Inhibitory Effect of MKL1 Knockdown on Myocardial Fibroblast Activation and Fibrotic Phenotype Through Activation of Autophagy Flow
Objective To investigate the effect of megakaryocytic leukemia factor 1(MKL1)knockdown on activation and fibrotic phenotype of myocardial fibroblasts(CFs)and its mechanism.Methods Human CFs were selected and MKL1 expression in CFs was knocked down to detect the knockdown effect.CFs were divided into control group,angiotensinⅡ(AngⅡ)group,shNC+AngⅡgroup and shMKL1+AngⅡ group.shNC +AngⅡ group and shMKL1 +AngⅡ group were transfected with shRNA NC and shRNA MKL1,respectively.CFs in AngⅡ group,shNC+AngⅡ group and shMKL1+AngⅡ group were re-stimulated by AngⅡ to establish myocardial fibrosis model.After treatment,the proliferative activity of CFs in each group was detected,the level of COL-Ⅰand COL-Ⅲ in the supernatant of CFs cell culture of each group was determined,and the expression of α-smooth muscle actin(α-SMA)in CFs of each group was observed.The expression levels of α-SMA,COL-Ⅰ and COL-Ⅲ in CFs were detected,and the auto-phagy flux of CFs in each group was observed.The ratio of LC3Ⅱ/LC3Ⅰ protein and the expression level of Beclin1 protein in CFs in each group were detected.Results The relative expression of MKL1 mRNA and protein in CFs transfected with shMKL1 were lower than those in CFs without transfection and in CFs transfected with shNC(P<0.05).Compared with the control group,the prolifer-ative activity of CFs at the same time point in AngⅡgroup was increased(P<0.05),and the levels of COL-Ⅰ and COL-Ⅲ in super-natant were increased(P<0.05);the α-SMA fluorescence staining intensity was increased in CFs,and the relative protein expres-sions of α-SMA,COL-Ⅰ and COL-Ⅲ were increased(P<0.05).The number of autophagosomes and autophagosomes was de-creased,the ratio of LC3Ⅱ/LC3Ⅰ protein was decreased(P<0.05),and the relative expression of Beclin1 protein was decreased(P<0.05).Compared with AngⅡ groupand NC+AngⅡ group,theproliferationactivityof CFsat thesametimepoint inshMKL1+ AngⅡ group was decreased(P<0.05),and the levels of COL-Ⅰ and COL-Ⅲ in supernatant were decreased(P<0.05).The α-SMA fluorescence staining intensity in CFs was decreased,and relative expression levels of α-SMA,COL-Ⅰ and COL-Ⅲ protein were down-regulated(P<0.05).Autophagosome and autophagosome were increased in CFs,LC3Ⅱ/LC3Ⅰ protein ratio was increased(P<0.05),and relative expression level of Beclin1 protein was up-regulated(P<0.05).Conclusion MKL1 knockdown can in-hibit CFs activation and myocardial fibrotic phenotype,which may be related to the activation of intracellular blocked autophagy flux.
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