醋氨酚诱导药物性肝细胞损伤中HNF-1调节GSTA1表达分析
发布时间：2018-09-17 13:31
【摘要】：随着药物滥用的现象日益严重,药物引起的肝脏问题也逐渐增加,成为临床上最常见的肝脏问题之一。醋氨酚(APAP)是常用的解热镇痛药,其造成的肝脏问题在药物性肝脏问题中比例最高。醋氨酚诱导的肝损伤模型是研究药物性肝损伤的经典模型。谷胱甘肽S转移酶A1(GSTA1)是机体的代谢酶系统之一,可保护细胞应对氧化应激,抵抗外来毒性物质和活性氧的产生而保护机体。肝细胞核因子-1(HNF-1)被确定具有与几种肝基因启动子相互作用的DNA结合活性,在肝特异性转录中起重要作用。因此,它在调节肝脏的生长发育和代谢发挥着不可或缺的作用。本课题在APAP诱导的肝细胞损伤模型中,通过C2-神经酰胺降低或奥替普拉升高HNF-1表达的两种情况下对GSTA1表达的影响,确定HNF-1对GSTA1表达的调节作用,为明确GSTA1在药物性肝细胞损伤中的重要作用,研究药物性肝损伤提供体外试验依据,并为深入研究其作用机制及保护肝损伤提供理论基础。本试验采用系列浓度的醋氨酚作用于肝细胞,通过检测培养上清液转氨酶(ALT、AST)活性、肝细胞指标(SOD、MDA、GSH、GSH-Px)水平来判断肝细胞的损伤程度,优化APAP作用浓度并复制肝细胞损伤模型。在此模型的基础上,通过检测转氨酶活性来确定C2-神经酰胺和奥替普拉的最适作用浓度。随后,进行HNF-1调节GSTA1表达的试验。本试验分为六组,即对照组、模型组、C2-神经酰胺组(C2组)、C2-神经酰胺+APAP组(C2+APAP组)、奥替普拉组(OL组)、奥替普拉+APAP组(OL+APAP组),应用试剂盒检测培养上清ALT、AST和肝细胞指标SOD、GSH-Px、MDA和GSH;采用Real-time RT-PCR方法检测肝细胞中HNF-1和GSTA1 m RNA的相对表达,应用Western blot方法测定肝细胞中HNF-1和GSTA1蛋白的相对表达,并应用试剂盒检测培养上清中GSTA1的含量变化。通过以上研究,明确在APAP诱导的药物性肝细胞损伤中,HNF-1对GSTA1表达是否具有调节作用。研究结果表明:1、采用系列浓度APAP作用10 h可对肝细胞产生不同程度的损伤。当APAP浓度为15 m M时,培养上清转氨酶活性显著升高(p0.05);肝细胞指标(MDA、GSH、GSH-Px)水平均有极显著性变化(p0.01),肝细胞SOD显著降低(p0.05);细胞状态不佳,出现变形,皱缩等,最终确定以15 m M APAP作为模型组浓度,并成功复制药物性肝细胞损伤模型。2、通过最适作用浓度筛选试验,确定C2-神经酰胺以6μM浓度、奥替普拉以8μM浓度作用细胞。3、C2-神经酰胺和奥替普拉作用于肝细胞损伤模型的结果显示,与对照组比较,C2组和OL组培养上清液转氨酶活性、肝细胞指标均无显著性变化,显示出6μM的C2-神经酰胺和8μM奥替普拉不会对肝细胞产生影响。与模型组比较,C2+APAP组中转氨酶活性极显著升高(p0.01),肝细胞指标均呈极显著变化(p0.01),显示出C2-神经酰胺会加剧APAP诱导的肝细胞损伤;OL+APAP组转氨酶活性极显著降低(p0.01),肝细胞指标也均呈极显著变化(p0.01),表明奥替普拉能减轻APAP诱导的肝细胞损伤。4、HNF-1的表达结果表明,与对照组比较,模型组HNF-1 m RNA和蛋白相对表达量均极显著降低(p0.01),而C2组和OL组均无明显变化;与模型组比较,C2+APAP组HNF-1的mRNA和蛋白相对表达水平极显著降低(p0.01),而OL+APAP组极显著升高(p0.01)。上述结果显示出,在APAP诱导的肝细胞损伤中,C2-神经酰胺降低了HNF-1的表达,奥替普拉升高了HNF-1的表达。5、GSTA1的表达及含量测定的结果显示,与对照组比较,模型组GSTA1 m RNA和蛋白相对表达量均极显著降低(p0.01),培养上清液GSTA1含量极显著升高(p0.01),而C2组和OL组均无显著性变化;与模型组比较,C2+APAP组中GSTA1 m RNA和蛋白的相对表达均极显著降低(p0.01),OL+APAP组均极显著升高(p0.01);C2+APAP组培养上清液GSTA1含量极显著增加(p0.01),OL+APAP组极显著降低(p0.01)。上述结果表明,在APAP诱导的肝细胞损伤中,GSTA1在基因和蛋白水平上的表达趋势均与HNF-1相一致。本研究成功复制APAP诱导的肝细胞损伤模型。确定C2-神经酰胺和奥替普拉的最适作用浓度。在APAP诱导的肝细胞损伤中,C2-神经酰胺可加重肝细胞损伤,奥替普拉可减轻肝细胞损伤。在肝细胞损伤的情况下,C2-神经酰胺可抑制HNF-1的表达,而奥替普拉可促进HNF-1的表达。确定HNF-1对GSTA1表达的调节作用,且基因和蛋白表达水平的趋势相同。HNF-1可通过调节GSTA1表达来保护肝细胞,但其机制尚需研究。
[Abstract]:Acetaminophen (APAP) is one of the most common antipyretic and analgesic drugs, which causes the highest proportion of liver problems in drug-induced liver injury. Acetaminophen-induced liver injury model is to study drug-induced liver injury. Classical model. Glutathione S-transferase A1 (GSTA1) is one of the metabolic enzymes in the body. It protects cells against oxidative stress and the production of toxic substances and reactive oxygen species. Hepatocyte nuclear factor-1 (HNF-1) has been identified as a DNA binding activity interacting with several liver gene promoters and is involved in liver-specific transcription. Therefore, it plays an indispensable role in regulating the growth, development and metabolism of the liver. In the APAP-induced hepatocyte injury model, through the reduction of C2-ceramide or the elevation of HNF-1 expression by otipra, we determined the regulatory effect of HNF-1 on GSTA1 expression, in order to clarify GSTA1 expression. TA1 plays an important role in drug-induced hepatocyte injury, and provides a theoretical basis for further study of its mechanism and protection against liver injury. In this study, a series of concentrations of acetaminophen were used to treat hepatocytes. The activity of ALT and AST in culture supernatant and hepatocyte index (SOD, M) were detected. DA, GSH, GSH-Px) levels were used to determine the degree of hepatocyte injury, optimize APAP concentration and replicate the hepatocyte injury model. On the basis of this model, the optimal concentration of C2-ceramide and otipra was determined by detecting transaminase activity. Model group, C2-ceramide group (C2 group), C2-ceramide+APAP group (C2+APAP group), Otipra group (OL group), Otipra+APAP group (OL+APAP group), using kits to detect ALT, AST and hepatocyte indicators SOD, GSH-Px, MDA and GSH; Real-time RT-PCR to detect the relative expression of HNF-1 and GSTA1 m RNA in hepatocytes, and Western RT-PCR to detect the expression of HNF-1 and GSTA1 m RNA in hepatocytes. Western blot was used to detect the relative expression of HNF-1 and GSTA1 proteins in hepatocytes, and a kit was used to detect the content of GSTA1 in culture supernatant. When the concentration of APAP was 15 m M, the activity of transaminase in the supernatant increased significantly (p0.05); the levels of hepatocyte markers (MDA, GSH, GSH-Px) changed significantly (p0.01); the SOD of hepatocytes decreased significantly (p0.05); the cells were in poor condition, deformed and shrunken. Finally, the concentration of 15 mMAP was determined as the model group. The model of drug-induced hepatocyte injury was successfully reproduced. 2. The optimal concentration of C2-ceramide was determined by screening test. Cell injury was induced by C2-ceramide at 6 mu M, cell injury was induced by otipra at 8 mu M, and transaminase activity in supernatant of C2 and OL groups was determined by C2-ceramide and otipra. Compared with the model group, the transaminase activity in C2 + APAP group was significantly increased (p0.01), and the hepatocyte index was significantly changed (p0.01), indicating that C2-ceramide could aggravate the hepatocyte injury induced by APAP. The activity of transaminase and hepatocyte index in L + APAP group were significantly decreased (p0.01), indicating that Otipra could alleviate APAP-induced hepatocyte injury. 4. The expression of HNF-1 m RNA and protein in model group were significantly decreased compared with control group (p0.01), but there was no significant change in C2 group and OL group. Compared with the model group, the expression of HNF-1 mRNA and protein in C2+APAP group was significantly decreased (p0.01), while that in OL+APAP group was significantly increased (p0.01). The results showed that the relative expression of GSTA1 m RNA and protein in model group was significantly lower than that in control group (p0.01), and the content of GSTA1 in culture supernatant was significantly higher (p0.01), but there was no significant change in C2 group and OL group. Compared with model group, the relative expression of GSTA1 m RNA and protein in C2 + APAP group was significantly lower (p0.01), and that in OL + APAP group was extremely high (p0.01). The results showed that the expression of GSTA1 at gene and protein levels was consistent with that of HNF-1 in APAP-induced hepatocyte injury. C2-ceramide can aggravate hepatocyte injury in APAP-induced hepatocyte injury, while otipra can alleviate hepatocyte injury. C2-ceramide can inhibit the expression of HNF-1 in the case of hepatocyte injury, while otipra can promote the expression of HNF-1. HNF-1 can protect hepatocytes by regulating the expression of GSTA1, but its mechanism needs to be studied.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575
本文编号：2246071
[Abstract]:Acetaminophen (APAP) is one of the most common antipyretic and analgesic drugs, which causes the highest proportion of liver problems in drug-induced liver injury. Acetaminophen-induced liver injury model is to study drug-induced liver injury. Classical model. Glutathione S-transferase A1 (GSTA1) is one of the metabolic enzymes in the body. It protects cells against oxidative stress and the production of toxic substances and reactive oxygen species. Hepatocyte nuclear factor-1 (HNF-1) has been identified as a DNA binding activity interacting with several liver gene promoters and is involved in liver-specific transcription. Therefore, it plays an indispensable role in regulating the growth, development and metabolism of the liver. In the APAP-induced hepatocyte injury model, through the reduction of C2-ceramide or the elevation of HNF-1 expression by otipra, we determined the regulatory effect of HNF-1 on GSTA1 expression, in order to clarify GSTA1 expression. TA1 plays an important role in drug-induced hepatocyte injury, and provides a theoretical basis for further study of its mechanism and protection against liver injury. In this study, a series of concentrations of acetaminophen were used to treat hepatocytes. The activity of ALT and AST in culture supernatant and hepatocyte index (SOD, M) were detected. DA, GSH, GSH-Px) levels were used to determine the degree of hepatocyte injury, optimize APAP concentration and replicate the hepatocyte injury model. On the basis of this model, the optimal concentration of C2-ceramide and otipra was determined by detecting transaminase activity. Model group, C2-ceramide group (C2 group), C2-ceramide+APAP group (C2+APAP group), Otipra group (OL group), Otipra+APAP group (OL+APAP group), using kits to detect ALT, AST and hepatocyte indicators SOD, GSH-Px, MDA and GSH; Real-time RT-PCR to detect the relative expression of HNF-1 and GSTA1 m RNA in hepatocytes, and Western RT-PCR to detect the expression of HNF-1 and GSTA1 m RNA in hepatocytes. Western blot was used to detect the relative expression of HNF-1 and GSTA1 proteins in hepatocytes, and a kit was used to detect the content of GSTA1 in culture supernatant. When the concentration of APAP was 15 m M, the activity of transaminase in the supernatant increased significantly (p0.05); the levels of hepatocyte markers (MDA, GSH, GSH-Px) changed significantly (p0.01); the SOD of hepatocytes decreased significantly (p0.05); the cells were in poor condition, deformed and shrunken. Finally, the concentration of 15 mMAP was determined as the model group. The model of drug-induced hepatocyte injury was successfully reproduced. 2. The optimal concentration of C2-ceramide was determined by screening test. Cell injury was induced by C2-ceramide at 6 mu M, cell injury was induced by otipra at 8 mu M, and transaminase activity in supernatant of C2 and OL groups was determined by C2-ceramide and otipra. Compared with the model group, the transaminase activity in C2 + APAP group was significantly increased (p0.01), and the hepatocyte index was significantly changed (p0.01), indicating that C2-ceramide could aggravate the hepatocyte injury induced by APAP. The activity of transaminase and hepatocyte index in L + APAP group were significantly decreased (p0.01), indicating that Otipra could alleviate APAP-induced hepatocyte injury. 4. The expression of HNF-1 m RNA and protein in model group were significantly decreased compared with control group (p0.01), but there was no significant change in C2 group and OL group. Compared with the model group, the expression of HNF-1 mRNA and protein in C2+APAP group was significantly decreased (p0.01), while that in OL+APAP group was significantly increased (p0.01). The results showed that the relative expression of GSTA1 m RNA and protein in model group was significantly lower than that in control group (p0.01), and the content of GSTA1 in culture supernatant was significantly higher (p0.01), but there was no significant change in C2 group and OL group. Compared with model group, the relative expression of GSTA1 m RNA and protein in C2 + APAP group was significantly lower (p0.01), and that in OL + APAP group was extremely high (p0.01). The results showed that the expression of GSTA1 at gene and protein levels was consistent with that of HNF-1 in APAP-induced hepatocyte injury. C2-ceramide can aggravate hepatocyte injury in APAP-induced hepatocyte injury, while otipra can alleviate hepatocyte injury. C2-ceramide can inhibit the expression of HNF-1 in the case of hepatocyte injury, while otipra can promote the expression of HNF-1. HNF-1 can protect hepatocytes by regulating the expression of GSTA1, but its mechanism needs to be studied.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575
【参考文献】
相关期刊论文 前5条
1 谢雅清;梁晓美;叶伟霞;;还原型谷胱甘肽的药理作用与临床应用研究进展[J];中国药业;2013年07期
2 乔玉成;;老龄大鼠脑组织SOD/MDA与NO/NOS的相关性研究[J];中国体育科技;2009年02期
3 章轶锋;唐善虎;秦文玲;张巍;谢芳;;铜锌超氧化物歧化酶的研究进展[J];四川畜牧兽医;2008年01期
4 袁平戈;张大志;;还原型谷胱甘肽的作用机制及临床应用[J];药品评价;2006年05期
5 迟乃玉,张庆芳,刘长江;SOD的化学特性及其应用[J];沈阳农业大学学报;1999年02期
相关博士学位论文 前2条
1 吴宇;药物性肝损伤体外筛选模型和何首乌致肝损伤的初步研究[D];北京协和医学院;2016年
2 詹亦贝;5'-AMP对哺乳动物急性肝损伤保护作用的研究[D];南京理工大学;2014年
相关硕士学位论文 前4条
1 马欣;醋氨酚诱导小鼠急性药物性肝损伤中HNF-1调节GSTA1表达分析[D];东北农业大学;2016年
2 荆孝东;三肽模拟谷胱甘肽过氧化物酶及其生物学效应研究[D];吉林大学;2014年
3 刘颖姝;急性肝损伤早期诊断指标GSTA1的研究及保肝药物初筛[D];东北农业大学;2013年
4 邓同兴;神经酰胺调节酒精诱导小鼠神经细胞增殖的实验研究[D];郑州大学;2011年
，本文编号：2246071
本文链接：https://www.wllwen.com/yixuelunwen/xiaohjib/2246071.html
最近更新
教材专著
