基于调控AMPK的丹参酮IIa保护腹膜间皮细胞的作用及机理研究

Peritoneal mesothelial cell (PMC) plays an important role on the maintenance of peritoneal structure and function especially in patients undergoing peritoneal dialysis. The long-term non-physiological stimulation induced by peritoneal dialysis fluid (PDF) usually results in PMC injury and leads to failure of peritoneal dialysis. Adenosine monophosphate-activated protein kinase (AMPK), a well-known cellular energy sensor, plays important roles in regulation of inflammation and fibrosis. In this study we will study the role and mechanism of Tanshinone IIa (Tan IIa) protects peritoneal mesothelial cell through regulation of AMPK. In vitro, primary cultured human peritoneal mesothelial cell (HPMCs) will be used to test whether and how PDF and Tan IIa regulate AMPK in HPMCs. We will first confirm the protection of Tan IIa on HPMCs stimulated by PDF. To reveal the mechanism by which Tan IIa regulates AMPK, we will downregulate the three AMPK upstream kinases, Liver kinase B1(LKB1), calcium/calmodulin-dependent kinase kinases (CaMKKs) and transforming growth factor-activated kinase-1 (TAK1), by transfection of HPMCs. Then we will study the regulation of AMPK on NF-κB and TGFβ through downregulation and upregulation of AMPK by transfection. In vivo, 60 SD male rats from total 65 rats will be randomly divided into peritoneal dialysis (PD) control, PD peritonitis, AMPK activator (metformin, 25mg/100g body weight) treatment, Tan IIa low(6ml Tan IIa and 20ml 4.25%PDF), middle(12ml Tan IIa and 20ml 4.25%PDF) and high dose((24ml Tan IIa and 20ml 4.25%PDF) groups. The other 5 rats will be sacrificed at the day before PD, the blood and peritoneal tissue samples will be collected as control. PD rat model will be established by peritoneally injection of 4.25% PDF once very day for 9 days. Then the peritonitis will be induced by injection of lipopolysaccharide (LPS, 0.06mg/100g bodyweight) at day 3, 5, 7 and 9 during peritoneal dialysis. 5 rats from each group will be sacrificed to get blood and peritoneal tissue samples at week 1 and week 4 after PD. The blood sample will be used to test glucose, insulin, albumin and lipid. The tissue samples will be subjected to pathological analysis to study the inflammation and fibrosis, and western blot to analyze the phosphorylated AMPK (activated form of AMPK).

腹膜间皮细胞在维持腹膜结构和功能方面具有重要的作用。腹膜透析液可造成腹膜间皮细胞损伤、炎症和纤维化。AMPK为一细胞能量感受器，在调控炎症和纤维化方面具有重要作用。本研究拟通过体内外研究明确丹参酮IIa通过调节AMPK保护腹膜间皮细胞的作用及机理。体外实验使用原代培养的人腹膜间皮细胞，研究在腹膜透析液刺激下AMPK的变化及丹参酮IIa对其调节作用。通过基因转染研究AMPK对炎症和纤维化的影响。体内研究，分组单纯腹透组、腹膜炎组、AMPK激动剂治疗组、丹参酮IIa低、中高剂量组。并于腹透开始前一天处死大鼠留取血液、腹膜标本。利用腹腔注射腹透液模拟腹透，于第3、5、7、9天腹腔注射脂多糖诱导腹膜炎。于腹透结束后第1和4周，每组处死5只大鼠留取血液及腹膜标本，血液行血糖、胰岛素、白蛋白、血脂检测。腹膜组织进行病理检测评估炎症和纤维化状况，并提取蛋白进行western blot分析AMPK变化。

丹参酮注射液临床上常用于治疗心脑血管疾病。既往也有研究提示丹参酮注射液可以通过减轻细胞凋亡，从而改善腹膜纤维化的进展。但是其作用机理尚未明确。我们在体内研究中发现，丹参酮注射液可以改善模型鼠的腹膜的病理损伤，上调IL-6、MMP2、TGF-β等炎症、纤维化指标蛋白的表达。在后期的深入研究中，我们建立了PDS诱导的人腹膜细胞株凋亡的细胞模型，发现高糖腹膜透析液（PDS）可以激活氧化应激，活化凋亡调节激酶1（ASK1）-p38 MPAK信号通路而诱发腹膜细胞凋亡，而给与丹参酮注射液以及N-乙酰半胱氨酸（NAC）后可以抑制PDS诱导的氧化应激，并且抑制ASK1-P38MAPK信号通路的活化，减轻细胞凋亡。应用ASK1 siRNA转染细胞后，也进一步证实了ASK1在PDS诱导的细胞凋亡中的重要作用。通过此研究我们证实了丹参酮磺酸钠注射液可以通过调控氧化应激MAPK信号通路等减轻细胞凋亡，为其在腹膜透析患者长期应用丹参酮来延缓腹膜纤维化，延缓腹膜功能衰竭提供了重要的依据。