Targeting Inhibition Of NF-κB And Endoplasmic Reticulum Stress Prevents Atherosclerosis Of ApoE^-/- Mice

Objective: To study the intervention about inflammatory NF-κB signaling pathway and endoplasmic reticulum stress on the role of atherosclerosis. 1) Though recombinant adeno-associated virus-mediated IκBα overexpression inhibited the activation of NF-κB signaling pathway to prevent Apo E^-/-mice atherosclerosis（AS） plaque and down regulate the NF-κB downstream cytokine expression; 2) Using the drug nicorandil to prevent the endoplasmic reticulum stress and NF-κB signaling pathway in atherosclerosis and the effects of nicorandil regulation on atherosclerosis mice. Methods: 1) The eight-week-old male Apo E^-/- mice were given high-fat diet and normal diet 8, 12, 16, 20, 24 weeks, the expression of lipids in aorta was detected by Oil Red O staining. AAV9-CMV-e GFP, 5.0×1011vg/per mice/100μl, was via the tail vein injection transfected into Apo E^-/-mice and the mice were observed at different time points after viral transfection to measure the transfection efficiency and safety. WB and laser confocal detection were used to detect the expression of green fluorescent protein in atherosclerotic plaque. Plasma from mice were collected from the adeno-associated virus-mediated gene therapy of Apo E^-/- mice to observe the enzymes of liver, renal, cardiac.Using the TUNEL to detect AAV9 transfected cardiomyocytes and hepatocytes apoptosis; 2) The recombinant adeno-associated virus-mediated IκBα overexpression in inhibiting AS of Apo E^-/- mice: The eight-week-old male Apo E^-/-mice were divided into Saline control group, AAV9-GFP group and AAV9-IκBα group, 25 mice in each group. The three groups of mice were fed with high-fat diet for 12 weeks, the Saline control group mice were injected saline（per mice/100μl） via tail vien, and the remaining two groups were given a tail vein injection for AAV9-CMV-GFP virus（5.0×1011vg/per mice/100μl） or IκBα adeno-associated virus vector（AAV9-CMV-IκBα, 5.0×1011vg/per mice/100μl）. After the intervention, three groups of mice were fed with high-fat continuely for five weeks, then all mice were sacrificed, whole blood and vascular tissues were collected. Full-automatic Biochemical Analyzer 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. Using the Oil Red O staining to detecte the lipids plaque area; HE staining for detecting mouse aortic plaque area; Sirius red staining for detecting the collagen of plaques; IHC were used to detect macrophages（MOMA-2） and smooth muscle cells（SMC）, inflammatory cytokines IL-6, TNF-α, chemokine MCP-1 and matrix metalloproteinases MMP-2 inplaques; the measurement of mouse aorta tissue used RT-PCR to detect the IL-6, TNF-α, chemotactic factor MCP-1 and matrix metalloproteinase MMP-2 m RNA expression; and the aortic of NF-κB pathway related proteins expression were detected by Western blot and Immunofluorescence. 3) The drug nicorandil through inhibition of endoplasmic reticulum stress and NF-κB pathway preventing AS progression in Apo E^-/-mice, the mice were divided into three groups: Saline control group, Nicorandil group and Atorvastatin group, each group with 25 mice. Three groups of Apo E^-/- mice were fed with high fat diet for 16 weeks, then were intragastric injection with saline, nicorandil and atorvastatin respectively, consecutive treated for 8 weeks, then mice were anesthetized and sacrificed, whole blood and vascular tissue were collected. Full-automatic Biochemical Analyzer 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. Using the Oil Red O staining to detecte the lipids plaque area; HE staining for detecting mouse aortic plaque area; Sirius red staining for detecting the collagen of plaques; IHC were used to detect endoplasmic reticulum stress marker GRP78 and CHOP, also the macrophages, smooth muscle cells and inflammatory factors in aortic plaque area; inflammation factor m RNA expression in mouse aorta tissues were measured in RT-PCR method; aortic endoplasmic reticulum stress apoptosis-related protein JNK, P-JNK, Caspase-12 and NF-κB pathway related proteins were detected by Western blot. TUNEL was used to detecte the apoptosis cells in plaques. Results: 1) Lipid test results showed high-fat diet group, total cholesterol（TC）, triglyceride（TG） and low density lipoprotein（LDL-C） levels were significantly higher than normal diet group（P<0.05）, Oil Red O staining the results showed that high-fat diet group plaque lesions faster than the normal diet group（P<0.05）, Apo E^-/-mice can form a stable model of atherosclerosis when fed high-fat diet for 16 weeks. AAV9-e GFP virus were transfected into AS mice, the aortic vasculars were detected by laser scanning confocal microscopy and Western blot. The results showed that r AAV9-e GFP can be transfected effectively and sustaining expression in atherosclerotic plaques for mice, the virus transfection reached the peak after transfected for 35 days. Apo E^-/- mice transfected with virus compared with control group of mice, the serum enzymes, liver function, renal function showed no significant difference（P>0.05）, AAV9-GFP virus transfected cardiomyocytes and hepatocytes, did not affect the cells apoptosis respectively（P>0.05）. AAV9 virus is an ideal, safe carrier for atherosclerosis gene therapy; 2) IκBα overexpression improved the atherosclerotic plaque stability in Apo E^-/- mice. Compared with the saline control group and empty viral group, IκBα intervention group were not decreased the arterial plaque area significantly（P>0.05）. But IκBα intervention group could improve the plaque stability significantly compared with the saline control group and the empty viral group. Saline control group, AAV9-GFP group and AAV9-IκBα group were all detected as follow: Sirius red staining for collagen in plaque, the percentage was 9.50±2.21; 9.31±2.62; 16.43±1.83, respectively; AAV9-IκBα group was significantly increased collagen content（P<0.01） than the other two groups, the percentage of macrophages were 27.91±3.32; 28.5±3.21; 17.84±3.35, respectively; percentage of smooth muscle cells were 9.32±2.36; 9.90±2.42; 16.83±2.21, respectively; the macrophage cells were significantly reduced and the content of smooth muscle in the plaque were increased significantly in AAV9-IκBα group than in the saline control group and empty virus vector group,（P<0.01）. The three group of mice plaque vulnerability index were 1.24±0.20; 1.25±0.18; 0.54±0.14, respectively; AAV9-IκBα group compared to the saline control group and empty virus group AAV9-GFP the vulnerability index decreased significantly and improved the plaque stability（P<0.01）. IκBα intervention group could reduce the plaque IL-6（P<0.05）, TNF-α（P<0.05）, the chemokine MCP-1（P < 0.05） and matrix metalloproteinase MMP-2（P < 0.05） inflammatory factors significantly in both the protein levels and m RNA levels, compared with the saline control group and viral empty group. IκBα overexpression could repress NF-κB pathway activity and inhibite P-P65 protein expression（P<0.05）; 3) Nicorandil group and atorvastatin group compared with the saline group, could inhibit atherosclerotic plaque progression. Weight, blood pressure, blood lipids showed no significant difference among the three groups of mice（P>0.05）. Three groups of mice aorta were detected by RT-PCR, nicorandil group and atorvastatin group significantly reduced the endoplasmic reticulum stress-related proteins GRP78 and CHOP m RNA expression than in the saline group（P < 0.05）; IHC results were shown: saline control group, nicorandil group, atorvastatin group, the percentage of GRP78 protein in plaque were 22.31±3.32; 15.33±3.21; 10.64±2.35（P < 0.01）, respectively; CHOP protein percentages were 12.40±2.57; 8.80±2.16; 4.90±2.20（P<0.01）. Oil Red O staining among the three groups of mice were 50.4±4.63, 43.4±3.41, 37.7±3.12（%）, respectively; nicorandil group or atorvastatin group compared with saline group could significantly reduce plaque deposition（P<0.05）; the three group of mice plaque vulnerability index was1.44±0.20; 0.77±0.14; 0.52±0.15, respectively; nicorandil group or atorvastatin statin group compared with saline group significantly reduced plaque vulnerability index（P<0.05）. Nicorandil group and atorvastatin group could reduce the plaque inflammatory factors from the gene and protein level（P<0.05）. Nicorandil group and atorvastatin group could inhibit the expression of endoplasmic reticulum stress-related proteins GRP78, CHOP, P-JNK, Caspase-12, and upregulate the apoptosis related protein Bcl-2/Bax（P<0.05）, also significantly raise IκBα（P<0.05）, inhibite P-P65 protein expression（P<0.05）, repress NF-κB pathway activity. TUNEL staining showed that the nicorandil group and atorvastatin group could reduce the apoptosis cells in plaques significantly（P<0.05）. Conclusion: 1) AAV9-CMV-GFP virus through tail vein injection transfected in Apo E^-/-mice AS model could be persistent expression in atherosclerotic plaques, the transfection peak nearly at 35 th day. Serological tests found no significant heart, liver, and kidney toxicity. AAV9 is an ideal and safe vetor for atheroscleros gene therapy; 2) Atherosclerotic disease process in Apo E^-/- mice, NF-κB signaling pathway was over-activated, IκBα gene was carried by AA9 virus transfected in Apo E^-/- mice AS model by targeting inhibit the overexpressing activation of NF-κB signaling pathway in mice aortic plaque, and down-regulated NF-κB signaling pathway target inflammatory genes such as IL-6, TNF-α, MCP-1 and MMP-2, thereby inhibited artery atherosclerosis progression; 3) In atherosclerosis process, endoplasmic reticulum stress and NF-κB activation is excessive, nicorandil could inhibite atherosclerosis by inhibiting endoplasmic reticulum stress related apoptosis proteins and upregulate IκBα expression level inhibiting NF-κB signaling pathway activation.Therefor nicorandil could reduce the inflammation in atherosclerotic plaque and the formation of plaque, remain plaque stable, could be an important role for inbibiting atherosclerosis.