【作者】 刘晓华；

【导师】 黎七雄；

【作者基本信息】 武汉大学 ， 药理学， 2005， 硕士

【摘要】 本研究通过建立大鼠顺铂（cisplatin,cis-diamminedichloroplatinum,DDP）肾毒性模型,观察川芎嗪（ligustrazine,tetramethypyrazine,TMP）对其保护作用,并从抗氧化损伤及细胞凋亡等方面探讨其作用机制。8mg·kg^-1DDP ip诱导大鼠肾损伤,于给DDP2d前开始大鼠分别ip50 mg·kg^-1·d^-1、100mg·kg^-1.d^-1TMP,连续5d,末次给药4h后处死大鼠,计算肾脏指数、测血清尿素氮（BUN）和肌酐（Scr）含量,测24h尿蛋白含量及尿N-乙酰-β-D-氨基葡萄糖苷酶（NAG）、γ-谷氨酸转换酶（GGT）、碱性磷酸酶（ALP）的活性:制备肾皮质匀浆,测定肾皮质中丙二醛（MDA）、还原型谷胱甘肽（GSH）含量、过氧化物歧化酶（SOD）、谷胱甘肽-S-转移酶（GST）活性及肾皮质一氧化氮（NO）含量、一氧化氮合酶（NOS）活性;大鼠肾脏用10%中性福尔马林溶液固定,石蜡包埋,HE染色,光镜观察肾组织形态学变化;采用原位缺口末端标记法（TUNEL）检测肾脏细胞凋亡,免疫组化SP法检测肾脏Bax和Bcl-2表达水平。 结果:50mg·kg^-1、100mg·kg^-1TMP给药组均能降低DDP所致大鼠24h尿蛋白及BNU、Scr含量的升高,分别下降41%、65%,18%、58%,14%、49%（P<0.05、p<0.01）;对DDP所致的尿NAG、GGT、ALP活性的升高,TMP两剂量组也可明显降低,分别下降17%、29%,46%、48%,18%、25%（P<0.05、p<0.01）;与DDP组比较,100mg·kg^-1TMP给药组可明显降低肾组织MDA、NO含量及NOS活力的升高,分别降低37%、45%、23%（p<0.05）,GSH含量、SOD、GST活性分别升高1.33、1.66、1.57倍（p<0.05）。肾脏病理组织学检查,DDP组肾小球结构清楚,但可见明显充血,近曲小管上皮细胞重度浊肿,管腔内可见明显蛋白管型和红细胞管型,间质呈炎症反应,远曲小管腔内可见充血及红细胞管型,TMP两剂量组地均可改善DDP引起的肾脏病理改变,但以100mg·kg^-1给药组明显。TUNEL检测结果显示,DDP组凋亡细胞以肾小管最明显,肾小球少见,TMP两剂量组可明显减少DDP引起的肾脏凋亡细胞数（P<0.01）,凋亡指数分别下降57%、91%（P<0.01）;免疫组化结果可见DDP组Bax表达与对照组比较明显增加（P<0.01）,而Bcl-2表达较弱（P<0.05）,TMP两剂量组显著增强肾脏凋亡相关蛋白Bcl-2的表达,而减少Bax表达,并明显降低Bax/Bcl-2比值,与DDP组比较分别下降40%、70%（P<0.01）。 研究提示:TMP对DDP所致大鼠肾毒性具有保护作用,其机制可能与增强机体的抗氧化能力、减少脂质过氧化水平及抑制DDP引起的肾脏细胞凋亡有一定关系。更多还原

【Abstract】 Nephrotoxicity induced by cisplatin was established in rats, observing the protective effects of Ligustrazine and investigate its action mechanism from the aspects of anti-oxidation damage and cell apoptosis. Nephrotoxicity in rats was induced to be impaired with single （ip） cisplatin dose of 8mgkg^-1 body weight. Ligustrazine (50 mg·kg^-1d^-1\、 100 mg·kg^-1d^-1 body weight) was given two days before the cisplatin injection for five days. Then the rats were sacrificed four hours after the last injection. After the kidneys were removed to calculate the renal index, Blood urea nitrogen（BUN） and serum creatinine （Scr） was tested , urine samples obtained to find the content of 24 hours urinary protein and the activity of N-acetyl-|3-D-glucosaminidase（NAG）, γ-Glutamyl-transpeptidase（GGT） and alkaline phosphatase（ALP）. The content of malondial dehyde （MDA） and glutathione （GSH） content, anti-oxidase activities of superoxide dismutase （SOD） and glutathione-S-transferase （GST） in renal were detected. The content of nitric oxide （NO） and the activity of nitric oxide synthase（NOS） in renal cortex were measured . Rat kidneys were soaked in 10% formaldehyde solution and then embedded in paraffin. The tissue sections were stained with hematoxylin and eosin and lesions of glomerulus and tubules were observed under Optical microscope. Apoptosis was evaluated by terminal dexoynucleotidl transferase mediated d-UTP nick and labeling（TUNEL）. Immunohis-tochemistry was used to detect the protein expression level of Bax and Bcl-2.The ligustrazine (50 mg·kg^-1d^-1、 100 mg·kg^-1d^-1 body weight) administration dose-dependently reduced content of 24h urinary protein, the content of BUN、 Scr which been raised by cisplatin, the content of 24h urinary protein, the content of BUN、 Scr was significantly decreased by 41%, 65%, 18% 、 58%, 14%、 49% （P<0.05、 p<0.01） respectively;compared with the DDP group, the ligustrazine 100 mg·kg^-1d^-1 body weight administration significantly decreased the activity of urinary NAG、 GGT、 ALP by 17 %、29%, 46%. 48%, 18%, 25% （P<0.05. p<0.01） respectively. The ligustrazine dose of 100 mg-kg’^d^body weight can significant reduced the content of MDA . NO and activity of NOS in kidney which can increased by cisplatin.The content of MDA > NO and activity of NOS in kidney was significantly decreased by 37%, 45%, 23%（ P < 0.05）respectively; the content of GSH and the activities of SOD. GST in TMP 100 mgkg’d"1 group were significantly increased by 1.33-fold, 1.66-fold , 1.57-fold （P <0.05） respectively compared with DDP group.The histopathological changes of kidney suggest that the serious congestion ofglomeruli > swelling of kidney tubules and the casts including protein casts and erythrocyte casts in DDP groups and ligustrazine can notably improve it. The results of TUNEL found more apoptotic cell in DDP group and the cells were focus on kidey tubules. Rats in DDP group show a much strong staining for Bax protein and a weaker staining of Bcl-2 than control group by means of .immunohistoc- hemistry .Ligstrazine can significant decling the number of apoptotic cells , increased the expression of Bcl-2 and decling the exprssion of Bax .And the Bax / Bcl-2 ratio was declined significantly by ligusrazine（p < 0.01）.The results suggested that the Ligstrazine can protect the nephrotoxisity induced by cisplatin in rats and the mechanism of the protective effect may have some relationship with increasing the anti-oxidation ability of body -. reducing the lipid peroxidation level and refraining cell apoptosis.更多还原