| Part Ⅰ:The effect of CLEC5A on myocardial infarction miceObjective:To explore the effect of CLEC5A on myocardial infarction(MI)in mice.Methods:C57BL/6 mice were randomly divided into 4 groups:Sham,MI,MI+Ad-NC,MI+Ad-sh-CLEC5A,6 mice in each group.After each mouse was anesthetized and fixed,the chest cavity was opened,and the left anterior descending coronary artery was ligated.In the Sham group,only the chest was opened without ligation.After ligation of MI+Ad-NC and MI+Ad-sh-CLEC5A mice,Ad-NC or Ad-sh-CLEC5A(CLEC5A inhibitor)was injected at 3 points near the site of myocardial necrosis,and mice in the control group and MI group were injected the equal dosage of PBS.Seven days after the injection,the LVESD,LVEDD,LVEDV,and LVESV of each group of mice were detected by echocardiography,and EF and FS were calculated.The mice of each group were killed by anesthesia,and myocardial tissue was taken.The expression of CLEC5A in the penumbra of infarction was detected by western blotting.The expression and distribution of CLEC5 A in the penumbra of infarction were detected by histochemistry,the area of myocardial infarction was detected by Triphenyltetrazolium chloride staining,and the apoptosis of myocardial cells in the penumbra of infarction was detected by TUNEL.Results:1.Compared with the sham operation group,the LVESD,LVEDD,LVEDV,and LVESV of the MI group were significantly increased,the EF and FS of the MI group were significantly reduced,and the heart function impairment in the Ad-sh-CLEC5A treatment group was less(P<0.001).2.Ad-sh-CLEC5A successfully silenced the expression of CLEC5A in model mice(P<0.001).3.The expression distribution of CLEC5A in the MI group was significantly increased,while the expression distribution of CLEC5A in the Ad-sh-CLEC5 A group was significantly reduced.4.The MI group had a larger area of myocardial infarction(P<0.001),but after using Ad-sh-CLEC5A to interfere with the mouse model,it was found that the area of myocardial infarction was significantly reduced(P<0.001).5.Compared with the Sham group,the density of TUNEL positive cells in the MI group was significantly increased,compared with MI group,the intervention of Ad-sh-CLEC5A significantly reduced the apoptosis of cardiomyocytes.Conclusion:1.CLEC5A is related to the impairment of cardiac function after MI,which may aggravate the degree of impairment.2.CLEC5A is related to MI infarct size.3.CLEC5A may be involved in the pathological process of MI myocardial damage.4.Knockdown of CLEC5A may reduce the impact of MI on the heart.Part Ⅱ The effect of CLEC5A on the activation of macrophages,inflammasome and pyrolysis and NF-κB signaling pathway in mice with myocardial infarctionObjective:To explore the effects of CLEC5A on the activation of macrophages,inflammasomes and pyrolysis in MI mice,and its effects on the NF-κB signaling pathway in MI mice.Methods:C57BL/6 mice were randomly divided into 4 groups:Sham,MI,MI+Ad-NC,MI+Ad-sh-CLEC5A,6 mice in each group.After each mouse was anesthetized and fixed,the chest cavity was opened,and the left anterior descending coronary artery was ligated.In the Sham group,only the chest was opened without ligation.After ligation of MI+Ad-NC and MI+Ad-sh-CLEC5A mice,Ad-NC or Ad-sh-CLEC5A(CLEC5A inhibitor)was injected at 3 points near the site of myocardial necrosis,and mice in the control group and MI group were injected the equal dosage of PBS.Seven days after the injection,the mice in each group were anesthetized and the myocardial tissue was taken.The levels of inflammatory factors IL-6 and TNF-α in the penumbra of the infarct were detected by enzyme-linked immunosorbent assay,and MAC-3 in the penumbra of the infarct was detected by immunofluorescence double staining.And iNOS,fluorescence quantitative PCR to detect the mRNA levels of iNOS and IL-6 in the infarct penumbra area,and Western blotting to detect NLRP3,ASC,caspase-1(pro and p10),IL-1β,IL-18,GSDMD and N-GSDMD in the myocardial tissue of the infarct penumbra area.Western blotting was used to detect the levels of NF-κB p65,p-NF-κB p65,NF-κB(nucleus)and NF-κB(cytoplasmic)in the myocardial tissue of the penumbra of infarction.Results:1.Compared with the Sham group,the levels of IL-6 and TNF-α in the MI group were significantly increased(P<0.001),after using Ad-sh-CLEC5A to intervene in model mice,the inhibition of IL-6(P<0.05)and TNF-α(P<0.01)were both significantly increased.2.iNOS can co-localize with MAC-3.Compared with the Sham group,the number of M1 macrophages per unit area in the MI group was significantly increased.After the intervention of Ad-sh-CLEC5A in model mice,the M1 macrophages per unit area is significantly reduced(P<0.001).3.Compared with the Sham group,the mRNA levels of iNOS and IL-6 in the infarct penumbra of the MI group were significantly higher(P<0.001),after the Ad-sh-CLEC5A intervention model mice,the levels of iNOS and IL-6 in the infarct penumbra of the mice mRNA levels were significantly suppressed(P<0.001).4.After the use of Ad-sh-CLEC5A intervention model mice,the protein expression level of NLRP3(P<0.001),ASC(P<0.001),cleaved caspase-1(45 kDa)(P<0.05),IL-1β(P<0.001),IL-18(P<0.001),N-GSDMD(53 kDa)(P<0.001)was reduced,and the protein expression levels of the inactive precursors of caspase-1(20 kDa)and GSDMD(35 kDa)proteins remained unchanged.5.p-NF-κB p65/NF-KB p65 in the myocardial tissue of the infarcted penumbra of mice was significantly increased(P<0.001),after Ad-sh-CLEC5A was used to intervene in model mice,p-NF-κB p65/NF-κB p65 was significantly inhibited in the myocardial tissue of the infarct penumbra area(P<0.05).6.The expression level of NF-κB p65 in the nucleus of mice in the MI group was higher(P<0.001),while the expression level in the cytoplasm was lower(P<0.001),indicating that NF-κB p65 translocated from the cytoplasm to the nucleus.However,the intervention of Ad-sh-CLEC5A significantly limited this phenomenon(P<0.01 in cytoplasm,P<0.001 in nucleus).Conclusion:1.CLEC5A is related to MI inflammation,and the relationship between the two is directly proportional.Knockdown can reduce inflammation.2.CLEC5A is related to the activation of macrophages after MI,and the relationship between the two is directly proportional.Knockdown can reduce the activation of macrophages.3.CLEC5A is related to the activation of NLRP3 inflammasomes after MI,and the relationship between the two is directly proportional.Knockdown can reduce the activation of NLRP3 inflammasomes.4.CLEC5A is related to the occurrence of myocardial cell pyrolysis after MI,and the relationship between the two is directly proportional.Knockdown can reduce myocardial cell pyrolysis after MI.5.The NF-κB signaling pathway may be involved in the inflammatory response after MI,and the activation of this signaling pathway is positively correlated with CLEC5A.Part Ⅲ:The effect of CLEC5A on macrophage polarization and inflammasome and pyrolysis after myocardial cell injury in miceObjective:To investigate the effect of CLEC5A on inflammasome and pyrolysis by LPS(Lipopolysaccharides)induced mouse macrophage polarization model.Method:The cultured RAW264.7 cells were randomly divided into 4 groups:Control,L/I,NC(normal saline)+L/I(100 ng/ml LPS+20 ng/ml IFN-γ),sh-CLEC5A+L/I.After co-culture of primary cardiomyocytes(cardiac myocyte,CMC)and RAW264.7 cells,they were randomly divided into 5 groups:CMC,CMC+RAW264.7,CMC+RAW264.7(L/I),CMC+RAW264.7(NC+L/I),CMC+RAW264.7(sh-CLEC5A+L/I).Cardiomyocytes are processed by isolation,culture,passage,resuscitation,and counting.RAW264.7 cells are cultured,transfected,polarized and stimulated,and then RAW264.7 cells are transfected with CLEC5A or NC shRNA and detected by western blot analysis.Knockout efficiency,fluorescence quantitative PCR to detect the mRNA levels of total iNOS and IL-6 after cell differentiation of MI type,immunofluorescence staining to detect the expression of cTnT,MTT to detect the cell viability of co-cultured cardiomyocytes,and western blotting to detect the levels of NLRP3,ASC,caspase-1(pro and p10),IL-1β,IL-18,GSDMD,and N-GSDMD in cells.Results:1.48 h after RAW264.7 cells were transfected with sh-CLEC5A,the level of CLEC5A decreased significantly(P<0.001),and the transfection effect was satisfactory.2.After the co-treatment of LPS and IFN-y,the levels of iNOS and IL-6 mRNA in RAW264.7 cells increased significantly,suggesting that macrophages appeared M1 polarization(P<0.001).However,down-regulation of the expression of endogenous CLEC5A in RAW264.7 cells significantly offset the M1 polarization of the treated cell method(P<0.001).3.Immunofluorescence staining showed clearly marked primary cardiomyocytes.4.Co-cultured Ml macrophages significantly reduced Cell viability of primary cardiomyocytes(P<0.05).However,knockdown of CLEC5A in polarized RAW264.7 cells finally significantly compensated for this effect(P<0.05).5.The level of protein NLRP3(P<0.001),IL-18(P<0.001),N-GSDMD(P<0.01)and p10-CASP1(P<0.001)in primary cardiomyocytes co-cultured with polarized RAW264.7 cells increased significantly,and decreased after CLEC5A was silenced.Conclusion:1.RAW264.7 cells can be successfully transfected with sh-CLEC5A RNA.2.LPS can successfully induce polarization of mouse macrophages.3.Macrophages can significantly reduce the cell viability of primary cardiomyocytes,and knocking down CLEC5A can eventually compensate for this effect.4.Macrophage polarization can lead to NLRP3 The activation of inflammasomes and the Pyrolysis of primary cardiomyocytes,and knockdown of CLEC5A can significantly compensate for these effects. |
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