C/EBPβ-血清反应因子复合体调控心脏成纤维细胞/肌成纤维细胞表型及功能转化的机制

Cardiac fibrosis is a common pathway through which myocardial inflammation progresses into heart failure. It significantly contributes to morbidity and mortality. Cardiac myofibroblasts which are absent from normal heart are the "culprit" cells in the pathogenesis of cardiac fibrosis. Myofibroblasts are responsible for the synthesis of extracellular matrix (ECM) which is associated with fibrosis. In response to tissue inflammation, fibroblasts differentiate into myofibroblasts, the "muscle-like" contractile cells characterized by synthesis and polymerization of α smooth muscle actin (α-SMA). The capacity of myofibroblasts to undergo de-differentiation as a potential mechanism for cardiac fibrosis reversion has not been examined till now. Moreover, the mechanisms involved in the CF/CMF transformation remains unclear. We found in previous studies that C/EBPβ played an important role in the modulation of CF/CMF phenotype transformation. Serum response factor (SRF) blocker could inhibit the pro-differentiation funciton of C/EBPβ. It has been well accepted that SRF is a central transcription factor that regulates the expression of α-SMA gene and actin depolymerization factor (ADF)/cofilin gene by formation of a complex with other tissue specific transcription factors or co-factors. SRF functions as a platform that generically binds to CArG box DNA, and then recruits accessory factors that either activate or repress transcription of target genes in response to various environmental factors. C/EBPβ is one of the co-factors which have the capability to interact with SRF. Thus, we deduced that C/EBPβ might function through SRF-dependent mechanisms. On the basis of the above observation, we hypothesize that ① C/EBPβ modulates the CF/CMF phenotypic and functional transformation through the SRF-dependent mechanisms. The activating isoform, LAP, and the inhibitory isoforms, LIP, functions as the co-activator and the co-inhibitor for SRF separately. LAP and LIP are involved in the modulation of actin expression and polymerization. ② Modulation of the LAP and LIP ratio, an indicator for C/EBPβ activation, might inhibit CF differentiation, or even induce cardiac myofibroblasts de-differentiation, through inhibition of actin expression and de-polymerization, and finally reverse cardiac fibrosis. In this study, we plan to modulate the transcriptional activation of C/EBPβ-SRF complex with genetically engineered mice model, accompanied with the lentivirus mediated C/EBPβ shRNA, or LAP and LIP overexpression, in order to elucidate the underlying mechanisms responsible for inflammation-induced cardiac fibrosis. This project would hopefully open a novel perspective for the prevention and treatment of heart failure.

心肌纤维化是炎症导致心力衰竭的必由之路。心脏肌成纤维细胞（CMF）是该过程的效应细胞。CMF"去分化"为前体细胞-心脏成纤维细胞（CF）是逆转纤维化的可能途径。但CF与CMF之间转化的机制尚未阐明。我们前期研究发现炎症转录因子C/EBPβ调控CF和CMF间表型转化，血清反应因子SRF的阻断剂可抑制C/EBPβ的促CF分化作用。推测C/EBPβ通过SRF发挥作用，在上述发现基础上提出假说①C/EBPβ以SRF依赖性方式调控CF/CMF转化，C/EBPβ两亚型LAP和LIP分别作为SRF的共激活子和共抑制子，参与平滑肌肌动蛋白表达和聚合/解聚调控；②调控C/EBPβ转录活性（LAP/LIP比率），可能实现CF活化的抑制；甚至CMF去分化，逆转心肌纤维化。拟以基因工程鼠、LAP及LIP过表达、RNA干扰调控C/EBPβ-SRF转录复合体活性，旨在阐明炎症性心肌纤维化的机制，为心衰防治开辟新思路。

目的：探讨炎症转录因子C/EBPβ在自身免疫性心肌炎心肌纤维化中的作用及可能机制。方法：体外实验中，原代培养心脏成纤维细胞，TGFβ诱导成纤维细胞分化为肌成纤维细胞。慢病毒介导C/EBPβRNA干扰，检测C/EBPβ蛋白不同亚型（LAP、LIP）的表达、比例及其对向肌成纤维细胞分化的影响，检测下游蛋白cofilin、FLNa的表达，探讨C/EBPβ影响心脏成纤维细胞分化的可能机制。在体实验中，将24只Lewis大鼠分为正常组、阴性对照组、阳性对照组和实验组。正常组无任何处置，其余各组分别于实验第1天和第7天给予猪心肌球蛋白（2 mg/只)后足垫注射两次，诱导实验性自身免疫性心肌炎模型。超声微泡法将对照和实验慢病毒导入大鼠心脏，观察超声心动图IVS、LVPW、LVEDD、LVESD、EF、FS等指标的变化，HE染色检测心肌炎症程度，Masson染色定量心肌纤维化程度，免疫组化测定α-SMA、C/EBPβ、MMP2、MMP9、collagen I、collagen III等表达，western blot定量C/EBPβ蛋白LAP和LIP亚型。结果：C/EBPβ基因沉默后，心脏成纤维细胞中LAP/LIP比值增加，下游蛋白cofilin、FLNa的表达均减少。实验组与对照组相比，炎症及纤维化程度减轻（P<0.01），α-SMA、C/EBPβ、MMP2、MMP9、collagen I、collagen III表达均减少（P<0.05）。结论：抑制炎症转录因子C/EBPβ的表达可减轻炎症介导性心肌纤维化程度，其机制可能通过调控cofilin、FLNa表达。