基于间隙连接介导的细胞间损伤信号传导调控机制的甘草解肾毒的机理研究

Gap junction-mediated intercellular communication plays a pivotal role on renal physiological and pathophysiological process. Connexin 43, one of the most widely expressed and deeply studied connexins, is abundantly expressed in kidney. Given that Glycyrrhetinic acid is one of the usually used gap junction blockers, such as Carbenoxolone(CBX), and the main extract from Glycyrrhizae herb, in this research we will study whether and how complex extracts of Glycyrrhizae protect renal cells from toxicity through regulation of gap junction mediated intercellular injury signal transduction. In vitro, the protection of Glycyrrhizae drug serum (GDS) on injured renal cell will be explored through puromycin hydrochloride -induced podocyte injury and gentamycin-induced NRK E52 renal tubular epithelium injury. To study whether and how GDS regulates Cx43, the effects of GDS both on mRNA and protein level will be studied. At the same the function of Cx43 gap junction channel will be analyzed by Lucifer yellow diffusion test in different treatment (GDS, GA and CBX) groups. Furthermore, to investigate the mechanism by which Cx43 mediates protection of GDS in renal injury, we will analyze different response of podocyte and NRK cells that expressing different level of Cx43, downregulation and upregulation of Cx43 by transfection of cells respectively, to treatment of GDS during injury. In vivo, the puromycin aminonucleoside-elicied nephropathy (PAN) and gentamycin-elicited nephropathy (GN) rat models will be established. All the rats will be randomly divided into 5 groups, control, model, GA treatment, Glycyrrhizae extract complex (GXC) low- and high- dose groups. After model establishment, the animals will be sacrificed to analysis weekly for 2 weeks. Urinary samples will be used to analyze the protein and the serum samples will be collected to serum creatine and blood urea nitrogen analysis. Some renal tissue samples will be subjected to pathological study. Some samples will be used to western blot and rt-PCR to evaluate the level of Cx43. Some injury related proteins, caspase 3 and p38, from samples will be subjected to western blot. Our study will complete the traditional Chinese medicine theory that Glycyrrhizae protects kidney from toxicity by block the diffusion. The regulation of gap junction mediated intercellular injury signal transduction underlies the protection of Glycyrrhizae on renal injury.

间隙连接介导的细胞间损伤信号转导在肾脏病的病理生理过程中具有重要作用，Cx43是分布较广、肾脏表达丰富的间隙连接蛋白。甘草成分复杂，有效成分之一甘草甜素具有抑制细胞间隙连接的作用。本研究利用体外嘌呤霉素诱导的足细胞损伤和庆大霉素诱导的肾小管上皮细胞损伤模型，研究甘草含药血清对肾脏细胞损伤的保护作用及对细胞间隙连接结构和功能的影响，并比较与单体甘草甜素和其他间隙连接抑制剂的作用差异。通过基因转染下调和上调Cx43，研究Cx43介导的间隙连接在甘草保护肾脏细胞中的重要作用及机理。在体内实验，利用嘌呤霉素肾损伤和庆大霉素肾损伤大鼠模型明确甘草复方提取物成分对大鼠血尿生化、肾脏病理、Cx43表达分布以及组织损伤相关蛋白的影响。通过本研究明确甘草复方成分对间隙连接的调节，及甘草通过调间隙连接介导的细胞间损伤信号发挥解肾毒、保护肾脏的机理。完善甘草解肾毒"阻邪入里"的中医理论。

间隙连接介导的细胞间损伤信号转导在多种疾病的病理生理过程中具有重要作用，其中连接蛋白43（Cx43）是分布较广、肾脏表达丰富的间隙连接蛋白。甘草次酸是甘草的主要成分之一，具有抑制间隙连接的作用。本研究利用体外阿霉素、嘌呤霉素、氨基糖苷、硫氧还蛋白抑制剂等诱导的肾脏细胞损伤模型，体内Cx43转基因小鼠阿霉素肾病和缺血再灌注肾损伤模型，研究了甘草次酸对肾脏细胞损伤的保护作用及其机理。. 体外细胞研究发现硫氧还蛋白结合蛋白（TXNIP）-硫氧还蛋白（Thioredoxin）-凋亡调节激酶1（ASK1）-p38信号转导在肾脏足细胞氧化损伤中具有重要作用，激活AMPK可调控TXNIP-Thioredoxin-ASK1-p38信号转导发挥保护足细胞的作用。抑制间隙连接可以减轻肾脏损伤，下调Cx43可以抑制TXNIP、激活Akt，从而减轻氨基糖苷、阿霉素和嘌呤霉素诱导的足细胞和肾小管细胞损伤。在硫氧还蛋白抑制剂（PX12）诱导的JNK介导的肾小管细胞氧化损伤中，上调Cx43可促进PX12的细胞毒性，抑制JNK可抑制Cx43蛋白，下调Cx43蛋白可抑制JNK。因此调控Cx43-TXNIP-AKT、Thioredoxin-Cx43-JNK信号转导，在保护肾脏细胞方面具有重要作用。. 在腹腔注射阿霉素诱导的Cx43转基因小鼠肾病模型中，Cx43+/-小鼠与Cx43+/+相比，尿蛋白和血肌酐水平更低，下调Cx43蛋白的小鼠对阿霉素毒性的耐受性更好，生存率更高。在单侧肾切除、单侧肾脏缺血再灌注诱导的Cx43转基因小鼠肾损伤模型中，Cx43蛋白表达与TXNIP表达想平行，Cx43+/-小鼠TXNIP蛋白表达更低。. 本研究明确了甘草通过调间隙连接介导的细胞间损伤信号发挥解肾毒、保护肾脏的机理，建立了甘草解肾毒“阻邪入里”的中医理论。. 本研究为肾脏病的治疗提供了新靶点。甘草次酸通过调控间隙连接，将在氧化应激、能量代谢等方面发挥重要的生物学效应。