| BackgroundFractalkine(FKN) is a membrane-tethered chemokine that functions as chemokines and adhesion molecules to capture and induce firm adhesion of a subset of leukocytes in a selectin- and integrin -independent manner.Soluble FKN can be released,presumably by proteolysis at a membrane-proximal dibasic cleavage site(Thr-Arg- Arg-Gln),and exhibits an efficient chemotactic activity for monocytes.The novel structure and functions of FKN suggest that it may play a key role in the multistep process of leukocyte trafficking,while raising several issues. Chronic inflammation is one of the mechanism of Atherosclerosis (AS).The migration of leukocyte through intima is an essential biological phenomena of inflammation.Monocytes are important cells for forming the foam cells.The primary step of this is to migrate through intima too.The molecular control of these essential trafficking events requires two broad classes of molecules:various cell adhesion molecules,as well as leukocyte chemotactic factors,the chemokine superfamily.Now we have increasingly more evidence that FKN participate in the formation and development of atherosclerosis(AS).In recent years,increasing evidences has emerged from experimental and epidemiological data that interleukin- 18(IL- 18),a pro-inflammatory cytokinee involved in both innate and acquired immune responses,plays a key role in the inflammatory response that contributes to AS.Baseline circulating IL-18 levels were strongly predictive of future cardiovascular disease.Most past studies suggest that IL-18 function mainly through interferon-γ(IFN -γ),and could not intervene AS without IFN-γ.Recent studies make it clear that IL-18 could also mediate several reactions in a IFN -γindependent manner.Banding of IL-18 and IL-18 receptor enhances the secretion of IL-6,IL-8,ICAM-1 and MMPs.However,it remains unclear whether IL-18 up-regulate the expression of FKN,and participate AS through the chemotactic and adhesive function of FKN.Simvastatin is the 3-hydroxy-3-methylglutanyl coenzyme A(HMG-CoA) reductase inhibitor lipid-lowering agents synthesized from degradation products of aspergillus terreus.It has been used as one of the important medicine for coronary heart diseases.Besides lipid-lowering function,Simvastatin could protect endothelial cells,improve myocardial ischemia,anti-inflammatory anti-thrombosis and stabilize atherosclerotic plaques.However,it is still unclear whether Simvastatin exert its role through effect the expression and chemotactic,adhesive function of FKN.Objective(1)To observe the expression of FKN induced by IL-18 in HUVECs, and to investigate its signal pathway.(2)Using Transwell to observe the chemotaxis of FKN and the effect of IL- 18 on monocyts migration; (3)Using in vitro Flow chamber mimic the vivo fluid dynamic environment to observe the adhesion between HUVECs and THP-lcells mediated by FKN and the effect of IL-18;(4)To investigate the effect of Simvastatine on expression of Fractalk -ine upregulated by IL-18,and the interventional effect on chemotaxis, adhesive function of FKN.Methods(1) HUVECs were cultured and treated with IL-18 of concentrations 100μg/L for 0,1,6,12,24 hours.FKN mRNA expressions were determined by semi-quantitative reverse-transcription polymerase chain reaction(RT-PCR).(2) HUVECs were cultured and treated with IL-18 of 4 different concentrations(5μg/L,25μg/L,50μg/L,and 100μg/L) for 24 hours.We determined mRNA expressions of FKN by semi-quantitative reverse-transcription polymerase chain reaction(RT-PCR).(3) Pretreated HUVECs with PDTC(A specific inhibitor of NF-KB) of 2 different concentrations(10umol/L,30umol/L),then cells were treated with IL-18(100ug/L) for 24 hours,mRNA expressions of FKN were determined by semi-quantitative RT-PCR.(4)Pretreated HUVECs with Simvastatine of 3 different concentrations (1 umol/L,10umol/L,100umol/L),then cells were treated with IL- 18 (100ug/L) for 24 hours.FKN mRNA expressions were determined by semi-quantitative RT-PCR.(5) HUVECs were treated as above 1,3,4 method.The other HUVECs were cultured and treated with IL- 18 of 100 ug/L for 24 hours, then cells were treated with neutralizing antibody of FKN(1 ug/L,5ug/L) for 2 hours.The migration of THP-1 cells mediated by FKN was odserved by Transwell.(6) HUVECs were treated as above 1,3,4 method.The other HUVECs were cultured and treated with IL- 18 of 100 ug/L for 24 hours, then cells were treated with neutralizing antibody of FKN(1ug/L,5ug/L) for 2 hours.The adhesion of HUVECs and THP-1 cells mediated FKN was odserved by In vitro Flow chamber assay.Results(1) HUVECs cultured and incubated with 100ug/L IL- 18 for 1 hour resulted in no difference of FKNmRNA expression as compared with control group(p＞0.05);HUVECs were cultured and incubated with IL-18 of concentrations 100μg/L for 6,12,24 hours resulted in upregulated expression of FKNmRNA,as compared with control group (p＜0.01);(2) HUVECs cultured and incubated with 5ug/L IL-18 for 24 hours resulted in no difference of FKNmRNA expression,as compared with control group(p＞0.05);HUVECs were cultured and incubated with IL-18 of concentrations 25,50,100μg/L for 24 hours resulted in upregulated expression of FKNmRNA,as compared with control group(p＜0.01);(3) Pretreated HUVECs with PDTC of 10umol/L,30umol/L and then incubated with IL-18(100ug/L) for 24h resulted in downregulated expression of FKN mRNA,as compared with the pure IL-18(100ug/L) group(p＜0.01);(4) Pretreated HUVECs with Simvastatin of 1umol/L and then incubated with IL-18(100ug/L) for 24h resulted in no difference of FKNmRNA expression,as compared with the pure IL-18(100ug/L) group (p＞0.05);Pretreated HUVECs with Simvastatin of 10umol/L and 100umol/L and then incubated with IL-18(100ug/L) for 24h resulted in down-regulated expression of FKN mRNA,as compared with the pure IL-18(100ug/L) group(p＜0.01).(5)The chemotactic assay was successful by using Transwell,FKN could chemotaxis the resting THP-1 cells;(6) After up-regulated FKN mRNA expression induced by IL-18 with different concentrations,the chemotactic effect of FKN to THP-1 cells were enhanced significantly,consistent to the expression of FKN mRNA.The chemotactic effect were inhibited if HUVECs pretreated with Simvastatin or PDTC,or treated with neutralizing antibody,as compared with the pure IL-18(100ug/L) group(p＜0.01)(7)Using in vitro flow chamber mimic the vivo fluid environment secessfully,FKN could mediate the adhesion in a Ca2+ independent maner;(8) After up-regulated FKN mRNA expression induced by IL- 18,the adhesion mediated by FKN was enhanced significantly,as compared with control group(p＜0.01).The adhesion were inhibited after HUVECs pretreated with PDTC,or treated with neutralizing antibody,as compared with pure IL-18(100ug/L) group(p＜0.01).Simvastatin with 10umol/ml concentration did not inhibit the adhesion,as compared with the pure IL-18(100ug/L) group(p＞0.05).Simvastatin with concentration of 100umol/L inhibated the adhesion,as compared with the pure IL- 18(100ug/L) group(p＜0.01).Conclusions(1) IL-18 could up-regulate the expression of FKN mRNA in a dose-and time-dependent manner,and this effect might be related to NF-KB.(2) IL-18 could enhance the chemotactic and adhesive effect of HUVECs to THP-1 cells significantly by up- regulation of FKN.(3) Simvastatin could down-regulated the expression of FKN mRNA induced by IL-18,and inhabit the chemotactic and adhesion. |
