Injuries Of Human Vascular Endothelial Cells Induced By Chlamydia Pneumonia And The Protection Of Baicalin

ObjectiveArteriosclerosis (AS) is an important basic pathologic process in cardiocerebrosvascular disease. In order to lower the mortality and disability of cardiocerebrosvascular disease, it is very important to study the reasons, mechanism, treatment and prevention of AS. It is not very clear the reason of AS. Recently, more and more facts indicate that AS is a chronic inflammatory disease. At the beginning of this century, somebody has posed the theory of infection of AS. Chlamydia pneumoniae (CPN) has recently been associated with the development of coronary heart diseases by sero-epidemiological studies and by direct detection of the organism in atherosclerotic tissues. The endothelium plays an important role in regulating vascular blood flow, and it is now apparent that endothelial dysfunction is an important contributor to the pathogenesis of arteriosclerosis. The objective of this study was focus on two aspects:(1)to investigate the harm of human vascular endothelial cells induced by Chlamydia pneumoniae and the moderation of Baicalin;(2)to study the inhibitory effect of Baicalin on the proliferation of CPN. Methodl.We established the cell modal of human umbilical endothelial cell infected Chlamydia pneumoniae. The growth of CPN was proved by way of Giemsa, electron microscope and genus specific FITC-labeled monoclonal antibody.2. We detected the expression of CD54, CD106 and E-selectin by flow cytometry (FCM).3.We evaluated the level of TNF-α , IL-8 IL-1 Soluble CD54(sCD54) Soluble E-selectin(sE-selectin) by enzyme linked immunosorbent assay (EL1SA).4. We evaluated the effect of Baicalin on the proliferation of HUVEC by MTT5.We investigated the expression of Toll-like receptor-2 and Toll-like receptor-4 mRNA by reversetranscription PCR (RT-PCR).6. We evaluated the Toll-like receptor-2 by FCM.7. We observed the expression of sphingosine kinase mRNA by real-time RT-PCR.8. We test the inhibitory effects of Baicalin by calculating the inclusion count of CPN. Results1 .The normal HUVEC has the typical "cobblestone"style,and is Triangle, polygon. After the cells were infected by CPN, morphologic changes were observed, including cell lysis or necrosis, shedding. We also found many abnormal grains in the cultured cells. We proved the capability of CPN to grow in the HUVEC through Giemsa and anti-chlamydia pneumoniae fluorescein stain. The inclusions were seen in the cytoplasm, purple, sesame-like. By fluorescein stain, we could see the EBs. These appeared as bright apple green fluorescent pin-point, smooth-edge, and could be seen against a background of reddish-brown counterstained cells. Through electron microscope, EBs could be seen like a pear. 2.Baicalin could inhibit the proliferation of HUVEC. The inhibition ratio was 70% by lmg/ml, 10% by 0.062mg/ml, 50% by 0.54mg/ml.3.The expression of CD54, CD 106 were increased after stimulation of CPN by 2 fold respectively. And the level of E-selectin was also higher than the normal cell by 1.5 fold. High-dose Baicalin could lower the level of CD54 CD 106. The inhibition ratio was 73% and 81% respectively, whereas it had no effect on the expression of E-selectin. High-dose Baicalin could lower the level of E-selectin induced by TNF- α and had no effect on the it in normal HUVEC.4.The level of TNF-α and IL-8 were increased after stimulated by CPN by 3.0 fold and 1.27 fold. Baicalin in high mid and low-dose could lowed the levels of TNF- α ; Baicalin in high dose couldlower the levels of IL-8.5. The level of soluble CD54 and E-selectin were increased after stimulated by CPN by 2.8 fold and 2.3 fold. Baicalin in high dose could low the levels of soluble CD54, whereas it had no effect on the expression of soluble E-selectin. Baicatin could lower the level of soluble E-selectin induced by TNF-α6. HUVECs infected with CPN had many vacuoles. The inclusion is purple, sesame-like. Baicalin in high dose could lower the inclusion count, and the vacuoles were diappeared in this group. 7.HUVECs were able to express TLR2mRNA, but not TLR4mRNA.CPN could upregulate the expression levels of TLR2mRNA and TLR2 obviously. The high-dose of Baicalin could lower the level of TLR2.8. Normal HUVECs were unable to express sphingosine kinase (SK) mRNA. Sphingosine kinase (SK) mRNA could be found after lh of infection of CPN. The highest level was in 2h. We could not detect the expression of SK mRNA after 4h. Baicalin in high dose could not change the expression of SK mRNA. Baicalin in high dose in cell for 4h before infected CPN could lower the expression of SK mRNA. ConclusionHUVECs would change in morphologic including the lysis, abnormal grains. On the other hand, the expression of CD54 CD106 and E-selectin were increased after stimulation of CPN, the level of TNF-α and IL-8 soluble CD54 and E-selectin were also increased, whereas, the level of IL-1 had no change. CPN could upregulate the expression levels of TLR2mRNA sphingosine kinase (SK) mRNA and TLR2 obviously. Our findings proved that CPN could upregulate the mediators of inflammation, TLR and SKmRNA.Baicalin could antiinflammation through lowing the proinflammatory cytokines, adhesion factors, the expression of SK) mRNA and TLR2. Baicalin could also inhibit the proliferation of CPN. Baicalin could potentially be used as a therapeutic strategy with which to inhibit the expression of adhesion factors, TLR in endothelial cells. Our findings encouraged the application of Baicalin for treatment of...