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ObjectiveTo explore the clinical efficacy of compound Wufengcao liquid （CWL） on tuberculous ulcer and the influence on macrophage polarization. Method① Clinical experiment： A total of 145 patients with tuberculous ulcer who were treated in Nanjing Integrated Traditional Chinese and Western Medicine Hospital were randomized into observation group， control group Ⅰ， and control group Ⅱ according to the random number table method. In addition to the basic anti-tuberculosis chemotherapy， CWL， Kangfuxin liquid， and isoniazid solution （local external application） were respectively used in the observation group， control group Ⅰ， and control group Ⅱ. The treatment lasted 4 weeks for each group. The total effective rate in wound healing， traditional Chinese medicine（TCM） syndrome score， and histopathological morphology of wound were observed and the expression of inducible nitric oxide synthase （iNOS） and arginase-1 （Arg-1） in wound tissue was measured. ② Cell experiment： RAW264.7 cells were cultured in DMEM （10% fetal bovine serum， 1% double-antibody solution） in a cell incubator （37 °C， 5% CO2）. Phorbol 12-myristate 13-acetate （PMA） was used to induce the differentiation of RAW264.7 cells into macrophages. Lipopolysaccharide （LPS） was employed to stimulate polarization of macrophages into M1 type and interleukin-4 （IL-4） to induce the polarization into M2 type. Kangfuxin solution， isoniazid solution， and CWL were respectively applied to the above cell model for 36 h. The cell supernatant was collected and centrifuged. Western blot was used to detect the protein expression of tumor necrosis factor-α （TNF-α）， transforming growth factor-β （TGF-β）， iNOS， and Arg-1， and flow cytometry （FCM） to detect the expression of CD86 and CD206. Result①Clinical experiment： The total effective rate in the CWL group ［98.0% （48/49）］ was higher than that in the control group Ⅰ ［87.5% （42/48）， χ2=3.962， P<0.05］ and control group Ⅱ ［83.3% （40/48）， χ2=6.162， P<0.05］. After 28 days of treatment， compared with control group Ⅰ and control group Ⅱ， CWL decreased the TCM syndrome score （P<0.05） and obviously improved the histopathological morphology of the wound. Immunohistochemistry results showed that the iNOS expression in local focus tissue was lower （P<0.05） and the expression of Arg-1 was higher （P<0.05， P<0.01） in the CWL group than in the control group Ⅰ and control group Ⅱ after 28 days of treatment. ② Cell experiment： Western blot assay showed that the expression of iNOS and TNF-α in LPS group increased compared with that in the M0 group （P<0.01） and the expression in the LPS+ isoniazid group， LPS+ Kangfuxin group， and LPS+CWL group was lower than that in the LPS group （P<0.05）. The expression of iNOS in LPS+Kangfuxin group and LPS+ CWL group was lower than that in the LPS+isoniazid group （P<0.05， P<0.01）， and the expression of TNF-α in LPS+ CWL group was lower than that in LPS+isoniazid group （P<0.01）. The expression of TNF-α in LPS+ CWL group decreased compared with that in the LPS+ Kangfuxin group （P<0.05）. The expression of Arg-1 and TGF-β in IL-4 group was higher than that in the M0 group （P<0.01）， and the expression in the IL-4+isoniazid group， IL-4+Kangfuxin group， and IL-4+ CWL group was higher than that in the IL-4 group （P<0.05）. The expression of Arg-1 and TGF-β in the IL-4+ Kangfuxin group and IL-4+CWL group was higher than that in the IL-4+isoniazid group （P<0.05， P<0.01）， and the expression was higher in the IL-4+CWL group than in the IL-4+Kangfuxin group （P<0.05， P<0.01）. The FCM result showed that the expression of CD86 and CD206 in LPS group and IL-4 group was higher than that in M0 group （P<0.01）. CD86 expression in LPS+isoniazid group， LPS+ Kangfuxin group， and LPS+CWL group was lower than that in the LPS group （P<0.01）. The expression of CD86 in LPS+Kangfuxin group and LPS+ CWL group increased compared with that in the LPS+isoniazid group （P<0.01）， and the expression was higher in the LPS+ CWL group than in the LPS+Kangfuxin group （P<0.01）. CD206 expression in IL-4+ isoniazid group， IL-4+Kangfuxin liquor group， and IL-4+ CWL group was increased compared with that in the IL-4 group （P<0.01）. CD206 expression in IL-4+Kangfuxin liquid group and IL-4+ CWL group was decreased compared with that in the IL-4+isoniazid group （P<0.01）. CD206 expression in IL-4+CWL group was lower than that in the IL-4+ Kangfuxin group （P<0.05）. ConclusionCWL can promote the healing of tuberculous ulcers， and the mechanism is that it inhibits the expression of iNOS， TNF-α， and CD86 and promotes the expression of Arg-1， TGF-β， and CD206， thereby regulating M1/M2 polarization balance.
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Objective To investigate the dynamic expression of cluster of differentiation 47 (CD47) and its ligands signaling regulatory protein α (SIRPα) and thrombospondin-1 (TSP-1) in mice infected with Toxoplasma gondii in the second and third trimesters.. Methods C57BL/6J mice (6 to 8 weeks old) were used for modeling T. gondii infection in the first trimester, and the pregnant mice were randomly divided into the normal control and infection groups, of 10 mice in each group. Pregnant mice in the infection group were intraperitoneally injected with 150 T. gondii tachyzoites on gestational day (Gd) 6.5, while pregnant mice in the normal control group were intraperitoneally injected with the same volume of physiological saline at the same time. The uterine and placental specimens were collected from all pregnant mice on Gd12.5 and Gd18.5, and the pregnant outcomes were recorded. The pathological damages of mouse uterine and placental specimens were observed using hematoxylin-eosin (HE) staining on Gd12.5 and Gd18.5. The relative expression of CD47, SIRPα, TSP-1, surface antigen 1 (SAG1), interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-4 and IL-13 mRNA was quantified in mouse uterine and placental specimens using real-time fluorescence quantitative PCR (qPCR) assay, and the CD47, SIRPα, TSP-1 expression was determined in mouse uterine and placental specimens using immunohistochemical staining. Results As compared with those in the normal control group, the pregnant mice in the infection group showed back arching, bristling, trembling and listlessness during pregnancy, and several mice presented virginal bleeding and abortion. Pathological examinations showed inflammatory cell infiltration, congestion and necrosis in uterine and placental specimens of pregnant mice in the infection group, a higher abortion rate of pregnant mice was seen in the infection group than in the normal control group on Gd12.5 (χ2 = 20.405, P < 0.001) and Gd18.5 (χ2 = 28.644, P < 0.001). qPCR assay showed significant differences in the expression of CD47, SIRPα, TSP-1, SAG1, INF-γ, IL-2, IL-4 and IL-13 genes in mouse placental specimens between the normal control and infection groups on Gd12.5 and Gd18.5 [F′ (F) = 37.511, 29.337, 97.343, 53.755, 67.188, 21.145, 8.658 and 13.930, all P values < 0.001]. Higher CD47, SIRPα and TSP-1 gene expression was quantified in mouse placental specimens in the infection group than in the normal control group on Gd12.5 (all P values < 0.01), and lower CD47, SIRPα and TSP-1 gene expression was quantified in the infection group than in the normal control group on Gd18.5 (all P values < 0.001), while higher SAG1 gene expression was detected in placental specimens of pregnant mice in the infection group than in the normal control group on Gd12.5 and Gd18.5 (both P values < 0.01). In addition, higher INF-γ and IL-2 expression and lower IL-4 and IL-13 expression was detected in mouse placental specimens in the infection group than in the normal control group on Gd12.5 and Gd18.5 (all P values < 0.001), and there were significant differences in the CD47, SIRPα, TSP-1, SAG1, INF-γ, IL-2, IL-4 and IL-13 gene expression in uterine specimens of pregnant mice between the normal control and infection groups on Gd12.5 and Gd18.5 [H(F′ and F) = 14.951, 25.977, 18.711, 48.595, 39.318, 14.248 and 15.468, all P values < 0.01], and higher CD47 and TSP-1 expression was detected in mouse uterine specimens in the infection group than in the control group on Gd12.5 and Gd18.5 (all P values < 0.01); however, no significant difference was found in the SIRPα expression (P > 0.05). Higher SAG1 expression was detected in uterine specimens of pregnant mice in the infection group than in the normal control group on Gd12.5 and Gd18.5 (both P values < 0.01), and higher INF-γ and IL-2 gene expression and lower IL-4 and IL-13 gene expression was found in the placental specimens of pregnant mice in the infection group than in the normal control group on Gd12.5 and Gd18.5 (all P values < 0.001). Spearman correlation analysis showed that the CD47 gene expression correlated positively with IFN-γ (rs = 0.735, P < 0.05) and IL-2 (rs = 0.655, P < 0.05) and negatively with IL-4 (rs = −0.689, P < 0.05) and IL-13 expression (rs = −0.795, P < 0.05) in the placental specimens of pregnant mice in the infection group on Gd12.5, and the CD47 gene expression correlated negatively with IFN-γ (rs = −0.745, P < 0.05) and IL-2 expression (rs = −0.816, P < 0.05) and positively with IL-4 (rs = 0.704, P < 0.05) and IL-13 (rs = 0.802, P < 0.05) in the placental specimens of pregnant mice in the infection group on Gd18.5. Immunohistochemical staining showed mild CD47, SIRPα and TSP-1 expression in uterine and placental specimens of pregnant mice in the normal control group on Gd12.5 and Gd18.5, strong CD47, SIRPα and TSP-1 expression in the placental specimens of pregnant mice in the infection group on Gd12.5 and strong CD47 and TSP-1 expression in the uterine specimens of pregnant mice in the infection group on Gd12.5. Conclusions T. gondii infection in the first trimester may cause abnormal expression of CD47 and its ligands SIRPα and TSP-1 in the maternal-fetal interface of pregnant mice in the second and third trimesters, which may be associated with the immune escape of T. gondii at the maternal-fetal interface.
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Objective To predict the structure and antigenic epitope of the Strongyloides stercoralis serine protease inhibitor 1 (Ss-SRPN-1) protein using bioinformatics tools, and to construct prokaryotic expression plasmids for expression of recombinant Ss-SRPN-1 protein, so as to provide the basis for unraveling the function of the Ss-SRPN-1 protein. Methods The amino acid sequence of the Ss-SRPN-1 protein was downloaded from the NCBI database, and the physicochemical properties, structure and antigenic epitopes of the Ss-SRPN-1 protein were predicted using bioinformatics tools, including ExPASy, SWISS-MODEL and Protean. Primers were designed according to the nucleotide sequences of Ss-SRPN-1, and the Ss-SRPN-1 gene was amplified, cloned and sequenced with genomic DNA extracted from the infective third-stage larvae of S. stercoralis as a template. The Ss-SRPN-1 protein sequence was cloned into the pET28a (+) expression vector and transformed into Escherichia coli BL21 (DE) cells for induction of the recombinant Ss-SRPN-1 protein expression. The recombinant Ss-SRPN-1 protein was then purified and identified using Western blotting and mass spectrometry. Results Bioinformatics analysis showed that the Ss-SRPN-1 protein, which was composed of 372 amino acids and had a molecular formula of C1948H3046N488O575S16, was a stable hydrophilic protein, and the subcellular localization of the protein was predicted to be extracellular. The Ss-SRPN-1 protein was predicted to contain 11 dominant B-cell antigenic epitopes and 20 T-cell antigenic epitopes. The Ss-SRPN-1 gene with a length of 1 119 bp was successfully amplified, and the recombinant plasmid pET28a (+)/Ss-SRPN-1 was constructed and transformed into E. coli BL21(DE) cells. The expressed recombinant Ss-SRPN-1 protein had a molecular weight of approximately 43 kDa, and was characterized as a Ss-SRPN-1 protein. Conclusions The recombinant Ss-SRPN-1 protein has been expressed successfully, and this recombinant protein may be a potential vaccine candidate against strongyloidiasis.
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OBJECTIVE:To prepare Levetiracetam soluble tablets and evaluate its quality. METHODS:The liquidity indexes of raw material(bulk density,tap density,etc.)and the particle size distribution,liquidity indexes,moisture of the intermediate,that was mixed granules,were investigated to establish preparation method of tablet. The content of levetiracetam was determined by HPLC,and quality indexes of soluble tablets were evaluated,such as appearance,disintegration time,the content of main com-ponent. RESULTS:The wet granulation method was established to prepare Levetiracetam soluble tablets. The specification was 100 mg/220 mg of prepared tablets,it was bright in appearance,and disintegration time was less than 1 min;average content of leveti-racetam was 100.1%,and rigidity was 7.5 kg;the friability of prepared tablets was up to the standard. CONCLUSIONS:The for-mulation and preparation technology of Levetiracetam soluble tablets are rational and controllable. The quality indexes of prepared tablets are all up to the requirements.
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OBJECTIVE:To study inhibitory effects in vivo and in vitro of gemcitabine on liver cancer. METHODS:MCF-7 cells and HepG2 cells were treated with different concentrations of gemcitabine solutions(5,10,20,30,50,70 and 90 μmol/L). The absorbance of cells was determined by MTT assay after treated for 24,48 and 72 h. The inhibition ratio and 50% inhibiting concentration(IC50) of cells were calculated. Tumor-bearing mice were established by inoculating 0.2 ml liver cancer H22 cell line via right anterior axillary,and then randomly divided into control group(normal saline)and gemcitabine group(40 mg/kg)with 5 mice in each group. They were given relevant medicine intravenously every 2 days,for 3 times. The changes of body weight and in-hibition ratios were recorded. RESULTS:Gemcitabine can inhibit MCF-7 cells and HepG2 cells,and IC50 of gemcitabine to them were 30 and >90 μmol/L within 24 h respectively,and those of gemcitabine to them were all lower than 5 μmol/L after 48 h and 72 h. There was no statistical significance in body weight of tumor-bearing mice between 2 groups,and inhibitory rate of gemcitabi-ne to H22 cell line was 75.76%. CONCLUSIONS:Gemcitabine can inhibit liver cancer in vivo and in vitro.