线粒体STAT3/p53通路参与alpha-2A受体调控肾缺血再灌注损伤的机制研究

Mitochondrial permeability transition pore (MPTP) opening is the critical reason of renal ischemia reperfusion (I/R) injury. Mitochondrial protein cyclophilin D (CypD) is the key factor in regulating MPTP opening. CypD, combined with mitochondrial signal transduction and transcription factor (STAT) 3 or its downstream p53, can affect MPTP opening and futher regulate renal I/R injury. Our previous studies have revealed that renal protective effect induced by alpha(α)2 adrenergic receptor is closely related to its α2A receptor subtype (α2A-AR), which may regulate MPTP opening through mitochondrial STAT3. However, the effects of mitochondrial STAT3 and p53 on MPTP changes induced by α2A-AR remain unknown. Our study is to up- or dowm-regulate α2A-AR protein and observe its effect on MPTP opening in vitro and vivo experiments, futher regulate mitochondrial STAT3 and p53 to determine their interrelation relationship and their effects on MPTP changes induced by α2A-AR, and finally investigate the mechanism of α2A-AR on regulating MPTP changes and renal I/R injury. There has no domestic and abroad reports similar to our relevent research here. This study will provide new molecular target and experimental evidences of protection against renal I / R injury clinically.

线粒体通透性转换孔（MPTP）开放是肾缺血再灌注（I/R）损伤的主要原因。线粒体亲环蛋白D（CypD）是调控MPTP开放的关键因子，它与线粒体信号转导和转录因子（STAT）3或其下游分子p53结合，均可影响MPTP开放，从而介导肾I/R损伤。我们的前期研究表明：alpha(α)2受体介导的肾保护与其α2A受体亚型（α2A-AR）密切相关，且α2A-AR通过线粒体STAT3调节MPTP开放。而线粒体STAT3和p53对α2A-AR介导的MPTP 变化的影响尚不清楚。本课题拟通过体外和体内实验，正负调控α2A-AR观察其对线粒体MPTP开放的影响，并进一步调控线粒体STAT3和p53确定两者相互关系及其对α2A-AR介导的MPTP变化的影响，探索α2A-AR调控肾I/R损伤诱导的MPTP变化的机制。相关方面的研究工作国内外尚未见报道。本研究将为临床防治肾I/R损伤提供新的分子靶点和实验依据。

背景：肾缺血再灌注(I/R)损伤是外科患者急性肾损伤的常见因素之一。右美托咪定具有肾保护作用，然而其机制尚不明确。本研究拟探讨SIRT3在右美托咪定减轻肾 I/R损伤中的作用及其分子机制。.方法和结果：本研究采用人近端肾小管上皮细胞(HK-2)建立缺氧复氧(H/R)模型，右美托咪定缺氧前预处理，质粒及siRNA正负调控SIRT3，流式细胞学检测细胞死亡率、线粒体膜电位（Δψm），Western blot检测SIRT3表达，免疫沉淀检测CypD 乙酰化。体内实验采用雄性成年C57BL/6J小鼠建立肾缺血再灌注（I/R）模型，右美托咪定缺血前预处理，SIRT3 siRNA负调控SIRT3，生化检测血清中肾功能指标，ELISA法测定血清Cys C和NGAL表达；取小鼠肾组织，显微镜下观察肾小管上皮细胞线粒体超微结构，TUNEL法检测细胞凋亡，Western blot检测SIRT3和CytC表达，免疫沉淀法测定CypD乙酰化。结果证实：右美托咪定显著抑制H/R诱导的细胞死亡、线粒体膜电位下降、SIRT3表达降低、CypD乙酰化水平和CytC表达增加；SIRT3质粒增强右美托咪定对H/R损伤细胞的保护作用；SIRT3 siRNA 逆转右美托咪定的细胞H/R损伤保护作用；右美托咪定抑制I/R引起的血清Scr、BUN、Cys C和NGAL水平升高，细胞凋亡增加，SIRT3表达和活性降低，CypD乙酰化水平和CytC表达增加；肾组织形态学改变和线粒体超微结构损伤；SIRT3 siRNA逆转右美托咪定对小鼠肾I/R损伤的保护作用。.结论：右美托咪定通过上调SIRT3表达减轻肾I/R损伤。