不同年龄和性别影响对乙酰氨基酚急性肝损伤敏感性差异的机制研究

发布时间：2018-01-05 17:25

本文关键词：不同年龄和性别影响对乙酰氨基酚急性肝损伤敏感性差异的机制研究　出处：《安徽医科大学》2017年博士论文　论文类型：学位论文

【摘要】：背景/目的对乙酰氨基酚(APAP)是临床上儿童常见的解热镇痛药物,其过量或不当使用引起的急性肝脏损伤是儿童药物性肝损伤的常见原因之一。然而,目前关于儿童和成人之间APAP急性肝损伤是否存在差异报道甚少。既往研究表明APAP在肝细胞内经P450代谢酶CYP2E1代谢生成有毒产物N-乙酰对苯醌亚胺(NAPQI),耗竭胞内还原型谷胱甘肽(GSH)进而引起氧化应激、线粒体功能障碍并最终导致肝细胞死亡。近期研究提示,APAP急性肝损伤过程发生二次打击,TLR4信号可能介导APAP急性肝损伤“二次打击”过程中肝细胞程序性坏死。本课题比较不同年龄和性别小鼠对APAP急性肝损伤的敏感性差异,为今后更好地指导不同人群APAP肝损伤的临床治疗提供理论依据;比较野生型(C3H/He N)与TLR4突变型(C3H/He J)小鼠APAP肝损伤的差异,初步探讨TLR4在APAP急性肝损伤不同阶段的作用。方法研究一、不同年龄和性别小鼠对APAP急性肝损伤的敏感性差异由两个实验组成:实验一、幼年(3周)CD-1小鼠(雌、雄各10只)和成年(8周)CD-1小鼠(雌、雄各10只)分成四组:幼年雄性小鼠、幼年雌性小鼠、成年雄性小鼠和成年雌性小鼠,每组各10只小鼠。所有小鼠统一禁食12小时后经腹腔注射给予单剂量APAP(300mg/kg),观察各组小鼠生存情况至APAP给药后的第7天。绘制各组生存曲线并比较各组小鼠生存率差异。实验二、幼年(3周)CD-1小鼠(雌、雄各40只)和成年(8周)CD-1小鼠(雌、雄各40只)各分成APAP给药组和对照组。所有小鼠统一禁食12小时。所有APAP给药组小鼠经腹腔注射给予APAP(300mg/kg),对照组给予生理盐水。给药后不同时点(第0,1,4和24小时及第7天)剖杀小鼠,比较各组肝指数,检测血清ALT和肝脏GSH、GSSG与GSSG/GSH水平,用HE染色以观察组织肝脏病理学损伤,用TUNEL检测肝细胞死亡,用免疫组化检测肝脏硝化酪氨酸(3-NT);用实时定量RT-PCR检测肝脏Cyp1a1、Cyp2e1、Cyp3a11、Ugt1a1、Gpx、Gr和Gst基因表达水平;用试剂盒检测肝脏GPX、GR和GST活性;用Western blot检测肝脏CYP2E1、p-JNK、JNK、RIP1和RIP3蛋白水平。研究二、TLR4在APAP急性肝损伤发生过程中的作用由两个实验组成:实验一、野生型(C3H/He N)小鼠和突变型(C3H/He J)小鼠各10只,统一禁食12小时后给予APAP(300mg/kg)单剂量腹腔注射,观察各组小鼠生存情况至APAP给药后72 h。绘制两组小鼠生存曲线并比较两组小鼠生存率的差异。实验二、野生型(C3H/He N)小鼠和突变型(C3H/He J)小鼠各分成APAP给药组和对照组。所有小鼠统一禁食12小时。APAP给药组小鼠经腹腔注射给予APAP(300mg/kg),对照组给予等体积生理盐水。给药后不同时点(第0,4和12 h)剖杀小鼠,比较两组小鼠肝指数,检测血清ALT水平,用HE染色以观察肝脏病理学损伤,用TUNEL检测肝细胞死亡,用Western blot检测肝脏p-JNK、JNK以及程序性坏死相关蛋白激酶RIP1、RIP3蛋白的表达。结果幼年小鼠APAP急性肝损伤较同性别成年小鼠更严重。小鼠生存实验结果提示,雄性或雌性幼年小鼠生存率(雄性50%、雌性60%)均显著低于成年小鼠(雄性80%、雌性90%)。进一步观察发现,与同性别成年小鼠相比,给药后4小时幼年小鼠肝指数和血清ALT水平上升更明显,组织病理学观察到肝脏充血明显;至给药后24小时雄性和雌性幼年小鼠血清ALT水平仍明显高于同性别成年小鼠,组织病理学观察到肝脏呈小叶中心性坏死,且幼年小鼠肝脏坏死面积明显大于同性别成年小鼠;TUNEL检测显示,幼年小鼠肝脏TUNEL阳性细胞数明显高于同性别成年小鼠;免疫印迹实验结果显示,APAP处理后幼年小鼠肝脏p-JNK和RIP3的蛋白水平均明显高于同性别成年小鼠。免疫组化显示,APAP处理后4小时和24小时幼年小鼠肝脏硝化酪氨酸(3-NT)阳性细胞数明显多于同性别成年小鼠。为探讨幼年小鼠对APAP肝损伤更敏感的机制,比较了不同年龄和性别小鼠肝脏APAP代谢酶水平和GSH代谢情况。结果显示,与同性别成年小鼠相比,幼年小鼠在APAP给药后肝脏GSH耗竭量更大,GSSG/GSH比值上升更明显;进一步分析发现,幼年小鼠肝脏Cyp2e1、Cyp3a11的m RNA水平和CYP2E1蛋白含量均较同性别成年小鼠更高。本课题发现,APAP急性肝损伤性别差异只存在于成年小鼠,成年雄性小鼠APAP急性肝损伤较成年雌性小鼠更严重。APAP处理后4小时和24小时成年雄性小鼠血清ALT水平均高于成年雌性小鼠;组织病理学观察到成年雄性小鼠肝脏坏死面积明显大于成年雌性小鼠;TUNEL检测提示,成年雄性小鼠肝脏TUNEL阳性细胞数明显高于成年雌性小鼠。进一步观察发现,APAP处理后4小时成年雄性小鼠肝脏p-JNK水平明显高于成年雌性小鼠;免疫组化显示,APAP处理后4小时和24小时成年雄性小鼠肝脏3-NT阳性细胞数明显多于成年雌性小鼠。APAP处理后4小时成年雄性小鼠肝脏GSH消耗量较成年雌性小鼠大,且GSSG/GSH比值恢复速度明显慢于成年雌性小鼠;成年雄性小鼠肝脏GPX和GST活性均低于成年雌性小鼠。野生型(C3H/He N)小鼠APAP急性肝损伤较突变型(C3H/He J)小鼠更严重。72h生存曲线结果提示野生型(C3H/He N)小鼠给药4小时后即发生死亡,72小时生存率为40%;(C3H/He J)小鼠至给药8小时才发生死亡,72小时生存率为70%。故野生型(C3H/He N)小鼠较突变型(C3H/He J)小鼠生存率低。APAP处理4 h后,C3H/He N小鼠肝指数较C3H/He J小鼠上升明显。给药4 h和12 h,C3H/He N小鼠血清ALT水平均较C3H/He J小鼠高。肝脏HE染色提示,C3H/He N小鼠肝脏坏死面积较大。肝脏TUNEL结果提示,C3H/He N小鼠肝脏TUNEL+阳性细胞数明显多于C3H/He J小鼠。C3H/He N小鼠在给药后的4 h和12 h,肝脏p-JNK的表达均高于C3H/He J小鼠。给药后4 h,C3H/He N小鼠肝脏RIP1的表达高于C3H/He J小鼠;给药后12 h,C3H/He N小鼠肝脏RIP1的表达与C3H/He J小鼠无差异,但肝脏RIP3的表达明显高于C3H/He J小鼠。结论不同年龄小鼠对APAP急性肝损伤存在敏感性差异,幼年小鼠更易于发生APAP急性肝脏损伤。其机制可能与幼年小鼠肝脏Cyp2e1、Cyp3a11的m RNA水平和CYP2E1蛋白含量均较同性别成年小鼠更高;幼年小鼠对于APAP引起的肝脏GSH耗竭更敏感;APAP处理后幼年小鼠肝脏P-JNK和RIP3蛋白表达更高有关。APAP急性肝损伤的性别差异只存在于成年小鼠,具体表现为成年雄性小鼠比成年雌性小鼠肝脏损伤更明显。其机制可能与成年雄性小鼠肝脏GSH代谢酶(GPX和GST)活性比成年雌性小鼠低,APAP处理后成年雄性小鼠肝脏GSH消耗多,进而抗氧化以及促进有毒代谢产物的排出能力降低,加剧肝细胞JNK持续性激活,继而导致肝脏损伤加重。在APAP急性肝损伤早期,野生型(C3H/He N)小鼠APAP肝脏损伤较突变型(C3H/He J)小鼠严重,提示TLR4基因突变在APAP急性肝损伤早期中起到保护作用,推测肝脏TLR4信号通路参与激活肝脏JNK、RIP1和RIP3,促进肝细胞程序性坏死。
[Abstract]:Background / purpose of acetaminophen (APAP) is a clinically common antipyretic analgesic drugs, the excessive or improper use of acute liver injury caused by children is one of the common causes of drug-induced liver injury. However, at present about between adults and children with acute liver injury APAP whether there are some differences are rarely reported. Previous study showed that APAP in the liver cells by P450 metabolic enzyme CYP2E1 metabolism of toxic products of acetyl N- quinone imine (NAPQI), depletion of intracellular glutathione (GSH) caused by oxidative stress, mitochondrial dysfunction and ultimately lead to the death of liver cells. Recent studies suggest that APAP induced acute liver injury during the two attack, TLR4 signaling could be mediated liver cell programmed necrosis APAP induced acute liver injury "two hit" in the process. The subject of sensitivity to APAP induced acute liver injury of mice of different age and gender differences, for this After in order to guide the clinical treatment of APAP in different populations of liver injury and provide a theoretical basis; compared to the wild type (C3H/He N type) and TLR4 (C3H/He J) mutation between APAP liver injury in mice, preliminary study on TLR4 damage in different stages in acute liver APAP. A research method, sensitivity of different age and gender on mice with acute APAP liver injury consists of two experiments: Experiment 1, young (3 weeks) CD-1 mice (female, male = 10) and adult (8 weeks) CD-1 mice (female, male = 10) were divided into four groups: young male mice, immature female mice, adult male mice and adult female mice. With 10 mice in each group. All mice were unified after fasting for 12 hours by intraperitoneal injection with a single dose of APAP (300mg/kg), to observe the survival rate of mice to APAP seventh days after dosing. The survival curves were compared and plotted for each group of mice survival rate difference. In experiment two, the young (3 weeks) CD-1 Mice (female, male = 40) and adult (8 weeks) CD-1 mice (female, male = 40) were divided into APAP treatment group and control group. All mice were fasted for 12 hours. All unified administration of APAP mice by intraperitoneal injection of APAP (300mg/kg), the control group received saline. After treatment at different time points (0,1,4 and 24 hours and 7 days) mice were killed, compared the liver index, serum ALT and liver GSH, GSSG and GSSG/GSH levels, HE staining was used to observe the pathology of liver tissue injury and death with liver cell TUNEL detection, detection of liver tyrosine nitration by immune group (3-NT) the liver was detected by real-time quantitative RT-PCR; Cyp1a1, Cyp2e1, Cyp3a11, Ugt1a1, Gpx, Gr and Gst gene expression levels in the liver; GPX kit, GR and GST activity; with Western blot detection of liver CYP2E1, p-JNK, JNK, RIP1 and RIP3 protein level. Two, TLR4 damage occurred in the process of the in acute liver APAP Composed of two experiments, the wild type (C3H/He N) mice and mutant mice (C3H/He J) 10, unified after 12 hours of fasting for APAP (300mg/kg) single dose intraperitoneal injection, the mice were observed to survival after administration of APAP 72 h. to draw the two groups of mice and survival curve differences in the survival rate of mice were compared between the two groups. In experiment two, wild type mice (C3H/He N) and mutant (C3H/He J) were divided into APAP treatment group and control group. All mice were fasted for 12 hours the unified administration of.APAP group were given intraperitoneal injection of APAP (300mg/kg), the control group received equal volume of physiological saline. After administration at different time points (0,4 and 12 h) mice were sacrificed and the liver index were compared between the two groups, the level of serum ALT detection, using HE staining to observe the pathological liver injury, liver cell death by detection of TUNEL, Western p-JNK and JNK blot detection of liver, programmed necrosis related protein kinase The enzyme RIP1, the expression of RIP3 protein. Results the juvenile mice APAP induced acute liver injury with the same sex of adult mice is more serious. The survival results suggest that male or female juvenile survival rate of mice (male 50%, female 60%) were significantly lower than those in adult mice (male 80%, female 90%). Further observation showed that, compared with the the sex of adult mice, 4 hours after administration of juvenile mice liver index and serum ALT levels increased more significantly, learn to observe liver hyperaemia obvious pathology; and 24 hours after administration of male and female juvenile mice serum ALT levels were still higher than those in same-sex adult mice, histopathological observation to the liver showed centrilobular necrosis, and the young mice liver necrotic area was significantly greater than the sex of adult mice; TUNEL showed that juvenile mice liver TUNEL positive cells was significantly higher than that of sex in adult mice; Western blotting results indicated that APAP After the treatment of protein levels in juvenile mice liver p-JNK and RIP3 were significantly higher than those in same-sex adult mice. Immunohistochemistry showed that APAP treatment after 4 hours and 24 hours of juvenile mice liver nitrotyrosine (3-NT) positive cells were significantly more than the number of same-sex adult mice. To explore the mechanism of juvenile mice are more sensitive to APAP liver injury. Comparison of different age and gender in mouse liver APAP metabolic enzyme levels and GSH metabolism. The results showed that compared with the sex of adult mice, juvenile mice after APAP administration of liver GSH depletion to be larger, the ratio of GSSG/GSH increased more significantly; further analysis found that young mice liver Cyp2e1, Cyp3a11 m RNA and CYP2E1 protein level the content of same sex were higher in adult mice. This study found that APAP induced acute liver injury of gender differences exist only in adult mice, adult male mice APAP acute liver injury compared with adult female mice Serious.APAP 4 hours after treatment and 24 hours of adult male mice serum ALT levels were higher than that of adult female mice; histological observation to adult male mice liver necrosis area was significantly greater than that of adult female mice tissues; TUNEL detection showed that adult male mouse liver TUNEL positive cell number was significantly higher than that of adult female mice. Further observation showed that after treatment with APAP 4 hours of adult male mice liver p-JNK levels were significantly higher than that of adult female mice; immunohistochemistry showed that APAP treatment after 4 hours and 24 hours of adult male mouse liver 3-NT positive cells were significantly more than the number of adult female.APAP mice 4 hours after the treatment of adult male mouse liver GSH consumption than adult female mice, and the ratio of GSSG/GSH recovery was slower in the adult female mice; GPX and GST activity in the liver of adult male mice were lower than adult female mice. The wild type mice (C3H/He N) A A mutant PAP of acute liver injury (C3H/He J) were more severe.72h results suggest that the survival curve of wild type (C3H/He N) mice were administered 4 hours after the occurrence of death, 72 hour survival rate was 40%; (C3H/He J) mice to administration 8 hours before the occurrence of death, 72 hour survival rate is 70%. so wild type (C3H/He N) in mice with mutant (C3H/He J) the survival rate of mice with low.APAP after 4 h treatment, the liver index of N mice C3H/He C3H/He J mice increased significantly. Administration of 4 h and 12 h, the level of serum ALT C3H/He N mice than C3H/He mice. J HE staining showed that the liver C3H/He, N liver necrosis area larger. Liver TUNEL results suggest that C3H/He mouse liver N TUNEL+ positive cells were significantly more than the number of C3H/He J.C3H/He in mice N mice after administration of 4 h and 12 h, the expression of liver p-JNK was higher than C3H/He in J mice. After Administration of 4 h C3H/He N mouse liver RIP1 expression was higher than that of C3H/He J mice; After administration of 12 h, C3H/He RIP1 expression in liver of N mice and C3H/He J mice had no significant difference, but the expression of hepatic RIP3 was significantly higher than that of C3H/He in J mice. Mice of different ages have different sensitivity to the conclusion of APAP induced acute liver injury, juvenile mice are more susceptible to APAP acute liver injury. The possible mechanism and juvenile mouse liver Cyp2e1. Cyp3a11 m RNA CYP2E1 level and protein content were lower than the same sex adult mice were higher; liver GSH depletion for juvenile mice caused by APAP are more sensitive; after APAP treatment, expression of RIP3 protein and P-JNK in mouse liver of juvenile sex differences in acute liver injury more about.APAP exists only in adult mice, the specific performance of adult male mice than adult female mice liver injury is more obvious. The mechanism may be related to adult male mice liver GSH metabolic enzymes (GPX and GST) activity than adult female mice, adult male mice liver after APAP treatment Dirty GSH consumption, and reduce the antioxidant and ability to promote the discharge of toxic metabolites, intensified hepatocyte persistent activation of JNK, and then lead to liver damage. In the early stage of acute liver injury APAP, wild type (C3H/He N) APAP mice liver injury than mutant mice (C3H/He J), suggesting that TLR4 gene mutation plays the protective effect in the early stage of acute hepatic injury in APAP, speculated that the liver TLR4 signaling pathway is involved in activation of liver JNK, RIP1 and RIP3, promote liver cell programmed necrosis.

【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R725.7

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