Study of Serum Metabonomics and Formula-Pattern Correspondence in Coronary Heart Disease Patients Diagnosed as Phlegm or Blood Stasis Pattern Based on Ultra Performance Liquid Chromatography Mass Spectrometry / 中国结合医学杂志

<p><b>OBJECTIVES</b>To study the characteristics of serum metabonomics in coronary heart disease (CHD) patients diagnosed as phlegm or blood stasis pattern and explore effects of formula-pattern correspondence treatment.</p><p><b>METHODS</b>A total of 102 stable CHD patients were enrolled and divided into phlegm group (P group, n=52) and blood stasis group (BS group, n=50) according to pattern identifification. Gualou Xiebai Banxia Decoction (, GXBD) and Xuefu Zhuyu Decoction (, XZD) were used as drug interventions. Relevant indicators of metabonomics were observed by ultra performance liquid chromatography mass spectrometry (UPLC-MS) and pattern recognition.</p><p><b>RESULTS</b>Levels of amino acids and phosphatidylethanolamine (PE) in the CHD group were much higher than those in healthy control group, while the levels of unsaturated fatty acids, sphingosine, Lyso, phosphatidylcholine (PC) were signifificantly lower (P<0.01). Most of the differential metabolites between the CHD and the healthy groups were also common metabolites of phlegm and blood stasis. 7(Z), 10(Z)-hexadecadienoic acid and DPA were decreased in the P group and increased in the BS group. According to the quantity of retraced metabolites, improvement in metabonomics by formula-pattern correspondence was superior to that without correspondence in the BS group. Based on the varieties of metabolites, GXBD could improve the levels of docosapentaenoic acid (DPA), sphingomyelin (SM) (d34:1), and L-Lactic acid and XZD could ameliorate the levels of sphingosine and Vit E in the P group. In the BS group, GXBD could improve vitamin E level and XZD could make improvements in the levels of octadecanoic acid, phosphoglycerol, and SM (d34:1).</p><p><b>CONCLUSIONS</b>Phlegm and blood stasis in CHD patients present specifific differential metabolites, and share common metabolites. Remarkable differences have been displayed in pathological properties and severity of phlegm and blood stasis. Patients with phlegm are more likely to have lipid metabolism disorders. However, in patients with blood stasis, problems mainly lie in glucose, protein and fat metabolism and the injury of vascular cell membrane is relatively severe. The metabolic disorder is more complicated in blood stasis pattern than that in phlegm pattern. Compared with non-correspondence, improvement of differential metabolites is more comprehensive and targeted in formulapattern correspondence with a better effect.</p>

Identification of in vivo-induced antigen Tp0462 of Treponema pallidum and evaluation of its application to serodiagnosis of syphilis / 中华皮肤科杂志

Objective To screen and identify the in vivo-induced antigen Tp0462 of Treponema pallidum (Tp),and to evaluate its value for clinical serological diagnosis of syphilis.Methods Genomewide DNA was extracted from the Tp Nichols strain,and polymerase chain reaction (PCR) was performed to amplify the Tp0462 gene.A recombinant plasmid pET30a (+)-Tp0462 was constructed and transfected into the Escherichia coli (E.coli) Rosetta (DE3) strain.Recombinant protein Tp0462 was abundantly expressed,purified and identified.A total of 18 New Zealand rabbits were randomly and equally divided into 3 groups:viable Tp-incubating group incubated with viable Tp in the testes,inactived Tp-incubating group incubated with ultraviolet irradiation-killed Tp in the testes,and control group receiving no treatment.After incubation,blood samples were collected at different time points,and the sera were isolated for identification of characteristics of in vivo-induced antigen Tp0462.Enzyme-linked immunosorbent assay for Tp0462 (Tp0462-ELISA),Treponema pallidum particle agglutination assay (TPPA),preliminary syphilis screening ELISA and rapid plasma reagin (RPR) card test were applied in 336 clinical serum samples from patients with syphilis,so as to preliminarily evaluate the value of Tp0462 for the diagnosis of syphilis.Results The optimum conditions for expression of the recombinant plasmid Tp0462-pET30a(+) in E.coli Rosetta (DE3) strains were the treatment with 0.5 mmol/L isopropyl thiogalactoside (IPTG) on a shaker at 180 rpm for 4 hours.In the viable Tp-incubating group,the serum level of specific anti-Tp0462 antibody sharply increased from week 2,and went steady after week 5.However,the specific anti-Tp0462 antibody maintained a low level in the inactived Tp-incubating group and the control group.The viable Tp-incubating group showed a significantly higher level of specific anti-Tp0462 antibody compared with the inactived Tp-incubating group and the control group (both P ＜ 0.05),while no significant difference was observed between the inactived Tp-incubating group and the control group (P =0.256).The level of anti-Tp92 antibody was significantly higher in the viable Tp-incubating group and the inactived Tp-incubating group than in the control group (P ＜ 0.05),while there was no significant difference between the viable Tp-incubating group and the inactived Tp-incubating group (P =0.127).Compared with TPPA,the sensitivity,specificity,consistency rate and area under the curve (AUC) of Tp0462-ELISA for the diagnosis of syphilis were 91.7％,98.8％,95.2％ and 0.997 respectively.Tp0462-ELISA was consistent to preliminary syphilis screening ELISA and RPR with a Kappa coefficient of 0.846 and 0.293,respectively.Conclusion Tp0462-ELISA has shown evidently higher sensitivity and specificity in the serodiagnosis of syphilis,and Tp0462 can serve as promising antigens for the diagnosis of syphilis.

Optimization of strategies for inoculation of Treponema pallidum pcD/Tp92 DNA vaccine / 中华皮肤科杂志

Objective To evaluate immune protective effects of Treponema pallidum(Tp)pcD/Tp92 DNA vaccine delivered through different inoculation routes against Tp-induced skin infection in New Zealand rabbits. Methods A total of 108 New Zealand rabbits were randomly and equally divided into 6 groups:A1 and A2 groups treated with intramuscular injection of empty plasmids pcD and pcD/Tp92 DNA vaccine respectively for 2 sessions, B1, B2, C1 and C2 groups firstly treated with intramuscular injection of the pcD/Tp92 DNA vaccine for 1 session for primary immunization, then receiving nasogastric feeding with pcD/Tp92 DNA vaccine, pcD/Tp92 DNA vaccine+cytosine-phosphate-guanine(CpG)oligodeoxynucleotide (ODN), and recombinant Tp92 protein, and recombinant Tp92 protein+CpG ODN respectively for booster immunization. Enzyme-linked immunosorbent assay(ELISA)was conducted to detect the serum level of anti-Tp92 IgG antibody at week 0, 2, 4, 6, 8 after immunization, the SIgA level in the nasopharyngeal region and vaginal mucosa at week 8 after immunization, as well as levels of interleukin-2(IL-2)and interferon-γ (IFN-γ)in the culture supernatant of rabbit spleen cells at week 8 after immunization, and methyl thiazolyl tetrazolium(MTT)assay was performed to estimate proliferative activity of rabbit splenic lymphocytes in three rabbits from each group. At week 10 after immunization, other 15 rabbits from each group were subcutaneously inoculated with Tp standard strain, and changes of skin lesions at the inoculation site during early-stage infection were observed and recorded. Results At week 8 after immunization, the C2 group showed significantly higher serum level of anti-Tp92 IgG antibody(1.825 ± 0.175), supernatant levels of IL-2 (154.7 ± 14.6)and IFN-γ(277.4 ± 24.4), and proliferative activity of T cells(3.57 ± 0.24)compared with the A2(1.372 ± 0.322, 112.3 ± 13.4, 232.8 ± 25.3, 3.08 ± 0.22, respectively, all P<0.05), B1(0.893 ± 0.297, 76.6 ± 21.5, 165.7 ± 22.6, 2.12 ± 0.14, respectively, all P<0.05)and B2(1.294 ± 0.124, 97.3 ± 18.7, 211.3 ± 24.6, 2.88 ± 0.18, respectively, all P<0.05)groups. In addition, effective immunoprotection was achieved in the C2 group with more production of mucosa-specific SIgA antibody, as well as the lowest Tp-positive rate (6.67%) and ulcer formation rate (6.67%) in skin lesions at the inoculation sites. Conclusion The effective vaccination strategy, namely intramuscular injection of the pcD/Tp92 DNA vaccine for primary immunization followed by nasogastric feeding with mucosal adjuvant CpG ODN combined with recombinant Tp92 protein for booster immunization, can induce the strongest mucosal immune responses and immune protective effects.

Study on correspondence between prescription and syndrome and the essence of phlegm and blood stasis syndrome in coronary heart disease based on metabonomics / 中国结合医学杂志

Studying the essence of a syndrome has been a key challenge in the field of Chinese medicine. Until now, due to limitations of the methods available, the progress towards understanding such complicated systems has been slow. Metabonomics encompasses the dynamics, composition and analysis of metabolites, enabling the observation of changes in the metabolic network of the human body associated with disease. Being from the point of view of the whole organism, metabonomics provides an opportunity to study the essence of a syndrome to an unprecedented level. Phlegm and blood stasis syndrome is the main syndrome associated with coronary heart disease (CHD), which bring difficulties in clinical treatment due to difficulties associated with differentiation of symptoms and signs. The fundamental differences of material between the two also need to be interpreted. The authors consider that we can use the method of combining a disease (in this case CHD) with associated syndromes (phlegm and blood stasis syndrome) to select patients with phlegm and blood stasis syndrome of CHD, and utilize metabonomics to explore the essence of the syndrome by difference analysis of metabolite spectra. Meanwhile, we can study the syndrome in CM, observe the change regularity of metabolism spectra after the treatment of corresponding and non-corresponding prescription and syndrome, in order to validate the material fundament in the progress of syndrome formation and their differences. This will not only have great significance in enhancing the ability to identify syndrome of phlegm and blood stasis in CHD and to establish the clinical curative criteria, but will also offer a new approach of studying the essence for a syndrome using metabonomics.

Fungal infection induced by Cryptococcus neoformans aerosol inhalation in immunosuppressed Balb/c mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the feasibility of inducing fungal infection by Cryptococcus neoformans aerosol inhalation in immunosuppressed Balb/c mice.</p><p><b>METHODS</b>Twenty-four Balb/c mice were randomized into cyclophosphamide (CTX) group and control group (n=12). The mice in CTX group were subject to inhalation of Cryptococcus neoformans aerosol prepared using a ultrasonic nebulizer 4 days after intraperitoneal CTX injection, and the control mice received the inhalation only. The leukocyte count and changes in appetite, body weight, and behaviors of the mice were observed after the treatments. On days 3 and 7 after the inoculation, the mice were sacrificed to analyze the fungal counts in brain and lung tissue homogenates and examine the pulmonary pathologies.</p><p><b>RESULTS</b>CTX injection caused lowered appetite and body weight loss in the mice, and significantly reduced the leukocyte counts (2.77∓0.45 vs 8.26∓0.56, P<0.05). At days 3 and 7 after inoculation, the Cryptococci load in the lungs increased obviously in CTX group with a colony-forming unit (CFU) of the lungs of 271.67∓122.22 and 41.67∓0.28, respectively, significantly higher than that in the control group (60.00∓43.36 and 3.00∓5.30, respectively, P<0.05). In CTX group, the CFU was 10.17∓5.42 and 9.17∓6.34 in the peripheral blood and 6.83∓4.92 and 11.00∓5.44 in the brain tissue at days 3 and 7, respectively, whereas no Cryptococci was detected in the peripheral blood or in the brain tissue in the control group. Pathological examination of the lungs revealed destruction of normal alveolar structure in CTX group, with numerous infiltrating inflammatory cells and visible yeast.</p><p><b>CONCLUSION</b>Inhalation of Cryptococcus neoformans aerosol can cause fungal infection in the lungs of immunosuppressed Balb/c mice.</p>

Construction and functional studies of uropathogenic E. coli strains with ompT gene knockout / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the role of ompT gene in uropathogenic E. coli (UPEC) CFT073 strain in urinary tract infection (UTI).</p><p><b>METHODS</b>An ompT deletion mutant (COTD) was generated by λ Red recombineering in the UPEC CFT073 strain, which was characterized by PCR and sequencing. C57B/L6 mouse models of acute UTI with the mutant and wild-type strains were established to compare the colonization abilities of the two strains in the bladder. The adhesion of CFT073 mutant to human unthelial 5637 cells was also investigated in vitro.</p><p><b>RESULTS</b>PCR and DNA sequencing confirmed the loss of ompT gene in the mutant COTD. The in vitro adhesion rate of the mutant strain COTD to 5637 cells was (6.7±2.2)%, significantly lower than that of (8.3±1.9)% of the wild-type strain (P<0.05). In the murine models of acute UTI, the mutant strain showed a mean colonization number of about (17±8)×10⁴ cfu, which was significantly lower than that of (7∓2)×10⁵ cfu of the wide-type CFT073 strain (P<0.05).</p><p><b>CONCLUSION</b>OmpT gene can be involved in the colonization of UPEC in the bladder tissue and plays an important role in the pathogenesis of UPEC-induced UTI.</p>

Preparation and dissolution of solid dispersion of Fructus Schisandrae Chinensis extracts / 中草药

Objective To prepare Wurenchun solid dispersion of alcohol extracts of Fructus Schisandrae Chinensis so as to improve the dissolution of its active compound in vitro.Methods The Wurenchun solid dispersions were prepared with various carriers and drug/carrier ratios by mixing the carrier in alcohol extractive solution of FSC directly,and the apparent solubility and dissolution of deoxyschisandrin in them were tested and compared.Results The apparent solubility and dissolution of deoxyschisandrin of Wurenchun solid dispersion(extracts: polyvinylpyrrolidone(PVP) K30=1∶3) were increased remarkably to 5.06 ?g/mL and 43.2% in water individually,including dispersed and dissolved drug whose particle size is below 0.22 ?m,compared with that of the self-prepared Wurenchun capsules.Conclusion Wurenchun solid dispersion made of PVP K30 can remarkably enhance apparent solubility and dissolution of the active compound in vitro.