The Impact Of Helicobacter Pylori Infection On Carotid Artery Plaque And The Role Of TLR2in Hp Lipopolysaccharide Promoting Foam Cell Formation

The inflammatory response plays a key role in the incidence and progression ofatherosclerosis, in which infection-induced inflammation and atherosclerosis relationshiphave received increasing attention. The latest studies suggest that Helicobacter pylori (Hp),Chlamydia pneumoniae, cytomegalovirus, herpes simplex virus infection of pathogenicmicroorganisms is one of the important risk factors for atherosclerosis, in which Hpinfection and atherosclerosis sclerosing disease is a hot topic in recent years. However,studies at domestic and international was restricted to observation and description of thephenomena that Hp promote atherosclerosis, lacking of mechanisms study. How Hppromote the development of atherosclerosis mechanism is still unclear. Hp is a type ofGram-negative bacteria, lipopolysaccharide (LPS) is an important pathogenic factor ofGram-negative bacteria. According the theory "atherosclerosis is an inflammatory disease"we conclude that Hp infection and LPS-induced inflammation and immune response mayplay a more important role in the development of atherosclerosis.The study is divided into the following aspects:(1)The body level: ultrasonographyobserve the size, nature and the carotid artery intima-media thickness (CIMT) of plaque incarotid atherosclerosis patients. Collect serum of carotid atherosclerosis patients and detectof Hp antibody and inflammatory factors related to the stability of atherosclerotic;(2) Thepathology level: obtain the atherosclerotic plaques by carotid endarterectomy surgery(CEA). Observe the changes in atherosclerotic plaque structure and expression ofinflammatory cytokines by immunohistochemical stained;(3) The cellular level: StimulateRAW264.7macrophages with Hp LPS, and observe the change of macrophagephagocytosis of oxidized low-density lipoprotein and the release of inflammatory factorsunder the influence of Hp LPS;(4) membrane receptor level: Through the knockoutanimals, clarify the type of Toll-like receptor on macrophages stimulated by Hp LPS. Thisstudy clarified the impact of Helicobacter pylori infection to carotid atherosclerotic plaquestability and the mechanism of Hp LPS promoting atherosclerosis. The stydy not onlyoffered new perspective for pathogenesis of atherosclerosis, but also offered newtheoretical basis and control strategies for stroke prevention. PART Ⅰ The Impact of Helicobacter Pylori Infection on UltrasoundMorphology of Carotid Atherosclerosis and SerumInflammatory CytokinesObjective: In this experiment, we observed the impact of H. pylori infection onarterial atherosclerotic plaque morphology, CIMT and serum inflammatory cytokines, bydetecting arterial atherosclerosis in patients with carotid ultrasound and serum MPO,MMP-9, LP-PLA2dencity, Analysis of Hp infection on atherosclerotic plaque stability,and to explore its possible mechanisms of infection lead to carotid artery atherosclerosisMethods:141cases of carotid artery ultrasonography in our hospital from April2011to September2011prompted atherosclerosis informed consent,85cases were malepatients,56cases of female patients. Line serum Helicobacter pylori (Hp IgG) weredetected in patients with Helicobacter pylori antibody results into the H. pylori-positivegroup and the H. pylori-negative group. Serum Helicobacter pylori antibodies (Hp-IgG)testing, carotid artery intima-media thickness detection, detection of carotid artery plaquemorphology and serum of MPO and MMP-9, LP-PLA2detected.Results:1.29unstable carotid plaque in Hp-positive group, including24soft plaquesand5ulcer plaques;43stable plaques, including flat plaques26and17hard plaques.17unstable carotid plaques in Hp-negative group, including14soft plaques and3ulcersplaques;52stable plaques, including38flat plaques and14hard plaques. WithHp-negative group compared to the Hp positive group of patients with unstable plaqueincidence increased, and there is a significant difference (P=0.0477).2. Observed throughthe carotid artery ultrasound Hp-positive group of patients in each group of bilateralcommon carotid artery CIMT values of the left common carotid artery CIMT value of0.096±0.0013, Hp-negative group, the left common carotid artery CIMT value of0.087±0.0019, positive group than in the negative The left common carotid artery CIMT groupsignificantly thicker (P=0.0003). Hp-positive group right common carotid artery CIMTvalue of0.093±0.0011, Hp-negative group, the right common carotid artery CIMT valueof0.086±0.0021, the positive group than negative group, the right common carotid arteryCIMT was significantly thickened (P=0.0041).3.Serum MPO concentration ofHp-positive group was767.6±11.06ng/ml compared to Hp-negative group,707.2±15.38ng/ml a significant increase (P=0.0017). Serum hs-CRP concentrations ofHp-positive group was4.257±1.102mg/dL, compared with Hp-negative group,4.408± 1.174mg/dl no significant difference.(P=0.0925). Hp-positive serum MPO concentrationof21.40±.2639ng/ml compared to Hp-negative group,19.80±0.4886ng/ml asignificant increase (P=0.0041). Hp-positive serum LP-of PLA2concentration was393.8±4.855ng/ml compared to Hp-negative group366.7±9.113ng/ml, a significantincrease (P=0.0090).Conclusion:1. In this study, confirmed by clinical, atherosclerotic plaque instability beincreased caused by Hp infection.2. Artery atherosclerosis patients serum MPO, MMP-9and LP-PLA2but not hs-CRP increased with Hp infection.PART Ⅱ Helicobacter Pylori infection and Carotid AtheroscleroticPlaque Results Changes of Structure and Expression ofInflammatory CytokinesObjective: In order to clarify Helicobacter pylori infection and carotid atheroscleroticplaque inflammation factors expression changes,this experiment treated surgery pointerpatients with CEA surgery.And we made pathological examination in atheroscleroticplaque tissue, by HE and immunohistochemical staining, to observe the morphology andthe expression of inflammatory factors.Methods:141cases from the first part of the research object,36patients of witchunderwent CEA surgery. Real-Time PCR detection of carotid atherosclerotic plaque withinthe organization of inflammatory factors mRNA, Immunohistochemical detection ofcarotid atherosclerotic plaques tissues, expression of inflammatory cytokines.Results:1.The Real-time PCR results showed that Hp-positive group of MPO mRNArelative GAPDH expression level was3.096±0.2846, significantly higher compared toHp-negative group,1.924±0.2535(P=0.0051); Hp positive MMP-9mRNA relativeGAPDH expression level was2.212±0.3147, significantly higher than Hp-negative group1.406±0.1907(P=0.0472); Hp positive group LP-PLA2mRNA relative GAPDHexpression level was7.013±0.9494, were significantly increased compared to Hp-negativegroup (3.614±0.8607).2., fibrous cap thickness of the Hp positive group was92.10±11.26μm, fibrous cap thickness of the Hp-negative group was130.9±12.43μm. Hpnegative group's fibrous cap thickness was significantly thicker than the positive group (P=0.0269). lipid pool area proportion of Hp positive group was0.6172±0.03054, lipidpool area proportion of the Hp negative group was0.5324±0.03702. lipid pool area increased in Hp-positive group, but the difference was not significant (P=0.0836).3.Hp-positive group of atherosclerotic plates in the LP-of PLA2, MPO and MMP-9expression were significantly increased compared with the Hp negative group.Conclusions:1. Further confirmed, compared Hp negative group, Hp-infected personswere suffering from a greater risk of carotid atherosclerosis.2.Hp infection may enhancingthe atherosclerotic plaque inflammatory response and immune response and increasedcarotid artery atherosclerosis.3.Hp infected with carotid artery atherosclerosis localinflammatory factors increased, thus promoting the formation of carotid atherosclerosis.PART Ⅲ Affection to Macrophages by Helicobacter PyloriLipopolysaccharideObjective: In order to clarify the mechanism of H. pylori infection to promoteatherosclerosis, we intervented vitro cultured macrophages with purified H. pylorilipopolysaccharide. Observed macrophage phagocytosis of oxidized low-densitylipoprotein and release of inflammatory cytokines.Methods: Interference cultured RAW264.7monocyte-macrophage cells with purifedHelicobacter pylori lipopolysaccharide. Observed tha change of macrophage phagocytosisox-LDL ability by fluorescent probes Dil, oil red O staining, enzyme fluorescent. Observerelease of macrophage inflammatory factor by detected IL-6, TNF-alpha mRNA, and at thesame time,we detect TLR2and TLR4mRNA to observe macrophage receptors.Results:1.macrophage fluorescence was significantly increased stimulated by Hp LPS,indicating that the increase of the phagocytosis of ox-LDL. And the macrophagecholesteryl ester (CE) in ox-LDL+Hp LPS group was significantly increased comparedwith ox-LDL group (P=0.0023).2.Real-time PCR results showed that at different times ofHp LPS stimulation of macrophage IL-6and TNF-alpha mRNA trends, the mostsignificant change was2-4hours (P <0.05).3. When macrophages was stimulated bydifferent dilutions of Hp LPS, TLR2receptor mRNA levels is an upward trend (P <0.01),and TLR4mRNA level rise trend was not significant (P>0.05).Conclusion: This study intervent vitro RAW264.7monocyte-macrophage cells bypurified H. pylori lipopolysaccharide.We found out that macrophage phagocytosis ofox-LDL increased, promoting the transformation of macrophages into foam cells, and cause macrophages release IL-6, TNF-alpha increased. Also observed that Helicobacterpylori lipopolysaccharide can promote the increase expression of TLR2receptors onmacrophage cell surface, indicating that H. pylori lipopolysaccharide may promote thetransformation of macrophages to foam cells and release of inflammatory mediatorsthrough TLR2.Part Ⅳ Hp LPS Promote Macrophage Foam Cells Affected by TLR2Gene KnockoutObjective: Intervent peritoneal macrophages of the TLR2gene knock out mice andC57BL/6J mouse by Hp LPS,and detect changes in phagocytosis ox-LDL and TLR2/TLR4,cholesterol metabolism, receptors and inflammatory cytokines expression. Clarify Hp LPSeffect on macrophage receptors and to explore the mechanism of its catalytic macrophagefoam cells.Methods: Lavage peritoneal macrophages from C57BL/6J and TLR2gene knock outmouse, dealed with(Dil-)ox-LDL,(Dil-)ox-LDL+Hp LPS,(Dil-)ox-LDL,(Dil-)ox-LDL+Hp LPS. Observed macrophage phagocytosis of ox-LDL ability by fluorescentprobe Dil, the statutory amount of the enzyme fluorescence. And detect IL-6, TNF-alphaand TLR receptors mRNA of macrophages and cholesterol metabolism in receptordetection macrophage CD36and of ABCG1mRNA.Results:1.It can be seen from the fluorescence imaging that fluorescence intensitydecreased in TLR2-/-cells compared with the C57group, indicating that TLR2-/-mouseperitoneal macrophage phagocytic activity lower than C57mice.2.The results showed thatcholesteryl ester (CE) in macrophages of ox-Hp LPS group increased significantly thanox-LDL group.(P=0.0023).3. Real-time PCR results showed that the IL-6and TNF-alphamRNA of C57mice were significantly increased than the TLR2knockout mice stimulatedby Hp LPS.4. Real-time PCR results showed that ABCG1mRNA decreased, and CD36mRNA levels increase stimulated by Hp LPS, in which LPS+LDL group weresignificantly different with the LDL group (P <0.05).Conclusion:1. Hp LPS can promote macrophage phagocytosis of ox-LDL, andrelease inflammatory mediators through TLR2.2. Hp LPS can promote ABCG1mRNAdecreased, with CD36mRNA levels increased through TLR.3. Hp LPS may activate PPARγ pathway to promote high expression of CD36, increased macrophage phagocytosisof oxidized LDL capacity, while inhibiting LXRα pathway, cause ABCG1low expression,and macrophage cholesterol efflux to reduce both coordination role in promoting thedevelopment of atherosclerosis.