The Regulatory Effect Of IKKε In High Fat Diet Induced Arterial Atherosclerosis

Background During the formation of atheroslerosis, the IKK/NF-κB pathway could be activated by high fat diet. Through which it could regulate the downstream target genes and take important regulatory effect in the development of chronic inflammation of atherosclerosis. During the formation of atheroslerosis, the IKK kinase complex combine with IκB to modulate its phosphorylation and ubiquitination, which finally lead to the degradation of IκB and the dissociation of NF-κB. The freed NF-κB would then translocate to the nucleus to take effect as an transcription factor and regulate the expression of the downstream target genes. Recent studies show that the severity of atherosclerotic lesions could not be fully illustrated by the canonic pathway upstream activator IKKβ. Being a newly discovered member of the IKK kinase complex family, IKKε could activate transcription factors such as NF-κB, interferon regulatory factor-3(IRF3) and CCAAA/enhancer-binding protein (C/EBPδ) to modulate such processes as inflammation, antiviral immunity and cell survival. While at the same time, recent studies also show that high fat diet could induce the activation of NF-κB in mice liver and adipose tissue, which could increase the expression level of IKKε. The knockout of IKKε could avoid mice liver and adipose tissue from chronic inflammation. In this study, we therefore investigated the regulatory role of IKKe in high fat diet induced atherosclerosis in mice and whether it’s through the activation of NF-κB signaling that modulate the development of high fat diet induced atherosclerosis.Methods Wild-type or IKKε knockout mice were bred into the ApoE knockout genetic background and were divided into4groups:(1) wild-type (WT),(2) ApoE knockout (AK),(3) IKKε knockout (IK),(4) or both ApoE and IKKε knockout (DK). Each group of mice were fed with a high fat diet (HFD) for12weeks from8weeks of age. We therefore first isolated the DNA of each group of mice and using the method of PCR and gel electrophoresis to justify the gene type of the DK group of mice. Through the application of immunohistochemistry and Western blotting analysis, the protein expression and localization of IKKε was studied among different groups. Through the application of immunoflurescence technique, the protein expression and localization of IKKβ was also studied among different groups. The metabolic index were measured through the measurement of body weight gain and lipid profiles. The morphological changes of the aortic root vessel wall were tested through the HE staining. Oil Red O staining was also applied to observe the lipid accumulation in aortic vessel wall. The scanning electron microscopy was introduced to have a brief view of the ultrastructure of the aortic vessel wall. The protein expression and localization of TNF-a and a-SMA was also measured in the aortic vessel wall of each group of mice through the application of immunohistochemistry. The interaction between IKKε and NF-κB pathway components P50and P65was studied through the immunoprecipitation method. Through the application of western blotting analysis, the protein expression of NF-κB signaling cascades were measured. The protein expression of VEGF and IL-18, two NF-κB signaling downstream factors was also measured. Through the application of immunoflurescence technology, the protein expression and localization of different NF-κB signaling cascades were also studied.Results Through the application of PCR and gel electrophoresis, we first justified the homozygosity of the both ApoE and IKKε knockout group of mice. Immunohistochemistry and Western blotting analysis demonstrated obvious increases in the expression of IKKε in the AK group compared with the WT group, especially in the intima. Immunoflurescence analysis illustrated that no significant differences in the expression of IKKβ.No significant differences were discovered between the IK and WT groups for body weight and serum lipid levels. Compared with that, the AK group of mice had a great increase in body weight and serum lipid levels. The DK group had a less weight gain than the AK group, and was similar with the WT and IK group. While at the same time, the DK group had similar lipid levels comparing with the AK group, which was significantly higher than the WT and IK group. HE staining revealed much more severe atherosclerotic lesions in the AK group when compared with other three groups. Oil Red O staining also revealed less lipid accumulation in the DK group compared with the AK group, but at the same time, more lipid accumulation in the DK group compared with the IK and WT group. Scanning electron microscopy revealed more integrity of the intima in the DK group than in the AK group. While at the same time, the adhering and migrating of monocytes to the intimal area of aortic vessel wall could be observed. Through the method of immunoprecipitation, we discovered a close interaction between IKKε and the NF-κB pathway components P50and P65. Western blotting analysis demonstrated obvious decrease in the protein expression of P50and P65in the aortic vessel wall of DK group when compared with the AK group. While no significant differences were found among the DK group with WT and IK group. The protein expression of phosphorylated P50and P65followed the same trend as that of total P65and P50. Through the application of immunofluorescence analysis, the expression levels of both P65and P50were proved to be low in the aorta of the DK group compared with the AK group. The localization of both P65and P50was widely spread throughout the cytoplasm of all different cell types of the aortic vessel wall in the DK group. At the same time, the localization of these proteins transferred to the nuclei, especially in the intima area of the AK group. The protein expression of IκB-α was highly increased in the DK group compared to the AK group, while that of the other two groups of mice stayed at the same level as the DK group. In addition, the expression levels of two NF-κB pathway downstream factors, IL-18and VEGF, were also downregulated in the DK group compared with the AK group using Western Blotting analysis. In addition, they were mainly localized in the cytoplasm rather than in the nucleus in the DK group compared with the AK group through the application of immunoflurescence analysis.Conclusion Through the construction of high fat diet induced atherosclerosis in mice, we investigated the expression of IKKε in different groups of mice and take a measurement of the atherosclerotic chronic inflammatory injury in all four different groups of mice. We investigated the interaction between IKKs and NF-κB signaling components. The protein expression and localization of different NF-κB signaling cascades in different groups of mice was also studied. Our study demonstrated the important regulatory effect of the activation of IKKε in high fat diet induced atherosclerotic lesions and that the regulatory effect mainly works through the activation of NF-κB signaling.