiltration. Some mucous acini are replaced by serous acini with dark stained cytoplasm; The palatoglossal muscle and palatopharyngeus muscle fibers can't be identified with disordered arrangement of structure, showing pleomorphic changes including swelling, atrophy and degeneration. Some of elastic fibers were disrupted and a lot of fat cells infiltration was observed. (2) The constituent ratios of the three kinds of tissues in soft palate from different degrees of OSAHS patients show that quantitative changes of glandular tissue and interstitial elements among the mild, moderate and severe OSAHS group patients exhibit statistically significant differences (P 0.05).@*CONCLUSION@#With the rising of severity of OSAHS, the soft palate squamous epithelial cells are swollen and irregular, exhibiting hyperkeratosis. Between acinar cell we could see a certain amount of fat cells infiltration. Some mucous acini are replaced by serous acini. Muscle fibers of palatopharyngeal tissue have pleomorphic changes of swelling, atrophy and degeneration.