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Objective:To investigate the role of TcpC in uropathogenic Escherichia coli (UPEC)-induced cystitis in mice and to preliminarily analyze the pathogenic mechanism. Methods:C57BL/6 mice were injected with 10 9 CFU wild-type UPEC CFT073 (CFT073 wt) or tcpc gene-deleted mutant (CFT073 Δ tcpc) from urethra into bladder to construct the mouse model of cystitis. The mice were sacrificed 3 d after infection and the bladders were taken to observe the gross pathological changes. Histopathological changes in bladder tissues were observed after HE staining. Immunohistochemistry was used to detect TcpC in bladder tissues. Bacterial loads in urine samples of UPEC-infected mice were counted by tenfold dilution method, and the presence of tcpc gene in the genomic DNA of bacteria from the bladder and urine samples of CFT073 wt-infected mice was measured by PCR. Real-time quantitative RT-PCR (qRT-PCR) and Western blot were performed to detect the expression of TcpC at mRNA and protein levels in macrophages after CFT073 wt infection. The influence of UPEC strains on the activation of NF-κB signaling pathway in macrophages were determined by Western blot. The levels of proinflammatory factors and the bacterial and cell activity after infecting macrophages with UPEC strains were detected by ELISA, laser confocal microscope and fluorescence microscope, respectively. Results:Compared with the mice with CFT073 Δ tcpc infection, CFT073 wt-infected mice had significantly enlarged bladder and severe neutrophil infiltration and abundant TcpC in bladder tissues. The number of bacteria in the urine of CFT073 wt-infected mice was significantly greater than that of the CFT073 Δ tcpc group. PCR results showed that the bacteria in bladder or urine were CFT073 wt. The expression of TcpC at both mRNA and protein levels in macrophages increased significantly after CFT073 wt infection. Moreover, in CFT073 wt-infected macrophages, the expression of IκBα was promoted and the phosphorylation of p65 and the production of proinflammatory factors were suppressed. TcpC was instrumental in the survival and invasion of CFT073 wt in macrophages. Conclusions:TcpC expression increased significantly in mice with CFT073 wt-induced cystitis. TcpC inhibited the activation of NF-κB signaling pathway and the production of proinflammatory factors in macrophages to improve the survival rate of CFT073 wt, which was closely related to the pathogenesis and immune evasion of UPEC.
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Objective:To investigate whether the hypervirulent Klebsiella pneumoniae (hvKP) induces liver abscess through activating NLRP3 inflammasome. Methods:K1-hvKP and K35-non-hvKP bacterial suspensions were intraperitoneally injected into C57BL/6 mice to establish the models of liver abscess. Human peripheral blood neutrophils were sorted by immunomagnetic beads with CD45 + and Gr-1 + , and the purity was detected by flow cytometry. The concentrations of capsular polysaccharide of K1-hvKP and K35-non-hvKP were detected by total carbohydrate assay kit. The expression of IL-18 and IL-33 by neutrophils at mRNA and protein levels was detected by real-time fluorescence quantitative PCR and ELISA, respectively. The activation of NLRP3 inflammasome in neutrophils was detected by Western blot. Neutrophil extracellular trap formation (NETosis) was observed under confocal laser scanning microscope. Results:The C57BL/6 mice with K1-hvKP infection had significantly serious liver abscess as compared with the K35-non-hvKP-infected mice. The purity of human neutrophils was more than 95%. The concentration of capsular polysaccharide in K1-hvKP was significantly higher than that in K35-non-hvKP. Compared with K35-non-hvKP, K1-hvKP significantly promoted the neutrophils to express IL-18 and IL-33 at both mRNA and protein levels, enhanced the activation of NLRP3 and induced NETosis.Conclusions:This study suggested that hvKP could promote NETosis by activating NLRP3 inflammasome to cause liver abscess.
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Objective:To investigate the signaling pathway of inhibiting macrophage phagocytosis of TIR domain-containing protein encoded by Escherichia coli (TcpC) N-terminal ubiquitin ligase active fragments of uropathogenic Escherichia coli. Methods:Bioinformatics software was used to analyze the amino acid sequences and the function of TcpC N-terminal ubiquitin ligase active fragments as well as the functional sites. PCR was performed to amplify tcpc-330, tcpc-450 and tcpc-510 genes and a prokaryotic expression system was constructed to express the target proteins. The recombinant proteins rTcpC-N110, rTcpC-N150 and rTcpC-N170 were purified by Ni-NTA affinity chromatography. LPS in the recombinant proteins was removed by Detoxi-gel chromatography. The expression of MyD88 at protein and mRNA levels in macrophages incubated with rTcpC-N110, rTcpC-N150, rTcpC-N170 or rTcpC-TIR was detected by Western blot and qRT-PCR. The activation of NF-κB signal pathway and the levels of proinflammatory factors in macrophages incubated with the above TcpC protein fragments were measured by Western blot and ELISA, respectively. Results:Cys12, Trp104 and Trp106 in the N-terminal fragment of TcpC were crucial amino acids in maintaining its ubiquitin ligase activity. The target recombinant proteins rTcpC-N110, rTcpC-N150 and rTcpC-N170 were successfully expressed and purified. After Detoxi-gel chromatography, rTcpC-N110, rTcpC-N150 and rTcpC-N170 extracts were undetectable for LPS. TcpC ubiquitin ligase fragments inhibited the expression of MyD88 at protein level, but not affect its expression at mRNA level in macrophages. LPS-induced phosphorylation of NF-κB signaling pathway-related proteins p50 and p65 was significantly inhibited in macrophages treated with TcpC ubiquitin ligase fragments. Moreover, LPS-induced production of pro-inflammatory factors was also significantly inhibited.Conclusions:The recombinant proteins rTcpC-N110, rTcpC-N150 and rTcpC-N170 could inhibit the expression of MyD88 at protein level and suppress the activation of NF-κB signaling pathway, suggesting that they were closely related to the inhibition of innate immune activity of macrophages.
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OBJECTIVE To study the quality difference of different specifications of Citri Grandis Exocarpium from different origins，and to provide reference for the orderly development of Citri Grandis Exocarpium industry. METHODS Different specifications [ Citrus grandis ‘Tomentos’young fruit ，Citrus grandis （L.）Osbeck young fruits ，exocarp] of 93 batches of Citri Grandis Exocarpium medicinal materials （decoction pieces ）from different origins [ Citrus grandis ‘Tomentosa’or Citrus grandis （L.）Osbeck] were taken as samples. The contents of naringin and rhoifolin in samples were determined by HPLC. Through pheatmap parameters of R language ，heatmap was drawn for the contents of naringin and rhoifolin according to origins and specifications （young fruit and exocarp ）. RESULTS Of 93 batches of samples ，the contents of naringin and rhoifolin were 16.52-214.64 and 1.03-10.96 mg/g，respectively. Among different specifications ，the contents of naringin and rhoifolin in the young fruit were the highest （their average contents were 108.96 and 6.30 mg/g respectively ）. Heatmap analysis of R language content showed that the contents of naringin and rhoifolin in Citri Grandis Exocarpium from origin of C. grandis ‘Tomentosa’were generally higher than those from origin of C. grandis （L.）Osbeck. Of different specifications of Citri Grandis Exocarpium from origins，the contents of naringin and rhoifolin were higher in KTP young fruit relatively. CONCLUSIONS The quality of Citri Grandis Exocarpium from origin of C. grandis ‘Tomentosa’with the young fruit as specification is the best.
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Objective:To preliminarily investigate the effect of circIGF2BP3 on autophagy in photoaged dermal fibroblasts.Methods:Human dermal fibroblasts were isolated from circumcised foreskin tissues from 6 children in the Department of Urological Surgery, Third Affiliated Hospital of Sun Yat-sen University. An ultraviolet A (UVA) -induced photoaged human dermal fibroblast model (UVA radiation group) was established by repeated UVA radiation at a dose of 10 J/cm 2 for 14 consecutive days, and human dermal fibroblasts receiving no treatment served as control group. The photoaged cell model was verified by β-galactosidase staining, Western blot analysis for determining P21 protein expression, and cell counting kit-8 (CCK8) assay for evaluating cell viability. Moreover, Western blot analysis was performed to determine the protein expression of autophagy-related proteins P62, microtubule-associated protein 1 light chain 3 (LC3) -Ⅰand LC3-Ⅱ in photoaged human dermal fibroblasts, and real-time quantitative RCR (qRT-PCR) to verify the differential expression of circIGF2BP3 between photoaged and normal human dermal fibroblasts. Furthermore, circIGF2BP3 was biologically annotated. Some cultured primary human dermal fibroblasts were divided into 4 groups: empty vector group transfected with an empty vector, UVA + empty vector group transfected with an empty vector followed by repeated UVA radiation, circIGF2BP3 group transfected with a circIGF2BP3-overexpressing lentiviral vector, UVA + circIGF2BP3 group transfected with a circIGF2BP3-overexpressing lentiviral vector followed by repeated UVA radiation. Western blot analysis was performed to determine the expression of autophagy-related proteins. Statistical analysis was carried out by using t test, one-way analysis of variance and least significant difference- t test. Results:Compared with the control group, the UVA radiation group showed significantly increased proportions of β-galactosidase-positive cells (61.33% ± 5.78% vs. 6.37% ± 0.32%, t = 9.49, P < 0.01) and P21 expression (1.25 ± 0.03 vs. 1.00 ± 0.05, t = 4.26, P < 0.05), but significantly decreased cell viability (74.33% ± 3.48% vs. 100%, t = 7.38, P < 0.01). Moreover, the P62 expression and LC3-Ⅱ/Ⅰ ratio were significantly higher in the UVA radiation group than in the control group (both P < 0.05). The relative expression of circIGF2BP3 was 0.72 ± 0.04 in the photoaged human dermal fibroblasts, which was significantly lower than that in the normal human dermal fibroblasts (1.00 ± 0.03, t = 5.46, P < 0.01). The P62 expression and LC3-Ⅱ/Ⅰ ratio were significantly lower in the circIGF2BP3 group (0.60 ± 0.01, 0.71 ± 0.01, respectively) than in the empty vector group (1.00 ± 0.02, 1.00 ± 0.01, t = 16.25, 2.75, P < 0.01, < 0.05, respectively), and lower in the UVA + circIGF2BP3 group (1.05 ± 0.02, 2.04 ± 0.05, respectively) than in the UVA + empty vector group (1.31 ± 0.02, 2.72 ± 0.14, t = 10.493, 6.472, respectively, both P < 0.01) . Conclusion:circIGF2BP3 can regulate autophagy in UVA-induced photoaged dermal fibroblasts, which provides a new potential therapeutic target for the prevention and treatment of skin photoaging.
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Objective To understand and determine the biological activity and pathogenicity of metalloendopeptidases encoded by LA2582 and LA2901 genes of Leptospira interrogans(L.interrogans) sero-group Icterohaemorrhagiaeserovar Lai strain Lai. Methods Structures and functions of LA2582 and LA2901 genes were analyzed by using bioinformatic software. Prokaryotic expression systems for expressing the extra-cellular regions of LA2582 and LA2901 genes were generated. The target recombinant expression products, rLA2582 and rLA2901,were extracted by Ni-NTA affinity chromatography. The Azo-casein-hydrolyzingactiv-ity of rLA2582 and rLA2901 was detected by spectrophotometry. Activities of rLA2582 and rLA2901 in the hydrolysis of Dabsyl-Leu-Gly-Gly-Gly-Ala-Edans, a fluorescence-labeling pentapeptide substrate, were de-tected by fluorospectrophotometry,and then the Km and Kcat values were determined. SDS-PAGE and spec-trophotometry were performed to detect the activities of rLA2582 and rLA2901 in hydrolyzing extracellular matrix molecules such as collagen type-Ⅰ (COL1), fibronectin (FN) and Congo red-labeling elastin (ELN). Real-time fluorescent quantitative RT-PCR(qRT-PCR) and Western blot were respectively used to measure the expression of LA2582 and LA2901 genes at mRNA and protein levels after infecting human um-bilical vein endothelial cells(HUVEC) with L. interrogans strain Lai. Results The gene products of LA2582 and LA2901 genes were identified as the signal peptide and matrix metalloproteinase motif HXH-containing Zn2+-dependent Gly-Gly metalloendopeptidases belonging to the M23 superfamily. rLA2582 and rLA2901 did not hydrolyze Azo-casein (Km=126.54 μmol/L, Kcat=4.67/s), but could hydrolyze the pentapeptide substrate (Km=190. 25 μmol/L, Kcat 4. 86/s). rLA2582 and rLA2901 could hydrolyze COL1, FN and ELN. Expression of LA2582 and LA2901 genes at both mRNA and protein levels was signifi-cantly increased after infection of HUVEC with L.interrogans strain Lai(P<0.05). Conclusion The prod-ucts of LA2582 and LA2901 genes of L.interrogans strain Lai are Zn2+-dependent M23 metalloendopeptidas-es, which can hydrolyze multiple ECM molecules and are closely associated with the leptospiral invasiveness.
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Objective To construct a fusion gene (h1a-spaO) encoding H1a-SpaO protein of Sal-monella paratyphi A ( S.paratyphi A) and to express it in prokaryotic expression system , then to further ana-lyze the immunoprotective effects of the expressed protein rH 1a-SpaO.Methods The h1a-spaO fusion gene formed from separate h1a and spaO genes was amplified by PCR using flexible peptide sequence-containing linking primers and then sequenced after T-A cloning.A prokaryotic expression system for expressing h1a-spaO fusion gene was constructed by using the genetic engineering technique .The expressed protein rH1a-SpaO was examined by SDS-PAGE.The antigenicity and immunoreactivity of rH1a-SpaO protein were deter-mined by Western blot assay .The ability of rH1a-SpaO antiserum agglutinating S.paratyphi A strains was detected by micro-Widal′s test.The immunoprotective effects of rH 1a-SpaO against the lethal dose challenge of S.paratyphi A strains were analyzed in a mouse model and that were compared with those by using equal dose of individual recombinant protein H1a and SpaO (rH1a and rSpaO) as the immunogens, respectively. Results The h1a-spaO fusion gene was 100%identical with the individual h1a or spaO gene in nucleotide and amino acid sequences .The constructed prokaryotic expression system could express the recombinant pro-tein rH1a-SpaO with an advantage of high efficiency .rH1a-SpaO protein was able to react with rH 1a or rSpaO antiserum.Moreover, rH1a-SpaO antiserum also could efficiently recognize rH 1a and rSpaO as well as agglutinate Salmonella paratyphi A strains by binding with H-antigen.The immunoprotective rate (93.3%) in mice pre-immunized with 100 μg of rH1a-SpaO protein was significantly higher than that in those pre-immunized with equal dose of rH1a (60.0%) protein or rSpaO protein(53.3%) (P<0.05).Conclusion The recombinant fusion protein rH 1a-SpaO showed more stronger immunoprotective function than the individ-ual rH1a or rSpaO protein , which could be used as an effective antigen for the development of bi -valent para-typhoid A vaccine or typhoid/paratyphoid capsular polysaccharide-protein combined vaccine .