| BackgroundAtherosclerosis is a systemic disease relating to lipid metabolismdisorder with the characteristics of the intimal lipid deposition, plaqueformation, fibrous hyperplasia and vessels sclerosis of large and mediumsized arteries. Atherosclerosis, as an important pathological basis of manycardiovascular and cerebrovascular diseases, seriously threats to humanhealth, however, the pathogenesis is still unclear. In recent years it isbelieved that the atherosclerosis is a sort of inflammatory disease and theinflammation reaction is accompanying with the occurrence anddevelopment of AS. Thus the inflammation hypothesis shows closerelationship to AS.Interleukin-8(IL-8) is one of the members of chemokine super familywith close relationship to inflammatory diseases. Studies have suggestedthat the chemokines such as IL-8/CXCL8and their specific receptors play a key role in atherosclerosis. Except for macrophages-mononuclear cells, theendothelial cells and vascular smooth muscle cells may also produceIL-8/CXCL8. The proliferation and migration of vascular smooth musclecells (VSMC) is one of the significant steps for the formation ofatherosclerosis.CXCL8K11R/G31P (3-72), a kind of IL-8analogue developed by pointmutations named G31P, can be effective competitive antagonist to combineCXCR1/CXCR2with high affinity, but no biological activities.Objective1. To investigate the regulation mechanisms of IL-8/CXCL8in a micemodel of hyperlipidemia and the preventive and therapeutic effects ofG31P in the hyperlipidemia mice model.2. To explore the regulation mechanisms of IL-8/CXCL8in AS micemodel and the preventive and therapeutic effects of G31P in the AS micemodel.3. To explore the impacts of IL-8/CXCL8on proliferation and migration ofvascular smooth muscle cells (A7r5), and to detect then inhibition of G31Pagainst IL-8/CXCL8induced proliferation and migration in vascularsmooth muscle cell.4. To explore the signal transduction pathways of G31P inhibitingIL-8/CXCL8induced proliferation and migration in of vascular smoothmuscle cell. Methods1. In vivo experiment:(1) To establish the hyperlipidemia mice model:male BALB/c mice (6weeks) were feed by high fat diet (ordinary feed78.8%, lard10%, egg yolk powder10%, cholesterol by1%, and the pigbile salt0.2%salt) for8months, the blood lipid and other biochemicalindicators were tested to determine the success of mice model.(2) Theestablishment of AS mice model: male APOE-/-mice (6weeks) were feedby high fat diet (ditto) for12weeks. The aortic pathology change wasobserved to determine the success of mice model.(3) ELISA detection ofanimal serum IL-8/CXCL8content.(4) The biochemical method detectionof the dynamic changes of serum high density lipoprotein (HDL-C),triglyceride (TG) and total cholesterol (TC).(5) RT-PCR andimmunohistochemical method were adopted to detect the expression of KC,CXCR2, TNF-α, γ-IFN, MMP-2, MMP-9, PCNA and mef2a in aorticblood vessels.2. In vitro experiment:(1) RT-PCR was adopted to detect the expression ofIL-8/CXCL8receptor in vascular smooth muscle cells (A7r5).(2) MTTwas adopted to detect the cell proliferation induced by IL-8/CXCL8and theinhibition of G31P.(3) Cell scratches and Bordern chamber method wasadopted to detect the cell proliferation induced by IL-8/CXCL8and theinhibition of G31P.(4) Immune protein imprinting technology was adoptedto detect the correlation of IL-8/CXCL8and ERKMAPK signal transduction pathways, and the cascade activation of ERKMAPK pathwayby the inhibition of G31P.ResultPart I(1) Weight: there was no difference in the weight of mice in each groupbefore experiment. The mice with high fat diet for one month showedhigher growth rate compared with the control group and significant highgaining rate of weight after the experiment (p<0.01). The G31P treatmentgroup showed significant lower weight gaining rate than the HFD group(p<0.01).(2) Blood Lipid: The blood was taken from inner canthus of the high-fatdiet mice after4months,6months and8months, respectively. The serumlipids including total cholesterol (TC), triglyceride (TG), low densitylipoprotein (LDL-C) and high density lipoprotein (HDL-C) were detected.HFD mice serum TC, TG, LDL-C were significantly higher than thecontrol group and HDL-C was lower than the control group, with statisticalsignificance (p<0.01), which was in line with hyperlipidemia diagnosticcriteria. With the G31P treatment, serum TC, TG, LDL-C levels decreasedsignificantly, while HDL-C levels increased, which suggested that G31Pmay down regulate the blood lipid.(3) After8months of high-fat diet induction, the proliferation andmigration of foam cells and vascular smooth muscle cells could not been seen in mice aorta under light microscope. The smooth muscle cells couldbe observed in the section of vascular endothelial cells in H.F.D mice underelectron microscopy, in which different sizes of fat cells were swallowedby the SMC, indicating early stages of atherosclerosis by the evidence ofthe migration of SMC and transformation of foma cell in high-fat diet micemodel.(4) Increased KC serum content was seen in HFD group with ELISA, butG31P-treated group decreased significantly (p<0.01).(5) RT-PCR resultsshowed significantly high expression of KC, CXCR2, TNF-α, γ-IFN,mef2a in HFD mice aorta tissue compared with the control group. Theexpressions of the above indicators statistically decreased after G31Ptreatment (p<0.01).(6)The immunohistochemical results showed significantly inhibitedexpression of MMP-2, MMP-9, PCNA in aortic tissues of mice in G31Ptreatment group and in hyperlipidemia-induced mice group (p<0.01).Part II(1) Blood Lipid: The blood of12-weeks high-fat diet mice were sacrificedafter taking blood sample from eyeball extraction. The collected serumappears different from control group. It was light yellow and transparent inserum of control, however milky white turbid, chylous-liked and opaque inAS group. The G31P treatment group showed the characters of white,slightly transparent. The serum TC, TG, LDL-C in AS group were significantly higher than the control group, while HDL-C was lower withstatistical significance (p<0.01), which according with hyperlipidemiadiagnostic criteria. The serum TC, LDL-C significantly reduced andHDL-C levels increased with the G31P treatment.(2) Pathological observation: In the control group, continuous, smooth andcomplete intima of the aorta could be observed under light microscopewithout foam cells and plaque formation. The course of smooth muscle wasclear in the aorta membrane layer. The aorta intima of AS and G31P groupsshowed significantly thicker with plaque formation, on which there was alayer of fibrous cap partially rupture visible. Deep to the plaque there werea large numbers of foam cells, cholesterol crystals and a great deal ofamorphous necrotic material that leads to the constriction of lumen, whichindicating atherosclerotic animal model was successfully established. Atthe root of aorta, there was no significant PA/LA ratio difference betweenAS and G31P group but coronary plaque area was reduced in G31P group.And the lumen showed significant stenosis in AS group (p <0.01).(3) KC serum content was measured with ELISA. The KC expressionincreased significantly in AS group compared with G31P-treated group (p<0.01).(4) qRT-PCR results showed that the expression of KC, CXCR2, TNF-α,γ-IFN, mef2a and PCNA were significantly increased in the AS group compared with the control. The expression of those indicators statisticallysignificantly decreased in G31P treatment group (p <0.01).(5)The expression of CXCR2, MMP-2, MMP-9, mef2a and PCNA in aortictissue of AS group was detected in immunohistochemical staining.G31Pmight significantly inhibited the expression of those factors inhyperlipidemia-induced mice (p <0.01).Part III(1) A7r5cells expressed IL-8/CXCL8receptor CXCR2.(2) IL-8/CXCL8could stimulate the proliferation and migration of A7r5cell in a dose-dependent manner.(3) G31P significantly inhibited IL-8/CXCL8induced the proliferation andmigration of A7r5cell.(4) IL-8/CXCL8induced A7r5cells expressed P-ERK. After1h ofIL-8/CXCL8treatment, the peak level of P-ERK expression could besignificantly inhibited by G31P.Conclusion1. G31P can improve the high-fat diet caused hyperlipidemia by loweringblood fat and inhibiting inflammation process.2. G31P might delay the formation of coronary atherosclerosis.3.G31P can inhibit the expression of inflammatory factors and proliferationand migration of vascular smooth muscle cells by competitively combinewith IL-8/CXCL8receptor during the process of atherosclerosis formation. 4. G31P may inhibit proliferation and migration of vascular smooth musclecell induced by IL-8/CXCL8.5. Proliferation and migration of vascular smooth muscle cell induced byIL-8/CXCL8relates to ERK signaling pathways.6. G31P may block cascade activation of ERK pathway to inhibitproliferation and migration of vascular smooth muscle. |
PDF Full Text Request