| Chapter1Specifically inhibit the expression of Act1in macrophageon the role in ApoE-/-mice atherosclerotic plaque formationPurpose:Atherosclerosis (AS) is a multifactorial disease that is characterized by the lipiddeposition, porridge minced lesions and fibrosis in arterial wall. It’s mainly due toendothelial damage caused by lipid deposition in the subendothelial layer,macrophage migration to the arterial wall remove these lipids result in foam cellsformation. There is evidence that nuclear factor kappa B activator1,(Act1) involvedin the IL-17mediated MAPKs/AP-1and IKK/NF-κB activation and the expressionof inflammatory genes. But it’s remain unclear that suppressing Act1in ainflammatory cells--macrophage whether it will affect the process of atherosclerosis.Therefore, our subject use the high-fat diet ApoE-/-mice and ApoE-/-; Anti-Act1mice as a AS model, to study the influence of inhibiting the expression of Act1inmacrophages on atherosclerosis.Method:Anti-Act1mice and ApoE-/-mice were hybridized to obtain24ApoE-/-; Anti-Act1mice, for experiments at it’s8weeks of age. Meanwhile,16ApoE-/-mice at8weeksof age were used as control group. High-fat diet for eight weeks, collecting tissuesamples and serum. Oil red O staining to observe mouse abdominal aorta and aorticroot plaque size; Immunohistochemical analysis of macrophages infiltration in ASplaque. Immunofluorescence analysis ACT1expression in atherosclerotic plaques.Results:1. High-fat diet for8weeks of ApoE-/-; Anti-Act1and ApoE-/-mice, as feedingtime increasing the weight of the two groups of mice were significantly increased, butthe two groups at each time point no significant difference in weight.ApoE-/-;Anti-Act1mice aortic root and aortic plaque area were significantly reduced thanApoE-/-mice.Compared to ApoE-/-mice, macrophages infiltration in atheroscleroticplaques of ApoE-/-; Anti-Act1mice were significant improved. ApoE-/-;Anti-ACT1 mice serum TC and LDL-C levels were significantly decreased, HDL-C andTG levels showed no markedly change.Conclusion:Specifically inhibit the expression of Act1in macrophage can inhibit the formation ofatherosclerotic plaques, reduce macrophage infiltration, significantly reduced TC,LDL-C serum level in mouse. ACT1is mainly expressed in macrophages ofatherosclerosis plaque. Chapter2specifically inhibit the expression of Act1in macrophageon the role in foam cells formationPurpose:Chapter1showed that specifically inhibit the expression of Act1in macrophagecan inhibit AS, and reduce macrophage infiltration and ACT1is mainly expressed inmacrophages of atherosclerosis plaque. The formation of AS plaque is closely relatedto foam cell formation.Therefor we hypothesis whether ACT1play a importantrole in foam cell formation. This section is to explain whether Specifically inhibitthe expression of Act1in macrophage affect foam cell formation, thereby exert itsanti-atherosclerotic effect.Method:Extracted eight weeks ApoE-/-and ApoE-/-; abdominal Anti-Act1micemacrophages, dil-oxLDL were treated in37℃6h and4℃2h, observed thebinding and phagocytosis with oxLDL of macrophages under fluorescent. Meanwhileoil red O staining after oxLDL treatment37℃24h, then observe foam cellformation.Result:1. After oxLDL treatment, Peritoneal macrophages of ApoE-/-; Anti-Act1miceuptake Dil-oxLDL i less than ApoE-/-mice’s. 2. After oxLDL treatment, the foam cell ratio of Peritoneal macrophages ofApoE-/-; Anti-Act1mice is lower than ApoE-/-mice’s.Conclusion:Specifically inhibit the expression of Act1in macrophage could reduce oxLDLuptaking and decrease foam cell formation. Chapter3the mechanism of specifically inhibit the expression ofAct1in macrophage on the role in foam cells formationPurpose:Generally considered transport protein in macrophages that mediate lipidendocytosis(CD36and SR-A) and efflux(ABCA1and ABCG1) play an important role inthe formation of foam cells, MAPKs (JNK and p38MAPK)and NF-κB is considered torelated to foam cell formation and the expression of Scavenger receptor. Act1asadapter mainly mediate IL-17downstream the activation of NF-κB, in the sametimeACT1also involve whit MAPKs signal pathway activation.based onpreceding two chapters’ result, this section is intended to elaborate themolecular mechanism of specifically inhibit the expression of Act1in macrophage onthe role in foam cells formation.Method:Extracted eight weeks ApoE-/-and ApoE-/-; abdominal Anti-Act1micemacrophages, treating oxLDL in37℃24h, detect the level of lipid endocytosisand efflux gene. THP-1cell culture, after PMA treatment, oxLDL-induced24h and48h, detection ACT1and phagocytosis-related gene and protein level changes. RNAinterference ACT1in THP-1cells, oxLDL induced48h, studying on the influence onNF-κB and MAPKs pathway. Adding NF-κB inhibitor, oxLDL-induced48hours toobserve the formation of foam cells, to study its effects on the MAPKs and CD36.Result： 1. After oxLDL treatment, the expression of CD36mRNA in Peritonealmacrophages of ApoE-/-; Anti-Act1mice is lower than ApoE-/-mice’s.2. After24h and48h oxLDL treatment, the protein and mRNA level of act1andcd36are significantly increased.3. After RNA interference ACT1in THP-1cells, the protein level of cd36, p-P65,P65, p-P38, P38, p-JNK and JNK2are significantly decreased.4. Comparing with oxLDL treat group, after adding NF-κB inhibitor, showed noeffect on foamcell formation. The expression of p-P65, p-JNK and JNK2is reduced,but the level of p-p38and cd36didn’t show markedly change.5.Elisa test showed, after high fat diet treatment, ApoE-/-mouse IL-17serum levelwas decreased accompany by b AS development.Conclusion:1. ACT1involve in oxLDL induced foam cell formation.2. the mechanism of specifically inhibit the expression of Act1in macrophage onthe role in foam cells formation possibly by regulating p38, JNK signaling pathwayaffecting the expression of CD36, and then impact macrophages uptake oxLDL.3. ACT1involve in oxLDL induced foam cell formation may not depend onIL-17/ACT1/NF-κB classic inflammation signal pathway， but directly regulateMAPKs phosphorylation regulate the expression of CD36impact foam cellformation。... |
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