本文選題：奧貝膽酸　切入點：法尼脂X受體(FXR)　出處：《安徽醫(yī)科大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的法尼脂X受體是一種配體依賴的轉(zhuǎn)錄因子,在調(diào)節(jié)膽汁酸代謝中發(fā)揮重要作用。本實驗的目的是探討新型法尼脂X受體激動劑--奧貝膽酸在四氯化碳誘導急性肝損傷中的保護作用及部分機制。方法6-8周雄性CD-1小鼠(28-30g)48只,隨機分為8組(每組6只),即CCl_4-0h組(即對照組),CCl_4-12h組,CCl_4-24h組,CCl_4-48h組;OCA+CCl_4-0h組(即OCA組),OCA+CCl_4-12h組,OCA+CCl_4-24h組,OCA+CCl_4-48h組。在CCl_4-12h、24h、48h組中,給予小鼠單次腹腔注射CCl_4(0.15 ml/kg,與橄欖油1:10混合),給藥劑量參照課題組既往試驗研究[1];在CCl_4-0h組中,給予腹腔注射相同劑量的PBS;在OCA+CCl_4-12h、24h、48h組中,所有小鼠在給予腹腔注射CCl_4(0.15 ml/kg)前1h,12h,24h分別經(jīng)灌胃給藥(OCA,5 mg/kg,溶于PBS中),OCA與CCl_4劑量參照相關研究[2.3];OCA+CCl_4-0h組中給予小鼠腹腔注射橄欖油前1h,12h,24h分別經(jīng)灌胃給藥(OCA,5 mg/kg)。所有小鼠經(jīng)腹腔注射CCl_4后分別在0,12,24和48 h麻醉后處死。稱量小鼠及肝臟重量;收集血清用于檢測生化指標;部分肝臟組織迅速放入液氮速凍后于-80℃儲存,用RT-PCR方法檢測相關炎癥基因的表達,用WB方法檢測相關蛋白質(zhì)的表達;另一部分肝臟組織使用4%的多聚甲醛固定后用于組織學檢查,HE染色觀察肝臟組織壞死及炎癥,TUNEL觀察肝臟組織凋亡。結(jié)果經(jīng)過OCA預處理的小鼠肝臟核蛋白FXR的水平明顯升高,表明OCA能夠激活FXR。(1)OCA預處理對小鼠肝重的影響:小鼠肝重及肝系數(shù)在給予CCl_412h后開始升高并持續(xù)至48h;OCA預處理后,小鼠肝重及肝系數(shù)明顯降低。(2)OCA預處理在CCl_4誘導的急性肝損傷中的作用:CCl_4處理后12h小鼠血清ALT開始輕微升高,24h升高最顯著,48h仍有升高;而OCA預處理后,血清ALT較單純CCl_4組明顯降低;CCl_4組中肝臟壞死面積在12h、24h、48h分別為22%、46%、26%,但在相應的OCA預處理組,肝臟壞死面積顯著減少。(3)OCA預處理抑制CCl_4誘導的肝細胞凋亡:在CCl_4組中,12h組可見少量肝細胞凋亡,24h組凋亡最明顯;OCA預處理后可顯著減少肝細胞凋亡。(4)OCA在CCl_4誘導的急性肝損傷中可以不同程度調(diào)控肝臟促炎因子、抗炎因子、趨化因子等相關炎癥因子基因的表達:在給予CCl_4 12h后TNF-α和Il-1β兩種促炎細胞因子mRNA水平較對照組明顯升高,抗炎因子-IL-4表達輕微上調(diào),Mcp-1,Mip-2和Kc三種細胞趨化因子表達明顯升高;OCA預處理后,促炎因子及細胞趨化因子較CCl_4組表達明顯下降,抗炎因子表達明顯升高。(5)OCA預處理可抑制CCl_4急性肝損傷中肝臟NF-κB信號通路的激活:肝臟胞漿pIκB在給予CCl_4 12h后開始明顯升高,與之相對應的胞核中的NF-κB p65和p50兩個亞基明顯升高;而在OCA預處理組胞漿中IκB磷酸化較CCl_4組明顯減少,胞核中NF-κB p65和p50兩個亞基明顯下降。(6)OCA預處理可抑制CCl_4急性肝損傷中肝臟AKT,ERK和p38的磷酸化:肝臟pAKT水平在所有時點較對照組均明顯升高,但OCA預處理后AKT的磷酸化明顯減少;pERK水平在給予CCl_4后12h開始升高并持續(xù)至24h,pp38水平在給予CCl_4后12h開始升高持續(xù)至48h;OCA預處理明顯抑制ERK和p38的磷酸化。結(jié)論OCA保護CCl_4誘導的肝細胞壞死及凋亡。OCA選擇性下調(diào)CCl_4急性肝損傷中肝臟炎性趨化因子與促炎性細胞因子的表達,同時上調(diào)抗炎性細胞因子表達;OCA激活肝臟FXR信號,同時抑制CCl_4所致的肝臟NF-κB信號通路的激活。本實驗研究表明:FXR是肝臟炎癥過程的重要調(diào)控因子。因此,OCA作為人工合成的FXR激動劑在急性肝損傷中發(fā)揮重要的保護作用。
[Abstract]:The purpose of farnesoid X receptor is a ligand dependent transcription factor, plays an important role in the regulation of bile acid metabolism. The purpose of this experiment is to explore the new farnesoid X receptor agonist was bile acid induced protective effects and mechanism of acute liver injury in carbon tetrachloride. Methods 6-8 week old male CD-1 mice (28-30g 48) rats were randomly divided into 8 groups (n = 6), group CCl_4-0h (control group), CCl_4-12h group, CCl_4-24h group, CCl_4-48h group; OCA+CCl_4-0h group (OCA group), OCA+CCl_4-12h group, OCA+CCl_4-24h group, OCA+CCl_4-48h group. In CCl_4-12h, 24h, 48h group, mice were given a single intraperitoneal injection of CCl_4 (0.15 ml/kg, mixed with olive oil, dosage 1:10) according to previous experimental study group [1]; in group CCl_4-0h, intraperitoneal injection of the same dose of PBS; in OCA+CCl_4-12h, 24h, 48h group, all mice received intraperitoneal injection of CCl_4 (0.15 ml/kg) Before 1H, 12h, 24h respectively by gavage (OCA, 5 mg/kg, dissolved in PBS, OCA and CCl_4) according to the related research of [2.3] dose; mice were given intraperitoneal injection of olive oil before 1H, OCA+CCl_4-0h group, 12h, 24h respectively by gavage (OCA, 5 mg/kg). All the mice were after intraperitoneal injection of CCl_4 at 0,12,24 and 48 h after anesthesia were killed. Weighing mice and liver weight; serum was collected for detection of biochemical indicators; part of liver tissue quickly put frozen by liquid nitrogen stored in -80 C, the expression of RT-PCR was used to detect the inflammation related genes, WB was used to detect the expression of related proteins; another part of the liver tissue the use of 4% paraformaldehyde fixed for histological examination, observation of inflammation and necrosis of liver tissue HE staining, TUNEL of liver tissue were observed. The apoptosis results after pretreatment of OCA mouse liver nuclear protein FXR levels were significantly increased, indicating OCA can activate FXR. (1) OCA Treatment of mice liver weight, liver weight and liver index in mice began to increase and continued to 48h in CCl_412h; OCA after pretreatment, liver weight and liver coefficient were significantly decreased. (2) effect of OCA pretreatment on acute liver injury induced by CCl_4 in CCl_4 treated 12h mice serum ALT slightly increased, 24h increased significantly, 48h still has increased; while OCA after pretreatment serum ALT than CCl_4 group obviously decreased; liver necrotic area in the CCl_4 group at 12h, 24h, 48h were 22%, 46%, 26%, but in the corresponding OCA pretreatment group, the liver necrosis area was significantly reduced. (3) liver cell apoptosis OCA pretreatment inhibited CCl_4 induced: in CCl_4 group, 12h group showed a small amount of liver cell apoptosis, 24h group was the most obvious apoptosis; after OCA pretreatment can significantly reduce the apoptosis of liver cells. (4) OCA can be different degrees of regulation of liver inflammatory cytokines in acute liver injury induced by CCl_4 because, anti-inflammatory 瀛，

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