| Objective: Atherosclerosis (AS) resulting cardiovascular and cerebrovasculardiseases are the strong killers of human health, which are chronic inflammatory diseasewith multiple contributing factors. The incidence rate in China was increased every year.Hyperlipidemia is a prerequisite for atherogenesis. The treatment of hyperlipidemia maybe an important component for the management of atherogenesis in early stages. IL-8,as an inflammatory factor, plays an important role in the development of atherosclerosis.G31P is a high affinity, non-active analogues of human IL-8screening through humanIL-8gene site-directed mutagenesis, which makes it an ideal IL-8receptorantagonist.Recent studies confirmed the G31P can bind to CXCR1/CXCR2effectively,and have a significant effect on treatment of neutrophil-related infections. It has notbeen studied whether this inhibition effect of G31P can be shown in vascular smoothmuscle cells (VSMCs). The aims of the study are the therapeutic efficacy of G31P, anantagonist of IL-8receptor, with a mouse model of hyperlipidemia and the potentialmechanisms of G31P through the vascular smooth muscle cell (VSMC) proliferationand migration in A7r5cell line.Methods: In vivo1）Model Building：30Male BALB/c mice (6-week-old) wererandomly divided into the high fat diet fed group (n=12, high fat diet, treated withnormal saline) and the G31P treatment group (n=12, high fat diet, treated with G31P)and the control group (n=6, standard show fed, treated with normal saline). Mice werefed for eight months, and we determined whether it was a successful model through thelipid prifile analysis.2)The lipid profile and IL-8content analysis: blood was collectedthrough eyeballs in the4th、6th and8th months from each mouse. IL-8levels in theserum were measured by ELISA and serum levels of HDL-C, LDL-C, triglycerid and total cholesterol were determined by selective precipitation or enzymatic method.3)The analysis of chemokine mRNAs levels in mouse root of aorta: We studied theinflammatory factors mRNA expressions in aorta by RT-PCR.4) The determination ofthe proliferation and migration factor expression in aorta: We detected the MMP-2,MMP-9levels related to migration and PCNA levels related to proliferation in the aortaof mice by immunohistochemical method.In vitro1) Cell proliferation was investigated by MTT assay and Flow cytometryassay.2) Cell migration was investigated by Boyden Chamber Assay, wound HealingAssay and Immunofluorescence Assay.Results: In vivo1) Model Building: the lipid profile analysis showed that H.F.D group’s CHO（127.30±11.87mg/dl，165.00±15.56mg/dl，147.40±14.33mg/dl）；TG（234.35±40.51mg/dl，252.63±41.78mg/dl，205.15±30.23mg/dl）；LDL（27.20±7.24mg/dl，34.90±6.51mg/dl，31.50±3.41mg/dl）.Control group’s CHO（72.70±4.77mg/dl，96.00±11.10mg/dl，84.20±10.39mg/dl）；TG（168.94±38.20mg/dl，168.42±16.00mg/dl，152.51±24.12mg/dl）；LDL（20.40±6.34mg/dl，21.50±8.70mg/dl，23.50±4.56mg/dl）.The content of CHO, TG, LDL in H.F.D group was higher than those in controlgroup(p＜0.05) and H.F.D group’s HDL content （60.00±26.32mg/dl,，85.83±17.52mg/dl，60.00±14.70mg/dl）was lower that that in control group（80.00±14.33mg/dl，155.80±11.19mg/dl，97.56±12.87mg/dl）, which showed the model of Hyperlipidemiaof mice fed by high-fat-diet for eight months was a success.2) The therapeutic effects of G31P to hyperlipidemia: G31P treatment group’sCHO （109.10±20.18mg/dl，136.00±26.56mg/dl，126.30±13.35mg/dl）、 TG（185.45±35.85mg/dl，201.24±67.63mg/dl，170.14±40.50mg/dl）、LDL（12.30±6.26mg/dl，22.50±4.56mg/dl，16.15±3.14mg/dl）were lower than those in H.F.D group(p＜0.05) and G31P treatment group’s HDL(100.00±19.63mg/dl，167.50±15.16mg/dl，104.32±24.09mg/dl）was obviously higher than that in H.F.D group(p＜0.05), whichshowed that G31P significantly suppressed hyperlipidermia-induced abnormal lipidprofile. The body weight in G31P group was evidently lower than that in H.F.D group(p＜0.05) and the IL-8content in serum in G31P group(38.04±8.81pg/ml) significantlydecreased compared with H. F. D group.3) The analysis of chemokine mRNAs levels in mouse root of aorta: the mRNAlevels of proinflammatory factor, MIP-2, KC, CXCR2, TNF-α, IFN-γ, in G31Ptreatment group respectively decreased by66.80%,54.41%,86.67%,51.43%and 72.08%(p<0.05) compared with those of the high fat diet fed group;4) The determination of the proliferation and migration factor expression in aorta:the MMP-2, MMP-9related to migration and PCNA related to proliferation in G31Ptreatment group respecitively decreased by45%(p<0.01),33%(p<0.01) and45%(p<0.01) compared with those of the high fat diet fed group.In vitro1) G31P inhibited the VSMCs proliferation by37.5%by MTT assay.2) Flow cytometry assay showed that G31P can reduce the IL-8（100ng/ml）-induced percentage of S phase. The IL-8-induced percentage of S phase in G31Pgroup decreased from23%to17%.3) Wound Healing Assay showed that the IL-8induced VSMCs migration ratewas0.347±0.022, while that in G31P group was0.280±0.029, which was16%lowerthan that of IL-8group (p<0.01). Boyden Chamber Assay showed that the IL-8inducedVSMCs migration cells were16.00±2.74, while the number in G31P group was10.60±3.21, which was33.75%lower than that of IL-8group (p<0.05).4) Immunofluorescence Assay showed that G31P could inhibit the increase ofpseudopodium induced by IL-8.Conclusions：1. Feeding the BALB/c mice with high fat diet for8months, thelipid profile analysis shows that we have succeeded in establishing an animal model ofhyperlipidemia.2. In vivo tests we have showed that the body weight of the mice，thecontents of lipid profile and IL-8in serums, the expressions of MIP-2, KC, CXCR2,TNF-α, IFN-γand the expressions of migration-related MMP-2and MMP-9factors andproliferation-related PCNA factors in the thoracic aorta of mice in G31P treatmentgroup all demonstrate significant differences compared with those of the high-fat dietgroup. G31P has improved hyperlipidemia of the mice in the treatment group.3. In vitrotests we have showed that G31P can inhibit proliferation and migration of vascularsmooth muscle cells (VSMCs). |
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