葡萄籽原花青素对鼠肾组织内质网应激保护作用的研究

发布时间：2018-06-05 04:54

  本文选题：葡萄籽原花青素 + 内质网应激　； 参考：《山东大学》2014年硕士论文

【摘要】：背景及目的：近年来一些研究发现内质网应激(endoplasmicreticulumstress, ERS)在顺铂(Cisplatin CP)诱导的肾损害中起着重要的作用。葡萄籽原花青素(grape seed proanthocyanidin extract,GSPE)作为天然的抗氧化剂对多种疾病具有保护及预防作用,然而,GSPE对CP导致的急性肾损伤是否具有保护作用尚不清楚。本实验通过给予小鼠腹腔注射CP,以诱导急性肾损害模型,并给予GSPE干预,观察其对肾损伤小鼠的一般情况,血肌酐水平,肾小管病理变化,肾组织细胞凋亡情况,以及肾组织葡萄糖调节蛋白(glucose-regulated protein78GRP78)、磷酸化细胞外信号调节酶(phosphorylated-extracellular signal-regulated kinase P-ERK)、半胱氨酸天冬氨酸蛋白酶12(Cysteine aspartate-specific proteases12Caspase-12)蛋白表达的影响,探讨GSPE对CP导致的急性肾损伤小鼠的保护作用及其可能机制,从而为深入阐明GSPE对CP导致的急性肾损伤的保护机制提供新的依据,为GSPE在临床上的应用开辟新的视野。 方法：雄性C57小鼠65只,适应性喂养1周后,随机分为四组：正常对照组(N组,n=10只),顺铂组(腹腔注射20mg/kg CP, CP组,n=20只),GSPE组(第一天及第三天灌胃GSPE500mg/kg, n=15只), CP+GSPE组(腹腔注射顺铂半小时前给予500mg/kg GSPE灌胃,第三天灌胃等量的GSPE,n=20只)。给药后第五天内眦取血测肌酐、尿素氮,取双肾并称重测肾脏系数,肾组织做PAS染色,应用TUNEL试剂盒检测小鼠肾脏细胞凋亡情况,应用蛋白印迹法、免疫组化方法检测小鼠GRP78、p-ERK和Caspase-12的蛋白表达。 结果：1.各组小鼠一般情况变化：与N组比较,CP组小鼠肾脏系数升高,血尿素氮、肌酐水平升高(P0.05), CP+GSPE组与CP组比较,以上指标均有明显下降。GSPE组与N组比较以上指标无明显变化。 2.各组小鼠肾脏病理改变：N组及GSPE组小鼠肾脏结构清晰,未见肾小管结构损伤,CP组可见肾小管上皮细胞肿胀,空泡样变性,大量管型形成,细胞核裸露,甚至可见细胞核坏死脱落,肾小管损伤明显。CP+GSPE组与CP组比较,肾小管损伤明显减轻。 3.TUNEL结果显示：与N组比较,CP组小鼠肾组织细胞可见明显的细胞凋亡,CP+GSPE组与CP组比较,小鼠细胞凋亡数量明显减少,GSPE组与N组细胞凋亡数量极少。 4.蛋白免疫印迹结果：Western blot结果显示,与N组比较,GRP78、p-ERI和Caspase-12蛋白的表达在CP组明显升高,CP+GSPE组三种蛋白表达较CP组明显降低,GSPE组与N组相比蛋白表达无明显差异。 5.免疫组化显示肾组织GRP78、p-ERK和Caspase-12蛋白的表达。与N组比较,CP组可见高表达的GRP78、p-ERK和Caspase-12蛋白,而CP+GSPE组GRP78、p-ERK和Caspase-12蛋白表达水平明显降低,GSPE组与N组比较无明显变化。 结论：GSPE能明显减轻CP导致的急性肾损伤,保护肾功能,其机制与GSPE下调Caspase-12通路调节的ERS介导的凋亡有关。 研究背景和目的：在发达国家,糖尿病肾病(diabetic nephropathy DN)已成为导致终末期肾脏病(endstage renal disease ESRD)最普遍的原因,在美国,糖尿病肾病(DN)占ESRD的45%,成为ESRD的首位原因。在我国,DN为ESRD的第二大病因,其高发病率及死亡率越来越受到人们的关注。 葡萄籽原花青素(GSPE)是强大的抗氧化剂,其抗氧化活性是维生素E的50倍,维生素C的20倍。有研究证明GSPE对DN有保护作用,前已介绍,GSPE具有预防和治疗多种疾病的功能,包括保护CP导致的肾损害,其机制可能与减轻ERS介导的凋亡有关。基于我们以上研究结果,本实验通过研究GSPE对DN的保护作用,探讨GSPE是否也是通过减轻ERS介导的凋亡来发挥对DN的保护作用的。 方法：健康雄性SD大鼠55只随机选取10只作为正常对照组(N),其余45只大鼠作为模型组给予高脂高糖饮食。喂养4周后,正常对照组给予柠檬酸盐缓冲液腹腔注射,模型组一次性腹腔注射STZ40mg/kg,72h后尾部采血测血糖,以空腹血糖水平≥16.7mmol/L为糖尿病模型成功。其中5只大鼠建模不成功,剔除实验,40只大鼠建模成功。建模成功后的大鼠随机分为糖尿病组(D,n=20),GSPE治疗组(G,n=20)。G组给予250mg/kg-d的GSPE灌胃,N组和D组给予生理盐水灌胃,持续灌胃12周。正常对照组大鼠给予喂养普通饲料,自由饮水,D组及G组给予喂养高脂高糖饲料。定期监测大鼠血糖,于第16周末测定各组大鼠的24尿蛋白定量、尿素氮(BUN)、血肌酐(SCr)、肾脏系数(RI),光学显微镜下观察大鼠肾组织PAS染色结果,并应用免疫组化方法,Western blotting实验技术,检测大鼠肾组织GRP78、p-ERK、Caspase-12的蛋白表达,应用TUNEL试剂盒检测大鼠肾脏细胞凋亡情况。 结果：1.各组大鼠一般情况变化：与N组比较,D组肾脏系数升高,24h尿蛋白定量明显升高,BUN、SCr无明显变化(P0.05),经GSPE治疗后肾脏系数、24h尿蛋白定量下降,BUN、SCr水平无明显改变(见表一),D组、G组大鼠血糖无明显变化。 2.各组大鼠肾脏病理改变：N组大鼠肾脏结构清晰,未见肾损伤,D组可见肾小球体积明显增大,肾小球基底膜增厚,系膜区增宽,系膜细胞增生,系膜基质增多。与D组相比GSPE治疗组肾损伤明显减轻。 3.TUNEL结果显示：与N组比较,D组大鼠细胞凋亡数明显增加,与D组比较,G组大鼠细胞凋亡数明显减少。 4.蛋白免疫印迹结果：蛋白免疫印迹显示,与N组比较,GRP78、p-ERK和Caspase-12蛋白的表达在D组明显升高,G组以上三种蛋白表达水平较D组明显降低。 5.免疫组化：肾组织GRP78、p-ERK和Caspase-12蛋白的表达。与N组比较,D组可见高表达的GRP78、p-ERK和Caspase-12蛋白,而G组GRP78、p-ERK和Caspase-12蛋白表达水平明显降低。 结论：GSPE能明显降低DN大鼠的肾脏系数,24h尿蛋白定量,减少肾脏细胞凋亡,改善肾脏病理变化,延缓DN的发展,其机制与GSPE下调Caspase-12通路调节的ERS介导的凋亡有关。
[Abstract]:BACKGROUND & OBJECTIVE : In recent years , it has been found that endoplasmic reticulum stress ( ERS ) plays an important role in the kidney damage induced by cisplatin ( CP ) .

Methods : Sixty - five male C57 mice were randomly divided into four groups : normal control group ( N group , n = 10 ) , cisplatin group ( intraperitoneal injection 20mg / kg CP , CP group , n = 20 ) , GSPE group ( GSPE 500mg / kg , n = 15 only ) , CP + GSPE group ( administration of GSPE 500 mg / kg GSPE for the third day , GSPE in the third day , n = 20 ) . The expressions of GRP78 , p - ERK and Caspase - 12 in mouse kidney were detected by using TUNEL assay and Western blotting . The expression of GRP78 , p - ERK and Caspase - 12 was detected by TUNEL .

Results : 1 . The changes of renal coefficient in group CP group were compared with those in group N , and the level of blood urea nitrogen and creatinine increased ( P0.05 ) . Compared with CP group , there was a significant decrease in the above indexes .

2 . The renal pathological changes of mice in each group were as follows : N group and GSPE group had clear renal structure , no renal tubular structure injury was observed . The CP group showed that tubular epithelial cells were swollen , vacuolar degeneration , large number of tubular forms , nuclear exposure , even nuclear necrosis and detachment , tubular injury was significant . CP + GSPE group was significantly reduced compared with CP group .

3 . TUNEL results showed that , compared with the N group , there were obvious apoptosis in the kidney tissues of CP group , compared with CP group , the number of apoptotic cells decreased obviously , and the number of apoptotic cells in GSPE group and N group was very little .

4 . Western blot analysis showed that the expression of GRP78 , pE and Caspase - 12 increased significantly in CP group compared with group N , and the expression of three proteins in CP + GSPE group was significantly lower than that in CP group , and there was no significant difference between the GSPE group and N group .

5 . The expression of GRP78 , p - ERK and Caspase - 12 protein in renal tissue was detected by immunohistochemistry . Compared with group N , the expression of GRP78 , p - ERK and Caspase - 12 in CP group was significantly lower than that of group N , while the expression level of GRP78 , p - ERK and Caspase - 12 in CP + GSPE group was significantly lower than that of N group .

Conclusion : GSPE can significantly reduce the acute renal injury induced by CP , protect renal function , and its mechanism is related to the regulation of apoptosis of Caspase - 12 pathway regulated by GSPE .

Background and Objective : In developed countries , diabetic nephropathy ( DN ) has become the most common cause of end - stage renal disease . In the United States , diabetic nephropathy ( DN ) accounts for 45 % of patients with end - stage renal disease . In our country , DN is the second major cause of end - stage renal disease , and its high morbidity and mortality rate are more and more attention .

Grape seed procyanidin ( GSPE ) is a powerful antioxidant whose antioxidant activity is 50 times that of vitamin E and 20 times of vitamin C . It has been introduced that GSPE has the functions of preventing and treating various diseases , including protecting the kidney damage caused by CP .

Methods : Fifty - five healthy male SD rats were randomly selected as normal control group ( N ) , and the other 45 rats were given high - fat and high - sugar diet as model group . After 4 weeks of feeding , the normal control group was given intraperitoneal injection of citrate buffer solution . After 4 weeks of feeding , the rats were injected intraperitoneally with STZ40mg / kg , and the blood glucose level 鈮，

本文编号：1980623