| Objective:To explore regulating effect of TLR4on intimal hyperplasia of vein graft viaConstruction of TLR4siRNA recombinant plasmid and transfection of rat vein graftbypass. To study the Effects of TLR4on inflammatory cytokines IL-1β、IL-6、TNF-a and the inhibitory effect of ICAM-1and VCAM-1expression. We illuminatedthe effects of TLR4gene silencing on the expression of Prx1induced cellproliferation, migration and invasion ability.Methods:The external jugular vein to carotid artery grafts model in rat was established.Four hundreds and fifty rats were randomly divided into five groups: theTLR4-siRNA solution group (n=90), the scramble siRNA group (n=90), theTLR4-siRNA fibrin glue (FG) group (n=90), the TLR4-siRNA solution+FG group(n=90), and the control group (n=90). The rats were sacrificed on day1,day3,week1,week2and week4after the operation (6rats per time point). The real time-PCRwas used to detect the IL-1β, IL-6, TNF-a, TLR4mRNA expression levels in samplesharvested on day1, day3, week1，week2and week4. The expression of Prx1in ratsof intimal hyperplasia was determined by western blot. Retrovirus infection techniquewas employed to up-regulate the expression of Prx1in VSMC cells. The cellproliferation and viability were measured by MTT assay. Transwell and woundinghealing assay were used to detect the cellular invasion and metastasis of VSMC cells.Then a recombinant plasmid for TLR4RNAi was constructed and transfected into theVSMC cell lines with Prx1stable over-expression. VSMC proliferation and cellviability were determined by MTT assay. The effect of TLR4RNA silencing onmetastasis of VAMCs cells was assayed by wound healing assay.Results:(l) Compared with normal veins, the neointimal thickness of vein grafting were significantly higher in the graft samples and peaked on the seventh day. Comparedwith the scramble siRNA group and control group, the neointimal thickness of veingrafting was markedly decreased in TLR4-siRNA solution group, TLR4-siRNA FGgroup and TLR4-siRNA solution+FG group.(2) Compared with normal veins, IL-1β,IL-6, TNF-a, ICAM-1, VCAM-1and TLR4mRNA and protein expression levelswere significantly higher in the graft samples within1week after surgery. Comparedwith the scramble siRNA group and control group, the IL-1β, IL-6, TNF-a, ICAM-1and VCAM-1and TLR4mRNA and protein level of grafted veins were markedlydown-regulated and decreased nearly to the baseline level in TLR4-siRNA solutiongroup, TLR4-siRNA FG group and TLR4-siRNA solution+FG group.(3) Comparedwith the TLR4-siRNA solution group, the RNA interference effect of TLR4-siRNAFG group and TLR4-siRNA solution+FG group. The treatment method did notinhibition the effects of TLR4siRNA on IL-1β、IL-6、TNF-a、ICAM-1and VCAM-1.Expression of Prx1was increased in rats of intimal hyperplasia compared withcontrol group. The numbers of cells penetrating through the membrane were countedin the transwell assays. The number of cells penetrating through the membrane ineach filed were150±17in stable Prx1cell lines, significantly higher than40±5incontrol groups. Also the average migration distance of VSMC cells with Prx1stableover-expression was significantly higher than control groups. The number of survivalcells in stable Prx1cell lines were respectively (5.625±0.1)×105,(8.9±0.737)×105,(10.635±0.065)×105in the1st,2nd and the3rd day, which present significantlyhigher than those in control group cells (3.0±0.025)×105,(4.1±0.035)×105and (5.06±0.023)×105. The result shows over-expression of Prx1promotes the cellsproliferation, metastasis and invasion of VSMC cells. After the TLR4-RNAirecombinant plasmid transfection, the average migration distance of VSMC cells withPrx1stable over-expression was significantly decreased than control groups. Thenumber of survival cells in TLR4-RNAi and Prx1co-transfected VSMC cell lines wasrespectively (3.824±0.43)×105,(5.395±0.6)×105and (6.456±0.28)×105,significantly lower than that in stable Prx1cell lines (5.825±0.12)×105,(9.1±0.5375)×105and (10.735±0.075)×105. Gene silencing of TLR4was investigated toinhibit the cell proliferation and metastasis of VSMC cells. Conclusions:These results imply that an acute inflammatory response emerged in the graftvein that grafted into the artery. Furthermore, local down-regulation of TLR4in thegraft vein can inhibit neointimal hyperplasia and VSMC proliferation bydown-regulating pro-inflammatory factors expression and alleviating inflammatoryresponse in the grafted veins, suggesting that TLR4may have a positive regulatoryrole in graft vein stenosis. Over-expression of Prx1promotes the cell proliferation,metastasis and invasion of VSMC cells. Gene silencing of TLR4was investigated toinhibit the cell proliferation and metastasis of VSMC cells. These results revealed thatTLR4and Prx1can be used as new targets for diagnosis, therapy and prognosis ofvascular intimal hyperplasia. |
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