| Atherosclerosis (AS), a chronic disease, which happens at large artery, is the primary cause of cardiovascular events and stroke, and seriously endangers man's health. In western developed countries, it has been one of the main causes of death. In recent years, there was significant increase in China. Its incidence and mortality rates rise year by year, younger by younger. Therefore, it is very important to study AS.AS is a complex multifactor disease, is now widely recognized as specific example of chronic inflammatory response mainly to dyslipidemia and other risk factors. It involved many cell types and biological response, such as lipid metabolism, cytoadherence, stress reactions, cell proliferation and apoptosis, blood agglutination. Each biological response involved multiple genes. Analysis expression patterns of those AS related genes is important to clarify pathogenesis of AS and to find new theory to prevent and treat AS. ApoE and LDLR double knockout (ApoE-/-/LDLR-/-) mice as AS model mice, with chow diet appears hyperlipidemia and AS lesion due to apoE and LDLR pathway block. In the past decades, most of the research of the mice and AS mainly focused on lipid metabolism. There are few on vascular inflammation impair and pathogenesis of inflammatory response of AS, especially in early states. We applied apoE-/-/LDLR-/- mice and C57BL/6J wild type (WT) mice at various ages of 14 days, 1, 2 and 3 months, total RNA was isolated from fresh aortas and livers of mice. Then RNA was reverse transcribed to first-strand cDNA by reverse transcriptase for reverse transcription polymerase chain reaction (RT-PCR) and real time RT-PCR assays. Primer pairs were designed using primer software and gene sequences available in Genbank.β-actin was used as an internal control. Then an RT-PCR assays have been used to analysis temporal expression patterns of C-reactive protein (CRP), mannose binding lectin (MBL), tumor necrosis factor-α(TNF-α) and Interleukin-1β(IL-1β) in livers, IL-1β, TNF-α, nuclear factor-kappa B (NF-κB), granulocyte-macrophage colony-stimulating factor (GM-CSF), CD36, endothelin-1 (ET-1), toll-like receptor-2 (TLR2), monocyte chemotacticprotein-1 (MCP-1), vascular adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1),Interleukin-6 (IL-6) and platelet derived growth factor-α(PDGF-α) in aortas. Relative amount of mRNA was evaluated by the ratio of band density of target gene/β-actin. We evaluated SYBR Green quantitative real-time RT-PCR to validate genes expression identified by RT-PCR. Serum levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL) and high density lipoprotein (HDL) were measured by using biochemical techniques. Serum concentrations of circulating TNF-α,IL-1βand oxidized LDL (OxLDL) were determined by ELISA. Aortic sinus and livers was fixed, cleaned and made into frozen section, latter stained with SudanⅣand oil red O to visualize intimal fatty lesions.The RT-PCR assays results in livers showed that: the comparative expression of CRP in the apoE-/-/LDLR-/- mice at the age of 14 days and 1 month was higher than that in the WT mice at the same age (P<0.05), and was down-regulated to the WT level progressing with age. The expression of IL-1βin 14-day old apoE-/-/LDLR-/- mice and TNF-αin the apoE-/-/LDLR-/- mice at the age of 1month was up-regulated compared to the WT mice at the same age (P<0.05), and continued to upgrade with time on. MBL had no significant changes in both two genotype mice at various ages. All the gene expression kept unchanged in WT mice at various stages.The RT-PCR assays results in aortas showed that the expression of IL-1β, GM-CSF, ET-1, TLR2, CD36, MCP-1, ICAM-1 and VCAM-1 in aortas at the age of 1 month were higher than those of age-matched WT mice (P<0.05, P<0.01), respectively. The expression of PDGF-αand TNF-αin the apoE-/-/LDLR-/- mice at the age of 2 months was up-regulated compared to the WT mice at the same age (P<0.05). All the target genes continued to upgrade (P<0.05, P<0.01) except that ET-1 at the age of 2 months. TLR2, VCAM-1and ICAM-1 at the age of 3 months were down-regulated to WT level. NF-κB had no significant changes in both two genotype mice at various ages. All the genes kept unchanged in WT mice at various stages, though IL-1βshowed a slight up-regulation in 2 and 3 months.The real time RT-PCR assays results in aortas showed that the expression of GM-CSF,ICAM-1,IL-1β,IL-6 and MCP-1 in aortas of 1-month old apoE-/-/LDLR-/- mice were up-regulated compared to age-matched WT mice, and all the genes in the 3-month old apoE-/-/LDLR-/- mice were down-regulated to WT level besides IL-1βand IL-6 continued to upgrade. NF-κB and I-κB had no significant change in both two genotype mice at the two ages. The real time RT-PCR assays results were consistent with the results of RT-PCR on the whole. Serum levels of TC, TG, LDL, HDL, and TNF-α, IL-1βand OxLDL in the apoE-/-/LDLR-/- mice at various stages were higher than those of WT mice (P<0.05, P<0.01) and all of them were progressing with age except OxLDL, and achieve the maxlmum at the age of 3 months. Plasma TC and LDL were higher than those in age-matched WT mice at 6- and 40-fold, TG,HDL,TNF-α,IL-1βand OxLDL were 2-fold higher than those in age-matched WT mice. Primary atherosclerotic lesions were observed from 1 month apoE-/-/LDLR-/- mice and were progressing with age. There was no observed lesion in the 14 days old apoE-/-/LDLR-/- mice and all WT mice of different ages.The data suggest that:The up-regulation of AS related genes temporal expression in the aortas and livers and the elevated TNF-α, IL-1βand OxLDL in serum in apoE-/-/LDLR-/- mice at various stages, may correlate to hyperlipidemia and AS. The hyperlipidemia due to apoE and LDLR genes mutant may stimulate the temporal expression of AS related genes and highly contribute to the process of primary atherogenetic lesions and vascular inflammation impair.
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