【作者】 孙妍；

【导师】 孙立荣；

【作者基本信息】 青岛大学 ， 儿科学， 2014， 博士

【摘要】 目的探讨藏红花素对顺铂致小鼠急性肝、肾损伤的保护作用及其机制。方法30只雄性昆明小鼠随机分为5组(每组6只),即空白对照组(生理盐水0.2ml/d).顺铂模型组(10mg/kg).低浓度藏红花素组(6.25mg/kg.d).高浓度藏红花素组(12.5mg/kg.d)及阳性对照组(水飞蓟素100mg/kg.d).除空白对照组外,其余各组均于实验第1天给予顺铂(10mg/kg腹腔注射,单剂1次),藏红花素给药组小鼠分别于实验每日腹腔注射不同剂量藏红花素(6.25mg/kg,12.5mg/kg)1次。阳性对照组小鼠分别于实验每日口服水飞蓟素(100mg/kg)1次。实验第7天断头处死所有动物。肝脏、肾脏组织切片HE染色观察肝脏病理组织学改变；常规分离血清后全自动生化仪检测小鼠血清肝功能指标(ALT.AST)及肾功能指标(BUN.Cr):肝脏、肾脏组织匀浆检测氧化应激指标丙二醛(MDA)及还原型谷胱甘肽(GSH)的含量,以及抗氧化酶指标超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-px)以及过氧化氢酶(CAT)的活性；免疫组化方法及Western blot分析定性及定量检测肝、肾匀浆中p-p38MAPK,p53, and caspase-3的表达差异。结果1.藏红花素对顺铂损伤小鼠肝、肾功能的影响空白对照组、顺铂模型组、低浓度藏红花素组、高浓度藏红花素组及阳性对照组小鼠血清ALT分别为(37.33±4.89),(138.83±11.25),(76.83±6.49),(71.33±8.66),(72.83±5.23)U/L。AST分别为(64.50±6.38),(242.00±14.94),(132.50±9.54),(126.67±11.69),(124.02±8.49)U/L。BUN分别为(20.94±1.92),(62.60±5.64),(32.41±2.97),(30.95±2.90),(32.83±3.11)mg/dl。Cr别为(0.49±0.10),(2.12±0.22),(0.98±0.16),(0.94±0.22),(0.95±0.18)mg/dl。顺铂模型组小鼠的血清ALT、AST、BUN及Cr明显高于空白对照组(p均<0.05)。然而,与顺铂模型组比较,低、高浓度藏红花素组小鼠血清ALT、AST、BUN及Cr水平均显著降低(p均<0.05)；低、高浓度藏红花素组与阳性对照组间无显著性差异(p均>0.05)。2.藏红花素对顺铂损伤小鼠肝、肾组织形态学的影响腹腔注射顺铂可导致肝脏形态组织学改变,包括小叶中心坏死,肝小叶结构改变,以及肝细胞气球样变性。然而,藏红花素给药后上述肝组织损伤及坏死程度均可见明显减轻。腹腔注射顺铂后,小鼠肾脏皮质肾小管可见上皮细胞肿胀、空泡变性,管腔可见脱落上皮细胞,细胞排列紊乱,出现蛋白管型。而给予藏红花素后,多数肾脏空泡变性、坏死明显减少,细胞排列较顺铂组规整,肾小球和肾小管结构基本完整,未见细胞坏死及管型。3.藏红花素对顺铂所致肝肾损伤小鼠抗氧化系统的影响空白对照组、顺铂模型组、低浓度藏红花素组、高浓度藏红花素组及阳性对照组小鼠肝脏MDA含量分别为(2.48±0.37),(5.82±0.72),(3.47±0.39),(3.14±0.38),(3.39±0.38)nmol/mg protein；GSH分别为(5.28±0.34),(2.35±0.27),(3.45±0.29),(3.69±0.36),(3.39±0.27)mg/g protein.1-5组小鼠肾脏MDA含量分别为(2.92±0.23),(6.87±0.37),(4.97±0.33),(4.64±0.32),(4.80±0.41)nmol/mg protein;GSH分别为(5.77±0.40),(2.31±0.28),(3.66±0.30),(3.83±0.33),(3.76±0.38)mg/g protein.顺铂模型组小鼠的肝肾组织中MDA含量显著高于空白对照组(p均<0.05),而GSH含量显著低于对照组(p均<0.05),但与顺铂模型组比较,低、高浓度藏红花素组小鼠肝肾MDA含量均显著降低(p均<0.05)；低、高浓度藏红花素组小鼠肝肾6SH含量均显著升高(p均<0.05)。低、高浓度藏红花素组与阳性对照组间MDA及GSH含量均无显著性差异(p均>0.05)。4.藏红花素对顺铂所致肝肾损伤小鼠抗氧化酶的影响空白对照组、顺铂模型组、低浓度藏红花素组、高浓度藏红花素组及阳性对照组小鼠肝脏SOD活性分别为(234.28±19.7),(118.66±13.93),(171.48±16.25),(177.50±15.21),(174.97±11.85)U/mg protein；GSH-px活性分别为(317.83±21.68),(171.33±21.95),(234.00±26.48),(241.33±25.22),(237.00±24.86)U/mg protein;CAT活性分别为(35.53±3.65),(16.16±2.46),(21.53±1.94),(24.81±2.41),(24.69±3.70)U/mg protein.肾脏SOD活性分别为(45.60±3.79),(20.96±2.56),(32.14±2.47),(33.27±2.82),(32.87±4.55)U/mg protein:GSH-px活性分别为(274.79±15.01),(163.06±11.45),(220.27±16.28),(213.89±25.08),(212.05±24.68)U/mg protein；CAT活性分别为(21.57±2.55),(9.97±1.90),(14.17±1.78),(15.08±1.67),(14.73±1.53)U/mg protein。顺铂所致急性肝肾损伤的小鼠,肝肾组织内上述酶活性与空白对照组小鼠相比,均明显降低(p均<0.05)。与顺铂模型组比较,低、高浓度藏红花素组小鼠肝肾内上述酶活性均显著升高(p均<0.05)。而低、高浓度藏红花素组与阳性对照组间上述各种酶活性无显著性差异(p均>0.05)。5.藏红花素对顺铂肝肾损伤小鼠p-p38MAPK、p53以及caspase3的影响免疫组化检测结果显示p_p38MAPK、p53定位于胞核,caspase3定位于胞浆,顺铂中毒小鼠肝肾p_p38MAPK、p53以及caspase3表达均增加,而低、高浓度藏红花素可减少中毒小鼠肝肾p-p38MAPK、p53以及caspase3的表达。Western Blot检测显示空白对照组、顺铂模型组、低浓度藏红花素组、高浓度藏红花素组及阳性对照组小鼠肝脏中p-p38MAPK蛋白表达：(0.05±0.03),(0.41±0.06),(0.21±0.04),(0.21±0.04),(0.22±0.04)；p53蛋白表达：(0.06±0.02),(0.73±0.06),(0.49±0.06),(0.50±0.07),(0.47±0.06); caspase3蛋白表达：(0.08±0.04),(0.83±0.05),(0.50±0.05),(0.50±0.04),(0.48±0.05)。小鼠肾脏中p-p38MAPK蛋白表达：(0.04±0.02),(0.45±0.04),(0.22±0.05),(0.21±0.04),(0.22±0.04)；p53蛋白表达：(0.05±0.02),(0.68±0.08),(0.49±0.06),(0.51±0.08),(0.46±0.05); caspase3蛋白表达：(0.09±0.04),(0.54±0.06),(0.36±0.04),(0.33±0.04),(0.31±0.03)。顺铂所致肝肾脏损伤的小鼠,肝肾组织中p-p38MAPK、p53及caspase3的表达明显高于空白对照组小鼠(p均<0.05)。而与顺铂模型组相比,低、高浓度藏红花素组小鼠肝肾组织中p-p38MAPK、p53及caspase3的表达表达均显著降低(p均<0.05)。而低、高浓度藏红花素组与阳性对照组间无显著性差异(p均>0.05)。结论1.藏红花素(6.25mg/kg. d、12.5mg/kg. d)可明显降低顺铂致急性肝、肾损伤小鼠的血清谷丙转氨酶、谷草转氨酶活性以及尿素氮、肌酐的水平,对顺铂引起的小鼠急性肝肾毒性具有良好的保护作用。2.藏红花素(6.25mg/kg. d、12.5mg/kg. d)可明显改善顺铂所致肝肾组织结构的改变,提示藏红花素对顺铂所致小鼠的肝肾组织损伤具有一定的保护作用。3.藏红花素(6.25mg/kg. d、12.5mg/kg. d)可通过减少MDA生成和GSH的消耗,并提高SOD, GSH-px及CAT抗氧化酶的活性而发挥其抗氧化作用,减轻了顺铂对小鼠肝肾的氧化损伤。4.顺铂对p38MAPK的激活是其导致肝肾毒性的主要信号转导通路,藏红花素(6.25mg/kg. d、12.5mg/kg. d)具有下调p-p38MAPK, p53,及caspase-3蛋白表达的作用,故藏红花素对顺铂所致肝肾组织细胞凋亡具有一定的抑制作用。更多还原

【Abstract】 ObjectiveTo investigate the protective effect of crocin on cisplatin(CDDP)-induced liver and kidney injury and its mechanismsMethods30male nude mice of5weeks were randomly divided into5groups, including the control (saline) group, CDDP group, the low and high concentration crocin group (6.25mg/kg·d,25mg/kg·d),and positive control group(silymarin100mg/kg·d). Crocin and silymarin was administered to the mice once daily for7consecutive days. On day1, a single intraperitoneal injection of CDDP was given at the dose of10mg/kg body weight. After7days, all the mice were executed. Blood was collected immediately on the day of sacrifice for alanine aminotransferase(ALT), aspartate aminotransferase(AST), creatinine(Cr), and blood urea nitrogen(BUN) assay. The paraffin sections of liver and kidney were stained with HE staining and then observed by light microscope. The level of malondialdehyde(MDA) and glutathione(GSH) were detected. Also, the activity of superoxide dismutase (SOD), glutathione peroxidase(GSH-px), and catalase(CAT) were detected. The p-p38MAPK, p53, and caspase-3expressions of the liver and kidney were detected by immunohistochemical technique and Western blot essay.Result1. The effects of crocin on the serum biochemistry index of the mice damaged by CDDPCompared with the control group, the level of ALT, AST, BUN and Cr in the CDDP group were significantly increased (p<0.05,totally). Compared with the CDDP group, the level of ALT, AST, BUN and Cr in the crocin and silymarin group were significantly decreased(p<0.05,totally). There was no significant difference between the crocin group and positive control silymarin group (p>0.05,totally)2. The effects of crocin on the histomorphology of the liver and kidney in the mice damaged by CDDPLiver and kidney histopathology indicated that crocin could alleviated CDDP-induced focal tissue damage.3. The effects of crocin on the antioxidant system of the mice damaged by CDDPCompared with the control group, the level of MDA of the liver and kidney in CDDP group were significantly increased (p<0.05,totally). Compared with the CDDP group, the level of MDA in the crocin and silymarin group were significantly decreased (p<0.05,totally). There was no significant difference between the crocin group and positive control silymarin group (p>0.05,totally)A significant reduction in GSH of the liver and kidney was found in CDDP group, compared to the control group (p<0.05, totally). Compared with the CDDP group, the level of GSH in the crocin and silymarin group were significantly decreased(p<0.05,totally). There was no significant difference between the crocin group and positive control silymarin group (p>0.05,totally)4. The effects of crocin on the antioxidant enzyme activity of the mice damaged by CDDPCompared with the control group, the anctivity of SOD, GSH-px, and CAT of the liver and kidney in CDDP group were significantly decreased (p<0.05,totally). Compared with the CDDP group, the anctivity of SOD, GSH-px, and CAT in the crocin and silymarin group were significantly increased (p<0.05,totally). There was no significant difference between the crocin group and positive control silymarin group (p>0.05,totally)5. The effects of crocin on the expression of p-p38MAPK, p53and caspase3The p-p38MAPK, p53, and caspase-3expressions of the liver and kidney were significantly increased (p<0.05,totally) in CDDP group, compared to the control group. Compared with the CDDP group, the expressions of p-p38MAPK, p53, and caspase-3in the crocin and silymarin group were significantly decreased (p<0.05,totally). There was no significant difference between the crocin group and positive control silymarin group (p>0.05,totally)Conclusion1. Crocin (6.25mg/kg·d、12.5mg/kg.d) treatment significantly improved CDDP-induced hepatic damage as indicated by serum ALT and AST. Crocin treatment also significantly improved CDDP-induced kidney damage as indicated by serum BUN and Cr.2. Liver and kidney histopathology indicated that crocin (6.25mg/kg·d、12.5mg/kg.d) alleviated CDDP-induced focal damage.3. Crocin relieved CDDP-induced oxidative stress by reducing MDA level and recovering the levels of GSH and antioxidant enzymes such as SOD, GHS-px, and CAT.4. Immunohistochemical staining and Western blot analysis showed that crocin (6.25mg/kg·d、12.5mg/kg.d) significantly decreased the levels of phospho-p38mitogen-activated protein kinase (MAPK), tumor protein53(p53), and caspase-3.更多还原