药物性肝损伤的临床特点、预后评估及内质网应激在利福平合用异烟肼致大鼠肝损伤中的作用

发布时间：2018-03-20 14:30

本文选题：药物性肝损伤　切入点：临床特点　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:(1)总结药物性肝损伤(DILI)的临床特点,探讨评估DILI预后的因素。(2)探讨内质网应激(ERS)在利福平(RFP)合用异烟肼(INH)致大鼠肝损伤中的作用及可能机制。方法:(1)回顾性分析近5年我院DILI的临床资料,运用Logistic回归分析筛选影响预后的指标。(2)将32只SPF级雄性Sprague-Dawley(SD)大鼠随机分成4组,每组8只。正常对照组(N组):予以等量的生理盐水每日定时空腹灌胃一次;RFP组(R组):给予RFP 50mg·kg-1·d-1定时空腹灌胃一次;模型组(M组):INH和RFP各50mg·kg-1·d-1定时空腹灌胃一次;PBA组:INH和RFP同M组,再给予4-苯基丁酸(PBA)160mg·kg-1·d-1腹腔注射一次;R组和M组给予等量溶剂腹腔注射。连续14d后处死大鼠。留取大鼠血液和肝脏组织标本。全自动生化分析仪检测大鼠血清中碱性磷酸酶(ALP)、总胆红素(TBIL)、直接胆红素(DBIL)、谷丙转氨酶(ALT)、谷草转氨酶(AST)、总胆汁酸(TBA)水平。HE染色观察大鼠肝脏病理学改变。免疫印迹法检测大鼠肝脏葡萄糖调节蛋白(GRP78)、P65、IκB及胆盐输出泵(Bsep)蛋白的表达变化。免疫组化法检测Bsep蛋白在大鼠肝细胞中的表达及定位变化。结果:DILI患者女性较多(57.5%),平均年龄52岁,用药以中药、抗菌类及解热镇痛类为主。肝细胞型DILl多见(81.1%),严重程度分级中以中度居多(54.7%),91.5%患者预后良好。不同严重程度肝细胞型DILI间Hy's法则符合率不同(P0.05),符合此法则的肝细胞型DILI患者肝衰竭的发生率为22.9%(11/48)。多因素回归分析显示:影响DILI预后的指标有ALT(OR=1.672,P=0.026)、国际标准化比值(INR)(OR=8.852,P=0.015)。(2)与N组相比,R组、M组大鼠血清TBIL、DBIL、TBA及ALP均升高(P0.05),ALT及AST升高不明显(P0.05);R组大鼠肝脏病理学检查可见肝细胞脂肪变性,M组可见肝细胞脂肪变性程度加重、部分可见炎细胞浸润和点灶状坏死;R组、M组大鼠肝脏中GRP78和P65蛋白的表达量明显上升(P0.05),IκB及Bsep蛋白的表达明显下降(P0.05)。R组、M组肝胞膜上Bsep的平均积分光密度(IOD)分别为3.56±1.18和2.35±1.18,较正常组(9.63±1.59)下降(P0.05),M组肝胞膜上Bsep分布不规则。与M组相比,PBA组大鼠TBIL、DBIL、ALP及TBA均降低(P0.05);肝脏病理学变化亦明显改善;GRP78、P65明显下调,IκB及Bsep明显上调(P0.05);肝胞膜上Bsep的平均IOD值为4.66±1.75,较M组上升(P0.05),肝胞膜上Bsep分布较M组规则。结论:(1)DILI患者大部分预后较好。发病时ALT及INR是预测DILI预后的独立指标。(2)RFP可导致胆汁淤积和肝细胞脂肪变性,上调GRP78、P65及下调Bsep、IκB蛋白的表达,减少肝细胞膜上的Bsep并改变其定位。(3)PBA可改善RFP合用INH所致的肝损伤,下调GRP78、P65及上调Bsep、IκB蛋白的表达,改善肝细胞膜上Bsep的表达和定位,提示ERS在RFP合用INH诱导的大鼠肝损伤中发挥重要作用,其可能机制是通过激活NF-κB,抑制Bsep表达和改变Bsep的定位。
[Abstract]:Objective to summarize the clinical features of drug induced liver injury (DILI). To evaluate the prognostic factors of DILI. (2) to explore the role and possible mechanism of endoplasmic reticulum stress (ERS) in liver injury induced by rifampicin and isoniazid in rats. Methods: 1) the clinical data of DILI in our hospital in recent 5 years were analyzed retrospectively. 32 SPF male Sprague-Dawley SD rats were randomly divided into 4 groups by Logistic regression analysis. Each group (n = 8): the control group (n = 8) was given the same amount of normal saline once a day, the group R was given RFP 50mg 路kg-1 路d -1, the model group was given RFP 50mg 路kg-1 路d -1, and the model group was given RFP 50 mg 路kg-1 路d -1 respectively. The rats in group R and group M were given intraperitoneal injection of the same amount of solvent once a time. The rats were killed after 14 days. Blood and liver tissue samples were collected from the rats. The serum alkaloids in rats were detected by automatic biochemical analyzer. The levels of ALPN, Tbilirubin, direct bilirubin, alanine aminotransferase (alt), glutamic oxalacetic transaminase (AST), total bile acid (TBA) in rats were observed by HE staining. The changes of liver pathology were observed by HE staining. The levels of Glucose-regulated protein GRP78P65I 魏 B in rat liver were detected by Western blot. The expression and localization of Bsep protein in rat hepatocytes were detected by immunohistochemical method. Results the average age was 52 years. Medicine with Chinese medicine, Antibacterial and antipyretic analgesic types were predominant. The majority of hepatocyte DILl was 81.1%, and 91.5% of the patients with moderate severity grade had a good prognosis. The coincidence rate of Hy's rule among DILI patients with different severity was different (P 0.05%), and the hepatocyte according with this rule was found. The incidence of liver failure in patients with type I DILI was 22.9 / 48. The multivariate regression analysis showed that the prognostic indexes of DILI were as follows: ALT OR1. 672% P0. 026, and international standard ratio INR OR8. 852% P0. 015. 0. 2) compared with group N, the serum levels of TBA and ALP in group R M were significantly higher than those in group N. The levels of serum TBA and AST in group R were not significantly higher than those in group R (P 0. 05 and P 0. 05). Histopathological examination of rat liver showed that the degree of fatty degeneration of hepatocytes was aggravated in group M. The expression of GRP78 and p65 protein in the liver of rats in group R and group R were significantly increased. The expression of I 魏 B and Bsep protein in P0.05 group was significantly decreased. The mean integrated optical density of Bsep on liver membrane in group R was significantly lower than that in group C (P 0.05). 3. 56 卤1. 18 and 2. 35 卤1. 18, compared with the normal group (9. 63 卤1. 59), the distribution of Bsep on liver membrane was irregular in P0. 05 + M group. Compared with the M group, the ALP and TBA of TBILD ILN in the PBA group decreased significantly (P 0. 05), and the pathological changes of the liver also improved obviously the downregulation of I 魏 B and Bsep in the liver membrane, and the up-regulation of Bsep on the membrane of the liver. The average IOD value was 4.66 卤1.75, which was higher than that in M group (P 0.05), and the distribution of Bsep on liver membrane was more regular than that in M group. Conclusion most of the patients with DILI have better prognosis. ALT and INR are independent markers for predicting the prognosis of DILI, which can lead to cholestasis and fatty degeneration of hepatocytes. Upregulation of GRP78 p65 and down-regulation of BsepI 魏 B protein, reduction of Bsep on liver cell membrane and alteration of its localization could improve liver injury induced by RFP combined with INH, down-regulate the expression of BsepI 魏 B protein, and improve the expression and localization of Bsep on liver cell membrane, and down-regulate the expression of BsepI 魏 B protein, down-regulate the expression of GRP78-p65 and up-regulate the expression of BsepI 魏 B protein, and improve the expression and localization of Bsep on liver cell membrane. The results suggest that ERS plays an important role in liver injury induced by RFP combined with INH in rats. The possible mechanism is to inhibit the expression of Bsep and change the location of Bsep by activating NF- 魏 B.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575

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