Ⅰ The Effects Of JAK2 Mutant In Atherosclerosis And Thrombosis Ⅱ The Effects Of ω3 Polyunsaturated Fatty Acid Metabolites In Early Nonalcoholic Fatty Liver Disease Progression

Part Ⅰ The Effects of JAK2 Mutant in Atherosclerosis and ThrombosisAims:Janus Kinase 2（JAK2）participates in the regulation of myeloid cell survival,proliferation,differentiation and function of mature cells.JAK2 point mutant at position 617 codon induced clonal hematopoiesis disease called myeloproliferative neoplasms.In addition to increasing the incidence of hematopoietic malignancies,whether it affects the occurrence and development of cardiovascular diseases is still unknown.Our study mainly focuses on assessing atherosclerosis and thrombosis and underlying mechanisms in hematopoietic Jak2 expression.Methods and results:We first construct hematopoietic system specific（Mx1-Cre）JAK2V617F mutant mice(Jak2^VF mice)by knock-in and LoxP-Cre system.Sub-lethally irradiated WT and Ldlr^-/-background mice were transplanted with bone marrow from WT or Jak2^VFF mice.After transplantion,these mice were divided into four groups which were WT/WT mice and Jak2^VF/WT mice or WT/Ldlr^-/-and Jak2^VF/Ldlr^-/-mice.After 6weeks,we fed the WT background mice with chow diet and Ldlr^-/-background mice with a high fat high cholesterol western diet（WD）for 7 weeks and 12 weeks to induce the early and advanced atherosclerosis.After 7 weeks of WD,we analyzed the composition and function of bone marrow cells by flow cytometry and found that Jak2^VF/Ldlr^-/-mice showing increased hematopoiesis stem cells expansion compared to WT/Ldlr^-/-mice.Moreover,the circulating neutrophils,monocyte,red blood cells and platelet were increased.The neutrophil showed higher rolling and adhesion activity.The red blood cells were abnormal because of microcytosis and anisocytosis.The platelets were morphologic abnormalities.By H&E staining,early lesion area of Jak2^VF/Ldlr^-/-significantly increased and mainly because of neutrophil infiltration.In advanced model,Jak2^VF/Ldlr^-/-mice compared to the WT/Ldlr^-/-mice,the area of plaques slightly increased,but necrotic core markedly increased.The iron staining indicated prominent iron deposition and immunohistochemistry staining showed co-staining of erythrocytes and macrophages suggesting increased erythrophagocytosis in the advanced lesion of Jak2^VF/Ldlr^-/-mice.Besides,we also found that decreased surface expression of CD47 on red blood cells,a"don’t eat me"signal and reduced protein levels of MerTK,an efferocytosis signal on macrophage.In addition,immunofluorescence staining showed the expression level of MerTK on macrophages was also reduced in lesions of Jak2^VF/Ldlr^-/-mice.When we challenged macrophage with LPS,Jak2^VF macrophages displayed increased expression of pro-inflammatory cytokines and chemokines.Further analyse found that the phosphorylation of P38 and JNK proteins also increased and treatment of P38 and JNK inhibitor can reverse this phenotype.Conclusion:In our model,hematopoietic JAK2V617F expression promotes thrombosis,early lesion formation and increased complexity in advanced atherosclerosis.The increase of early lesion was mainly due to abnormal neutrophils induced by JAK2 mutation.The increased necrotic core of advanced lesion were mainly induced by（1）increased iron deposition in the lesion;（2）decreased surface expression of CD47 on red blood cells surface,a"don’t eat me"signal and reduced protein levels of MerTK,an efferocytosis signal on macrophage surface,both contributed to the increased free apoptotic cells in the lesion of Jak2^VF/Ldlr^-/-mice;（3）Jak2^VF macrophages displayed increased expression of pro-inflammatory cytokines and chemokines.The collective effects promoted increase of necrotic core and instability of advanced lesion in Jak2^VF/Ldlr^-/-mice.Part Ⅱ The Effects of ω3 Polyunsaturated Fatty Acid Metabolites in Early Nonalcoholic Fatty Liver Disease ProgressionAims: The development of many metabolic diseases is often accompanied by the occurrence of obesity,including diabetes,atherosclerosis,and nonalcoholic fatty liver disease（NAFLD）.Recent studies found that a week of high-fat diet is sufficient to induce adipose inflammation and NAFLD.While the early pathogenesis of NAFLD and other metabolic diseases caused by high-fat diet is still unknown.Omega-3 polyunsaturated fatty acids（PUFAs）containing eicosapentaenoic acid（EPA）and docosahexaenoic acid（DHA）have protective effects in NAFLD.This study aims to reveal the role and mechanisms of omega-3 PUFA and its metabolites in the early development of NAFLD.Methods and results: 6-8 weeks wild-type mice C57BL/6 were chosen and given normal diet（Ctrl）,high-fat diet（HFD）,and high-fat diet mixed with omega-3 PUFA diet（ω3 HFD）.The progress of NAFLD was evaluated after 4 days.The H&E staining showed that there was no significant difference in liver morphology between the HFD and the ω3 HFD group.Oil red O staining and hepatic lipid extraction showed that ω3 PUFA significantly improved the HFD-induced hepatic steatosis.Real-time PCR showed that ω3 PUFA reduced hepatic lipogenesis genes and increased beta-oxidation-related genes expression.Immunohistochemistry of adipose tissue showed that ω3 HFD significantly reversed the infiltration of inflammatory cells caused by HFD diet.UPLC-MS/MS platform was used to detect the concentration of PUFA and its metabolites in plasma and liver.The results show that metabolites HEPEs and EEQs of EPA decreased in HFD,while significantly increased in the ω3 HFD group,suggesting that HEPEs and EEQs may play a key role in the protective effect of ω3 PUFA on hepatic steatosis.We found that mixture of EPA and DHA and HEPEs,EEQs have no effectcs on palmitic acid-induced hepatocyte lipid deposition.But the supplement of ω3 PUFA significantly improved HFD-induced adipose tissue macrophage infiltration and inflammatory factors in plasma.In order to further screen the active ingredients in HEPEs and EEQs,we treated macrophages with single metabolite and found that 17,18-EEQ,5-HEPE and 9-HEPE reduced palmitic acid induced inflammation,chemokine expression and JNK signaling pathway activation.In vivo,we injected intraperitoneally with 17,18-EEQ,5-HEPE,and 9-HEPE along with HFD in mice.We found that this mixture significantly improved hepatic lipid deposition and adipose tissue inflammation.Therefore,17,18-EEQ,5-HEPE and 9-HEPE mediated protective effect of ω3 PUFA on short-term HFD induced hepatic lipid deposition by improving adipose tissue inflammation.Conclusion: ω3 PUFA significantly protect short-term HFD induced hepatic lipid deposition by improving adipose tissue inflammation.Based on UPLC-MS/MS we found that the metabolites HEPEs and EEQs of EPA were the effective ingredients candidates.It was further confirmed that 17,18-EEQ,5-HEPE and 9-HEPE inhibited palmitic acid induced macrophage inflammatory factor expression and JNK signaling pathway activation,thereby improving short-term HFD induced hepatic lipid deposition and adipose tissue inflammation.