| Objective: Atherosclerosis(AS) is a common pathological basis of cardiovascular disease. Adiponectin(APN) has been shown to have an anti-AS effect, and the underlying mechanisms, however, are largely unknown. Nuclear factor κB(NF-κB) has also been regarded as a proatherogenic factor, mainly because of its regulation of a variety of the proinflammatory genes linked to AS. It was hypothesized that the inhibitory effects of APN on the AS is through the inhibition of NF-κB signaling pathway. This study sought to test the hypothesis via(1) investigate and validate the inhibitory effect of APN on AS in Apo E-/-mice and on AS-like injury in Human umbilical vein endothelial cells(HUVEC);(2) delineate the roles of NF-κB signaling pathway in modulating the APN effect in in vivo AS and in vitro AS-like injury.Methods:(1) APN overexpression in Apo E-/- mice and in HUVECs were mediated by transfecting adenovirus bearing a vector encoding for APN and enhanced Green Fluorescent Protein(Ad-APN-e GFP). The AS in Apo E-/- mice was induced by feeding a high-fat diet, and the AS-like injury in HUVECs was induced by adding oxidized low density lipoprotein(ox-LDL) into the culture medium.(1) To assess the transfection efficiency and the safety of the adenovirus in mice 48 male Apo E-/- mice aged 12-week were randomly and evenly assigned into two groups(24 mice per group), and were injected tail-intravenously with either 100 μl of saline(control group) or Ad-APN-e GFP virus(3.0×108 p.f.u, APN group). At 0 day, 1, 2, and 4 weeks of post-injection, blood samples and aortic vascular tissues were executed from the 2 groups of mice. Enzyme-linked immunosorbent assay(ELISA) was used to detect serum APN concentration; Western blot(WB) was conducted to assess the transfection efficiency of Ad-APN-e GFP virus, as indicated by the expression of e GFP in vascular tissues; Full automatic biochemical analyzer(FABA) was used to analyze blood conventional indexes, cardiac enzymes, and blood urea nitrogen(BUN).(2) To validate the inhibitory effect of the adenovirus mediated APN overexpression on the AS in Apo E-/- mice 120 male Apo E-/-mice aged 12-week were randomly and evenly assigned into two groups(60 mice per group), and were fed with a high-fat diet to induce AS. At 0 day, 2, 4, and 6 week of high-fat diet feeding the 2 groups of mice were injected tail-intravenously with either 100 μl(3.0×108 p.f.u) of Ad-e GFP virus(control group) or the same amount of Ad-APN-e GFP virus(APN group). Blood samples and aortic vascular tissues were executed at 0 day, 4, and 8 week of high-fat diet feeding. For blood samples, FABA was used to analyze the concentrations of total cholesterol(TC), triglyceride(TG), High-density lipoprotein cholesterol(HDL-C), and Low-density lipoprotein cholesterol(LDL-C), respectively; ELIZA was used to test the concentrations of serum APN and matrix metalloproteinase-9(MMP-9); Colorimetry was used to determine the concentrations of superoxide dismutase(SOD) and malondialdehyde(MDA); For the aortic tissues, Oil red O staining was used to detect the surface lesion percentage; Hematoxylin-eosin(HE) staining was used to detect the pathological alterations; Masson staining was used to evaluate the collagen content and fibrous cap thickness of the plaque area; Immunohistochemical approach was used to detect the proportion of vascular macrophages and smooth muscle cells; Real-time fluorescence quantitative PCR was used to test the m RNA expressions of APN, Endothelial nitric oxide ribozymes(e NOS), Interleukin-6(IL-6), Monocyte chemotactic protein 1(MCP-1), vascular cell adhesion molecule-1(VCAM-1), and Intercellular cell adhesion molecule-1(ICAM-1); Immunofluorescence method was used to detect APN,Nuclear factor kappa B(NF-κB) p65 and protein expressions; WB was used to detect the expressions of APN and the downstream factors of NF-κB pathway.(3) To delineate the roles of NF-κB pathway in modulation of inhibitory effect of APN on AS-like injury in HUVECs induced by ox-LDL The cultured HUVECs were assigned into 4 groups and treated with conventional culture medium for 48 h(normal control); 30 μg/m L of ox-LDL for 48 h(ox-LDL intervention); 100 μmol/L of NF-κB antagonist pyrrolidine dithiocarbamate(PDTC) for 1h, and then 30 μg/m L of ox-LDL for 48 h(PDTC + ox-LDL intervention); and Ad-APN-e GFP(MOI=100 p.f.u/cell) for 2 h, then 30 μg/m L of ox-LDL for 48 h(APN + ox-LDL intervention). The all treated HUVECs were assessed with a variety of approaches: Cell Counting Kit-8(CCK-8) colorimetric assay for cell proliferation; Flow cytometry for apoptosis; ELISA for the expression of the inflammatory factors ICAM-1, VCAM-1 and MCP-1, and anti-inflammatory factor e NOS in the supernatant of HUVEC culture; Real-time fluorescence quantitative PCR for the m RNA expression of e NOS, ICAM-1, VCAM-1, and MCP-1; WB for the protein expression of e NOS, ICAM-1, VCAM-1, MCP-1,, APN, and nuclear NF-κB p65.Results:(1) Adenovirus had no significant effect on blood routine indexes and myocardial enzyme expression, had significant effect on the liver function of ALT(P<0.01), renal function indexes BUN(P<0.01). r Ad-APN-e GFP vector in vivo efficiently mediated exogenous APN gene expression, serum APN concentration in serum reached the highest value was 78.28±15.02 vs 23.42±5.35(μg/ml) after 1 week transfection, 2 weeks stable expression. At 1 week, the expression of GFP in vascular tissue was the highest, GFP/GAPDH was 83.63±1.50%, then decreased gradually, and GFP/GAPDH was 54.00±4.00 % in 2 weeks, GFP/GAPDH was 22.00±3.99 % in 4 weeks. Adenovirus vector has the characteristics of high efficiency transfection and stable expression and safety in 2 weeks. It is an ideal gene therapy vector for vascular disease.(2) APN inhibited the formation of atherosclerotic plaque in Apo E-/-mice. The lesion formation in aortic sinus was inhibited significantly(P<0.01). Compared with the control group, plaque lesion area of the Ad-APN group in the 4 weeks was 1.35±0.17 vs 1.50±0.74(×104μm2); 8 weeks was 1.70±0.34 vs 2.21±0.28(×104μm2) lession lumen ratio decreased significantly(P<0.001), lession lumen ratio in 4 weeks was 28.36±3.22 vs 35.61±7.43(%), and weeks was 38.25±6.30 vs 56.89±6.21(%); compared with the control group, the oil red O staining, surface of atherosclerotic lesion rate decreased significantly in Ad-APN group(P<0.001), the percentage of surface lession in 4 weeks was 27.78±8.64 vs 33.02±5.18(%); 8 weeks was 31.58±5.87 vs 52.16±5.79(%). As serum adiponectin increased, SOD activity increased(P<0.05) and the concentration of MDA decreased(P<0.001). TC(P<0.001)、TG(P<0.001)、LDL-C(P<0.001) levels decreased, slow down the growth of body weight(P<0.05). Compared with the control group, the levels of MMP-9 were significantly decreased in Ad-APN group(P<0.05), 50.23±14.24 vs 51.33±15.66(ng/ml) in 4 weeks,55.99±11.74 vs 80.50±14.54(ng/ml) in 8 weeks. With the increase of serum adiponectin, inflammatory state animal model of whole body were reduced, arterial plaque stability were increased, the oxidative stress degree were reduced. Exogenous adiponectin increased anti-inflammatory factor m RNA expression e NOS(P<0.05) and IL-10(P<0.001), reduce the m RNA expression of inflammatory factor TNF-α (P<0.001)、IL-6(P<0.001)、VCAM-1(P<0.05). Adiponectin effectively inhibits the activation of NF-κB p65 pathway, inhibiting the expression of NF-κB nuclear protein p65. In control group with prolonged high fat diet, NF-κB p65 expression in aorta gradually increased, decreased levels of adiponectin expression in vascular endothelial cells. To elucidate the adiponectin to reduce this inflammatory response through effectively inhibiting the NF-κB pathway.(3) Flow cytometry analysis showed that the transfection rate of r Ad-APN-e GFP in HUVEC was 85.03±6.77(%)when the MOI was 100 p.f.u/cel1 at 48 h. The transfection rate of r Ad-APN-e GFP in HUVEC was 84.26±7.16% at 72 h. Ox-LDL could activate the NF-κB signal pathway, and the nuclear protein levels of NF-κB p65 was notably increased after ox-LDL induced HUVEC(P<0.001). WB showed that overexpression of APN gene could inhibit the nuclear NF-κB p65, decrease the expression of NF-κB downstream molecules VCAM-1, ICAM-1, MCP-1(P<0.001), increase the expressionof e NOS(P<0.001). The results showed that APN can effectively inhibit ox-LDL induced NF-κB pathway activation in HUVEC, and PDTC intervention has similar effect.Conclusion:(1) Adenovirus injection has a transient liver toxicity and renal toxicity, but has no significant effect on the physiological state of the animal. r Ad-APN-e GFP is a high efficiency, stable expression and safety vector in vascular in 2 weeks.(2) The expression of adiponectin demonstated a protective effect on atherosclerosis in Apo E-/-mice. Adiponectin may protect the aorta from atherosclerotic injury by reducing inflammation.(3) r Ad-APN-e GFP vector can be efficiently and stably transfected into HUVEC, which effectively inhibits NF-κB activation in HUVEC. The molecular mechanism may involve inhibitation of NF-κB and its transcription factors in downstream. |
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