| Background Atherosclerosis(AS)is the underlying cause of myocardial infarction(MI).However,even with the development of medical science and technology have made MI a better diagnosis in recent decades,but the mortality of MI patients in and out of hospital has not been significantly depressed.Therefore,it is of great significance to study the pathogenesis of AS and find new therapeutic intervention targets.AS is a chronic inflammatory disease.Macrophages are the main players in the entire stage of AS inflammatory lesions and innate immune responses,playing a vital role in all stages of AS.Polarization activation of macrophages is usually defined as two states,M1 and M2.M1 phenotype macrophages are abundant in the developed lipid core of unstable plaques,which promote the progression of AS lesions.In stable AS plaques,the number of M2 phenotype macrophages is highe,and M2 phenotype macrophages which promotes plaque stability and has potential protective effect.Therefore,regulation of M1 phenotype macrophages to M2 phenotype in plaque has become an important target for anti-AS treatment.The Janus kinase-signal transduction and Janus activator of transcription(JAK-STAT)signaling pathway are involved in a variety of cytokine-induced biological responses.Macrophage phenotypic activation is mainly regulated by JAK-STAT signaling pathway cytokines,and JAK2-STAT3 is a key factor determining macrophage polarization to M2 phenotype.Hepatocyte growth factor(HGF)and its specific receptor mesenchymal-epithelial transition factor(Met,also known as c-Met)are considered to be the important molecules regulating the cardiovascular system.Increased expression of HGF/c-Met in the endothelium of AS can promote the repair of inflammatory damage.HGF has a strong anti-inflammatory effect,and its production is closely related to the activation of M2 phenotype macrophages.In this thesis,we investigate whether HGF combined with c-Met promotes M1 phenotype macrophages to M2 polarization via JAK2-STAT3 signaling pathway.The results will provide new ideas for the prevention and drug treatment of AS.Objective In this thesis,we investigate the effects of hepatocyte growth factor(HGF)with different concentrations on the polarization of RAW264.7 macrophages in vitro,and explore to promote M1 phenotype macrophages to M2 polarization and its possible mechanisms by using of HGF combined with c-Met via Janus Kinase 2-Signal transduction and activator of transcription 3(JAK2-STAT3)signaling pathway.Methods 1.The mouse peritoneal macrophage cell line RAW264.7 cells were cultured in vitro.The RAW264.7 cells were used as M0 control group;RAW264.7 cells which were induced to be M1 phenotype after 12 hours of intervention with IFN-γ and LPS,were used as M1 group;M1 phenotype macrophages interfered with different concentrations of HGF(5,10,20 ng/ml)for 12 hours were used as 5M1 group,10M1 group,and 20M1 group,respectively;M1 phenotype macrophages interfered with 50 μmol/L JAK2 specific blocker(AG490)and 10 ng/ml HGF were AG490+10M1 group.2.To observe morphological characteristics of normal macrophages,M1 macrophages,the macrophages treated with HGF and their combination with AG490 by using of the inverted microscope.3.The effect of HGF on the proliferation of macrophages was detected by CCK8 method.4.The nitrite concentration of culture supernatant(NO%)in each group was measured by the Griess reagents kit.5.Expression of P-c-Met in macrophages cytoplasm of M0 control group,M1 group and 10M1 group which was interfered with HGF was observed by immunofluorescence assay.6.Western Blot was used to detect the protein expressions of marker for M1 group,including nitric oxide synthase(iNOS),interleukin-6(IL-6),and that for M2,including arginase I(Arg I),interleukin-10(IL-10).The protein expressions of JAK2,P-JAK2,STAT3,P-STAT3 involved in JAK2-STAT3 signaling pathway were also detected using Western blot.Results 1.Under the inverted microscope,the normal macrophages were round and small in shape;M1 phenotype macrophages protruded with pseudopodia,like dendritic or finger;the pseudopodia of M1 phenotype macrophages retreated after the HGF,and the cells became larger and round;M1 phenotype macrophages interacted with HGF and AG490,the cells showed various forms.HGF did not significantly inhibited the proliferation of macrophages.2.The results of the Griess reagents kit showed that the nitrite concentration of culture supernatant(NO%)in the M1 group was obviously higher than that in the M0 or HGF intervention groups;the nitrite concentration of culture supernatant(NO%)in the HGF intervention groups,compared with M1 group,was decreased explicitly in a dosedependent manner(P < 0.05).3.The results of immunofluorescence showed that c-Met was phosphorylated in the cytoplasm of M1 phenotype macrophages,which were treated with HGF.4.Western Blot analysis showed that,compared with M0 group,the protein expressions of iNOS and IL-6 were obviously increased,while the protein expressions of Arg I and IL-10 were significantly decreased in M1 group(P<0.05).Compared with M1 group,the protein expressions of iNOS and IL-6 in HGF intervention group were decreased in a dose-dependent manner,while the protein expressions of Arg I and IL-10 were increased in a dose-dependent manner(P<0.05).Compared with M0 or M1 group,the phosphorylation of JAK2-STAT3 signaling pathway in HGF intervention group was increased in a dose-dependent manner(P<0.05).Compared with the 10M1 group,the protein expression of M2 phenotype-associated Arg I and IL-10 in the AG490+10M1 group were decreased after blocking the JAK2-STAT3 signaling pathway(P<0.05).Conclusion 1.In vitro,RAW264.7 macrophages could be induced to M1 phenotype macrophages by IFN-γ and LPS.2.In vitro,HGF with different concentrations could inhibite the polarization of M1 phenotype macrophages,and promote the polarization of macrophages from M1 to M2 phenotype.3.HGF acts on c-Met receptor and promotes the polarization from M1 phenotype macrophages to M2 phenotype by activating JAK2-STAT3 signaling pathway. |
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