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To investigate the role and mechanisms of transcription factor CCAAT/enhancer binding protein beta (C/EBPβ) in myosin-induced myocardial fibrosis.C/EBPβ consists of LAP, LAP* and LIP. We intend to find the expression of LAP, LAP* and LIP and their ratio during the regulation.
In vitro, we culture fibroblasts from SD rat, then use TGFβ1 to stimulate the fibroblasts, three days later, detect the expression of cytokines of myofibroblasts, finally transfer the control lentivirus and experimental lentivirus into cells, then evaluated the expression and influence of C/EBPβ to myocardial fibrosis, assess the proportion of different subtypes of C/EBPβ(LAP, LAP* and LIP) and the ratio between them, assess the degree of fibrosis in each group, detected the expression of downstream proteins cofilin and FLNa, and investigate the relationship of C/EBPβ and cofilin, FLNa. In vivo, 24 Lewis rats were randomly divided into the control group, the myosin group, myosin+ control lentivirus group and the myosin+ experimental lentivirus group. The control group did not receive any disposal, other groups were given footpad injection of porcine cardiac myosin 1mg/per rat twice, on the first day and the seventh day respectively. Then import the control lentivirus and experimental lentivirus fused ultrasound microbubble via femoral vein injection combined with local ultrasonic irradiation. The ultrasound indicators of heart were observed, mainly include IVS, IVPW, LVEDD, LVESD, EF, FS and so on. The degree of inflammation in the myocardium was quantified by HE staining, and cardiac fibrosis was quantified by Masson staining. We also measured the expression of α-SMA, C/EBPβ, MMP2, MMP9, collagen I, collagen III by immunohistochemistry, the expression of C/EBPβ in each group by western blot.
When C/EBPβ was silenced, the myocardial fibrosis, the ratio between LAP and LIP was increased, at the same time the expression of downstream protein cofilin and FLNa were reduced. Compared with the control group, the experimental group has a lower inflammation and fibrosis (P<0.01) level. The expression of α-SMA, C/EBPβ, MMP2, MMP9, collagen I, collagen III were reduced simultaneously in the experimental groups (P<0.05).
Inhibition of the expression of transcription factor C/EBPβ could reduce myocardial fibrosis and inflammation. Inhibition the expression of cofilin and FLNa might be the underlying mechanism.