【作者】 周俪姗；

【导师】 陆付耳；

【作者基本信息】 华中科技大学 ， 中西医结合临床， 2013， 博士

【摘要】 第一部分从PKCα-NADPH氧化酶信号通路探讨胡芦巴丸及其单味药治疗大鼠糖尿病肾病的分子机制目的探讨胡芦巴丸及其单味药抗氧化应激治疗大鼠糖尿病肾病(DN)的分子机制。方法采用高糖高脂饮食和尾静脉小剂量链脲佐菌素(STZ)的方法建立大鼠DN模型,将大鼠随机分为模型组、胡芦巴组、补骨脂组、胡芦巴丸组、卡托普利组,另设正常对照组,给予相应药物灌胃治疗16周,检测血糖、血脂、血尿素氮(BUN)、血肌酐(SCr)、尿24小时总蛋白、尿24小时白蛋白水平,光镜下观察肾脏形态学改变(HE染色、PAS染色、Masson染色),电镜下观察肾脏超微结构改变,DHE染色法评估肾脏组织超氧阴离子水平,浓度比色法检测肾组织烟酰胺腺嘌呤二核苷酸磷酸(NADPH)活性,Western blot检测肾脏组织p47phox、磷酸化蛋白激酶C-α(PKC-α)、纤粘蛋白(FN)的蛋白表达水平,实时荧光定量PCR(RT-PCR)检测肾脏组织p47phox、 PKC-α、的mRNA表达水平。结果与正常组比较,模型组大鼠血糖升高、血脂紊乱,24小时尿总蛋白及白蛋白水平均明显升高(P<0.01),肾小球体积增大、肾小球纤维化、细胞外基质增生、足细胞足突融合；与模型组比较,胡芦巴、补骨脂、胡芦巴丸组大鼠血糖水平下降,血脂紊乱改善,24小时尿总蛋白及白蛋白水平均明显下降(P<0.01),上述肾脏病理学改变明显减轻。与正常组比较,模型组大鼠肾组织DHE荧光水平明显升高,肾组织NADPH氧化酶活性、p47phox蛋白及mRNA表达水平、磷酸化PKC-α、FN蛋白表达水平均明显升高(P<0.01)；与模型组比较,胡芦巴、补骨脂、胡芦巴丸组大鼠肾组织DHE荧光水平明显降低,肾组织NADPH氧化酶活性、p47phox蛋白及mRNA表达水平磷酸化PKC-α、FN蛋白表达水平均明显降低(P<0.01)。与胡芦巴丸组比较,胡芦巴、补骨脂组大鼠肾组织NADPH活性、p47phox蛋白及mRNA表达水平、肾组织磷酸化PKC-α、FN蛋白表达均增加(P<0.01,P<0.05)结论胡芦巴丸及其单味药均可有效治疗大鼠糖尿病肾病,其机制可能与抑制肾组织PKCα-NADPH氧化酶信号通路减轻氧化应激有关,胡芦巴丸改善肾组织氧化应激优于单味药。第二部分灰兜巴抑制肾组织NADPH氧化酶活性治疗大鼠糖尿病肾病的机理研究目的探讨灰兜巴对大鼠糖尿病肾病的防治作用及机制。方法采用高糖高脂饮食和小剂量链脲佐菌素(STZ)尾静脉注射建立大鼠糖尿病肾病模型,将大鼠随机分为模型组、灰兜巴组、卡托普利组,另设正常对照组,给予相应药物灌胃治疗12周,检测血糖、血脂、尿素氮(BUN)、肌酐(SCr)、血清丙二醛(MDA)、24小尿微量白蛋白(UALB)水平,观察肾组织形态学改变,浓度比色法检测肾组织烟酰胺腺嘌呤二核苷酸磷酸(NADPH)(?)舌性,Western blot检测肾组织NADPH氧化酶亚单位p47phox蛋白的表达。结果与正常组比较,模型组大鼠空腹及餐后血糖、血脂紊乱,血清MDA、BUN、SCr、24hUALB均明显升高(P<0.01),肾小球体积增大,系膜区及细胞外基质增生(P<0.01),肾组织NADPH活性、p47phox蛋白表达明显升高(P<0.01)；与模型组比较,灰兜巴组大鼠空腹及餐后血糖明显降低,血脂紊乱改善,血清MDA、BUN、SCr和24hUALB明显降低(P<0.01),肾小球体积近乎正常,系膜区、基质未见增生,肾组织NADPH(?)舌性、p47phox蛋白表达明显降低(P<0.01)。结论灰兜巴可明显改善糖尿病肾病大鼠糖脂代谢紊乱,对糖尿病肾病具有防治作用。其机制可能与下调肾组织NADPH(?)舌性减轻氧化应激有关。第三部分绝经后骨质疏松症患者骨密度与性激素水平、氧化应激相关指标的相关性分析目的分析绝经后骨质疏松症(PMOP)患者骨密度(BMD)与性激素水平、氧化应激相关指标的相关性。方法入选58例PMOP患者,行双能X线吸收仪(DXA)检测骨密度(BMD),同时进行人体学特征、血清性激素水平和氧化应激相关指标丙二醛(MDA)、超氧化物歧化酶(SOD)、总抗氧化能力(T-AOC)的检测,分析BMD与上述各指标的相关性。结果PMOP患者BMD仅与身高呈正相关,相关系数分别为腰椎r=0.390、髋关节r=0.357(P<0.01),BMD与血清性激素水平、氧化应激相关指标均无相关性。结论PMOP患者骨密度(BMD)与性激素水平、氧化应激相关指标之间无明显相关性,需寻找更有价值的相关联因素。更多还原

【Abstract】 Section Ⅰ Hu-lu-ba-wan and its Single Components Attenuate Rat Diabetic Nephropathy through PKCa-NADPH Oxidase Signaling PathwayObjectiveThe aim of this study was to explore the effect of Hu-lu-ba-wan (HLBW) and its single components on diabetic nephropathy (DN) in type2diabetic rats and to investigate its possible oxidative stress-against mechanism.MethodsThe rat model of DN was established by feeding with a high-fat diet and intravenous injection of small doses of streptozotocin. DN rats were randomized into the diabetic control group, TFG treated group, PC treated group, HLBW treated group and Captopril treated group. Meanwhile, eight rats were selected randomly as the normal group. Rats in the above treatment groups were administered with corresponding therapy intragastrically for16weeks. Blood glucose level, lipid profile, blood urea nitrogen (BUN), serum creatinine (SCr) concentrations, and total urinary protein and albumin content for24hours were examined. Renal morphology changes were evaluated by the staining with hematoxylin and eosin (HE), periodic acid-schiff (PAS), aniline blue (AB) and the observation under transmission electron microscope. Renal superoxide anion levels were assessed by dihydroethidium (DHE) fluorescence staining. The activity of nicotinamide-adenine dinucleotide phosphate (NADPH), the protein expressions of p47phox, phosphorylated PKC-a and fibronectin, the mRNA expressions of p47phox and PKC-a in renal tissue were also determined.ResultsCompared with the normal group, the rats in the model group showed remarkable glomerular expansion and fibrosis. extracellular matrix (ECM) accumulation, effacement of the foot processes of podocytes. Blood BG, TG, TC, LDL-C, BUN, SCr concentrations and urinary total protein and albumin content were also significantly increased. Meanwhile, diabetic rats showed the elevation of DHE fluorescence level, the NADPH oxidase activity, the P47phox protein and mRNA expression, and the phosphorylated PKC-a and fibronectin protein expression in renal tissues (P<0.01). However. HLBW and its single components treatment effectively reversed the above biochemical changes in diabetic rats (P<0.01). The addition of HLBW and its single components also reduced DHE fluorescence level, NADPH oxidase activity, the protein and mRNA expression of P47phox, and the protein expression of phosphorylated PKC-a and fibronectin in renal tissues (P<0.01). Compared with HLBW treated group, rats in TFG treated group and PC treated group showed elevated NADPH oxidase activity, the protein and mRNA expressions of p47phox. and the protein expression of phosphorylated PKC-a in renal tissues (P<0.01,P<0.05).ConclusionThese results suggest that HLBW and its single components are effective in the treatment of DN in type2diabetic rats. The mechanism may be related to the reduction of renal oxidative stress via PKCa-NADPH oxidase signaling pathway on the administration of HLBW and its single components. The attenuating renal oxidative stress efficacy of composite prescription (HLBW) is superior to that of simple prescription (TFG or PC). Section Ⅱ Huidouba Attenuates Rat Diabetic Nephropathy by Inhibition of NAPDH Oxidase ActivityObjectiveTo investigate the mechanism and effect of Huidouba in the treatment of diabetic nephropathy in rats.MethodsThe rat model of diabetic nephropathy (DN) was established by feeding with a high-fat diet and intravenous injection of small doses of streptozotocin. DN rats were randomized into the diabetic control group, Huidouba polysaccharides treated group and Captopril treated group. Meanwhile, seven rats were selected randomly as the normal group. Rats in the above treatment groups were administered with corresponding therapy intragastrically for12weeks. Renal histological assessment was observed. Blood glucose (BG), blood lipid (TG, TC, LDL-C, HDL-C), malondialdehyde (MDA), blood urea nitrogen (BUN), serum creatinine (SCr) concentrations, and24hours urinary microalbumin (UALB) content were examined. The activity of nicotinamide-adenine dinucleotide phosphate (NADPH) and the protein expressions of P47phox in renal tissues were also determined.ResultsCompared with the normal group, the rats in the model group showed remarkable glomerular hypertrophy. Blood BG, TG, TC, LDL-C, MDA, BUN, SCr concentrations and UALB content were also significantly increased with the elevation of the NADPH oxidase activity and the P47phox protein expression in renal tissues (P<0.01). However, Huidouba treatment effectively reversed the above biochemical changes in diabetic rats. The addition of Huidouba also reduced NADPH oxidase activity and the protein expression of P47phox in renal tissues (P<0.01).ConclusionIt is concluded that Huidouba is effective on ameliorating glucose and lipid metabolism disturbances and renal injury in diabetic rats. The mechanism may be related to reducing oxidative stress via inhibiting the activity of NADPH oxidase. Section Ⅲ Correlation of Bone Mineral Density with Sexual Hormones and Oxidative Stress in Patients with Postmenopausal OsteoporosisObjectiveTo analyze the correlation of bone mineral density (BMD) with sexual hormones and oxidative stress in Patients with postmenopausal osteoporosis (PMOP).MethodsFifty-eight postmenopausal osteoporosis patients were selected. BMD was quantified by dual energy X-ray absorptiometry (DXA). Anthropometric features and serum sexual hormones and oxidative stress parameters were tested. Correlations between BMD and other items were analyzed.ResultsWe found that only height was significantly correlated with BMD (r=0.390, r=0.357)(P<0.01). Serum sexual hormones and oxidative stress parameters were not correlated with BMD(P>0.05).ConclusionIt is concluded that there is no correlation of BMD with sexual hormones and oxidative stress in patients with PMOP. More effective related factors are needed to be identified.更多还原