| Background and objective:Hypercholesterolemia is one of the high-risk factors of arterial thrombotic diseases,hypercholesterolemia can significantly increase the risk of arterial thrombosis.Some retrospective and prospective clinical trials have proved that lipid cholesterol is closely related to peripheral blood cells.Therefore,the study on the mechanism of lipid cholesterol regulation the proliferation and activation of blood cells,will help to better identify the risk factors of MPNs complicated with arterial thrombosis and prevent its occurrence.First of all,we studied the mechanism of lipid cholesterol promoting platelet activation in mice with LNK gene mutation.Secondly,we applied JAK2 inhibitor TG101348 to further study the mechanism of lipid cholesterol promoting the proliferation of neutrophils and monocytes.Finally,for macrophage,the mechanism of uptaking lipid cholesterol is still a challenge.We will further study the mechanism of lipid cholesterin mediating macrophage.Methods:Part 1,In this part,we first used Lnk-/-mouse model to investigate whether the mutation of Lnk gene has synergy with lipid cholesterol on platelet activity.1.Lnk-/-→WT and Lnk-/-→Ldlr-/-mice models were established by bone marrow cell transplantation,and compared with WT→WT,WT→Ldlr-/-mice fed with normal diet and western diet respectively.2.Comparing the number and activity of platelets among four groups3.To construct cholesterol-rich liposomes co-incubate with platelet4.Further analyze whether lipid cholesterol has an effect on platelet proliferation and activation pathway.Part 2,In this part,we fed JAK2 inhibitor TG101348（Fedratinib）through oral gavage to WT mice and APOE-/-mice to explore whether JAK2 inhibitor could reverse the effect of high lipid cholesterol on peripheral blood cells and reduce the risk of arterial thrombosis.1.WT mice and APOE-/-mice fed with WTD,TG101348 or vehicle were administered orally for 30 days.The numbers of neutrophils,monocytes,hemoglobin and platelets in peripheral blood were monitored.The numbers of Ly 6c/g hi or Ly6c/g Lo monocytes were also monitored by flow cytometry.2.After 60 days of oral gavage,peripheral blood,bone marrow and spleen were taken from mice after euthanasia to measure spleen size,platelet activity and the proportion of bone marrow hematopoietic stem cells.3.After euthanasia,the descending aortas of mice were stained with oil red O to measure lipid cholesterol deposition.Part 3,In this part,we labeled the native LDL which can carry lipid cholesterol through fluorescence staining and co-incubated it with the macrophages of mice in vitro to explore whether modified LDL could promote the uptake of natural LDL in mouse macrophages,and the gene expression of macrophages was analyzed to explore the potential Mechanisms of macrophage uptake native LDL.1.Bone marrow cells of WT,CD36-/-and Ldlr-/-mice were isolated and cultured by cell culture medium.2.Macrophages of WT,CD36-/-,Ldlr-/-mice were incubated with fluorescent labeled natural LDL and modified oxLDL.The fluorescence intensity of macrophages was measured at time course.3.Collect the macrophages after incubation,extract the RNA,reverse transcript the DNA,and measure the expression of related genes by the method of RT-qPCR.4.SREBF2 gene was knocked out by SREBF2 SiRNA in mouse macrophages,and then the expression of related genes was measured by RT-qPCR.Results:Part 11.Hypercholesterolemia increased platelet activity in WT and Lnk-/-mice.The expression of P-selectin and PKC increased significantly in Lnk-/-mice compared with WT mice.2.High lipid increases platelet AKT（PKB）（p-AKT）activity in WT and Lnk-/-mice.The level of p-AKT in Lnk-/-mice is much more than that in WT mice3、High lipid cholesterol decreases SHIP1 phosphorylation,Part 21.After oral gavage,TG101348 reversed the proliferation of high monocytes and neutrophils in APOE-/-mice fed with WTD.2.TG101348 inhibited the proliferation of myeloid hematopoietic stem cells in APOE-/-hypercholesterolemic mice.3.After oral gavage,lipid cholesterol deposited on artery in mice fed with APOE-/-by TG101348 was reduced by 74%compared with mice fed with vehicle by oil red O staining.Part 31.OxLDL promotes macrophage uptake natural LDL in WT mice,the peaking was from 48 to 72 hours,and gradually declining after 96 hours.2.The uptake of natural LDL by macrophages of CD36-/-and Ldlr-/-mice was not significantly different with or without oxLDL.3.After 48 hours of incubation of oxLDL with natural LDL,the gene expression of IL-6 and IL-1βin macrophages of WT mice significantly increased compared with that of natural LDL.In CD36-/-mice,the gene expression of IL-6 and IL-1βdid not change significantly.In the absence of natural LDL（nLDL）,incubation of oxLDL did not increase the gene expression of IL-6 and IL-1βin macrophages.4.In WT mice,when oxLDL was co-incubated with natural LDL in macrophages,the expression of Ldlr in macrophages increased by 5 to 10 times compared with that in non-oxLDL macrophages.There was no significant difference in the expression of Ldlr in CD36-/-mice.When SREBF2 transcription was blocked,the increased expression of Ldlr in macrophages of WT mice after co-incubated oxLDL and natural LDL was inhibited.Conclusions:1.Lipid cholesterol and Lnk gene mutation have synergistic effects in platelet activity.AKT pathway is enhanced by SHIP1 protein depletion,which further enhances platelet activity and thus increases the risk of artery thrombosis.2.In the model of APOE-/-mice,hypercholesterolemia increase the number of neutrophils and monocytes,the application of JAK2 inhibitor TG101348（Fedratinib）can inhibit the IL-3/GM-CSF pathway,reverse increased numbers of precursor cells of myeloid hematopoietic stem cells induced by hypercholesterolemia,thereby inhibiting the levels of neutrophils and monocytes.Finally,TG10138 have prevention for arterial thrombosis.3.Lipid-cholesterol-bound LDL is accelerated by mouse monocyte-macrophage system under oxLDL stimulation.oxLDL may interact with bone marrow-derived macrophage CD36 receptor and natural LDL in vivo to increase inflammation stress in macrophages,thus affecting SREBP2 activity and further upgrade the expression of Ldlr in macrophage.The expression of Ldlr enhanced macrophage phagocytosis,then promoted the formation of macrophage foam cells. |
