Effect And Mechanism Of LCK-ZAP70 On Reverse Cholesterol Transport And Atherosclerosis

BackgroundNowadays,atherosclerosis is harmful to human health.However,the pathogenesis of atherosclerosis still remains unknown.Recently,some researches consider T lymphocyte play an important role in the development of atherosclerosis.In the early stage of development of atherosclerosis,T cells infiltrates in artery,and identify and present specific antigens from human arterial plaque such as oxidatively modified low density lipoprotein(ox-LDL).After that,T cells produce and secrete inflammatory factors to induce foam cells formation.Lots of study prove that inhibiting activation of T cells could reduce the development of atherosclerosis.LCK is a tyrosine kinase of Src family expressed in T cell cytoplasm.When T cell receptor recognize specific antigen,LCK play a pivotal role in signaling cascade,proliferation,differentiation and activation of T cells.PP2 is an inhibitor of LCK.PP2 could inhibit activation of T cell and reduce inflammatory immune response.In our previously study,we found that inhibiting LCK with siRNA improve the cholesterol efflux capacity and increase expression of cholesterol transportation regulatory proteins.The intracellur cholesterol was reduced.The secretion of inflammatory factors was also decresed after LCK inhibition.However,these studies are limited to the cellular level.the effect and mechanism of LCK on the formation of AS plaques in animals are not clear.Our study intends to illuminate the effect of inhibiting LCK on the formation stability of AS plaques in vitro and vivo.We also explore the effect of LCK on T cell differentiation and HDL function which are related to the development of AS.On the mechanism,we try to identify which downstream pathways of LCK play important roles in T cell differentiation and reverse cholesterol transport.We pretend to clarify the effect and mechanism of LCK on atherosclerosis and provide new direction for treatment of atherosclerosis.MethodsIn vivo,Different concentrations of LCK inhibitor PP2 were injected intraperitoneally to ApoE-/-mice fed with chow diet and high fat diet.Oil red O staining,Masson trichrome staining,immunofluorescence,flow cytometry,Elisa,RT-PCR and Western Blot were used.We used these experimental methods to detect the effects of PP2 on plaque formation,lipid content and plaque stability in ApoE-/-mice after inhibiting LCK,and explore the possible mechanism of these effects.In vitro,Jurkat cells and ZAP70 deficient cell lines were used to investigate the effect of ZAP70 downstream of LCK on reverse cholesterol transport by 3[H]-cholesterol,RT-PCR,Western Blot et al.We also used specific inhibitor of ERK,JNK or p38 MAPK to detect the effector downstream pathway of LCK-ZAP70 which influenced cholesterol efllux and its' possible mechanism.Res?ltsThe study found that:1.PP2 could reduce aortic plaque formation and increase plaque stability in a concentration-dependent manner.2.PP2 could decrease the secretion of serum IFN-? and TNF-? in a concentration-dependent manner.3.PP2 reduced the infiltration of CD4+ T cells in aortic root in a concentration-dependent manner.PP2 also regulated spleen T cell differentiation,decreases the ratio of Thl cells and increased the ratio of Treg cells.4.PP2 could improve cholesterol efflux capacity of HDL and increase expression of cholesterol transportation regulatory proteins in a concentration-dependent manner.5.PP2 inhibited the activation of PI3K/AKT/mTOR pathway which were related to T cell differentiation.PP2 also Inhibited phosphorylation of ZAP70.6.Deficiency of ZAP70 which is downstream protein of LCK can improve cholesterol efflux capacity of HDL and increase expression of cholesterol transportation regulatory proteins.7.ZAP70 deletion can inhibit the secretion of inflammatory factors IL-2,IL-6,IFN-? and TNF-?.Deficiency of ZAP70 also inhibited phosphorylation of ERK,JNK and p38 MAPK.8.ERK,JNK and p38 MAPK inhibitors could inhibit the secretion of IL-6,IFN-? and TNF-?,but only ERK inhibitors increased the cholesterol efflux rate of T cells and Up-regulate the expression of cholesterol-related proteins.9.ERK inhibitors can promote the binding of LXR-? to LXRE-A on ABCG1 promoter,thus promoting the expression of ABCG1.ConclusionLCK inhibitor PP2 regulate T cell differentiation,reduced the ratio of Thl/Treg and secretion of inflammatory factors probably by inhibiting the activation of PI3K/AKT/mTOR pathway.PP2 also increased the reverse cholesterol transport of HDL and expression of RCT regulatory proteins through the LCK downstream ZAP70-ERK pathway.Consequently,LCK inhibitor PP2 decreased the development of AS.