The Effect And Mechanism Of Insulin On Unstable Plaque Formation

Background:Acute coronary syndrome is the leading cause of coronary heart disease mortality.Many studies showed that plaque unstability is the key factor of ACS, and the unstableplaque is the key pathology of ACS. Erosion or rupture of unstable plaque can induced theacute thrombosis, which leads to malignant cardiovascular events eventually. In-depthstudy of the unstable plaque formation and rupture is vital to reduce the mortality of ACS.Recent studies showed that the inflammatory response can change the structure of animalatherosclerotic plaque, and can involve in the atherosclerotic plaque rupture. Effectiveintervention on inflammatory response mediated signaling pathway should be taken toprevent the formation of unstable plaques. As a key regulator of inflammation, toll-likereceptor4(TLR4) signaling pathway participates in the development of atherosclerosis,which is the key to ACS onset. Insulin is considered to be an important hormone to theregulation of glucose. In recent years, however, more and more evidence showed that insulin exerted anti-inflammatory effects through phosphatidylinositol3′-kinase(PI3K)-Akt-dependent (PI3K-Akt) pathway, In vivo, the PI3K-AKT pathway activity wasinhibited by the TLR-NF-kappa B signaling pathway mediated inflammatory response, butto strengthen the PI3K-Akt pathway activity to the TLR-NF-kappa B signaling pathwayhave not yet been reported. Based on the above we design and completion of theexperiment.Methods:1.40male ApoE‐/‐mice，6-8weeks of age, were fed on diet of0.25%cholesterol and15%fat for1week. All mice were randomly divided into two groups:(1) control group;(2) operated group. Unstable atherosclerotic plaque were induced by perivascularcollar placement on the right common carotid arterye in the operated group.8weeksafter surgery, operated group mice were sacrificed by over-dose anesthesia randomly,the carotid artery were processed and examined by HE stain.2. Operated group were treated with/without insulin randomly.2weeks after thetreatment, all mice were sacrificed by over-dose anesthesia. TNF-α and IL-6wereexamined by ELISA. The carotid artery were processed and examined by HE stain, redoil O stain and immunohistochemical stain of macrophage.3. In vitro, RAW264.7macrophages were randomized into5groups:(1) co ntrol group;(2) ox-LDL incubation;(3) ox-LDL+insulin;(4) ox-LDL+insulin+wortmannin;(5)ox-LDL+wortmannin. Intracellular lipid accumulation was determined by Oil Red Ostaining. Apoptosis were detected by TUNEL method. TNF-α and IL-6were examinedby ELISA.The expression of TLR4、Myd88、MMP9mRNA were analyzed byreal-time PCR and the expression of TLR4、Myd88、NF-κB were detected by Westernblot.Results:1.8weeks after the surgery, the atherosclerotic plaque were formation on the carotidartery of the operated group mice.2. Compared with control group, insulin treatment obviously decreased the TNF-α andIL-6(P <0.05) and plaque stability, especially in lipid content and macrophageinfiltration(P <0.05). 3. In vitro, compared with control group, ox-LDL incubation increased the formation offoam cells and the apoptosis index (P <0.01). Insulin significantly blunted the effectsof ox-LDL. The intracellular lipid content and the apoptosis index were significantlyreduced(P <0.01). Moreover, wortmannin, which is the inhibitor of PI3K, could blockthe effects of insulin (P <0.01vs ox-LDL+insulin).4. Compared with control group, ox-LDL incubation increased TNF-α and IL-6(P <0.01). The mRNA and protein level of TLR4and its downstream Myd88, NF-κBincreased significantly detected by real-time PCR and Western blot respectively (P <0.05vs control). In the meanwhile, mRNA and protein level of MMP9were alsoincreased remarkedly(P <0.01vs control). Insulin significantly blunted the effects ofox-LDL. Not only the TNF-α and IL-6were significantly reduced (P <0.01vsox-LDL group), but the mRNA and protein level of TLR4, Myd88, NF-κB and MMP9decreased significantly (all P <0.05vs ox-LDL group). Moreover, wortmannin, whichis the inhibitor of PI3K, could block the effects of insulin (P <0.05vs ox-LDL+insulin).Conclusion:1. Unstable atherosclerotic plaque model were successfully established on ApoE-/-mice.Insulin can effectively reduce the secretion of TNF-α and IL-6and increase thestability of atherosclerotic plaques;2. Insulin could reduce the formation of macrophage foam cells and macrophageapoptosis and the expression of TNF-α, IL-6;3. Insulin could inhibit the expression of pro-inflammatory cytokines by blocing theTLR4-MyD88-NF-κB signaling pathway in the PI3K-AKT-dependent way, and playanti-inflammatory effect in the atherosclerotic.