本文選題：利拉魯肽 + 胰島素抵抗��； 參考：《承德醫(yī)學(xué)院》2017年碩士論文

【摘要】：目的:利拉魯肽是目前治療2型糖尿病的新上市降糖藥物,不僅可以促進(jìn)胰島素分泌,還可增加胰腺外組織對(duì)胰島素作用的敏感性。胰島素抵抗(insulin resistance,IR)是2型糖尿病及代謝綜合征的發(fā)病機(jī)制之一。既往有研究發(fā)現(xiàn)利拉魯肽可改善IR,但具體機(jī)制尚未明確。本實(shí)驗(yàn)通過(guò)使用不同濃度的利拉魯肽對(duì)高脂飼料喂養(yǎng)的大鼠進(jìn)行干預(yù),觀(guān)察其在IR大鼠肝臟的脂質(zhì)沉積以及對(duì)胰島素的敏感性的影響;本研究經(jīng)檢測(cè)肝臟內(nèi)質(zhì)網(wǎng)應(yīng)激分子伴侶GRP78,內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)分子JNK,胰島素信號(hào)傳導(dǎo)通路相關(guān)分子GLUT-2、AKT表達(dá)的情況,探究利拉魯肽改善IR,減少肝臟脂肪沉積的機(jī)制,為其在臨床的應(yīng)用提供理論支持。方法:Wistar雄性4月齡大鼠,體重250g左右,54只,經(jīng)過(guò)3天適應(yīng)性的喂養(yǎng),隨機(jī)將其分為對(duì)照組(NC,n=16)和高脂組(n=38),分別以普通和高脂飼料喂養(yǎng)。8周末,兩組在隨機(jī)下取5只大鼠在其清醒狀態(tài)下進(jìn)行高胰島素-正葡萄糖鉗夾試驗(yàn),然后計(jì)算兩組大鼠的葡萄糖輸注率(glu cose infusion rate,GIR),判定其IR狀態(tài)。判定造模成功后,將高脂大鼠分為三組即高脂對(duì)照組(HFD)、利拉魯肽低劑量組(LL)、利拉魯肽高劑量組(HL),每組11只。分別以0.5ml的滅菌生理鹽水對(duì)NC組及HFD組進(jìn)行皮下注射;以100ug/kg的利拉魯肽對(duì)LL組日一次進(jìn)行皮下注射;以200ug/kg的利拉魯肽對(duì)HL組日一次行皮下注射。藥物干預(yù)2周后,四組分別隨機(jī)選取5只大鼠,后進(jìn)行口服糖耐量(oral glucose tolerance test,OGTT)實(shí)驗(yàn)和鉗夾試驗(yàn),以算出大鼠葡萄糖曲線(xiàn)下面積(Area Un der Curve,AUC)及GIR。對(duì)各個(gè)組中剩余的6只大鼠分別稱(chēng)體重(body weight,BW),然后進(jìn)行麻醉,腹主動(dòng)脈取血,-80℃保存,測(cè)定甘油三酯(triglyceride,TG)、游離脂肪酸(free fatty acids,FFA)、高密度脂蛋白膽固醇(high density lipoprotein cholesterol,HDL-C)、總膽固醇(tot al cholesterol,TCH)以及低密度脂蛋白膽固醇(low density lipoprotein c holesterol,LDL-C)、肝轉(zhuǎn)氨酶學(xué)改變。分別制作肝臟標(biāo)本并保存,測(cè)定肝組織甘油三酯(LTG),經(jīng)透電子顯微鏡察看肝細(xì)胞內(nèi)質(zhì)網(wǎng)結(jié)構(gòu)是否發(fā)生改變,應(yīng)用Western blot技術(shù)對(duì)肝臟中GRP78、GLUT-2、p-AKT、p-JN K蛋白表達(dá)情況進(jìn)行檢測(cè),應(yīng)用Realtime-PCR對(duì)GRP78、GLUT-2 m RNA表達(dá)情況進(jìn)行測(cè)定。結(jié)果:1四組大鼠一般情況的比較與NC組相較,HFD組中BW、血FFA以及LTG均顯著增高(P0.05)。與HFD組相比較,HL組中BW、血FFA、TG、LTG、肝轉(zhuǎn)氨酶學(xué)指標(biāo)均顯著降低(P0.05);LL組中僅有FFA、肝轉(zhuǎn)氨酶學(xué)指標(biāo)的下降(P0.05)。與LL組相比,HL組血液指標(biāo)均有明顯改善,血FFA、TG、LTG也均有顯著減少(P0.05)2四組大鼠GIR的比較喂養(yǎng)第8周時(shí),高胰島素-正葡萄糖鉗夾實(shí)驗(yàn)結(jié)果示:與NC組相比,HFD組葡萄糖輸注率GIR下降(30.21±2.51vs20.41±3.64mg/kg.min)(P0.05)。藥物干預(yù)2周后,與HFD組相比,HL組GIR升高(18.83±1.95)vs(25.80±1.40)mg/kg.min(P0.05),LL組GIR升高(18.83±1.95)vs(21.55±2.46)mg/kg.min(P0.05);與LL組相比,HL組GIR亦升高(21.55±2.46)vs(25.80±1.40)mg/kg.min(P0.05)。3四組大鼠OGTT的比較與NC組相比較,HFD組大鼠血糖及葡萄糖曲線(xiàn)下面積AUC于0min、30min、60min、120min四個(gè)點(diǎn)處均顯著升高(P0.05);與HFD組相比較,HL組大鼠血糖及AUC在四個(gè)點(diǎn)處均降低(P0.05),LL組大鼠雖四個(gè)點(diǎn)處血糖及AUC均有所降低,然而僅在120min時(shí)血糖變化有顯著性差異(P0.05);與LL組相比較,HL組大鼠血糖及AUC于0min、120min兩個(gè)點(diǎn)處均顯著降低(P0.05)。4四組大鼠肝臟形態(tài)學(xué)變化(光學(xué)顯微鏡)在NC組中,肝細(xì)胞細(xì)胞壁及內(nèi)部細(xì)胞器結(jié)構(gòu)完整,肝小葉和中央靜脈結(jié)構(gòu)正常,可在中央靜脈周?chē)逦^(guān)察到呈放射狀排布的肝細(xì)胞索,HFD組肝細(xì)胞體積顯著擴(kuò)大,胞內(nèi)存在大小不等的脂滴,細(xì)胞核移向細(xì)胞邊緣,LL組肝細(xì)胞體積有所減小,胞內(nèi)脂滴數(shù)目減少,體積縮小,HL組大鼠的肝細(xì)胞排列規(guī)整,胞內(nèi)可見(jiàn)少量脂滴,其肝小葉及肝索結(jié)構(gòu)完整。5四組大鼠肝細(xì)胞內(nèi)質(zhì)網(wǎng)變化(透射電子顯微鏡)與NC組相比,HFD組肝細(xì)胞核周?chē)螖?shù)量多,體積大,內(nèi)質(zhì)網(wǎng)明顯擴(kuò)張,脫顆粒明顯;與HFD組相比,LL組肝細(xì)胞內(nèi)質(zhì)網(wǎng)輕度擴(kuò)張,脫顆粒輕微緩解;與HFD組相比,HL組肝細(xì)胞核周?chē)鸁o(wú)明顯脂滴存在,內(nèi)質(zhì)網(wǎng)擴(kuò)張及脫顆粒得到顯著緩解。6四組大鼠肝臟GRP78、GLUT-2 m RNA表達(dá)水平比較與NC組大鼠相比較,HFD組大鼠的GRP78 m RNA的表達(dá)顯著增高,GLUT-2 m RNA表達(dá)顯著減低(P0.05);與HFD組大鼠相比較,LL組及HL組大鼠GRP78 m RNA的表達(dá)均顯著降低(P0.05),與LL組大鼠相比較,HL組減低更為明顯(P0.05);與HFD組大鼠相比較,LL組及HL組GLUT-2 m RNA的表達(dá)均顯著上調(diào)(P0.05),但HL組上調(diào)程度顯著高于LL組(P0.05)。7四組大鼠肝GRP78、GLUT-2、p-AKT和p-JNK蛋白表達(dá)水平比較與NC組相比,HFD組GRP78蛋白表達(dá)上調(diào),GLUT-2蛋白表達(dá)下調(diào),磷酸化JNK(p-JNK)/JNK明顯升高,磷酸化AKT(p-AKT)/AKT明顯下降(P0.05)。與HFD組相比,HL組、LL組GRP78蛋白表達(dá)均下調(diào),GLUT-2蛋白表達(dá)均上調(diào),p-JNK/JNK均下降,p-AKT/AKT均升高(P0.05)。與LL組相比,HL組GRP78蛋白表達(dá)下調(diào),GLUT-2蛋白表達(dá)上調(diào),p-JNK/JNK下降,p-AKT/AKT增加(P0.05)。8相關(guān)分析研究結(jié)果:GIR與BW,血FFA、TG及LTG呈負(fù)相關(guān)(P0.05或P0.01)。GRP78蛋白表達(dá)量與血FFA、TG,LTG呈正相關(guān)(P0.05或P0.01),同時(shí)與GIR呈負(fù)相關(guān)(P0.01)。GLUT-2蛋白表達(dá)量與血FFA、TG,LTG呈負(fù)相關(guān)(P0.01),并且與GIR呈正相關(guān)(P0.01)。JNK磷酸化水平與血FFA、TG,LTG呈正相關(guān)(P0.01),且與GIR呈負(fù)相關(guān)(P0.01)。AKT磷酸化水平與與血FFA、TG,LTG呈負(fù)相關(guān)(P0.05或P0.01),且與GIR呈正相關(guān)(P0.01)。結(jié)論:1高脂飲食可使大鼠出現(xiàn)糖脂代謝紊亂,并增加肝臟脂肪沉積,出現(xiàn)糖耐量的降低及IR。2利拉魯肽對(duì)改善糖脂代謝紊亂、減少肝臟脂質(zhì)蓄積以及改善IR和糖耐量減低呈濃度依賴(lài)性。3利拉魯肽呈濃度依賴(lài)性對(duì)GRP78/JNK/AKT/GLUT-2通路進(jìn)行調(diào)控,與緩解肝臟內(nèi)質(zhì)網(wǎng)應(yīng)激,減少肝臟脂質(zhì)沉積,促進(jìn)肝細(xì)胞對(duì)葡萄糖攝取,改善IR相關(guān)。
[Abstract]:Objective: it is a new hypoglycemic drug for the treatment of type 2 diabetes, which not only promotes insulin secretion, but also increases the sensitivity of external pancreatic tissue to insulin. Insulin resistance (insulin resistance, IR) is one of the pathogenesis of type 2 diabetes and metabolic syndrome. Good IR, but the specific mechanism is not clear. In this experiment, the rats fed with high fat diet were treated with different concentrations of lieragin, and the lipid deposition in the liver of IR rats and the effect on the sensitivity of insulin were observed. The study was conducted to detect the liver endoplasmic reticulum stress sub chaperone GRP78, the endoplasmic reticulum stress related molecule JNK, and the pancreas. The expression of GLUT-2 and AKT in the signal transduction pathway of ISO signal transduction pathway, explore the mechanism of liararu peptide to improve IR, reduce the liver fat deposition, and provide theoretical support for its clinical application. Methods: Wistar male 4 month old rats, weight 250g and 54, were randomly divided into control group (NC, n=16) and high fat group after 3 days of adaptive feeding. (n=38).8 weekend was fed with ordinary and high fat diet, and the two groups were randomly selected to take 5 rats at random to carry out high insulin positive glucose clamp test, and then calculate the glucose infusion rate (Glu cose infusion rate, GIR) in two groups of rats and determine their IR like state. After the model was successful, the high fat rats were divided into three groups, that is, high fat fat. The control group (HFD), the low dose group of Liraru peptide (LL), the high dose of Liraru peptide group (HL), 11 rats in each group, were subcutaneously injected with the NC and HFD group with 0.5ml sterilized saline, and a subcutaneous injection of the 100ug/kg's Liraru peptide to LL group daily, and a subcutaneous injection of the 200ug/kg's Liraru peptide to the HL group. In the four groups, 5 rats were randomly selected, then the oral glucose tolerance (oral glucose tolerance test, OGTT) experiment and clamp test were carried out to calculate the area of the glucose curve (Area Un der Curve, AUC) and GIR. on the remaining 6 rats in each group. Triglyceride, TG, free fatty acids (free fatty acids, FFA), high density lipoprotein cholesterol (high density lipoprotein cholesterol, HDL-C), total cholesterol (TOT), and low density lipoprotein cholesterol (LDL), liver transaminase change, were made respectively. The liver specimens were preserved, the liver tissue triglyceride (LTG) was measured, and the changes in the structure of the endoplasmic reticulum of the liver cells were examined through electron microscopy. The expression of GRP78, GLUT-2, p-AKT, p-JN K protein in the liver was detected by Western blot technique. The expression of GRP78 and GLUT-2 m was measured by Realtime-PCR. Results: 1 groups of four groups were large Compared with NC group, BW, blood FFA and LTG in group HFD were significantly higher (P0.05). Compared with group HFD, BW, FFA, TG, LTG, liver transaminase index in HL group were significantly decreased. Also significantly decreased (P0.05) 2 four groups of rats in the comparative feeding of GIR eighth weeks, the results of high insulin positive glucose clamp showed that compared with the NC group, the glucose infusion rate in the HFD group decreased (30.21 + 2.51vs20.41 + 3.64mg/kg.min) (P0.05). After 2 weeks of drug intervention, the GIR increased (18.83 + 1.95) vs (25.80 + 1.40) (25.80 + 1.40) compared with the HFD group. 05), GIR in group LL increased (18.83 + 1.95) vs (21.55 + 2.46) mg/kg.min (P0.05). Compared with group LL, HL group GIR also increased (21.55 + 2.46) vs (25.80 + 1.40) mg/kg.min (P0.05).3 four rats. Compared with group HL, blood glucose and AUC decreased at four points (P0.05). The blood glucose and AUC were decreased at four points in group LL rats, but there was a significant difference (P0.05) at 120min only (P0.05). Compared with group LL, the blood sugar and AUC of rats in group HL and 120min two points were significantly reduced in the four groups of rat liver morphology. In the NC group, the cell wall and internal organelle structure of the liver cells were complete, the hepatic lobule and the central vein were normal, and the hepatic cell cord was clearly observed around the central vein. The volume of the hepatocytes in the HFD group was significantly enlarged, the fat droplets of different sizes were found in the cell, the nucleus moved to the edge of the cell and the liver of the LL group. The cell volume decreased, the number of intracellular lipid droplets decreased and the volume was reduced. The liver cells in the HL group were arranged regularly and a small amount of lipid droplets were visible. The changes in the endoplasmic reticulum of the hepatocytes in the group of hepatic lobules and the complete.5 four groups of the hepatic cord (transmission electron microscope) were compared with that of the NC group. The number of lipid droplets around the nucleus of the liver of the group HFD was large and the endoplasmic reticulum was obvious. Compared with the HFD group, the endoplasmic reticulum in the LL group was slightly dilated and the degranulation was slightly relieved. Compared with the group HFD, there was no obvious lipid droplet around the nucleus of the liver in the group HL, and the endoplasmic reticulum dilation and degranulation significantly alleviated the GRP78 in the liver of.6 four rats, and the RNA expression level of GLUT-2 m was compared with the NC group rats, and G in HFD group rats. The expression of RP78 m RNA was significantly higher and the expression of GLUT-2 m RNA decreased significantly (P0.05). Compared with group HFD, the expression of GRP78 m in LL group and HL group decreased significantly. Compared with group NC, the expression level of GLUT-2, p-AKT and p-JNK protein in group.7 four was significantly higher than that of group.7 (P0.05). The expression level of GRP78 protein in HFD group was up, the expression of GLUT-2 protein was down, phosphorylation JNK was obviously increased and phosphorylation was obviously decreased. The expression of GLUT-2 protein increased, p-JNK/JNK decreased, and p-AKT/AKT increased (P0.05). Compared with the LL group, the expression of GRP78 protein in the HL group was down regulated, the expression of GLUT-2 protein was up, p-JNK/JNK decreased, and p-AKT/AKT increased (P0.05). TG, LTG was positively correlated (P0.05 or P0.01), and negative correlation with GIR (P0.01).GLUT-2 protein expression was negatively correlated with FFA, TG and LTG (P0.01). 01) and positive correlation with GIR (P0.01). Conclusion: 1 high fat diet can cause metabolic disorder of glycolipid in rats, increase liver fat deposition, decrease glucose tolerance, and IR.2 liololu peptide to improve glycolipid metabolic disorder, reduce liver lipid accumulation, and improve IR and impaired glucose tolerance in a concentration dependent.3 liololu peptide concentration dependence on G Regulation of RP78/JNK/AKT/GLUT-2 pathway is associated with alleviation of endoplasmic reticulum stress, reduction of hepatic lipid deposition, promotion of glucose uptake by hepatocytes, and improvement of IR.
【學(xué)位授予單位】：承德醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R587.1

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