Brg1/lncCBRF调控necroptosis恢复糖尿病心肌对七氟烷后处理敏感性的作用及机制

Subjects with diabetes are more vulnerable to ischemic myocardial injury (IRI) and less or not sensitive to anesthetic sevoflurane postconditioning (SevoPC) mediated cardioprotection, and the underlying mechanism is unclear. Reduction in cardiac expression of Brg1 in diabetes has been shown to be associated with increased vulnerability to IRI but the exact role of Brg1 in this pathology is unknown. We have identified a specific long non-coding RNA (lncRNA), which we named lncCBRF, that was significantly downregulated in transgenic mice with cardiac Brg1 overexpression. This lncCBRF has tight links with genes related to necroptosis. Further, either Brg1 gene knock-down or lncCBRF overexpression can cancel SevoPC-mediated protection against hypoxia/reoxygenation in cultured cardiomyocytes. Our preliminary studies showed that SevoPC cardioprotection was lost in diabetes, accompanied with reduced cardiac Brg1, increased lncCBRF and necroptosis. The above preliminary findings prompted us to postulate that hyperglycemia-induced reduction in Brg1 and enhancement of lncCBRF and necroptosis are responsible for the loss of SevoPC cardioprotection in diabetes. We, therefore, aim to examine the role of Brg1 in SevoPC cardioprotection against myocardial IRI and to explore the underlying mechanism in relation to lncCBRF and necroptosis in diabetic rodents. We will conduct series experiments using in vivo and in vitro cell models of myocardial IRI in normal and diabetic mice with Brg1 gene knock out, incorporating the use of Brg1 and lncCBRF adenoviruses to address mechanisms. This study will provide new insights regarding the molecular mechanism whereby Brg1/lncCBRF restores diabetic hearts sensitivity to SevoPC, and facilitate the development of effective therapy to combat ischemic heart diseases.

糖尿病心脏对缺血再灌注损伤（IRI）耐受性降低，并对麻醉剂七氟烷后处理（SevoPC）不敏感，与心肌Brg1减少有关，但其调控机制不详。我们前期发现心肌Brg1高表达下调与细胞程序性坏死（necroptosis）关系密切的长链非编码RNA（lncCBRF），而在离体心肌细胞上Brg1基因沉默或lncCBRF过表达均取消SevoPC对缺氧复氧损伤的保护作用。糖尿病心肌Brg1下降，lncCBRF和necroptosis上调。我们推测糖尿病中降低的Brg1上调lncCBRF，增加necroptosis，加重IRI并取消SevoPC。我们拟建立Brg1基因敲除鼠2型糖尿病，心肌IRI及心肌细胞缺氧复氧模型，结合应用Brg1或lncCBRF腺病毒转染或necroptosis诱导，探讨Brg1/lncCBRF调控对糖尿病心肌IRI及SevoPC敏感性的影响。为糖尿病患者心脏IRI防治提供实验依据。

糖尿病心脏对缺血再灌注损伤（IRI）耐受性降低，并对麻醉剂七氟烷后处理（SevoPC） 不敏感，与心肌Brg1减少有关，但其调控机制不详。本课题研究发现，（1）在正常大鼠（非糖尿病）中，七氟烷后处理（SevoPC）上调Brg1，下调程序性坏死（necroptosis），并减轻心肌IRI。（2）在糖尿病中SevoPC保护作用消失，同时心肌Brg1下调，necroptosis上调。（3）在链脲佐菌素 Streptozotocin（STZ）诱 导在体大鼠糖尿病模型中，心肌肥厚且心功能受损，同时心肌Brg1表达下调伴随着心肌necroptosis上调，而这些变化可通过过抑制 PKCb逆转。（4）通过生物信息学分析发现Brg1可能与necroptosis关键调节蛋白RIP3上游区域结合进而抑制RIP3基因转录并抑制其蛋白表达，此结果通过染色质免疫沉淀验证。（5）在正常（非糖尿病）大鼠中，缺血后处理通过脂联素进而通过 AMPK 依赖途径激活细胞核内 STAT3，同时通过 非 AMPK 依赖途径激活线粒体 STAT3，通过这两个途径共同实现心肌保护作用。综述，以上结果表明，缺血后处理及七氟烷后处理通过Brg1及脂联素调控STAT3并抑制necroptosis，起心肌保护作用。而在糖尿病心肌中，Brg1及脂联素的下调，可抑制STAT3激活，进而上调necroptosis，从而加重糖尿病心肌损伤并减弱后处理及七氟烷后处理心肌保护作用。本课题研究成果可为糖尿病心肌缺血再灌注损伤提供理论依据。