Objective:To investigate the production of cytokines from macrophages induced by Treponema pallidum membrane proteins Tp0971.Methods: The Tp0971 was amplified by PCR from a preparation of T.pallidum genomic DNA and then sub-cloned into the prokaryotic expression vector pET28a(+)to construct the recombinant plasmid pET28a(+)/Tp0971.The recombinant plasmids were transfected into E.coli Rosseta strain to express recombinant protein Tp0971 by IPTG induction.The expression products were purified by Ni-NTA affinity chromatography,and the concentration was determinated by BCA method.Detoxi-Gel was used to remove endotoxin contamination in during the protein preparation.After induced by PMA,cells were incubated with various concentrations of recombinant proteins Tp0971.The expression levels of IL-8,IL-1β and IL-6 were detected by ELISA.Cells were pretreated with anti-TLR2 antibody or TLR2 siRNA,or pyrrolidine dithiocarbamate,an inhibitor of NF-κB,for evaluation of the role of TLR2 and NF-κB in the production of cytokines by ELISA.Results: Tp0971 gene were amplified successfully by PCR,and the recombinant plasmids were confirmed by enzyme digestion and sequencing.SDS-PAGE results showed three recombinant proteins were expressed as the soluble with a relative molecular weight of 29 kD.0.5-10 μg/ml of Tp0971 could stimulate macrophages to produce IL-8,IL-1β and IL-6 dose-dependently.After cells were pretreated with siRNA or neutralizing antibody targeting TLR2 or the PDTC,the Tp0971 induced protein expression levels of proinflammatory cytokines were significantly reduced in macrophages.Conclusion: Tp0971 induces macrophages to produce proinflamatory cytokines via TLR2/NF-κB pathway.

Objective:To observe the molecular mechanism involved in expression of hemeoxygenase -1 (HO-1) induced by a macrophage-activating lipopeptide-2 (MALP-2).Methods:THP-1 cells were cultured in vitro and stimulated by MALP-2 for 12 h, expression of HO-1 was detected by Western blot .TLR2 and TLR6 neutralizing antibodies incubation , dominant negative plasmids transfection were used to assess the functional of TLR 2,6 in mediating HO-1 expression.Phosphorylation of c-Src and Akt were detec-ted by Western blot, and c-Src siRNA and PI3K inhibitor LY294002 were used to investigate the role of c-Src and PI3K in HO-1 ex-pression.Results:MALP-2 induced c-Src phosphorylation , and TLR2 and TLR6 neutralizing antibodies , or their dominant negatively plasmids could abrogate this effect .In addition, siRNA of c-Src could decrease the phosphorylation level of Akt , and the PI3K inhibi-tor could inhibit HO-1 expression.Conclusion: MALP-2 can induce THP-1 cells expression of HO-1 through TLR2,6/c-Src/PI3K pathways .

Objective To investigate whether macrophage-activating lipopeptide-2 ( MALP-2) in-duces the expression of heme oxygenase-1 ( HO-1 ) in THP-1 cells and its possible mechanism .Methods Human monocyte cells THP-1 were cultured in vitro and then were incubated with various concentrations (0, 0.01, 0.1, 1.0 or 5.0 ng/ml) of MALP-2 for 16 h, or were stimulated by 5.0 ng/ml MALP-2 for different length of time (0 h, 4 h, 8 h, 12 h, 16 h or 24 h).The expression of HO-1 at mRNA and protein levels were detected by real-time PCR analysis and Western blot assay .The enzyme activity of HO-1 was detected by colorimetric analysis.THP-1 cells were pre-incubated with 30 μmol/L of SB203580, PD98059 and SP600125 for 30 min and then were cultured with 5.0 ng/ml MALP-2 for 16 h to investigate the role of mito-gen-activated protein kinases (MAPKs) signaling pathway in HO-1 production.After incubating THP-1 cells with 5.0 ng/ml MALP-2 for different periods of time, NF-E2-related factor 2 (Nrf2) protein was detected by Western blot assay to study the effects of Nrf2 pathway on MALP-2-induced HO-1 expression.Nrf2 and HO-1 proteins were measured by Western blot assay after transfecting THP-1 cells (1×106/well) with Nrf2 siRNA at a final concentration of 100 nmol/L.Results MALP-2 enhanced the expression of HO-1 at mRNA and protein levels as well as the enzyme activity of HO-1 in THP-1 cells in a concentration-dependent manner.The expression of HO-1 protein induced by MALP-2 was significantly inhibited by 30 μmol/L MAPKs specific inhibitors ( SB203580 , PD98059 and SP600125 ) .MALP-2 induced Nrf2 translocation at a concentration of 5.0 ng/ml.The expression of Nrf2 and HO-1 proteins were significantly decreased in Nrf 2 siRNA-transfected THP-1 cells.Conclusion MAPKs and Nrf2 signaling pathways were involved in the MALP-2 induced HO-1 expression .

Objective To investigate whether macrophage-activating lipopeptide-2 ( MALP-2) in-duces the expression of hemoxygenase-1 ( HO-1 ) in THP-1 cells and to further elucidate its possible regulatory mechanism for a better understanding of protective response upon mycoplasma infection .Methods THP-1 cells were cultured in vitro and stimulated by MALP-2 at different concentrations for 12 h.THP-1 cells were incubated with TLR 2 or TLR6 neutralizing antibodies , or transfected with their dominant negative plasmids to evaluate the effects of TLR 2 and TLR6 on HO-1 expression .Phosphorylation of Akt was detected by Western blot.PI3K inhibitor LY294002 was used to investigate the role of PI3K in HO-1 expression.Im-munofluorescence and electrophoretic mobility shift assay ( EMSA ) were performed to observe the nuclear translocation and DNA-binding activity of nuclear factor Nrf 2.Small interfering RNA ( siRNA) was used to silence the genes encoding Nrf2 and HO-1.Cobalt protoporphyrin (CoPP), an inducer of HO-1, was used to treat THP-1 cells.The expression of HO-1 was detected by Western blot .The secretion of TNF-αand IL-1βby THP-1 cells were measured by ELISA .Results MALP-2 induced the expression of HO-1 in THP-1 cells.However, the expression of HO-1 was inhibited by TLR2 and TLR6 neutralizing antibodies and expres-sion of their dominant negative plasmids .Moreover, PI3K pathway was activated by MALP-2, and with the use of PI3K inhibitor, the expression of HO-1 was decreased.The translocation of Nrf2 to the nucleus and itsDNA-binding activity were enhanced by MALP-2, but were inhibited by the treatment of PI3K inhibitor.Theexpression of HO-1 was significantly down-regulated upon the interference of Nrf2 gene expression withsiRNA.Silenced expression of HO-1 increased the level of TNF-αand IL-1β, while CoPP treatment decreasedthe secretion of MALP-2-induced cytokines.Conclusion MALP-2 might induce the expression ofHO-1 in THP-1 cells through TLR2,6/PI3K/Nrf2 pathways.The expression of HO-1 could negatively regulatethe hyper-secretion of cytokines.

Objective To investigate the relationship between the composition of vaginal microbiota and the course of cervical precancerous lesion.Methods A total of 64 vaginal swabs were collected from 22 healthy women, 18 CINⅠ patients and 24 CINⅡ/Ⅲ patients who visited Obstetrics and Gynecology of the Second Xiangya Hospital of Central South University during July 2014 and July 2015.The Bacterial genomic DNA was extracted and the V3 and V4 hypervariable regions of 16S rRNA were amplified and high-throughput sequenced.The abundance and composition of vaginal microbiota were analyzed by Uparse, Mothur and LefSe statistical software.Results There was no significant difference in Alpha diversity index between CINⅡ/Ⅲ group(Chao:63±32;ACE:72±38;Simpson:0.70±0.27;Shannon:0.70±0.63) and control group ( Chao:48±24;ACE:54±25;Simpson:0.71±0.27;Shannon:0.65±0.58)(W=192,P=0.11;W=189,P=0.10;W=281,P=0.72;W=241,P=0.62).The ACE(85±37) and Chao(66±25) values of CINⅠgroup were significantly different from those of the control group (ACE:54±25;Chao:48±24)(W=99,P=0.006;W=113,P=0.02).At the phylum level, 78.69%(309 020/392 722) of the vaginal microbiota in the control group was Firmicutes, 16%(62 846/392 722) was Actinobacteria.Firmicutes was reduced to 64.86%(208 422/321 318) and Actinobacteria increased to 27.71%(89 040/321 318) in CINⅠgroup.The composition of vaginal microbiotain in CINⅡ/Ⅲ group was similar to those of control group.At the genus level, the composition of vaginal microbiota were similar between CINⅡ/Ⅲ group and control group, with Lactobacillus as predominant genus[71.81%(307 658/418 424)], Gardnerella[12.91%(55 299/428 424)], others such as Prevotella, atopobium were less.In the CINⅠ group, the abundance of Lactobacillus was decreased to 56.26%(180 787/321 318), Gardnerella was increased to 19.62%(63 057/321 318), and Listeria was increased to 7.7%(24 746/321 318).The composition of vaginal microbiota in the most samples was classified as CSTⅢ and CSTⅠ, with Lactobacillus inersand and Lactobacillus crispatus were dominant respectively.There was no significant difference in the composition of vaginal microbiota between the three groups(χ2=2.72, P=0.949).LEfSe analysis showed that the abundance of bacteria in CIN group and control group were varied.At the genus level, there were significant differences in the abundance of Geobacter, Atopobium and Ureaplasma (P<0.05, P<0.05, P<0.01, respectively).At the species level, there was significant difference in the abundance of Ureaplasma urealyticum serotype 9 (P<0.01).Conclusion The diversity and the composition of vaginal microbiota were similar between CIN patients and healthy women, but the abundances of some bacteria were varied, with Ureaplasma increased in patients with CIN.

Objective: To study the prevalence and antimicrobial susceptibility of Ureaplasma urealyticum (Uu) and Mycoplasma hominis (Mh) in Changsha, and provide laboratory evidence for clinical drug use. Methods: A retrospective study was conducted to analyze 53 006 specimens of suspected genital mycoplasma infection in Xiangya Second Hospital of Changsha District and Hunan Wangwang Hospital from 2010 to 2017, and to analyze the infection rate and drug resistance rate of Uu and Mh. Results: From 2010 to 2017, a total of 53 006 specimens were detected, where there were 16 830 cases of Uu infection, the infection rate was 31.75%; 2 471 cases of Mh infection, the infection rate was 4.66%; and 1 071 cases of Uu and Mh mixed infection, the infection rate was 2.02%. Male Uu infection rate was 19.48%(5 989/30 749), which was lower than the female infection rate 48.71%(10 841/22 257) (χ²=5 091, P<0.001); male Mh infection rate was 3.16%(973/30 749), lower than female infection rate 6.73%(1 498/22 257) (χ²=369,P<0.001). The population of genital mycoplasma infection is concentrated between 20 and 40 years old, accounting for 71.76% (12 077/16 830). The drug resistance rates of Uu and Mh to doxycycline and minocycline were less than 2%, while the drug resistance rate to quinolones was higher; The resistance rate of Uu to macrolide antibiotics such as erythromycin, josamycin and clarithromycin were less than 2%, while the resistance rate to azithromycin, erythromycin and roxithromycin were higher, 29.74%(5 006/16 830) and 53.74%(9 045/16 830), respectively, and the resistance rate of Mh to macrolide antibiotics (except josamycin) was higher than 90%.Between 2010 and 2017, a gradually increasing resistance of ureaplasmas to azithromycin, from 3.81% (46/1 206) in 2010 to 53.15% (1 503/2 828) in 2017, and decreasing resistance to gatifloxacin and thiamphenicol were observed, from 76.78% (926/1 206) and 60.28% (727/1 206) in 2010 decreased to 34.23% (968/2 828) and 37.87% (1 071/2 828) in 2017, respectively. The resistance rate of Mh to gatifloxacin and thiamphenicol were decreased, from 68.93% (122/177) and 41.81% (74/177) in 2010 to 53.54% (159/297) and 21.21% (63/297) in 2017, respectively. Conclusions Doxycycline, minocyclinum and josamycin are good treatment options for genital mycoplasma in Changsha. The resistance rate of Uu to azithromycin is increasing, suggesting that the abuse of azithromycin is present in Changsha, and indicating that better management of antibiotics is necessary.