Table 5_Quercetin reverses β1-adrenoceptor autoantibody-induced heart failure by promoting MDM2-mediated ubiquitination and degradation of p53 in cardiomyocytes.xlsx

IntroductionCardiomyocyte autophagy is essential for preserving cardiac homeostasis. Previous studies revealed that β1-adrenergic receptor autoantibody (β1-AA) suppressed cardiomyocyte autophagy, triggering cell death and heart failure (HF). Qiliqiangxin capsule enhances autophagy and mitigates HF through multiple pathways, but its complex composition complicates mechanistic clarity. Network pharmacology identified quercetin as a pivotal autophagy-inducing component in Qiliqiangxin, yet its role in counteracting β1-AA-induced autophagy impairment remains unvalidated. In this study, quercetin’s therapeutic potential and mechanisms in restoring autophagy in β1-AA-associated HF were investigated.
MethodsBioinformatics methods, including a STRING database analysis, PPI network construction, and Cytoscape-based pathway mapping, were used to delineate quercetin’s autophagy-related targets. The in vivo efficacy was assessed in β1-AA-positive mice treated with quercetin (100 mg/[kg·d], intraperitoneal). The in vitro validation used H9c2 cardiomyocytes pretreated with quercetin (100 μM) prior to β1-AA exposure. Autophagy markers, p53 signaling, and ubiquitination pathways were analyzed by immunoblotting and functional enrichment analysis using the GOrilla database. A p53 knockdown and overexpressing cardiomyocyte model confirmed pathway specificity.
ResultsQuercetin administration significantly restored myocardial autophagy levels in β1-AA-positive mice, which improved cardiac function and survival rates. In H9c2 cells, quercetin pretreatment reversed β1-AA-induced autophagy suppression. Bioinformatics linked quercetin to p53 pathway modulation, with experimental validation showing quercetin downregulated p53 expression via MDM2-mediated ubiquitination. p53 knockdown enhanced autophagy, while its overexpression blocked quercetin’s effect, indicating quercetin restores autophagy in a p53-dependent manner. GO enrichment highlighted the association between quercetin and ubiquitin-dependent protein degradation, which was corroborated by elevated MDM2 levels and accelerated p53 degradation in quercetin-treated cells.
DiscussionQuercetin rescues β1-AA-impaired cardiomyocyte autophagy by activating MDM2-dependent p53 ubiquitination and degradation, thereby attenuating HF progression. These findings establish quercetin as the mechanistic basis of the cardioprotective effects of Qiliqiangxin and provide preclinical evidence for targeting autophagy by regulating p53 in β1-AA-induced cardiac dysfunction.

引言
心肌细胞自噬（cardiomyocyte autophagy）对于维持心脏内稳态至关重要。既往研究显示，β1-肾上腺素能受体自身抗体（β1-adrenergic receptor autoantibody，β1-AA）可抑制心肌细胞自噬，进而引发细胞死亡与心力衰竭（heart failure，HF）。芪苈强心胶囊（Qiliqiangxin capsule）可通过多条通路增强自噬并缓解心力衰竭，但由于其成分复杂，具体作用机制难以明确。网络药理学研究证实，槲皮素（quercetin）是芪苈强心胶囊中关键的自噬诱导成分，但其能否拮抗β1-AA诱导的自噬损伤尚未得到验证。本研究旨在探究槲皮素在β1-AA相关心力衰竭中恢复心肌细胞自噬的治疗潜力与作用机制。

方法
本研究采用生物信息学方法，包括STRING数据库分析、蛋白质相互作用（protein-protein interaction，PPI）网络构建以及基于Cytoscape的通路注释，以明确槲皮素相关的自噬作用靶点。体内实验以β1-AA阳性小鼠为模型，给予槲皮素（100 mg/[kg·d]，腹腔注射）以评估其药效。体外实验则采用H9c2心肌细胞，在暴露于β1-AA前先以100 μM槲皮素进行预处理。通过免疫印迹法以及基于GOrilla数据库的功能富集分析，对自噬标志物、p53信号通路以及泛素化通路进行检测分析。通过构建p53敲低与过表达的心肌细胞模型，验证了该通路的特异性。

结果
给予槲皮素可显著恢复β1-AA阳性小鼠的心肌自噬水平，进而改善心脏功能并提高生存率。在H9c2细胞中，槲皮素预处理可逆转β1-AA诱导的自噬抑制。生物信息学分析将槲皮素与p53通路调控关联起来，实验验证结果显示，槲皮素通过鼠双微体2（murine double minute 2，MDM2）介导的泛素化途径下调p53的表达。敲低p53可增强自噬水平，而过表达p53则会阻断槲皮素的作用，表明槲皮素通过p53依赖的方式恢复自噬。基因本体（Gene Ontology，GO）富集分析显示，槲皮素与泛素依赖的蛋白质降解通路密切相关，这一结果得到了实验验证：经槲皮素处理的细胞中MDM2水平升高，p53降解加速。

讨论
槲皮素通过激活MDM2依赖的p53泛素化与降解通路，挽救β1-AA诱导的心肌细胞自噬损伤，进而延缓心力衰竭的进展。本研究证实槲皮素是芪苈强心胶囊发挥心脏保护作用的机制基础，并为通过调控p53靶向干预自噬以治疗β1-AA诱导的心脏功能障碍提供了临床前证据。