本文選題：姜黃素衍生物 + 非酒精性脂肪肝��； 參考：《重慶醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:非酒精性脂肪與二型糖尿病、肥胖癥、胰島素抵抗等代謝綜合癥患者最常見(jiàn)的肝病,目前尚無(wú)有效治療NAFLD的藥物。姜黃素是姜黃中提取的天然多酚化合物,研究證實(shí)其對(duì)非酒精性脂肪肝具有明顯改善,但是姜黃素水溶性差、生物利用度低限制了其臨床應(yīng)用。低分子PEG修飾后形成的聚乙二醇姜黃素衍生物(Curc-mPEG454),顯著提高母本姜黃素生物活性,組織分布更為廣泛,前期研究證實(shí)其抗炎、抗氧化能力顯著高于普通姜黃素。本研究采用高脂飲食喂養(yǎng)C57BL/6J小鼠復(fù)制NAFLD小鼠模型,用Curc-mPEG454進(jìn)行干預(yù),以探討Curc-mPEG454對(duì)高脂飲食誘導(dǎo)的小鼠非酒精性脂肪肝脂肪變性的改善作用及可能的機(jī)制,為NAFLD的治療提供新的思路。方法:1.40只6-8周齡雄性C57BL/6J小鼠飼養(yǎng)在SPF級(jí)別屏障中心動(dòng)物房,適應(yīng)一周后,隨機(jī)分為4組,對(duì)照組、模型組、Curc-mPEG454低劑量組、Curc-mPEG454高劑量組,每組10只。對(duì)照組給予低脂飼料,其余各組均給以高脂飼料喂養(yǎng),自由攝取水和食物,高脂飲食16周構(gòu)建NAFLD小鼠模型。造模期間每周測(cè)小鼠體質(zhì)量。對(duì)照組和模型組給予腹腔注射生理鹽水(0.9%NS 50mg/kg體重)、Curc-mPEG454低劑量組給予姜黃素衍生物50mg/kg/d腹腔注射、Curc-mPEG454高劑量組給予姜黃素衍生物100mg/kg/d腹腔注射,造模期間每周測(cè)小鼠體質(zhì)量,根據(jù)小鼠體質(zhì)量調(diào)整Curc-mPEG454注射劑量。2.Curc-mPEG454治療16周后,禁食12小時(shí)后稱(chēng)小鼠體質(zhì)量、心包采血、取肝臟組織標(biāo)本。稱(chēng)肝質(zhì)量,計(jì)算肝指數(shù)。檢測(cè)血清中肝功能及血脂水平:谷丙轉(zhuǎn)氨酶(ALT)、谷草轉(zhuǎn)氨酶(AST)、甘油三酯(TG)、總膽固醇(TC)、低密度脂蛋白膽固醇(LDL-C)、高密度脂蛋白膽固醇(HDL-C)、游離脂肪酸(FFA);肝組織HE染色觀察脂肪變程度;熒光定量PCR(Real-Time PCR)檢測(cè)肝臟組織中參與脂肪生成、脂肪酸攝取、脂肪酸β氧化、TG分泌及膽汁酸代謝的細(xì)胞因子。Western Blot及免疫組化檢測(cè)小鼠肝組織中CD36、PPARγ、CREB的蛋白表達(dá)水平。3.所有實(shí)驗(yàn)數(shù)據(jù)均以平均數(shù)±標(biāo)準(zhǔn)差表示,組間數(shù)據(jù)比較采用SPSS20.0軟件中單因素方差分析程序進(jìn)行,當(dāng)P0.05認(rèn)為組間差異具有統(tǒng)計(jì)學(xué)意義。結(jié)果:1.Curc-mPEG454對(duì)小鼠體質(zhì)量、肝質(zhì)量及血清脂質(zhì)的影響實(shí)驗(yàn)結(jié)束時(shí),各組小鼠體質(zhì)量均顯著增高(P0.05)。對(duì)照組、模型組、Curc-mPEG454低劑量治療組、Curc-mPEG454高劑量治療組體質(zhì)量增加值分別為(9.51±2.45)g、(22.95±5.14)g、(16.26±3.42)g、(17.12±4.56)g。與模型組相比,Curc-mPEG454低劑量(50mg/kg/d)與Curc-mPEG454高劑量(100mg/kg/d)治療后,顯著降低體質(zhì)量及體質(zhì)量增加值(P0.05),Curc-mPEG454低劑量治療組與Curc-mPEG454高劑量組間體質(zhì)量增加值無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);與模型組相比,Curc-mPEG454治療組血清TG水平顯著降低(P0.05),但治療組間差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。2.Curc-mPEG454對(duì)肝臟脂質(zhì)沉積的影響肝臟HE及油紅O染色可見(jiàn)對(duì)照組肝細(xì)胞形態(tài)正常,排列整齊,無(wú)脂肪變性;模型組可見(jiàn)彌漫性肝脂肪變性,肝細(xì)胞內(nèi)大量大小不等的脂肪空泡,未見(jiàn)明顯的炎癥細(xì)胞浸潤(rùn)及纖維化,模型組脂肪變分級(jí)處于最高級(jí),主要分布于3-4級(jí);Curc-mPEG454治療后,肝臟脂肪變性程度較模型組明顯減輕,脂肪空泡明顯減少,脂肪變分級(jí)顯著降低,主要分布于1-2級(jí),差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。Curc-mPEG454治療后顯著降低肝臟TG水平,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。3.Curc-mPEG454對(duì)脂代謝相關(guān)因子m RNA及蛋白表達(dá)的影響高脂飲食喂養(yǎng)后,與對(duì)照組相比,模型組肝臟脂肪酸轉(zhuǎn)運(yùn)蛋白CD36及其上游PPARγ的m RNA及蛋白表達(dá)均顯著升高,差異具有統(tǒng)計(jì)學(xué)意義(P0.05);Curc-mPEG454治療后,肝臟CD36及PPARγ水平與模型組相比均顯著降低,差異具有統(tǒng)計(jì)學(xué)意義(P0.05);治療組間肝臟CD36及PPARγ的表達(dá)差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。Curc-mPEG454治療對(duì)參與脂肪酸合成SREBP-1C、ACC1、FAS、SCD-1基因,脂肪酸氧化PPARα、CPT1α基因,TG分泌DGAT2、apo B、MPT基因及膽汁酸合成代謝FXR及CYP7a1基因與模型組相比差異均無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。4.Curc-mPEG454對(duì)肝臟CREB及P-CREB m RNA及蛋白表達(dá)的影響與對(duì)照組相比,模型組肝臟CREB的m RNA及蛋白水平均顯著降低(P0.05),Curc-mPEG454治療后,與模型組相比,顯著上調(diào)CREB及磷酸化CREB的表達(dá)(P0.05)。結(jié)論:1.Curc-mPEG454能夠減輕小鼠體質(zhì)量及肝質(zhì)量,降低血清及肝臟TG含量,減少肝臟脂質(zhì)積聚,具有減肥降脂,改善非酒精性脂肪肝的作用。2.Curc-mPEG454通過(guò)激活CREB的磷酸化,抑制肝臟PPARγ/CD36信號(hào)路,從而減少肝臟脂肪酸攝取和甘油三酯的合成改善肝臟脂質(zhì)沉積。
[Abstract]:Objective: Nonalcoholic Fat is the most common liver disease in patients with type two diabetes, obesity, insulin resistance and other metabolic syndrome. There is no effective treatment for NAFLD. Curcumin is a natural polyphenol compound extracted from turmeric. Research has proved that it has obvious improvement on nonalcoholic fatty liver, but the water solubility of curcumin is poor and biological. Low degree of use limits its clinical application. The polyethylene glycol curcumin derivative (Curc-mPEG454), formed by low molecular PEG modification, significantly improves the biological activity of the mother curcumin, and the tissue distribution is more extensive. Earlier studies have confirmed that its anti-inflammatory and antioxidant capacity is significantly higher than that of ordinary Jiang Huang. This study used high fat diet to feed C57BL/6J mice to copy N The AFLD mouse model, with Curc-mPEG454 intervention, was used to explore the effect and possible mechanism of Curc-mPEG454 on nonalcoholic fatty liver degeneration induced by high fat diet in mice. Methods: 1.40 6-8 weeks male C57BL/6J mice were fed in the SPF level barrier center animal room for a week, Randomly divided into 4 groups, the control group, the model group, the Curc-mPEG454 low dose group, the Curc-mPEG454 high dose group, 10 in each group. The control group was given low fat diet, the other groups were fed with high fat diet, free intake of water and food, and the high fat diet for 16 weeks to construct the NAFLD mice model. Intraperitoneal injection of normal saline (0.9%NS 50mg/kg body weight), Curc-mPEG454 low dose group given curcumin derivative 50mg/kg/d intraperitoneal injection, Curc-mPEG454 high dose group to give curcumin derivative 100mg/kg/d intraperitoneal injection, test the body mass per week of mice, according to the constitution of mice adjusted Curc-mPEG454 injection dose.2.Curc-mPEG454 treatment 16 After 12 hours of fasting, the body mass of the mice was called the body mass, the pericardium was collected and the liver tissue specimens were collected. The liver quality and the liver index were calculated. The serum liver function and blood lipid levels were measured: ALT, AST, triglyceride (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), and free of high density lipoprotein cholesterol (HDL-C). Fatty acid (FFA); liver tissue HE staining to observe the degree of adipose degeneration; fluorescence quantitative PCR (Real-Time PCR) detection of lipid formation, fatty acid uptake, fatty acid beta oxidation, TG secretion and bile acid metabolism of cytokine.Western Blot and immunohistochemical detection of CD36, PPAR gamma, CREB protein expression level.3. all.3. all The experimental data were expressed with mean standard deviation, and the data were compared with the single factor variance analysis program in SPSS20.0 software. When P0.05 thought the difference between groups was statistically significant. Results: the effect of 1.Curc-mPEG454 on the body mass, liver quality and serum lipid of mice was increased significantly (P0.05 The body mass of the control group, the model group, the Curc-mPEG454 low dose treatment group and the high dose Curc-mPEG454 treatment group were (9.51 + 2.45) g, (22.95 + 5.14) g, (16.26 + 3.42) g and (17.12 + 4.56) g., compared with the model group, and the body mass and body mass were significantly reduced after Curc-mPEG454 low dose (50mg/kg/d) and Curc-mPEG454 high dose (100mg/kg/d) treatment. The value of added value (P0.05), Curc-mPEG454 low dose treatment group and Curc-mPEG454 high dose group had no statistical significance (P0.05). Compared with the model group, the serum TG level of Curc-mPEG454 treatment group was significantly decreased (P0.05), but there was no significant difference between the treatment group (P0.05).2.Curc-mPEG454 on liver lipid deposition in the liver HE and Oil red O staining showed that the liver cells in the control group were normal, orderly and without fatty degeneration. The model group showed diffuse hepatic steatosis, fat vacuoles with large numbers of different sizes in the liver cells, no obvious inflammatory cell infiltration and fibrosis. The model group was in the highest grade, mainly in grade 3-4; after Curc-mPEG454 treatment, the liver was in the liver. The degree of visceral adipose degeneration was significantly lower than that in the model group, the fat vacuoles were significantly reduced and the fat classification was significantly reduced, mainly in the 1-2 level. The difference was statistically significant (P0.05).Curc-mPEG454 treatment significantly reduced the liver TG level, the difference was statistically significant (P0.05).3.Curc-mPEG454 on lipid metabolism related factors m RNA and protein expression After the high fat diet, the liver fatty acid transporter CD36 and its upstream PPAR gamma m RNA and protein expression were significantly increased in the model group, and the difference was statistically significant (P0.05). After Curc-mPEG454 treatment, the liver CD36 and PPAR gamma levels were significantly lower than those in the model group, and the difference was statistically significant (P0.05). There was no significant difference in the expression of CD36 and PPAR gamma (P0.05) between the treatment groups (P0.05), and there was no significant difference in the difference of.Curc-mPEG454 in fatty acid synthesis SREBP-1C, ACC1, FAS, SCD-1 gene, fatty acid oxidation PPAR alpha, CPT1 alpha gene, TG secretory DGAT2, and bile acid synthesis and metabolism. 05) the effect of.4.Curc-mPEG454 on the expression of CREB and P-CREB m RNA and protein in the liver was significantly lower than that of the control group. The level of M RNA and protein in the liver of the model group decreased significantly (P0.05). After Curc-mPEG454 treatment, the expression of CREB and phosphorylated CREB was significantly up to be compared with the model group. Conclusion: the results can reduce the body mass and liver of mice. Quality, reducing TG content in serum and liver, reducing lipid accumulation in liver, reducing fat and reducing fat, improving non-alcoholic fatty liver function,.2.Curc-mPEG454 can inhibit the liver PPAR gamma /CD36 signaling by activating CREB phosphorylation, thus reducing liver fatty acid intake and triglyceride synthesis to improve liver lipid deposition.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R575

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