The Effect Of TLR4 Down-regulation In Preventing Restenosis And Comparison With Pioglitazone

Objectives:Recent years PTCA and PCI have gradually become the important method for reascularization among patients with coronary heart disease and acute coronary syndrome. However, restenosis(RS) restricts the application and development of the technology. Although the advent of drug-eluting stents(DES) reduced the incidence of RS to some extent, more and more studies have indicated the disadvantage of DES, such as long-term inflammatory state, stent thrombosis and RS. Based on these, we intended to explore whether a better means of preventing RS exists, with which we could combined the traditional protecting approach to further reduce the incidence of RS.Toll like receptor 4(TLR4) is the first recognized membrane receptor from TLR family of mammals, the activation of TLR4 could activate nuclear factor κB(NF-κB), then induce a serious of local inflammatory response, result in the neointimal hyperplasia in lesion. Based on these, we speculated the potential role of TLR4 in RS after vessel injury, while whether TLR4 inhibition could decrease the formation of RS remained uncertain.In our research, we down-regulated the TLR4 gene through RNA interference, transfected the plasmid packed with virus particles into the RS model and human aortic vascular smooth muscle cells(Ha VSMC) stimulated with lipopolysaccharide(LPS). A serious of inflammatory and proliferative molecules expression associated with TLR4 were measured, the inhibition effect on neointimal formation and proliferative, migratory ability were respectively observed to discuss the mechanism of preventing RS via inhibiting TLR4 expression. Besides, the inhibition efficacy of RNA interference on proliferative and migratory ability was compared with different concentrations of pioglitazone.Methods:1. In vivo experiments: according to the preliminary work in our lab, the rabbit model of restenosis was established via carotid artery balloon injury: 50 healthy rabbits were randomly divided into five groups, respectively named the normal group(NC), the shame group(S), the model group(M), negative plasmid transfection group(N), and positive plasmid transfection group(P). Normal control group was just set to be controlled, the shame group received incision of neck area and 30 minutes’ occlusion of right common carotid artery, the model group suffered the balloon injury of right common carotid artery, N group received the same surgeon and injected with 30 ul adenovirus uploaded with negative plasmid in the lesion artery segment for 30 minutes’ local incubation in warm and moist environment, the only difference between P group and N group is the injection substance: 30 ul adenovirus uploaded with si RNA-TLR4 plasmid. The rabbits were all fed normally and executed 14 days after various intervention.(1) the plasma level of TNF-α, IL-6 were detected with enzyme-linked immunosorbent assay(ELISA);(2) the pathologic section of carotid arterial specimens was reserved to proceed HE stain, measure thickness and area of the tunica intima and tunica media, additionally, the immunohistochemisty of tlr4 expression in injured artery from each group was observed.(3) m RNA expression of TNF-α、IL-6 and VCAM-1 was examined in lesion artery specimens from each group;(4) protein level of TLR4, inflammatory factors NF-κB、VCAM-1、MCP-1, and proliferative factors t-AKT、p-AKT、t-m TOR、p-m TOR from individual injured arterial segment were determined by Western blotting.2. In vitro experiments: The Ha VSMC were treated with 1μg/ml LPS respectively at 0 h、6h、12h、24h and 48 h before cells were collected and lysized to determine the time point of the TLR4 peak expression. Design and construct 2 recombinant plasmids targeting TLR4, packaging with lentivirus, then transfect the sh RNA- TLR4-1, sh RNA TLR4-2 and negative control plasmid, which carried with green fluorescent protein, into human VSMC. Transfection success was confirmed through the detection of green fluorescent with inverted microscope. The plasmid with higher inhibition efficiency was regarded as positive group(P), in addition, negative group(N), non-LPS group(NL) and only LPS group(L) were preserved, another three group were respectively pretreated with pioglitazone at 10μmol/L(Pio10), 50μmol/L(Pio50) and 100μmol/L(Pio100), all above groups were stimulated with LPS at 1μg/ml to the peak expression of TLR4 except NL group. The m RNA expression of TLR4 were examined by RT-PCR while protein level of TLR4, NF-κB, VCAM-1, MCP-1 were determined by Western blotting, expression of t-AKT, p-AKT in these groups after 1 hour’s stimulation were also determined; the proliferation state of VSMC from each group was determined by MTT colorimetric method; wound healing test was conducted to judge the healing proportion at 24 h and 48h; the cell cycle distribution analysis was performed with the flow cytometry.Results:1. Results of in vivo experiments:(1) trauma operation succeeded and survival rate was 83.3%(40/48);(2) ELISA showed plasmic IL-6、TNF-α of group S significantly elevated compared with group NC(p<0.05), group M、N and P elevated more apparently, and no significant difference was found among these three groups(p>0.05);(3) HE stain showed compared with group NC and S, group M、N and P appeared incrassate tunica intima(IT) and tunica media(MT), increased IT/MT depth and area(p<0.05); above indexes of group P significantly decreased compared with group M and N(p<0.05); immunohistochemisty showed no TLR4 expression was found in group NC and S, while positive expression of TLR4 was found in neointimal of group M、N and P;(4) Western blot prompt content of local tissue TLR4、NF-κB、MCP-1、VCAM-1、p-AKT、p-m TOR increased in group M、N and P, expression of TLR4、NF-κB、MCP-1 and VCAM-1 in group P decreased, while p-AKT、p-m TOR remained compared with group M and N;(5) q RT-PCR showed compared with group NC and S, m RNA expression of TLR4、IL-6、TNF-α、VCAM-1 in group M、N and P obviously increased(p<0.05), group P presented significant down-regulation of above gene expression.2. Results of in vivo experiments:(1)1μg/mL LPS stimulated Ha VSMC could activate TLR4, the activation performed time-dependent effect, peak value of TLR4 expression occurred in 24 h after stimulation;(2) two recombinant plasmids sh RNA-TLR4 transfected into Ha VSMC, inhibition efficiency reached 61% and 55% respectively;(3) q RT-PCR showed group P and pretreated with pioglitazone both down-regulated m RNA expression of TLR4;(4) Western blot showed TLR4、NF-κB、MCP-1 and VCAM-1 expression among separate groups were similar with the result of q RT-PCR. 1 hour’s stimulation with LPS could up-regulate p-AKT expression, which could be inhibited by sh RNA-TLR4 and pioglitazone;(5) LPS could enhance proliferative ability of Ha VSMC, while sh RNA-TLR4 and pioglitazone could inhibit;(6) flow cytometry found LPS could increase the proportion of G2 and S in Ha VSMC, while sh RNA-TLR4 and pioglitazone could attenuate the change in distribution;(7) LPS promote migration of Ha VSMC, which could be suppressed by sh RNA-TLR4 and pioglitazone;(8) above effects of inhibiting proliferation、migration and affectting cycle distribution by pioglitazone performed dose-dependent, the down-regulation of inflammatory factors induced by pioglitazone also manifested dose-dependent effect;(9) The inhibition effect of sh RNA-TLR4 on proliferative and migratory ability performed the similar efficacy as pioglitazone at 50μM-100μM.Conclusions:TLR4 down-regulation could inhibit the RS incidence after carotid artery balloon injury in rabbits, which maybe related with the expression inhibition of TLR4 and TLR4-mediated inflammatory cytokines such as NF-κB、TNF-α、IL-6 、MCP-1、VCAM-1 in local tissue. TLR4 down-regulation could inhibit the LPS-induced proliferation and migration of Ha VSMC, and performed the similar efficacy as pioglitazone at 50μM-100μM, which prompt a potential target in RS prevention on gene level.