Abstract:Aim To explore the role of C-X-C motif chemokine receptor 4 (CXCR4) in the formation of atherosclerosis (As) induced by Chlamydia pneumoniae (C.pn) infection. Methods The As model in mice of ApoE－/－, ApoE－/－＋TLR2－/－ and ApoE－/－＋Toll-like receptor 2 (TLR2)－/－＋AMD3100 induced by C.pn infection was established on the basis of high fat diet. C.pn IgG and IgM antibody levels were detected by ELISA, and C.pn specific DNA was detected by PCR. Lipid deposition and As lesion area in aorta and aortic root were observed by oil red O and HE staining. Serum levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDLC) and high density lipoprotein cholesterol (HDLC) were analyzed by colorimetry, and ELISA was used to measure the contents of serum interleukin-1β (IL-1β) and interleukin-6 (IL-6). Results The ApoE－/－ mice model of C.pn infection was established successfully. Compared with the control group, lipid deposition in aorta and aortic root of ApoE－/－ mice increased by 89.08% and 71.83%, and As lesion area increased by 34.12% after C.pn infection (all P＜0.05). Compared with the C.pn infection group, lipid deposition in aorta and aortic root reduced by 46.16% and 75.73%, and the lesion area of As decreased by 63.37% in the TLR2－/－＋C.pn infection group (all P＜0.05). Compared with the TLR2－/－＋C.pn infection group, lipid deposition in aorta and aortic root decreased by 26.19% and 56.94%, and the lesion area of As decreased by 22.24 % in the TLR2－/－＋AMD3100＋C.pn infection group (all P＜0.05). Compared with the control group, serum levels of TC, TG and LDLC increased by 0.62 times, 1.43 times and 1.34 times after C.pn infection, respectively, while serum contents of IL-1β and IL-6 increased by 4.10 times and 6.00 times, respectively (all P＜0.05). Compared with the C.pn infection group, serum levels of TC, TG and LDLC in the TLR2－/－＋C.pn infection group decreased by 56.96%, 50.41% and 66.64%, and serum contents of IL-1β and IL-6 also decreased by 66.72% and 69.54% respectively (all P＜0.05). Compared with the TLR2－/－＋C.pn infection group, serum levels of TC, TG and LDLC in the TLR2－/－＋AMD3100＋C.pn infection group decreased by 52.18%, 58.56% and 60.61%, and serum contents of IL-1β and IL-6 and reduced by 28.84% and 43.18%, respectively (all P＜0.05). Conclusion CXCR4 enhances the roles of TLR2 in increasing the serum lipid levels and the contents of inflammatory factors, and then participates in the formation of As lesions induced by C.pn infection.

Abstract:Aim To explore the pyroptosis of vascular smooth muscle cell (VSMC) induced by Chlamydia pneumoniae (C.pn) infection and its possible mechanisms. Methods Primary rat VSMC were cultured by explant method. After the model of VSMC infected with C.pn was established, the changes in morphology of VSMC were observed under an inverted phase microscope, the lactic dehydrogenase (LDH) content was detected by the kit, and the expression levels of GSDMD and Caspase-1 were determined by Western blot, the changes in mitochondrial oxidative phosphorylation and the expression of complex-related proteins were measured by quantitative proteomic analysis by tandem mass tag technology and gene ontology. Results Compared with the control group, bubble-like vesicles were found outside the membrane of VSMC after C.pn infection under an inverted phase microscope. After C.pn infection of VSMC for 36 h and 48 h, LDH content increased by 38.92% and 79.54% (P＜0.001), respectively, and the expression of pyropotosis-related protein GSDMD increased by 1.74 times and 1.67 times (P＜0.001). After C.pn infection of VSMC for 48 h, the expression(pro-Caspase-1) and activity(Caspase-1 p12/p10)of Caspase-1 increased by 2.69 times and 3.47 times (P＜0.001), respectively. The mass spectrometry results showed that there were 20 differentially expressed proteins enriched in the oxidative phosphorylation pathway after C.pn infection, and at the same time, ComplexⅠubiquinone oxidoreductase iron-sulfur protein 4 (NDUFS4) decreased significantly. Further Western blot results showed that the expression level of NDUFS4 decreased by 57.5% and 57% (P＜0.001) after C.pn infection of VSMC for 36 h and 48 h respectively. Conclusion C.pn infection may induce VSMC pyroptosis by affecting mitochondrial function through downregulating NDUFS4 expression.

Abstract:Aim To investigate the roles of N-WASP in angiogenesis induced by Chlamydia pneumoniae (C.pneumoniae) infection and its possible mechanism. Methods After proliferation culture, C.pneumoniae infected human vascular endothelial cell (VEC), and immunofluorescence staining confirmed successful infection. The phosphorylation level of N-WASP was detected by Western blot in VEC infected by C.pneumoniae. The effect of N-WASP specific inhibitor Wiskostain on VEC viability was detected by CCK-8. The effect of Wiskostain working concentrations on C.pneumoniae infection rate was detected by immunofluorescence assay. After C.pneumoniae infected VEC with 5 μmol/L Wiskostain pretreatment, capillary tube formation assay was performed to observe the changes of VEC angiogenesis ability. Results Under fluorescence microscope, typical C.pneumoniae inclusions were found in infected VEC cytoplasm. After VEC was infected with C.pneumoniae for 10 and 24 hours, the level of N-WASP phosphorylation was significantly higher than that in the control group. Capillary tube formation assay showed that after VEC was infected with C.pneumoniae for 16 hours, the number of capillary tube node was significantly higher than that in the control group (P＜0.05). After the pretreatment of VECs with Wiskostain, the role of C.pneumoniae infection in promoting the formation of capillary tube node was significantly weakened, and it was almost impossible to form capillary tube structure (P＜0.05). Conclusion C.pneumoniae infection may promote the formation of new blood vessels possibly through N-WASP.

Abstract:Aim To prepare and identify the monoclonal antibodies against recombinant protein of chlamydial protease-like activity factor from chlamydia pneumoniae for clinical diagnosing C.pneumoniae infection. Methods The chlamydial protease-like activity factor recombinant protein was used as antigen for the immunization of female BALB/c mice, and the spleen cells of mice were fused with SP2/0 myelomas. Indirect ELISA and Western blot analysis were used to screen the hybridomas secreting antibodies and then subcloning positive clones were carried out to establish stable cell lines by limiting dilution. Ascites were induced to produce MAbs and their specificity were identified by indirected immunofluorescence (IIF) test and Western blot analysis based C.pneumoniae type strain. The new indirect ELISA was estabilished to examine coronary artery plaques from patients and the results were analyzed by statistical method. Results Four hybridoma cell lines secreted the monoclonal antibodies stably were finally obtained and named as 3F8、7B9、8C4 and 11B5, respectively the subtype of the monoclonal antibodies secreted by 3F8 and 7B9 strain was IgG2b, the other two monoclonal antibodies were IgG1 and their titers in ascites were 1∶28000, 1∶16000, 1∶38000 and 1∶12000, respectively. The Western blot and IIF analysis showed that these monoclonal antibodies have excellent specificity to C.pneumoniae. The results of detecting clinical samples by the new indirect ELISA based on self-produced the monoclonal antibodies had better consistency with the results of PCR kits for C.pneumoniae infection. Conclusion The specific monoclonal antibodies against chlamydial protease-like activity factor recombinant protein of C.pneumoniae were obtained.All the 4 monoclonal antibodies belonged to IgG, which can react with native chlamydial protease-like activity factor from C.pneumoniae. The self-producted monoclonal antibodies are suitable for C.pneumoniae antigen diagnosis to coronary arteriosclerosis patients.

Abstract:AimTo investigate the role of PI3K/Akt in vascular smooth muscle cell (VSMC) migration induced by Chlamydia pneumoniae (C.pn) infection.MethodsThe successful infection of rat VSMCs with C.pn was identified by transmission electron microscope; After VSMCs were pretreated with the specific PI3K inhibitor LY294002, wound-healing assay and Transwell assay were performed to observe the changes in the migration ability of VSMCs；The phosphorylation level of Akt was detected by Western blot.ResultsThe typical C.pn elementary bodies were observed in the infected VSMCs under the transmission electron microscope; The migration ability of VSMCs infected with C.pn was enhanced and significantly higher than that of control group at 24 h postinfection in the cell migration assay (p<0.05); Western blot results showed that the phosphorylation level of Akt was up-regulated and also higher than that of control group at 24 h after infection（p<0.05）.The effects of C.pn infection on the VSMC migration and the phosphorylation level of Akt in the VSMCs were significantly inhibited by the specific PI3K inhibitor LY294002.ConclusionC.pn infection may promote VSMC migration via activation of PI3K/Akt.

Abstract:Aim To observe the effects of Chlamydia pneumoniae infection on migration of endothelial cells,to investigate the interventive effect of Berberine on Chlamydia pneumoniae induced-endothelial cell migration and to explore its possible mechanism. Methods Endothelial cells pretreated with Berberine at different concentration(0,100,150 and 200 mmol/L) were infected by Chlamydia pneumoniae AR-39 in vitro.At 24,48 and 72 h after Chlamydia pneumoniae infection,the wound healing assay and Transwell assay were performed to observe the effects of Berberine on migration of endothelial cells.The mRNA expression and enzymatic activity of PI3K were detected by RT-PCR and ELISA at the corresponding time point. Results Migration of endothelial cells,the mRNA expression and enzymatic activity of PI3K increased significantly at 24,48 and 72 h after Chlamydia pneumoniae infection in comparison with the control group(P<0.01).Migration of endothelial cells,the mRNA expression and enzymatic activity of PI3K decreased markedly after administration of the PI3K inhibitor of LY294002 compared with the Chlamydia pneumoniae infection group(P<0.01).Migration of endothelial cells,the mRNA expression and enzymatic activity of PI3K decreased markedly after administration of the middle-dose and high-dose Berberine compared with the Chlamydia pneumoniae infection group(P<0.01),and migration of endothelial cells was positively correlated with the mRNA expression(r1) and enzymatic activity of PI3K(r2)(r1=0.841,r2=0.832,P<0.01). Conclusions Chlamydia pneumoniae may induce endothelial cell migration via the activation of PI3K.Berberine can antagonize Chlamydia pneumoniae induced-endothelial cell migration possibly through down-regulating the PI3K mRNA expression and inhibiting the activation of PI3K.

Abstract:Aim To investigate the mechanisms of chlamydia pneumoniae (Cpn)-induced human monocytic cell line (THP-1)-derived foam cell formation, the expression of scavenger receptor A (SR-A1) and CD36 were examined. Methods THP-1-derived macrophages were incubated with or without increasing concentrations of Cpn (1×105 to 1×106 IFU) for 0 to 72 h. Lipid droplets in cytoplasm were observed by oil red O staining. The contents of intracellular cholesterol ester were detected by enzyme-fluorescence. The expression of SR-A1 and CD36 at mRNA and protein levels were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western-Blot, respectively. Results Higher concentrations of Cpn infection (5×105 and 1×106 IFU) for 48 h result in the large accumulation of lipid droplets and the ratio of cholesteryl ester to total cholesterol was much higher than 50% in THP-1-derived macrophages when co-cultured with low density lipoprotein (LDL). Although Cpn infection had no effect on CD36 mRNA and protein expression, it up-regulated the expression of SR-A1 mRNA and protein in concentration-and time-dependent manner in THP-1 macrophages when co-cultured with LDL. Conclusions Cpn induces THP-1-derived foam cell formation by up-regulating the expression of SR-A1, which may provide a new evidence for the development and progression of atherosclerosis initiated by Cpn infection.

Abstract:Aim To explore the relation between serum levels of C-reactive protein(CRP) and soluble intercellular adhesion molecule-1(sICAM-1) and Chlamydia pneumoniae(Cpn) infection and coronary heart disease(CHD).Methods Serum levels of CRP,sICAM-1 and IgG,IgM antibodies to Cpn were detected by enzyme-lined immunosorbent assay(ELISA) in 60 patients with CHD and 40 healthy subjects.These patients were divided into four groups:acute myocardial infarction(AMI),unstable angina pectoris(UAP),old myocardial infarction(OMI) and stable angina pectoris(SAP).Results Cpn-IgG positive rates and levels in patients with CHD were significantly elevated compared with control group(p<0.01);Cpn-IgG positive rates between each group werent different significantly(p>0.05);Cpn-IgG levels in AMI were higher than in OMI,UAP and SAP(p<0.05);Cpn-IgM positive rates in patients with CHD werent different compared with control group(p>0.05).The levels of CRP and sICAM-1 in patients with CHD were significantly elevated compared with control group(p<0.01);the levels of CRP and sICAM-1 in AMI were significantly higher than in UAP,OMI and SAP(p<0.01);The levels of CRP and sICAM-1 in UAP were higher than in SAP.There was correlation between the levels of CRP,sICAM-1 and Cpn-IgG in patients with CHD(p<0.05).Conclusion The levels of CRP and sICAM-1 may reflect the severity of CHD at some degree.There is correlation between Chlamydia pneumoniae infection and CHD.Both inflammation and infection are revelant to CHD.

Abstract:Aim To investigate the effect of C.pneumoniae infection on the expression of peroxisome proliferator-activated receptor γ(PPARγ),nuclear factor-κB(NF-κB) and activated protein-1(AP-1) in aortic endothelial cell in hyperlipidemia mice. Methods Forty-eight,8-week-old female C57BL/6J mice were divided into 4 groups: control group,infected group,atherogenic diet group and infected atherogenic diet group(each twelve).14 weeks later,the expression of PPARγ,P50(subunit of NF-κB) and cFos(subunit of AP-1) was determined by indirect immunofluorescence in the aortic endothelial cell.Slides of aortic sinus were prepared by cryosection,and stained with Sudan Ⅳ for examination of atherosclerotic plaque.The score of atherosclerotic plaque was determined by microscopy. Results The score of atherosclerotic plaque in infected group was not increased,while it was significant higher in atherogenic diet group and infected atherogenic diet group(p<0.01),still the score in infected atherogenic diet group was higher than in atherogenic diet group(p<0.01).The expression of PPARγ,NF-κB and AP-1 in endothelial cell in aortic sinus was upregulated in infected group,atherogenic diet group and infected atherogenic diet group,in comparison with that in control group(p<0.05).There was no significant difference among infected group,atherogenic diet group and infected atherogenic diet group. Conclusion The inflammatory process was already initiated in the aortic endothelial cell in C57BL/6J mice at the early stage of Chlamydia pneumoniae infection and hyperlipidemia.Chlamydia pneumoniae infection alone would not accelerate the process of atherosclerosis.But Chlamydia pneumoniae infection could accelerate the process of atherosclerosis.

Abstract:Aim To investigate the relationship between atherosclerosis(As) and inflammation and the effect of azithrumycin on As rabbits. Methods 45 rabbits were divided into control group,high lipid group and azitheromycin group.The latter 2 groups were made as modle by high lipid feeding [cholesterol 1.5 g/(kg·d)] and vascular intimal injury.Azithromycin group were given azithromycin 30 mg/(kg·d) 3 days 4weeks.High sensitivity C-reactive protein(hs-CRP),Chlamydia pneumoniae immunoglobulin G(CPIgG) were detected at 0,4th,8th,and 12th week.The damaged segments of abdominal artery were harvested for morphometry and reverse transcription polymerase chain reaction evaluated the mRNA expression of vascular cell adhesion molecule-1(VCAM-1). Results High-lipid As rabbit had higher level of hs-CRP(212.8±5.3 g/L),CPIgG(2.03±0.08 g/L) and mRNA expression of VCAM-1(1.102±0.108) than azithromycin group(hs-CRP: 123.9±2.1 g/L,CPIgG: 1.40±0.09 g/L,VCAM-1 mRNA: 0.978±0.135).Azithromycin could reduce the levels of these inflammation indexes. Conclusion As had relationship with inflammation,azithromycin could inhibit inflammation and inhibit As.