芳香烴受體及肝臟代謝酶在飲用水有機提取物染毒大鼠肝臟中的表達及意義
發(fā)布時間：2018-07-23 20:55
【摘要】：目的:利用課題組前期制備的飲用水有機提取物致大鼠肝損傷動物模型,對芳香烴受體及下游調(diào)控基因(肝臟代謝酶)的表達及活性進行觀察,探討飲用水有機污染物的異常肝臟生物轉(zhuǎn)化在肝損傷中的作用。方法:于2012年7-9月(豐水期)采集該地的末梢水水樣并制備有機提取物。將50只清潔級SD大鼠隨機分為5組,分別為空白對照組、溶劑對照(玉米油)組和飲用水有機提取物低中高3個染毒組(劑量分別為5L/kg、20L/kg、80L/kg),每組10只,雌雄各半。采用經(jīng)口灌胃方式進行染毒,每天1次,連續(xù)染毒12周。制備大鼠血清,采用分光光度法檢測天冬氨酸轉(zhuǎn)氨酶(Aspartate aminotransferas,AST)、丙氨酸轉(zhuǎn)氨酶(Alanine amunotransferase,ALT)、膽堿酯酶(Cholinesterase,CHE)的活力及總蛋白(Total protein,TP)、白蛋白(Albumin,ALB)的含量;提取肝組織總RNA,采用實時熒光定量PCR(RT-q RCR)法檢測芳香烴受體(aryl hydrocarbon receptor,Ah R)、熱休克蛋白90(heat shock protein 90,HSP90)、芳香烴受體核轉(zhuǎn)位蛋白(aryl hydrocarbon nuclear translocator,ARNT)、細胞色素1A2(CYP1A2)、細胞色素2E1(CYP2E1)、谷胱甘肽-S-轉(zhuǎn)移酶A1(glutathione-S-transferase A1,GSTA1)的m RNA表達情況;提取肝組織總蛋白,采用蛋白印記(Western Blot)法檢測Ah R、HSP90、ARNT、CYP1A2、CYP2E1、GSTA1的蛋白表達情況;制備肝勻漿,采用分光光度法檢測谷胱甘肽-S-轉(zhuǎn)移酶(glutathione-S-trans ferase,GSTs)活力;制備肝微粒體,熒光分光光度法和活性比色法分別測定CYP1A2與CYP2E1酶活性;采用Spearman相關對芳香烴受體和肝臟代謝酶及肝損傷的相關性進行分析。結果:(1)隨著染毒劑量的增加,大鼠血清CHE活力在中、高劑量組明顯升高,而ALT、AST活力僅在高劑量組升高(P0.05);與對照組相比,TP和ALB水平在高劑量組明顯下降有統(tǒng)計學意義(P0.05)。(2)與對照組相比,HSP90的m RNA和蛋白表達水平在低、中、高劑量組都明顯升高,而Ah R與ARNT的m RNA和蛋白表達水平僅在中、高劑量組升高(P0.05)。(3)與對照組相比,CYP1A2與GSTA1的m RNA和蛋白表達水平在中、高劑量組明顯升高,而CYP2E1的m RNA和蛋白表達水平僅在高劑量組升高(P0.05);隨著染毒劑量的增加,高劑量組GSTA1 m RNA和蛋白表達水平比中劑量組有所降低,差異具有統(tǒng)計學意義(P0.05);CYP1A2和GSTs酶活性在中劑量與高劑量組顯著增加,而CYP2E1酶活性僅在高劑量組增加(P0.05);與中劑量組相比,高劑量組GSTs的酶活性則明顯降低(P0.05)。(4)肝臟代謝酶CYP1A2、GSTs酶活性與Ah R蛋白表達水平均呈正相關關系;染毒大鼠血清CHE水平與Ah R的蛋白表達水平及CYP1A2、CYP2E1、GSTs酶活性均呈正相關關系。結論:(1)在本實驗條件下,較高劑量飲用水有機提取物暴露可活化芳香烴受體Ah R,從而誘導其配體HSP90及ARNT的轉(zhuǎn)錄表達和翻譯表達。(2)芳香烴受體通路的異常上調(diào),調(diào)控下游基因代謝酶CYP1A2與GSTA1的m RNA和蛋白表達增強,誘導代謝酶活性升高。(3)肝臟代謝酶活性異常升高,在對飲用水有機污染物解毒代謝的過程中,誘導自由基和毒性中間代謝物的產(chǎn)生,破壞細胞結構,造成肝細胞損傷。
[Abstract]:Objective: To observe the expression and activity of aromatic hydrocarbon receptor and downstream regulatory gene (liver metabolic enzyme), and to explore the role of abnormal liver biotransformation of drinking water organic pollutants in the liver damage caused by the organic extracts of drinking water prepared by the group in the previous period. Methods: in 7-9 months of 2012 (Fengshui period) 50 clean SD rats were randomly divided into 5 groups: blank control group, solvent control (corn oil) group and drinking water organic extract in 3 low middle and high toxic groups (dose of 5L/kg, 20L/kg, 80L/kg), each group of 10 rats and male and male. 1 times, the rat serum was prepared for 12 weeks. Aspartate aminotransferas (AST), alanine aminotransferase (Alanine amunotransferase, ALT), the activity of cholinesterase (Cholinesterase, CHE), the total protein (Total protein, TP), the content of albumin (Albumin,) were extracted by spectrophotometric method. The detection of aromatic hydrocarbon receptor (aryl hydrocarbon receptor, Ah R), heat shock protein 90 (heat shock protein 90, HSP90), aromatic hydrocarbon receptor nuclear transposition protein, cytochrome aryl, glutathione transferase (Ah) were detected by real time fluorescence quantitative PCR (RCR) method. The expression of M RNA in one-S-transferase A1, GSTA1, the total protein of liver tissue was extracted, and the protein expression of Ah R, HSP90, ARNT, CYP1A2, and Western Blot method was used to detect the protein expression of Ah, and the liver homogenate was prepared by spectrophotometric method to detect the activity of glutathione transferase, and the preparation of liver microsomes was made. The activity of CYP1A2 and CYP2E1 enzyme were measured by fluorescence spectrophotometry and activity colorimetry, and the correlation of Spearman correlation to the metabolic enzymes of liver and liver was analyzed by Spearman correlation. Results: (1) with the increase of dose, the activity of CHE in the serum of rats increased significantly, and the activity of ALT and AST was only in high dose group. Higher (P0.05). Compared with the control group, the level of TP and ALB decreased significantly in the high dose group (P0.05). (2) compared with the control group, the m RNA and protein expression level of HSP90 increased significantly in low, middle and high dose groups, while Ah R and ARNT m RNA and protein expression levels were only in the middle, high dose group increased. (3) compared with the control group, the level of Ah R and ARNT increased. With the level of M RNA and protein expression of GSTA1, the high dose group increased obviously, while the m RNA and protein expression level of CYP2E1 increased only in the high dose group (P0.05). With the increase of the dose, the GSTA1 m RNA and protein expression level in the high dose group was lower than that in the middle dose group, and the difference was statistically significant (P0.05); CYP1A2 and enzyme activity were in the high dose group. The medium dose and high dose group increased significantly, while the activity of CYP2E1 enzyme increased only in the high dose group (P0.05). Compared with the medium dose group, the activity of GSTs in the high dose group decreased significantly (P0.05). (4) the liver metabolic enzyme CYP1A2, the activity of GSTs enzyme and the expression level of Ah R protein were all Cheng Zhengxiang; the serum CHE level and Ah R protein expression water in the rats were infected. The activities of CYP1A2, CYP2E1 and GSTs enzyme were positively correlated. (1) under this experimental condition, the exposure of organic extracts from high doses of drinking water can activate the aromatic hydrocarbon receptor Ah R, thus inducing the transcription and expression of the ligand HSP90 and ARNT. (2) the abnormal up regulation of the aromatic hydrocarbon receptor pathway and the regulation of the downstream gene metabolic enzyme CYP1A2 and G The expression of M RNA and protein in STA1 is enhanced, and the activity of induced metabolic enzyme is increased. (3) the activity of metabolic enzymes in the liver is abnormal. It induces the production of free radicals and toxic intermediate metabolites in the process of detoxification and metabolism of organic pollutants in drinking water, destroys the structure of cells and causes damage to the liver cells.
【學位授予單位】：貴陽醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R114
[Abstract]:Objective: To observe the expression and activity of aromatic hydrocarbon receptor and downstream regulatory gene (liver metabolic enzyme), and to explore the role of abnormal liver biotransformation of drinking water organic pollutants in the liver damage caused by the organic extracts of drinking water prepared by the group in the previous period. Methods: in 7-9 months of 2012 (Fengshui period) 50 clean SD rats were randomly divided into 5 groups: blank control group, solvent control (corn oil) group and drinking water organic extract in 3 low middle and high toxic groups (dose of 5L/kg, 20L/kg, 80L/kg), each group of 10 rats and male and male. 1 times, the rat serum was prepared for 12 weeks. Aspartate aminotransferas (AST), alanine aminotransferase (Alanine amunotransferase, ALT), the activity of cholinesterase (Cholinesterase, CHE), the total protein (Total protein, TP), the content of albumin (Albumin,) were extracted by spectrophotometric method. The detection of aromatic hydrocarbon receptor (aryl hydrocarbon receptor, Ah R), heat shock protein 90 (heat shock protein 90, HSP90), aromatic hydrocarbon receptor nuclear transposition protein, cytochrome aryl, glutathione transferase (Ah) were detected by real time fluorescence quantitative PCR (RCR) method. The expression of M RNA in one-S-transferase A1, GSTA1, the total protein of liver tissue was extracted, and the protein expression of Ah R, HSP90, ARNT, CYP1A2, and Western Blot method was used to detect the protein expression of Ah, and the liver homogenate was prepared by spectrophotometric method to detect the activity of glutathione transferase, and the preparation of liver microsomes was made. The activity of CYP1A2 and CYP2E1 enzyme were measured by fluorescence spectrophotometry and activity colorimetry, and the correlation of Spearman correlation to the metabolic enzymes of liver and liver was analyzed by Spearman correlation. Results: (1) with the increase of dose, the activity of CHE in the serum of rats increased significantly, and the activity of ALT and AST was only in high dose group. Higher (P0.05). Compared with the control group, the level of TP and ALB decreased significantly in the high dose group (P0.05). (2) compared with the control group, the m RNA and protein expression level of HSP90 increased significantly in low, middle and high dose groups, while Ah R and ARNT m RNA and protein expression levels were only in the middle, high dose group increased. (3) compared with the control group, the level of Ah R and ARNT increased. With the level of M RNA and protein expression of GSTA1, the high dose group increased obviously, while the m RNA and protein expression level of CYP2E1 increased only in the high dose group (P0.05). With the increase of the dose, the GSTA1 m RNA and protein expression level in the high dose group was lower than that in the middle dose group, and the difference was statistically significant (P0.05); CYP1A2 and enzyme activity were in the high dose group. The medium dose and high dose group increased significantly, while the activity of CYP2E1 enzyme increased only in the high dose group (P0.05). Compared with the medium dose group, the activity of GSTs in the high dose group decreased significantly (P0.05). (4) the liver metabolic enzyme CYP1A2, the activity of GSTs enzyme and the expression level of Ah R protein were all Cheng Zhengxiang; the serum CHE level and Ah R protein expression water in the rats were infected. The activities of CYP1A2, CYP2E1 and GSTs enzyme were positively correlated. (1) under this experimental condition, the exposure of organic extracts from high doses of drinking water can activate the aromatic hydrocarbon receptor Ah R, thus inducing the transcription and expression of the ligand HSP90 and ARNT. (2) the abnormal up regulation of the aromatic hydrocarbon receptor pathway and the regulation of the downstream gene metabolic enzyme CYP1A2 and G The expression of M RNA and protein in STA1 is enhanced, and the activity of induced metabolic enzyme is increased. (3) the activity of metabolic enzymes in the liver is abnormal. It induces the production of free radicals and toxic intermediate metabolites in the process of detoxification and metabolism of organic pollutants in drinking water, destroys the structure of cells and causes damage to the liver cells.
【學位授予單位】：貴陽醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R114
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