【摘要】：目的觀察高熱量高膽固醇飲食（high-calorie and high-cholesterol diet, HCD）對(duì)C57BL/6小鼠肝臟的影響，初步探討非諾貝特（fenofibrate）對(duì)非酒精性脂肪性肝病（non-alcoholic fatty liver disease, NAFLD）小鼠肝臟損傷的保護(hù)作用機(jī)制。 方法將C57BL/6小鼠隨機(jī)分為正常飲食（SCD）組、高熱量高膽固醇飲食（HCD）組和非諾貝特治療（HCF）組。用高熱量高膽固醇飲食喂養(yǎng)3月，建立非酒精性脂肪性肝病小鼠模型，3月后，用非諾貝特（40mg/kg/d）治療4周。葡萄糖耐量試驗(yàn)（GTT）和胰島素耐量試驗(yàn)（ITT）測(cè)定胰島素敏感性，檢測(cè)TG、TC、LDL-C、HDL-C、ALT、AST等血清生化指標(biāo)以及TNF-α、IL-6、MCP-1等炎性指標(biāo)水平，HE、油紅及Masson染色觀察肝臟組織的病理學(xué)變化，制備10%肝組織勻漿測(cè)定MDA、GSH-Px、T-AOC和SOD等氧化應(yīng)激指標(biāo)及TG含量情況，免疫組化檢測(cè)肝臟組織中TNF-α、CD68炎性分子表達(dá)，TUNEL法檢測(cè)小鼠肝細(xì)胞的原位凋亡，Real-time RT-PCR和RT-PCR測(cè)定肝組織PPARα、SREBP1-c、PTP1B、IRE-α、CHOP和GRP78mRNA表達(dá)，Western-blot檢測(cè)肝組織GRP78、JNK、ERK蛋白表達(dá)量。 結(jié)果①GTT試驗(yàn)和ITT試驗(yàn)表明，HCD組小鼠胰島素敏感性降低，存在胰島素抵抗，而非諾貝特治療后能夠增加胰島素敏感性，改善胰島素抵抗。②與SCD組小鼠相比，HCD組小鼠血清TG、TC、LDL-C水平顯著升高，HDL-C水平明顯下降（P0.01或P0.05）。非諾貝特治療后能夠顯著降低血清TG水平（P0.05）。③HCD組小鼠肝細(xì)胞內(nèi)可見(jiàn)脂滴形成，氣球樣變的肝細(xì)胞及炎性細(xì)胞浸潤(rùn)，但尚未進(jìn)展至纖維化階段，而HCF組肝臟脂肪變性程度及浸潤(rùn)的炎細(xì)胞明顯減少。④HCD組小鼠血清TNF-α、MCP-1等促炎因子明顯升高，而IL-6等抗炎因子降低（P0.05），HCF組小鼠血清促炎因子MCP-1明顯降低（P0.05）。免疫組化結(jié)果表明，與SCD組相比，HCD組小鼠肝臟TNF-α和CD68的表達(dá)量均增加。非諾貝特治療后能夠降低炎性分子表達(dá)量，減輕炎癥反應(yīng)。⑤TUNEL結(jié)果顯示HCD組小鼠肝細(xì)胞出現(xiàn)明顯凋亡，而HCF組小鼠體內(nèi)發(fā)生凋亡的肝細(xì)胞明顯減少。⑥與SCD組相比，HCD組小鼠肝組織內(nèi)MDA明顯增加，T-SOD和GSH-Px明顯降低（P0.05）。與HCD組相比，HCF組小鼠肝組織內(nèi)T-SOD、T-AOC明顯增加，，MDA明顯降低（P0.05）。⑦HCD組小鼠肝組織內(nèi)SREBP-1c、PTP1B、IRE-α、XBP-1s和CHOP mRNA的表達(dá)增加，而PPARα、GRP78mRNA的表達(dá)降低，非諾貝特治療后能降低PTP1B、IRE-α、XBP-1s和CHOP mRNA的表達(dá)量，增加PPARα、GRP78mRNA的表達(dá)量；此外，HCD組小鼠肝組織內(nèi)GRP78、JNK蛋白表達(dá)量明顯降低，而ERK蛋白表達(dá)量明顯升高，非諾貝特治療后，下調(diào)的GRP78、JNK蛋白表達(dá)量顯著升高，而上調(diào)的ERK蛋白表達(dá)量顯著降低。 結(jié)論①高熱量高膽固醇飲食能夠成功構(gòu)建NAFLD小鼠模型；②非諾貝特治療后可降低模型小鼠血脂水平、增加胰島素敏感性、減輕炎癥反應(yīng)、減少氧化應(yīng)激和內(nèi)質(zhì)網(wǎng)應(yīng)激水平，改善肝臟病理?yè)p傷；③非諾貝特可能是通過(guò)減輕內(nèi)質(zhì)網(wǎng)應(yīng)激中的IRE-α—XBP-1通路發(fā)揮肝臟保護(hù)作用的。
[Abstract]:Objective to observe the effect of high calorie and high cholesterol diet (high-calorie and high-cholesterol diet, HCD) on liver of C57BL/6 mice and to explore the protective mechanism of fenofibrate (fenofibrate) on liver injury in non-alcoholic fatty liver disease, NAFLD) mice. Methods C57BL/6 mice were randomly divided into normal diet (SCD) group, high calorie high cholesterol diet (HCD) group and fenofibrate treated (HCF) group. Mice were fed with high-calorie and high-cholesterol diet for 3 months to establish a model of non-alcoholic fatty liver disease. After 3 months, the mice were treated with fenofibrate (40mg/kg/d) for 4 weeks. Glucose tolerance test (GTT) and insulin tolerance test (ITT) were used to detect insulin sensitivity, serum biochemical indexes such as TGG TCU, LDL-C, HDL-C, and inflammatory indexes such as TNF- 偽, IL-6 and MCP-1, oil red and Masson staining were used to observe the pathological changes of liver tissue. 10% liver tissue homogenate was prepared to determine the oxidative stress indexes such as GSH-PxCT-AOC and SOD and the content of TG in the liver tissue homogenate. Expression of TNF- 偽 CD68 in liver tissue was detected by immunohistochemistry and Tunel method was used to detect in situ apoptosis of mouse hepatocytes. Real-time RT-PCR and RT-PCR were used to detect the expression of PPAR 偽 -SREBP1-cPTP1-PTP1BnIRE- 偽 and Western-blot to detect the expression of GRP78-JNKERK protein in liver tissue. Results 1GTT test and ITT test showed that insulin sensitivity was decreased and insulin resistance was found in HCD group, but fenofibrate could increase insulin sensitivity after treatment. Compared with SCD group, the improvement of insulin resistance was significantly higher than that of SCD group (P0.01 or P0.05). After treatment with fenofibrate, serum TG level (P0.05) .3HCD group could significantly reduce lipid droplet formation, balloon like hepatocyte and inflammatory cell infiltration, but did not progress to fibrosis stage. However, the degree of hepatic steatosis and the infiltration of inflammatory cells in HCF group were significantly decreased. The serum TNF- 偽, MCP-1 and other anti-inflammatory factors such as TNF- 偽 MCP-1 in HCF group were significantly increased, while the anti-inflammatory factors such as IL-6 were significantly decreased (P0.05). The immunohistochemical results showed that the expression of TNF- 偽 and CD68 in liver of SCD group was higher than that of SCD group. After fenofibrate treatment, the expression of inflammatory molecules was decreased, and the inflammatory response. 5 Tunel showed that the hepatocytes in HCD group showed obvious apoptosis. The number of apoptotic hepatocytes in HCF group was significantly lower than that in SCD group (P0.05). Compared with HCD group, T-SOD T-AOC in liver tissue of HCF group increased and MDA decreased significantly (P0.05). 7 HCD group increased the expression of SREBP-1cPTP1BnPTP1- 偽 IRE- 偽 XBP-1s and CHOP mRNA, and decreased the expression of PPAR 偽 -pGRP78mRNA. After fenofibrate treatment, the expression levels of PTP1BU IRE- 偽 XBP-1s and CHOP mRNA were decreased. In addition, the expression of GRP78 JNK protein in liver tissue of PPAR group decreased significantly, while the expression of ERK protein increased significantly. After fenofibrate treatment, the down-regulated expression of GRP78 JNK protein increased significantly in the liver tissue of the mice treated with fenofibrate, and the expression of GRP78 JNK protein decreased significantly after fenofibrate treatment, and the expression of GRP78 JNK protein increased significantly after fenofibrate treatment. The up-regulated expression of ERK protein decreased significantly. Conclusion 1 High calorie and high cholesterol diet can successfully construct NAFLD mice model of fenofibrate, which can decrease the level of blood lipids, increase insulin sensitivity, alleviate inflammatory reaction and decrease the levels of oxidative stress and endoplasmic reticulum stress after treatment with fenofibrate. The improvement of liver pathological injury by fenofibrate may play a protective role by alleviating the IRE- 偽 -XBP-1 pathway in endoplasmic reticulum stress.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R575.5

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