大黄酚改善缺血再灌注诱导的急性肾损伤的机制研究

Mechanism of chrysophanol in improving acute kidney injury induced by ischemia reperfusion

肾脏缺血再灌注损伤(ischemic reperfusion injury,IRI)是导致急性肾损伤(acute kidney injury,AKI)的主要原因,预后不良且死亡率高。有报道提示大黄酚具有肾脏保护作用,但其对IRI的影响及机制尚不清楚。本研究旨在探索大黄酚对IRI诱导的AKI的影响及其作用机制。通过建立小鼠单侧肾脏IRI模型,观察肾脏组织病理变化,检测血清中肌酐、尿素氮水平及肾组织中细胞凋亡、线粒体自噬相关蛋白的表达;建立肾小管上皮细胞(human kidney-2,HK-2)缺氧复氧(hypoxia/reoxygenation,H/R)模型,测定其线粒体膜电位水平及活性氧含量;筛选大黄酚与AKI的相关作用靶点并进行功能富集分析,对核心靶点、关键通路进行验证。动物实验经北京大学实验动物伦理委员会批准(编号:LA2021503)。结果显示,与假手术组相比,IRI组小鼠血清肌酐、尿素氮水平升高,肾组织结构明显遭到破坏,肾损伤分子(KIM1)、凋亡相关蛋白(cleaved-caspase 3、caspase 3、cytochrome C)、线粒体自噬蛋白(PINK1)表达增加,而大黄酚可呈剂量依赖性地改善上述病理变化,并能够显著提高H/R条件下HK-2细胞线粒体膜电位,抑制活性氧产生;通过网络药理学分析发现HSP90AA1和PIK3R1为关键靶点,主要富集于磷脂酰肌醇3激酶/蛋白激酶B(phosphoinositide 3 kinase/protein kinase B,PI3K/Akt)通路,验证发现大黄酚能够显著降低H/R条件下HK-2细胞HSP90AA1和PIK3R1 mRNA水平,并增加p-PI3K、PI3K、p-Akt和Akt蛋白表达水平。综上,大黄酚可能通过靶向调节HSP90AA1、PIK3R1,并活化PI3K/Akt通路,减少细胞凋亡,调节线粒体自噬,提高线粒体膜电位,抑制活性氧产生,进而改善由IRI诱导的AKI,为急性肾损伤提供了潜在治疗方案。

Kidney ischemia reperfusion injury(IRI)is a leading cause of acute kidney injury(AKI)with a poor prognosis and high mortality rate.Recent studies have reported that chrysophanol may have a renal protective effect,but its specific impact and mechanism on IRI remain unclear.This study aimed to explore the effects and mechanisms of chrysophanol on AKI induced by IRI.By utilizing a unilateral kidney IRI mouse model,histopathological changes in the kidney,serum levels of creatinine and urea nitrogen,and protein expressions of apoptosis and mitophagy in kidney tissue were examined.Additionally,a hypoxia/reoxygenation(H/R)model of human kidney-2(HK-2)cells was established to measure mitochondrial membrane potential levels and reactive oxygen species(ROS).Functional enrichment analysis was performed to screen relevant targets of chrysophanol and AKI,and to verify key targets and pathways.The animal experiments conducted in this study were ethically approved by the Experimental Animal Ethics Committee of Peking University(No.LA2021503).The findings indicate that the IRI group exhibited elevated levels of creatinine and urea nitrogen in serum,significant renal tissue damage,and increased expression of renal injury markers(KIM1),apoptosis-related proteins(cleavedcaspase 3,caspase 3,cytochrome C),and mitochondrial autophagy protein(PINK1)compared to the sham surgery group.Chrysophanol treatment ameliorated the aforementioned pathological changes in a dose-dependent manner in an IRI model.Additionally,it exhibited significant improvements in mitochondrial membrane potential and inhibition of ROS production in HK-2 cells subjected to H/R conditions.Through network pharmacological analysis,HSP90AA1 and PIK3R1 were identified as key targets primarily enriched in the phosphoinositide 3 kinase/protein kinase B(PI3K/Akt)pathway.Real-time quantitative PCR(qPCR)validation confirmed that chrysophanol significantly decreased HSP90AA1 and PIK3R1 mRNA levels in HK-2 cells under H/R conditions,while also enhancing the protein expressions of p-PI3K,PI3K,p-Akt,and Akt.In conclusion,chrysophanol has the potential to enhance AKI by selectively modulating HSP90AA1 and PIK3R1,activating the PI3K/Akt pathway,decreasing apoptosis,regulating mitochondrial autophagy,enhancing mitochondrial membrane potential,and suppressing ROS production.These findings suggest that chrysophanol could serve as a promising therapeutic option for the treatment of AKI.