本文關(guān)鍵詞： 高脂膳食 雙酚A 染料木黃酮 葡萄糖代謝 脂代謝　出處：《華中科技大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：目的：(1)觀察BPA和染料木黃酮干預(yù)對成年雄性Wistar大鼠糖脂代謝的影響。(2)探索BPA暴露導(dǎo)致血葡萄糖平衡紊亂的可能機(jī)制；觀察父代大鼠長期“安全劑量”BPA暴露對子代大鼠出生結(jié)局及子代大鼠成年后糖脂代謝的影響。(3)探索BPA和染料木黃酮對大鼠肝臟脂代謝影響的可能機(jī)制。 方法：體重為150-180g的雄性Wistar大鼠,適應(yīng)性喂養(yǎng)一周后,隨機(jī)分為：普通飼料組(STD),普通飼料+50μg/kg BPA組(STD-BPA50),普通飼料+50μg/kg BPA+10mg/kg染料木黃酮組(STD-(BPA50+G)),普通飼料+10mg/kg染料木黃酮組(STD-G)；高脂對照組(HFD),高脂飼料+50μg/kg BPA組(HFD-BPA50),高脂飼料+50μg/kg BPA+10mg/kg染料木黃酮組(HFD-(BPA50+G)),高脂飼料+10mg/kg染料木黃酮組(HFD-G)。在干預(yù)的第0周和第21周末測定血糖、胰島素、血清TG和TC水平；在干預(yù)的30周末進(jìn)行腹膜內(nèi)葡萄糖耐量試驗(yàn)(IPGTT),在干預(yù)的35周末,處死大鼠,測定生化指標(biāo)。干預(yù)21周末,選取STD、STD-BPA50、HFD和HFD-BPA50組大鼠作為父代,與健康成年雌性Wistar大鼠合籠。子代大鼠出生后計(jì)算性別比、記錄出生窩重和每窩子代個數(shù)。子代斷奶后給予普通飼料直至10周齡后處死。用免疫熒光法測定胰腺β細(xì)胞面積；用實(shí)時熒光定量PCR檢測大鼠胰腺組織LC3、Beclin-1和Bip的(?)mRNA表達(dá)水平；采用免疫組化和蛋白免疫印跡法檢測胰腺LC3的蛋白表達(dá)水平；用激光共聚焦顯微鏡檢測p細(xì)胞上的LC3蛋白表達(dá)。測定子代大鼠血清生化指標(biāo),計(jì)算HOMA指數(shù)和胰島素敏感性指數(shù)。用HE染色觀察父代大鼠肝臟組織的病理變化；用實(shí)時定量PCR檢測肝臟中脂代謝關(guān)鍵基因的表達(dá)水平；用蛋白免疫印記法檢測肝臟中PPARα、PPARγ和LC3蛋白表達(dá)水平。 結(jié)果：(1)干預(yù)35周后,HFD-BPA50組大鼠血糖水平在明顯高于HFD組(P0.01)；STD-BPA50組胰島素水平明顯高于STD組(P0.05)和STD-G組(P0.01)。在IPGTT實(shí)驗(yàn)中,HFD-BPA50組在給予葡萄糖后的第15min和30min血糖水平和血糖曲線下面積明顯高于HFD組(P0.05)。此外,在干預(yù)35周后STD-BPA50組的HOMA指數(shù)明顯高于STD組和STD-G組(P0.01),并且胰島素敏感指數(shù)明顯低于STD組(P0.05)。在干預(yù)21周后HFD-(BPA50+G)和HFD-G組大鼠的HOMA指數(shù)明顯低于HFD-BPA50組(P0.05和P0.01)。在干預(yù)35周末,HFD-G組的血清TG水平明顯低于HFD-BPA50組(P0.05)；并且HFD-G組血清TC水平明顯低于HFD組和HFD-BPA50組(P0.05)。在干預(yù)35周末,與HFD組相比,HFD-(BPA50+G)組和HFD-G組肝臟中TG和TC水平明顯降低(P0.05)。(2)高脂飲食和BPA干預(yù)明顯增加了父代大鼠的胰腺β細(xì)胞數(shù)量(P0.01)；高脂飲食和BPA干預(yù)還明顯增加了父代大鼠胰腺LC3、Beclin-1和Bip的mRNA表達(dá)水平；并且高脂喂養(yǎng)和BPA干預(yù)的大鼠胰腺LC3蛋白表達(dá)水平也明顯高于各自的對照組(P0.01)；高脂飲食和BPA干預(yù)增加了胰腺β細(xì)胞上的LC3表達(dá)水平(P0.01)。子代大鼠的出生性別比、窩重和窩別大小、血糖和血脂無明顯差別(P0.05)。(3)HFD組、HFD-(BPA50+G)組和HFD-G組大鼠肝臟SREBP-1C的mRNA表達(dá)水平明顯高于STD組(P0.05)；HFD組大鼠肝臟PPARγ的mRNA表達(dá)水平明顯高于STD組、HFD-(BPA50+G)組和HFD-G組(P0.05),并且HFD組大鼠肝臟的PPARγ蛋白質(zhì)表達(dá)水平明顯高于STD組、HFD-(BPA50+G)組和HFD-G組(P0.05)；HFD組大鼠肝臟LC3的mRNA和蛋白質(zhì)表達(dá)水平低于STD組(P0.01,P0.05)。 結(jié)論：(1)長期BPA暴露導(dǎo)致了大鼠的胰島素抵抗和葡萄糖不耐受；染料木黃酮干預(yù)能明顯改善高脂喂養(yǎng)大鼠的血脂紊亂。(2)父代大鼠BPA暴露導(dǎo)致的胰腺β細(xì)胞功能紊亂可能與上調(diào)的內(nèi)質(zhì)網(wǎng)應(yīng)激和自噬水平相關(guān)；父代大鼠長期BPA暴露并沒有導(dǎo)致子代大鼠的出生結(jié)局及成年后糖脂代謝發(fā)生改變。(3)BPA干預(yù)并沒有影響肝臟脂代謝相關(guān)基因和自噬水平；染料木黃酮干預(yù)可能通過下調(diào)肝臟PPARγ的表達(dá)進(jìn)而緩解高脂造成的脂代謝紊亂。
[Abstract]:Objective: To observe the BPA (1) and genistein intervention effects on lipid metabolism of adult male Wistar rats. (2) to explore the possible mechanisms leading to BPA exposure to blood glucose balance disorders; observe parent rats long-term safe dose BPA exposure affects lipid metabolism in offspring rats and offspring birth outcome adult rat. (3) the possible mechanism of BPA and explore the effects of genistein on liver lipid metabolism in rats.
Methods: male Wistar rats weighing 150-180g, feeding a week, were randomly divided into normal diet group (STD), +50 g/kg BPA group with normal diet (STD-BPA50), +50 g/kg BPA+10mg/kg normal diet genistein group (STD- (BPA50+G)), normal diet + 10mg/kg genistein group (STD-G); high fat control group (HFD), high fat diet +50 g/kg BPA group (HFD-BPA50), high fat diet +50 g/kg BPA+10mg/kg genistein group (HFD- (BPA50+G)), high fat diet +10mg/kg genistein group (HFD-G). Insulin in zeroth weeks and twenty-first weeks of intervention measure blood glucose, serum TG and TC levels; intraperitoneal glucose tolerance test in the 30 week intervention (IPGTT), in the 35 week intervention, the rats were sacrificed and the determination of biochemical indicators. At the end of the 21 week intervention, including STD, STD-BPA50, HFD and HFD-BPA50 rats as the parent, and healthy adult female Wistar rats rats were mated. The calculation of rats after birth sex ratio, litter birth weight and position of each record generation number. The offspring after weaning was given normal feed until 10 weeks of age were determined. The area of pancreatic beta cells by immunofluorescence; using real-time fluorescence quantitative PCR detection of pancreatic tissue of rats with LC3, Beclin-1 and Bip (?) mRNA expression; expression of pancreatic LC3 detected by immunohistochemistry and Western blotting of protein; expression of P cells was detected by laser confocal microscope. The LC3 protein determination of offspring rats serum biochemical index, HOMA index and insulin sensitivity index. HE staining was used to observe the pathological changes of parent rat liver the expression levels of key genes; using real-time quantitative PCR detection of liver lipid metabolism; PPAR alpha protein by Western blot in the liver, the level of PPAR gamma and LC3 protein expression.
Results: (1) after 35 weeks of treatment, blood glucose levels in rats of HFD-BPA50 group was significantly higher than HFD group (P0.01); insulin levels were significantly higher in STD-BPA50 group than in STD group (P0.05) and STD-G group (P0.01). In IPGTT experiment, HFD-BPA50 group was given glucose after 15min and 30min blood glucose levels and blood glucose curve the next area was significantly higher than HFD group (P0.05). In addition, in the 35 weeks after the intervention group STD-BPA50 HOMA index was significantly higher than that of STD group and STD-G group (P0.01), and the insulin sensitivity index was significantly lower than that of STD group (P0.05). In 21 weeks after the intervention of HFD- (BPA50+G) and HFD-G group rats HOMA index was significantly lower than that of group HFD-BPA50 (P0.05 and P0.01). After 35 weeks, the serum TG level of HFD-G group was significantly lower than that of HFD-BPA50 group (P0.05); and the level of serum TC in HFD-G group was significantly lower than that of HFD group and HFD-BPA50 group (P0.05). After 35 weeks, compared with group HFD, HFD- (BPA50+G) group and HFD-G group of liver TG 鍜孴C姘村鉤鏄庢樉闄嶄綆(P0.05).(2)楂樿剛楗鍜孊PA騫查鏄庢樉澧炲姞浜嗙埗浠ｅぇ榧犵殑鑳拌吅尾緇嗚優(yōu)鏁伴噺(P0.01)錛涢珮鑴傞ギ椋熷拰BPA騫查榪樻槑鏄懼鍔犱簡鐖朵唬澶ч紶鑳拌吅LC3,Beclin-1鍜孊ip鐨刴RNA琛ㄨ揪姘村鉤錛涘茍涓旈珮鑴傚杺鍏誨拰BPA騫查鐨勫ぇ榧犺儼鑵篖C3铔嬬櫧琛ㄨ揪姘村鉤涔熸槑鏄鵑珮浜庡悇鑷殑瀵圭収緇，

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