本文選題：硫辛酸 + 雷帕霉素靶蛋白　； 參考：《河北醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:糖尿病腎臟病(diabetic kidney disease,DKD)是2型糖尿病(type 2 diabetes mellitus,T2DM)常見微血管并發(fā)癥之一,并已成為導(dǎo)致患者終末期腎臟疾病甚至死亡的主要原因。DKD的發(fā)病機(jī)制尚未系統(tǒng)闡明,目前認(rèn)為糖脂代謝紊亂、血流動力學(xué)改變、氧化應(yīng)激、炎癥反應(yīng)、腎臟自噬抑制等與DKD發(fā)生發(fā)展有著密切聯(lián)系,其中氧化應(yīng)激是DKD重要發(fā)病機(jī)制,還可以介導(dǎo)炎癥反應(yīng)進(jìn)一步加速腎臟損傷。8-羥基脫氧鳥嘌呤(8-hydroxy-2-deoxygnanosine,8-OHdG)是一種目前反映DNA氧化損傷及體內(nèi)氧化應(yīng)激的敏感指標(biāo)。近年來,隨著對雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信號通路研究的逐漸深入,發(fā)現(xiàn)mTOR通路的激活促進(jìn)腎臟細(xì)胞肥大、蛋白質(zhì)合成增加,參與氧化應(yīng)激、炎癥反應(yīng)并抑制腎臟細(xì)胞自噬,在局灶節(jié)段性腎小球硬化、膜性腎病等慢性腎臟疾病的發(fā)病中起重要作用。腺苷酸活化蛋白激酶(adenosine5’-monophosphate-activate protein kinase,AMPK)是調(diào)控mTOR的上游信號之一,其主要通過抑制mTOR信號通路發(fā)揮作用。硫辛酸作為抗氧化劑,可以抑制氧自由基的生成,臨床上用于糖尿病周圍神經(jīng)病變治療收到了良好療效,但對DKD有無作用尚不明確。本實(shí)驗(yàn)通過復(fù)制T2DM大鼠模型,測定各組大鼠腎臟中AMPK、p-AMPK、mTOR、p-mTOR的表達(dá)情況及體內(nèi)8-OHdG水平,以探討mTOR信號通路、氧化應(yīng)激與T2DM腎臟病的關(guān)系及硫辛酸對腎臟mTOR信號通路及氧化應(yīng)激的影響。方法:22只雄性清潔級Sprague-Dawley(SD)大鼠,鼠齡6周,體重為180g±20g,清潔級環(huán)境飼養(yǎng)。應(yīng)用excel隨機(jī)數(shù)字表法將大鼠隨機(jī)分為正常對照組(NC組,n=6)、糖尿病組(DM組,n=8)、硫辛酸干預(yù)組(LA組,n=8)。正常對照組大鼠繼續(xù)予以普通飼料喂養(yǎng),其余兩組大鼠予以高糖高脂飼料(蔗糖20%、豬油10%、膽固醇2.5%,膽酸鹽1%,基礎(chǔ)飼料66.5%)喂養(yǎng)。4周后,糖尿病組及硫辛酸干預(yù)組大鼠給予30mg/kg鏈脲佐菌素腹腔注射,正常組大鼠腹腔注射等體積檸檬酸緩沖液,注射2周后測定大鼠空腹血糖(fastingbloodglucose,fbg),fbg7.8mmol/l視為t2dm造模成功。隨后硫辛酸干預(yù)組予以100mg/kg硫辛酸腹腔注射,正常對照組和糖尿病組均以等體積生理鹽水腹腔注射,每日給藥1次,連續(xù)給藥8周。實(shí)驗(yàn)結(jié)束前一日收集各組大鼠24小時(shí)尿液,用于測定24小時(shí)尿蛋白;當(dāng)日留取血清用于測定腎功能和8-ohdg等指標(biāo);取左腎稱重后,4%多聚甲醛固定,行he染色和pas染色觀察腎臟組織形態(tài)學(xué)變化,行masson染色觀察腎臟組織纖維化情況。免疫組織化學(xué)染色法檢測大鼠腎臟ampk、p-ampk、mtor、p-mtor的表達(dá)。結(jié)果:1各組大鼠體重及腎臟肥大指數(shù)的比較正常組大鼠體重為(455.17±22.46)g,腎臟肥大指數(shù)為(3.65±0.24)mg/g;糖尿病組大鼠體重為(250.67±26.56)g,腎臟肥大指數(shù)為(7.12±0.28)mg/g;硫辛酸組大鼠體重為(259.20±15.48)g,腎臟肥大指數(shù)為(6.10±0.45)mg/g。與正常組相比,糖尿病組和硫辛酸組大鼠體重減輕,腎臟肥大指數(shù)升高(p0.01);與糖尿組相比,硫辛酸組大鼠腎臟肥大指數(shù)降低,差異具有統(tǒng)計(jì)學(xué)意義(p0.01),兩組大鼠體重?zé)o統(tǒng)計(jì)學(xué)差異(p0.05)。2各組大鼠生化指標(biāo)的比較正常組大鼠血糖為(5.17±0.71)mmol/l,尿蛋白為(15.27±1.45)mg/24h,血肌酐為(37.23±4.97)μmol/l,血尿素氮為(6.38±1.18)mmol/l;糖尿病組大鼠血糖為(21.38±1.66)mmol/l,尿蛋白為(314.00±16.14)mg/24h,血肌酐為(36.70±4.80)μmol/l,血尿素氮為(7.52±1.21)mmol/l;硫辛酸組大鼠血糖為(20.60±0.60)mmol/l,尿蛋白為(249.88±28.61)mg/24h,血肌酐(37.80±4.03)μmol/l,血尿素氮(7.76±1.24)mmol/l。與正常組相比,糖尿病組和硫辛酸組大鼠的血糖和24小時(shí)尿蛋白均顯著升高(p0.01),血肌酐和血尿素氮無明顯差異(p0.05);與糖尿病組相比,硫辛酸組大鼠24小時(shí)尿蛋白降低(p0.01),血糖、血肌酐和血尿素氮無明顯差異(p0.05)。3各組大鼠8-ohdg的比較正常組大鼠8-ohdg為(1.63±0.62)ng/ml,糖尿病組大鼠8-ohdg為(5.09±0.89)ng/ml,硫辛酸組8-ohdg為(2.84±0.64)ng/ml。與正常組相比,糖尿病組和硫辛酸組大鼠的8-ohdg明顯升高(p0.05);與糖尿病組相比,硫辛酸組8-ohdg水平明顯降低,差異有統(tǒng)計(jì)學(xué)意義(p0.01)。4各組大鼠腎臟形態(tài)學(xué)改變腎臟he染色:正常組大鼠腎臟各組織形態(tài)正常、結(jié)構(gòu)清晰,無炎性細(xì)胞浸潤。糖尿病組大鼠腎小球毛細(xì)血管袢開放不良,系膜區(qū)擴(kuò)張,部分腎小管細(xì)胞出現(xiàn)空泡變性,間質(zhì)有較多炎性細(xì)胞浸潤。與糖尿病組相比,硫辛酸組上述病理變化減輕。腎臟pas染色:正常組系膜基質(zhì)指數(shù)為(0.1754±0.0149),糖尿病組系膜基質(zhì)指數(shù)為(0.4052±0.0523),硫辛酸組系膜基質(zhì)指數(shù)為(0.2259±0.0103);與正常組相比,糖尿病組及硫辛酸組大鼠基底膜增厚,系膜基質(zhì)明顯增多(p0.05);與糖尿病組相比,硫辛酸組大鼠系膜基質(zhì)明顯減少,差異有統(tǒng)計(jì)學(xué)意義(p0.01)。腎臟masson染色:與正常組相比,糖尿病組及硫辛酸組大鼠腎小球藍(lán)色膠原纖維明顯增多;與糖尿病組相比,硫辛酸大鼠膠原纖維沉積減少。5免疫組化檢測結(jié)果各組大鼠腎臟mtor和ampk均有表達(dá),差異無統(tǒng)計(jì)學(xué)意義(p0.05)。正常組大鼠腎臟p-mtor蛋白少量表達(dá),iod/area為(0.0230±0.0034);糖尿病組和硫辛酸組大鼠腎臟p-mtor蛋白表達(dá)明顯增加(p0.05),iod/area分別為(0.0547±0.0089)和(0.0316±0.0039);與糖尿病組相比,硫辛酸組大鼠腎臟p-mtor表達(dá)減少,差異有統(tǒng)計(jì)學(xué)意義(p0.01)。正常組大鼠腎臟p-ampk蛋白表達(dá)呈棕黃色顆粒沉著,iod/area為(0.0317±0.0040);與正常組相比,糖尿病組大鼠腎臟p-ampk蛋白表達(dá)減少,iod/area為(0.0183±0.0024)(p0.01);與糖尿病組相比,硫辛酸組大鼠臟p-ampk表達(dá)增加,iod/area為(0.0273±0.0026),差異均有統(tǒng)計(jì)學(xué)意義(p0.01)。結(jié)論:1糖尿病組大鼠腎臟組織出現(xiàn)典型病理改變,腎臟肥大指數(shù)和24小時(shí)尿蛋白升高,并且8-ohdg水平和mtor蛋白磷酸化明顯升高,說明氧化應(yīng)激增加及mtor信號通路激活與糖尿病腎臟病變有關(guān)。2硫辛酸組大鼠腎臟肥大指數(shù)和24小時(shí)尿蛋白降低,8-ohdg水平明顯下降,且腎臟病理改變減輕,說明硫辛酸可以降低糖尿病大鼠體內(nèi)氧化應(yīng)激,延緩糖尿病大鼠腎臟病變進(jìn)展。3硫辛酸組大鼠mTOR蛋白磷酸化水平明顯降低,AMPK蛋白活化增加,說明硫辛酸可激活A(yù)MPK蛋白,抑制mTOR信號通路,在糖尿病腎臟病變中起到的保護(hù)作用。
[Abstract]:Objective: diabetic kidney disease (DKD) is one of the common microvascular complications of type 2 diabetes mellitus (type 2 diabetes mellitus, T2DM), and has become the main cause of end-stage renal disease and death. The pathogenesis of.DKD is not systematically explained. Chemical stress, inflammatory response, and autophagy inhibition are closely related to the development of DKD. Oxidative stress is an important pathogenesis of DKD, and it also mediates the inflammatory reaction to further accelerate renal injury,.8- hydroxyl deoxy guanine (8-hydroxy-2-deoxygnanosine, 8-OHdG), which is a present reflection of DNA oxidative damage and oxidative stress in the body. Sensitive indicators. In recent years, with the research of the mammalian target of rapamycin (mTOR) signaling pathway, the activation of mTOR pathway promotes renal cell hypertrophy, protein synthesis, oxidative stress, inflammatory reaction and inhibition of autophagy of renal cell, in focal segmental glomerulosclerosis and membranous kidney Disease and other chronic renal diseases play an important role. Adenosine5 '-monophosphate-activate protein kinase (AMPK) is one of the upstream signals for the regulation of mTOR. It plays a role mainly by inhibiting the mTOR signaling pathway. As an antioxidant, lipoic acid can inhibit the formation of oxygen free radicals and be used in clinical application. The treatment of diabetic peripheral neuropathy has received good curative effect, but the effect on DKD is not clear. By replicating the T2DM rat model, the expression of AMPK, p-AMPK, mTOR, p-mTOR and the level of 8-OHdG in the kidneys of the rats were measured to explore the relationship between the mTOR signaling pathway, the oxidative stress and T2DM kidney disease and the renal m of the lipoic acid. Methods: the effect of TOR signaling pathway and oxidative stress. Methods: 22 male clean Sprague-Dawley (SD) rats, 6 weeks of age and a weight of 180g + 20g, were kept in a clean environment. The rats were randomly divided into normal control group (NC group, n=6), diabetes group (DM group, n=8), lipoic acid intervention group (LA group, n=8). The normal control group was followed. The other two groups of rats were fed with high sugar and high fat diet (sucrose 20%, lard 10%, cholesterol 2.5%, cholate 1%, and basal diet 66.5%) after.4 weeks, the diabetic group and the rats in the thioctyl acid group were given 30mg/kg streptozotocin intraperitoneal injection, and the normal group rats were injected with equal volume citric acid buffer, after 2 weeks of injection. The rat fasting blood glucose (fastingbloodglucose, FBG) was measured and fbg7.8mmol/l was regarded as a successful model of T2DM. Then 100mg/kg lipoic acid was intraperitoneally injected with lipoic acid in the intervention group. The normal control group and the diabetic group were intraperitoneally injected with equal volume physiological saline, 1 times a day for 8 weeks. The 24 hour urine of rats in each group was collected one day before the end of the experiment. The liquid was used to determine the 24 hour urine protein; on the same day, the serum was used to determine the renal function and 8-OHdG. After the left kidney was weighed, 4% polyformaldehyde was fixed. The changes of renal histomorphology were observed by HE staining and PAS staining. The renal tissue fibrosis was observed by Masson staining. The kidney AMPK, p-ampk, mTOR in rat kidneys was detected by the immunostaining method. P-mTOR expression. Results: 1 the body weight and renal hypertrophy index of rats in each group were compared to (455.17 + 22.46) g and renal hypertrophy index (3.65 + 0.24) mg/g; the weight of the diabetic rats was (250.67 + 26.56) g and the renal hypertrophy index was (7.12 + 0.28) mg/g; the weight of the rats in the sulphur octanic acid group was (259.20 + 15.48) g, and the renal hypertrophy index was the index of renal hypertrophy. (6.10 + 0.45) mg/g. compared with the normal group, the weight loss and renal hypertrophy index of rats in the diabetic group and the lipoic acid group increased (P0.01). Compared with the diabetic group, the renal hypertrophy index of the rats in the sulphur octanic acid group decreased, and the difference was statistically significant (P0.01). The body weight of the two groups was not statistically different (P0.05), the biochemical indexes of the rats in each group of.2 were compared to the normal group. The blood sugar of rats was (5.17 + 0.71) mmol/l, urine protein was (15.27 + 1.45) mg/24h, blood creatinine was (37.23 + 4.97) mu mol/l, blood urea nitrogen was (6.38 + 1.18) mmol/l, blood sugar of diabetic rats was (21.38 + 1.66) mmol/l, urinary protein was (314 + 16.14) mg/24h, serum creatinine was (36.70 + 15.27) mu mol/l, blood urea nitrogen was mmol/l, and lipoic acid group rats Blood sugar was (20.60 + 0.60) mmol/l, urine protein was (249.88 + 28.61) mg/24h, serum creatinine (37.80 + 4.03) mu mol/l, blood urea nitrogen (7.76 + 1.24) mmol/l. compared with normal group, the blood sugar and 24 hourly proteinuria of rats in diabetic group and lipoic acid group increased significantly (P0.01), blood creatinine and blood urea nitrogen had no significant difference (P0.05); compared with diabetic group, The 24 hour urinary protein decreased (P0.01), blood sugar, blood creatinine and blood urea nitrogen (P0.05) in rats of.3, 8-OHdG of rats in each group of 8-OHdG was (1.63 + 0.62) ng/ml, 8-OHdG in diabetic rats was (5.09 + 0.89) ng/ml, and 8-OHdG was (2.84 + 0.64) ng/ml. in the group of lipoic acid (2.84 + 0.64), compared with normal group, diabetic group and lipoid The 8-OHdG of rats in the acid group was significantly increased (P0.05), and the 8-OHdG level of the lipoic acid group was significantly lower than that in the diabetic group. The difference was statistically significant (P0.01) the renal morphological changes of the kidney in each group of.4 rats were changed by HE staining: the normal group of kidney tissues in the normal group was normal, the structure was clear, and the inflammatory cells were not infiltrated. The glomerular capillaries in the diabetic rats The loop opening was bad, the mesangial region expanded, some renal tubular cells appeared vacuolated degeneration, and there were more inflammatory cell infiltration in the interstitium. Compared with the diabetic group, the pathological changes of the lipoic acid group were reduced. The renal PAS staining was (0.1754 + 0.0149), the mesangial matrix index of the diabetic group was (0.4052 + 0.0523), and the mesangial base of the sulphur octanic acid group The mass index was (0.2259 + 0.0103). Compared with the normal group, the basement membrane of the diabetic group and the lipoic acid group was thickened and the mesangial matrix increased significantly (P0.05). Compared with the diabetic group, the rat mesangial matrix of the lipoic acid group was significantly reduced, the difference was statistically significant (P0.01). The renal Masson staining was compared with the normal group, the diabetic and lipoic acid group rats. The glomerular blue collagen fibers increased significantly; compared with the diabetic group, the collagen deposition in the rats with lipoic acid was reduced by.5 immunohistochemistry. The renal mTOR and AMPK were expressed in each group, and the difference was not statistically significant (P0.05). A small amount of p-mTOR protein in the normal group of rats, iod/area was (0.0230 + 0.0034), and the diabetic group and lipoic acid were found. The expression of p-mTOR protein in the kidneys of the rats was significantly increased (P0.05), iod/area was (0.0547 + 0.0089) and (0.0316 + 0.0039), respectively. Compared with the diabetic group, the expression of p-mTOR in the kidney of the rats with lipoic acid was decreased (P0.01). The expression of p-ampk protein in the kidney of the normal group was brown and yellow granules, and iod/area was (0.0317 + 0.0040); Compared with the normal group, the expression of p-ampk protein in the kidney of diabetic rats decreased and iod/area was (0.0183 + 0.0024) (P0.01). Compared with the diabetic group, the expression of p-ampk in the rats of the lipoic acid group increased and the iod/area was (0.0273 + 0.0026). The difference was statistically significant (P0.01). Conclusion: the renal tissue of the 1 diabetic rats showed a typical pathological change of kidney tissue and kidney fertilizer. The high index and 24 hour urine protein increased, and the level of 8-OHdG and the phosphorylation of mTOR protein increased obviously, indicating that the increase of oxidative stress and the activation of mTOR signaling pathway were associated with the decrease of renal hypertrophy index and 24 hour urinary protein in the rats with diabetic renal disease, the level of 8-OHdG decreased significantly, and the renal pathological changes were reduced, indicating thioxin. Acid can reduce oxidative stress in diabetic rats and delay the progression of renal diseases in diabetic rats. The phosphorylation of mTOR protein in.3 thioctanoic acid group is significantly reduced, and the activation of AMPK protein is increased. It shows that thioctanoic acid activates AMPK protein and inhibits the mTOR signaling pathway and plays a protective role in diabetic renal disease.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R587.2;R692.9

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 黃宏;邱偉;朱明;張婭;崔文慧;邢偉;李向云;安天琛;陳民佳;郭j:;徐祥;;蛋白激酶B/哺乳動物雷帕霉素靶蛋白信號通路關(guān)鍵蛋白在糖尿病大鼠皮膚組織和創(chuàng)面組織中的表達(dá)研究[J];中華燒傷雜志;2016年10期

2 蘇杭;周健;賈偉平;;2016年ADA糖尿病醫(yī)學(xué)診療標(biāo)準(zhǔn)解讀[J];中國醫(yī)學(xué)前沿雜志(電子版);2016年01期

3 楊晶晶;黃燕如;萬毅剛;沈山梅;毛志敏;吳薇;姚建;;mTOR信號通路調(diào)控糖尿病腎病腎小球肥大的機(jī)制及中藥的干預(yù)作用[J];中國中藥雜志;2015年16期

4 盧新星;范秋靈;徐莉;李琳;徐艷艷;張東成;王力寧;;烏索酸改善高糖誘導(dǎo)系膜細(xì)胞損傷的機(jī)制[J];中華腎臟病雜志;2015年01期

5 張巍;于為民;任小軍;;糖尿病腎病的炎癥反應(yīng)[J];中國現(xiàn)代醫(yī)生;2014年09期

6 漆洪波;;關(guān)于妊娠期糖尿病國際新標(biāo)準(zhǔn)的爭議[J];中華婦幼臨床醫(yī)學(xué)雜志(電子版);2013年03期

7 楊楠楠;剛曉坤;劉青;;糖尿病腎病與氧化應(yīng)激[J];中國老年學(xué)雜志;2013年05期

8 賈振華;劉毅;曹銘鋒;徐靜;完強(qiáng);王榮;;AMPK在高糖誘導(dǎo)大鼠腎小球系膜細(xì)胞Ⅳ型膠原過度表達(dá)中的作用[J];基礎(chǔ)醫(yī)學(xué)與臨床;2010年11期

9 李艷英;耿厚法;班博;;α-硫辛酸對糖尿病大鼠血糖、血脂及肝臟內(nèi)AMPKα表達(dá)及活性的影響[J];中國生物制品學(xué)雜志;2010年09期

10 奚苗苗;文愛東;梁欣;陳宏莉;劉瑞;柏樺;海春旭;;2型糖尿病大鼠模型的建立及其氧化應(yīng)激特征分析[J];中國藥物與臨床;2010年01期

，

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