褪黑素和吡菲尼酮对大鼠腹膜透析相关性腹膜纤维化的影响

发布时间：2018-01-08 10:38

本文关键词：褪黑素和吡菲尼酮对大鼠腹膜透析相关性腹膜纤维化的影响　出处：《安徽医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:腹膜透析(Peritoneal Dialysis,PD)是终末期肾病ESRD的有效治疗方法之一,但长期腹膜透析治疗可出现腹膜纤维化,导致腹膜结构和功能的丧失,由腹膜纤维化最终导致的腹膜超滤衰竭是腹膜透析患者退出治疗的主要原因,因此预防和减缓腹膜纤维化是保证腹膜透析患者长期成功透析的关键。褪黑素是人体松果体分泌的主要激素,具有抗炎、抗氧化、抗纤维化作用;吡菲尼酮(pirfenidone,PFD)是一种新型的广谱抗纤维化药物,肾纤维化、肝纤维化模型中表现出明显的抗纤维化作用。目前尚未发现褪黑素和吡菲尼酮应用于腹膜透析相关性腹膜纤维化防治,本研究利用高糖腹腔注射诱导腹膜纤维化,分别给予褪黑素、吡菲尼酮干预,观察他们对腹膜透析相关性腹膜纤维化的影响,为腹膜透析相关性腹膜纤维化的防治提供新思路。方法:随机将46只雄性SD大鼠分为6组。(1)正常组(n=6):每日腹腔内注射0.9%生理盐水20ml+治疗药物等体积的三蒸水稀释的无水乙醇;(2)模型组(n=8):每日腹腔内按体重100ml/kg注射4.25%腹膜透析液+治疗药物等体积的0.9%NS;(3)低剂量褪黑素组(n=8):每日腹腔内按体重100ml/kg注射4.25%腹膜透析液+褪黑素5mg/kg;(4)中剂量褪黑素组(n=8):每日腹腔内按体重100ml/kg注射4.25%腹膜透析液+褪黑素10mg/kg;(5)高剂量褪黑素组(n=8):每日腹腔内按体重100ml/kg注射4.25%腹膜透析液+褪黑素20mg/kg;(6)吡菲尼酮组(n=8):每日腹腔内按体重100ml/kg注射4.25%腹膜透析液+胃管灌注吡菲尼酮500mg/kg。实验第28天行4小时腹膜平衡实验(PET),量取超滤量(UF),检测腹透液尿素氮浓度(D)、血浆尿素氮浓度(Purea)、初始腹透液葡萄糖浓度(D0)、4小时后透出液葡萄糖浓度(D4),并计算D/Purea、D4/D0。处死大鼠后取大鼠壁层腹膜组织行HE和Masson染色,在光镜下观察形态学改变。并通过免疫组织化学方法检测壁层腹膜中转化生长因子β1(TGF-β1),胶原I(Col-I)和α-平滑肌肌动蛋白(α-SMA)的表达情况。结果:1、4小时PET结果显示:模型组与正常组相比,UF(超滤量)和D4/D0(4小时后透出液葡萄糖浓度/初始腹透液葡萄糖浓度)减少(P0.05),D/Purea(腹透液尿素氮浓度/血浆尿素氮浓度)增加(P0.05);高、中、低剂量褪黑素组与模型组比较,UF(超滤量)和D4/D0(4小时后透出液葡萄糖浓度/初始腹透液葡萄糖浓度)增加(P0.05),D/Purea(腹透液尿素氮浓度/血浆尿素氮浓度)减少(P0.05);吡菲尼酮组与模型组相比,差异无统计学意义;吡菲尼酮组与高、中、低剂量褪黑素组相比,UF(超滤量)和D4/D0(4小时后透出液葡萄糖浓度/初始腹透液葡萄糖浓度)减少(P0.05),D/Purea(腹透液尿素氮浓度/血浆尿素氮浓度)增加(P0.05);2、HE和Masson染色结果显示,正常组腹膜薄,间皮细胞连续,模型组腹膜明显增厚,部分可见间皮细胞缺失,间皮下可见胶原沉积,高、中、低剂量褪黑素组上述腹膜病理改变较模型组明显减轻;吡菲尼酮组上述病理改变较模型组无明显改善;3、免疫组化显示:TGF-β1、Col-I、α-SMA在正常组大鼠中几乎无表达,在模型组中表达最强烈,模型组与正常组相比,TGF-β1、Col-I、α-SMA表达增加(P0.05);高、中、低剂量褪黑素组与模型组比较,TGF-β1、Col-I、α-SMA表达减少(P0.05);吡菲尼酮组与模型组相比,差异无统计学意义;吡菲尼酮组与高、中、低剂量褪黑素组相比,TGF-β1、Col-I、α-SMA表达增加(P0.05),差异无统计学意义。结论:1、本实验证明了通过腹腔注射高糖可建立大鼠腹膜纤维化模型;2、褪黑素能通过改善腹膜的结构和功能来发挥其对抗大鼠腹膜纤维化的作用;3、褪黑素抗腹膜纤维化的作用可能是通过降低TGF-β1、α-SMA的表达和减少Col-I的合成来实现的;4、吡菲尼酮对大鼠腹膜纤维化无明显影响。
[Abstract]:Objective: peritoneal dialysis (Peritoneal Dialysis PD) is one of the end-stage renal disease ESRD and effective treatment, but long-term peritoneal dialysis can appear peritoneal fibrosis, resulting in the loss of peritoneal structure and function, peritoneal ultrafiltration failure eventually induced by peritoneal fibrosis is the main cause of peritoneal dialysis patients quit the treatment, therefore the prevention and alleviation of peritoneum fibrosis is the guarantee of peritoneal dialysis patients dialysis. The key to the long-term success of melatonin is the main hormone secretion of human pineal gland has anti-inflammatory, antioxidant, anti fibrosis effect; pirfenidone (pirfenidone, PFD) is a novel broad-spectrum anti fibrosis drugs, renal fibrosis, showed obvious anti fibrosis liver fibrosis model. Has not yet found that melatonin and pirfenidone used in peritoneal dialysis related peritoneal fibrosis prevention, the use of high glucose induced by intraperitoneal injection of peritoneal fibrosis D, were treated with melatonin, pirfenidone intervention, to observe their effects on peritoneal dialysis related peritoneal fibrosis, and provide new ideas for the prevention and treatment of peritoneal dialysis related peritoneal fibrosis. Methods: 46 male SD rats were divided into 6 groups. (1) normal group (n=6): three ethanol distilled water injection 0.9% 20ml+ normal saline treatment volume daily intraperitoneal dilution; (2) the model group (n=8): daily intraperitoneal injection of 4.25% 100ml/kg according to the weight of Liquor Dialysisintraperitoneus + drugs volume 0.9%NS; (3) low dose melatonin group (n= 8): daily intraperitoneal injection of 4.25% 100ml/kg according to the weight of Liquor Dialysisintraperitoneus + melatonin 5mg/kg; (4) in a dose of melatonin group (n=8): daily intraperitoneal injection of 4.25% 100ml/kg according to the weight of Liquor Dialysisintraperitoneus + melatonin 10mg/kg; (5) the high dose melatonin group (n=8): daily intraperitoneal injection of 4.25% 100ml/kg according to the weight of Liquor Dialysisintraperitoneus + melatonin 20mg/kg; (6) pirfenidone group (n=8): daily intraperitoneal injection of 4.25% 100ml/kg according to the weight of Liquor Dialysisintraperitoneus + intragastric infusion pirfenidone 500mg/kg. experiment twenty-eighth days 4 hour peritoneal equilibration test (PET), amount of ultrafiltration volume (UF), detection of dialysate urea nitrogen concentration (D), plasma urea nitrogen concentration (Purea), the initial glucose concentration of peritoneal dialysate (D0), 4 hours after the dialysate glucose concentration (D4), and calculate D/Purea, D4/D0. rats were sacrificed after rat parietal peritoneum tissue HE and Masson staining, the morphological changes were observed under light microscope. And by immunohistochemical method to detect the parietal peritoneum transforming growth factor beta 1 (TGF- beta 1), collagen I (Col-I) and alpha smooth muscle actin (alpha -SMA). Results: the expression of 1,4 PET showed that: the model group compared with normal group, UF and D4/D0 (UF) (4 hours after the dialysate glucose concentration / initial dialysate glucose concentration ) (P0.05), D/Purea (reducing dialysate urea nitrogen concentration / plasma urea nitrogen concentration) increased (P0.05); high, low dose of melatonin group compared with model group, UF (UF) and D4/D0 (4 hours after the dialysate glucose concentration / initial dialysate glucose concentration increased (P0.05)) (D/Purea, dialysate urea nitrogen concentration / plasma urea nitrogen concentration) decreased (P0.05); pirfenidone group compared with model group, the difference was not statistically significant; pirfenidone group and high, compared with the low dose of melatonin group, UF and D4/D0 (UF) (4 hours after the dialysate glucose concentration / the initial glucose concentration of peritoneal dialysate (P0.05), D/Purea (reduced) dialysate urea nitrogen concentration / plasma urea nitrogen concentration) increased (P0.05); 2, HE and Masson staining showed that the normal group of peritoneal mesothelial cells in peritoneal thin, continuous, model group was thickened, partially visible mesothelial cell loss, collagen deposition under mesothelium high, In the low dose of melatonin group peritoneal pathological changes significantly reduced compared with the model group; pirfenidone group the pathological changes compared with the model group had no obvious improvement; 3, immunohistochemistry showed that TGF- beta 1, Col-I, alpha -SMA in normal rats, almost no expression in the model group were strongest model compared with the normal group, Col-I, alpha TGF- beta 1, increase the expression of -SMA (P0.05); high, low dose of melatonin group compared with model group, TGF- beta 1, Col-I alpha, -SMA expression decreased (P0.05); pirfenidone group compared with model group, the difference was not statistically significant; pirfenidone the group with high, low dose group, compared with melatonin, Col-I, alpha TGF- beta 1, increase the expression of -SMA (P0.05), the difference was not statistically significant. Conclusion: 1. This experiment proved that high glucose can be established by intraperitoneal injection of peritoneal fibrosis model in rats; 2, melatonin can through improving the structure and function of peritoneal to play the anti rat peritoneal fibrosis Dimensional effect; 3, effect of melatonin on peritoneal fibrosis effect may be through reducing the expression of alpha TGF- beta 1, -SMA and reduce the synthesis of Col-I to realize; 4, pirfenidone has no obvious effect on rat peritoneal fibrosis.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R692.5

【参考文献】