奥贝胆酸对四氯化碳急性肝损伤的保护作用

发布时间：2018-03-11 14:41

  本文选题：奥贝胆酸　切入点：法尼脂X受体(FXR)　出处：《安徽医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的法尼脂X受体是一种配体依赖的转录因子,在调节胆汁酸代谢中发挥重要作用。本实验的目的是探讨新型法尼脂X受体激动剂--奥贝胆酸在四氯化碳诱导急性肝损伤中的保护作用及部分机制。方法6-8周雄性CD-1小鼠(28-30g)48只,随机分为8组(每组6只),即CCl_4-0h组(即对照组),CCl_4-12h组,CCl_4-24h组,CCl_4-48h组;OCA+CCl_4-0h组(即OCA组),OCA+CCl_4-12h组,OCA+CCl_4-24h组,OCA+CCl_4-48h组。在CCl_4-12h、24h、48h组中,给予小鼠单次腹腔注射CCl_4(0.15 ml/kg,与橄榄油1:10混合),给药剂量参照课题组既往试验研究[1];在CCl_4-0h组中,给予腹腔注射相同剂量的PBS;在OCA+CCl_4-12h、24h、48h组中,所有小鼠在给予腹腔注射CCl_4(0.15 ml/kg)前1h,12h,24h分别经灌胃给药(OCA,5 mg/kg,溶于PBS中),OCA与CCl_4剂量参照相关研究[2.3];OCA+CCl_4-0h组中给予小鼠腹腔注射橄榄油前1h,12h,24h分别经灌胃给药(OCA,5 mg/kg)。所有小鼠经腹腔注射CCl_4后分别在0,12,24和48 h麻醉后处死。称量小鼠及肝脏重量;收集血清用于检测生化指标;部分肝脏组织迅速放入液氮速冻后于-80℃储存,用RT-PCR方法检测相关炎症基因的表达,用WB方法检测相关蛋白质的表达;另一部分肝脏组织使用4%的多聚甲醛固定后用于组织学检查,HE染色观察肝脏组织坏死及炎症,TUNEL观察肝脏组织凋亡。结果经过OCA预处理的小鼠肝脏核蛋白FXR的水平明显升高,表明OCA能够激活FXR。(1)OCA预处理对小鼠肝重的影响:小鼠肝重及肝系数在给予CCl_412h后开始升高并持续至48h;OCA预处理后,小鼠肝重及肝系数明显降低。(2)OCA预处理在CCl_4诱导的急性肝损伤中的作用:CCl_4处理后12h小鼠血清ALT开始轻微升高,24h升高最显著,48h仍有升高;而OCA预处理后,血清ALT较单纯CCl_4组明显降低;CCl_4组中肝脏坏死面积在12h、24h、48h分别为22%、46%、26%,但在相应的OCA预处理组,肝脏坏死面积显著减少。(3)OCA预处理抑制CCl_4诱导的肝细胞凋亡:在CCl_4组中,12h组可见少量肝细胞凋亡,24h组凋亡最明显;OCA预处理后可显著减少肝细胞凋亡。(4)OCA在CCl_4诱导的急性肝损伤中可以不同程度调控肝脏促炎因子、抗炎因子、趋化因子等相关炎症因子基因的表达:在给予CCl_4 12h后TNF-α和Il-1β两种促炎细胞因子mRNA水平较对照组明显升高,抗炎因子-IL-4表达轻微上调,Mcp-1,Mip-2和Kc三种细胞趋化因子表达明显升高;OCA预处理后,促炎因子及细胞趋化因子较CCl_4组表达明显下降,抗炎因子表达明显升高。(5)OCA预处理可抑制CCl_4急性肝损伤中肝脏NF-κB信号通路的激活:肝脏胞浆pIκB在给予CCl_4 12h后开始明显升高,与之相对应的胞核中的NF-κB p65和p50两个亚基明显升高;而在OCA预处理组胞浆中IκB磷酸化较CCl_4组明显减少,胞核中NF-κB p65和p50两个亚基明显下降。(6)OCA预处理可抑制CCl_4急性肝损伤中肝脏AKT,ERK和p38的磷酸化:肝脏pAKT水平在所有时点较对照组均明显升高,但OCA预处理后AKT的磷酸化明显减少;pERK水平在给予CCl_4后12h开始升高并持续至24h,pp38水平在给予CCl_4后12h开始升高持续至48h;OCA预处理明显抑制ERK和p38的磷酸化。结论OCA保护CCl_4诱导的肝细胞坏死及凋亡。OCA选择性下调CCl_4急性肝损伤中肝脏炎性趋化因子与促炎性细胞因子的表达,同时上调抗炎性细胞因子表达;OCA激活肝脏FXR信号,同时抑制CCl_4所致的肝脏NF-κB信号通路的激活。本实验研究表明:FXR是肝脏炎症过程的重要调控因子。因此,OCA作为人工合成的FXR激动剂在急性肝损伤中发挥重要的保护作用。
[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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