本文選題：葡萄籽原花青素 + 內(nèi)質(zhì)網(wǎng)應(yīng)激��； 參考：《山東大學(xué)》2014年碩士論文

【摘要】：背景及目的：近年來(lái)一些研究發(fā)現(xiàn)內(nèi)質(zhì)網(wǎng)應(yīng)激(endoplasmicreticulumstress, ERS)在順鉑(Cisplatin CP)誘導(dǎo)的腎損害中起著重要的作用。葡萄籽原花青素(grape seed proanthocyanidin extract,GSPE)作為天然的抗氧化劑對(duì)多種疾病具有保護(hù)及預(yù)防作用,然而,GSPE對(duì)CP導(dǎo)致的急性腎損傷是否具有保護(hù)作用尚不清楚。本實(shí)驗(yàn)通過(guò)給予小鼠腹腔注射CP,以誘導(dǎo)急性腎損害模型,并給予GSPE干預(yù),觀察其對(duì)腎損傷小鼠的一般情況,血肌酐水平,腎小管病理變化,腎組織細(xì)胞凋亡情況,以及腎組織葡萄糖調(diào)節(jié)蛋白(glucose-regulated protein78GRP78)、磷酸化細(xì)胞外信號(hào)調(diào)節(jié)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)蛋白表達(dá)的影響,探討GSPE對(duì)CP導(dǎo)致的急性腎損傷小鼠的保護(hù)作用及其可能機(jī)制,從而為深入闡明GSPE對(duì)CP導(dǎo)致的急性腎損傷的保護(hù)機(jī)制提供新的依據(jù),為GSPE在臨床上的應(yīng)用開辟新的視野。 方法：雄性C57小鼠65只,適應(yīng)性喂養(yǎng)1周后,隨機(jī)分為四組：正常對(duì)照組(N組,n=10只),順鉑組(腹腔注射20mg/kg CP, CP組,n=20只),GSPE組(第一天及第三天灌胃GSPE500mg/kg, n=15只), CP+GSPE組(腹腔注射順鉑半小時(shí)前給予500mg/kg GSPE灌胃,第三天灌胃等量的GSPE,n=20只)。給藥后第五天內(nèi)眥取血測(cè)肌酐、尿素氮,取雙腎并稱重測(cè)腎臟系數(shù),腎組織做PAS染色,應(yīng)用TUNEL試劑盒檢測(cè)小鼠腎臟細(xì)胞凋亡情況,應(yīng)用蛋白印跡法、免疫組化方法檢測(cè)小鼠GRP78、p-ERK和Caspase-12的蛋白表達(dá)。 結(jié)果：1.各組小鼠一般情況變化：與N組比較,CP組小鼠腎臟系數(shù)升高,血尿素氮、肌酐水平升高(P0.05), CP+GSPE組與CP組比較,以上指標(biāo)均有明顯下降。GSPE組與N組比較以上指標(biāo)無(wú)明顯變化。 2.各組小鼠腎臟病理改變：N組及GSPE組小鼠腎臟結(jié)構(gòu)清晰,未見(jiàn)腎小管結(jié)構(gòu)損傷,CP組可見(jiàn)腎小管上皮細(xì)胞腫脹,空泡樣變性,大量管型形成,細(xì)胞核裸露,甚至可見(jiàn)細(xì)胞核壞死脫落,腎小管損傷明顯。CP+GSPE組與CP組比較,腎小管損傷明顯減輕。 3.TUNEL結(jié)果顯示：與N組比較,CP組小鼠腎組織細(xì)胞可見(jiàn)明顯的細(xì)胞凋亡,CP+GSPE組與CP組比較,小鼠細(xì)胞凋亡數(shù)量明顯減少,GSPE組與N組細(xì)胞凋亡數(shù)量極少。 4.蛋白免疫印跡結(jié)果：Western blot結(jié)果顯示,與N組比較,GRP78、p-ERI和Caspase-12蛋白的表達(dá)在CP組明顯升高,CP+GSPE組三種蛋白表達(dá)較CP組明顯降低,GSPE組與N組相比蛋白表達(dá)無(wú)明顯差異。 5.免疫組化顯示腎組織GRP78、p-ERK和Caspase-12蛋白的表達(dá)。與N組比較,CP組可見(jiàn)高表達(dá)的GRP78、p-ERK和Caspase-12蛋白,而CP+GSPE組GRP78、p-ERK和Caspase-12蛋白表達(dá)水平明顯降低,GSPE組與N組比較無(wú)明顯變化。 結(jié)論：GSPE能明顯減輕CP導(dǎo)致的急性腎損傷,保護(hù)腎功能,其機(jī)制與GSPE下調(diào)Caspase-12通路調(diào)節(jié)的ERS介導(dǎo)的凋亡有關(guān)。 研究背景和目的：在發(fā)達(dá)國(guó)家,糖尿病腎病(diabetic nephropathy DN)已成為導(dǎo)致終末期腎臟病(endstage renal disease ESRD)最普遍的原因,在美國(guó),糖尿病腎病(DN)占ESRD的45%,成為ESRD的首位原因。在我國(guó),DN為ESRD的第二大病因,其高發(fā)病率及死亡率越來(lái)越受到人們的關(guān)注。 葡萄籽原花青素(GSPE)是強(qiáng)大的抗氧化劑,其抗氧化活性是維生素E的50倍,維生素C的20倍。有研究證明GSPE對(duì)DN有保護(hù)作用,前已介紹,GSPE具有預(yù)防和治療多種疾病的功能,包括保護(hù)CP導(dǎo)致的腎損害,其機(jī)制可能與減輕ERS介導(dǎo)的凋亡有關(guān)�；谖覀円陨涎芯拷Y(jié)果,本實(shí)驗(yàn)通過(guò)研究GSPE對(duì)DN的保護(hù)作用,探討GSPE是否也是通過(guò)減輕ERS介導(dǎo)的凋亡來(lái)發(fā)揮對(duì)DN的保護(hù)作用的。 方法：健康雄性SD大鼠55只隨機(jī)選取10只作為正常對(duì)照組(N),其余45只大鼠作為模型組給予高脂高糖飲食。喂養(yǎng)4周后,正常對(duì)照組給予檸檬酸鹽緩沖液腹腔注射,模型組一次性腹腔注射STZ40mg/kg,72h后尾部采血測(cè)血糖,以空腹血糖水平≥16.7mmol/L為糖尿病模型成功。其中5只大鼠建模不成功,剔除實(shí)驗(yàn),40只大鼠建模成功。建模成功后的大鼠隨機(jī)分為糖尿病組(D,n=20),GSPE治療組(G,n=20)。G組給予250mg/kg-d的GSPE灌胃,N組和D組給予生理鹽水灌胃,持續(xù)灌胃12周。正常對(duì)照組大鼠給予喂養(yǎng)普通飼料,自由飲水,D組及G組給予喂養(yǎng)高脂高糖飼料。定期監(jiān)測(cè)大鼠血糖,于第16周末測(cè)定各組大鼠的24尿蛋白定量、尿素氮(BUN)、血肌酐(SCr)、腎臟系數(shù)(RI),光學(xué)顯微鏡下觀察大鼠腎組織PAS染色結(jié)果,并應(yīng)用免疫組化方法,Western blotting實(shí)驗(yàn)技術(shù),檢測(cè)大鼠腎組織GRP78、p-ERK、Caspase-12的蛋白表達(dá),應(yīng)用TUNEL試劑盒檢測(cè)大鼠腎臟細(xì)胞凋亡情況。 結(jié)果：1.各組大鼠一般情況變化：與N組比較,D組腎臟系數(shù)升高,24h尿蛋白定量明顯升高,BUN、SCr無(wú)明顯變化(P0.05),經(jīng)GSPE治療后腎臟系數(shù)、24h尿蛋白定量下降,BUN、SCr水平無(wú)明顯改變(見(jiàn)表一),D組、G組大鼠血糖無(wú)明顯變化。 2.各組大鼠腎臟病理改變：N組大鼠腎臟結(jié)構(gòu)清晰,未見(jiàn)腎損傷,D組可見(jiàn)腎小球體積明顯增大,腎小球基底膜增厚,系膜區(qū)增寬,系膜細(xì)胞增生,系膜基質(zhì)增多。與D組相比GSPE治療組腎損傷明顯減輕。 3.TUNEL結(jié)果顯示：與N組比較,D組大鼠細(xì)胞凋亡數(shù)明顯增加,與D組比較,G組大鼠細(xì)胞凋亡數(shù)明顯減少。 4.蛋白免疫印跡結(jié)果：蛋白免疫印跡顯示,與N組比較,GRP78、p-ERK和Caspase-12蛋白的表達(dá)在D組明顯升高,G組以上三種蛋白表達(dá)水平較D組明顯降低。 5.免疫組化：腎組織GRP78、p-ERK和Caspase-12蛋白的表達(dá)。與N組比較,D組可見(jiàn)高表達(dá)的GRP78、p-ERK和Caspase-12蛋白,而G組GRP78、p-ERK和Caspase-12蛋白表達(dá)水平明顯降低。 結(jié)論：GSPE能明顯降低DN大鼠的腎臟系數(shù),24h尿蛋白定量,減少腎臟細(xì)胞凋亡,改善腎臟病理變化,延緩DN的發(fā)展,其機(jī)制與GSPE下調(diào)Caspase-12通路調(diào)節(jié)的ERS介導(dǎo)的凋亡有關(guān)。
[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，

本文編號(hào)：1980623