核苷类似物对小鼠肝细胞线粒体DNA ND1和ND4的影响
|
摘要
目的探讨长期使用核苷类似物(NAs)是否导致小鼠肝脏线粒体DNA(mt DNA)ND1和ND4区损伤。方法 7周龄雌性Balb/c小鼠25只,采用简单随机分组法分为5组,对照组和4种核苷类似物组,每组各5只。实验组司他夫定(D4T)50 mg/kg,齐多夫定(AZT)100 mg/kg,拉米夫定(3TC)50 mg/kg和去羟肌苷(DDI)50 mg/kg,对照组为双蒸水,分别腹腔内注射,每周5次,连续3个月。留取各组肝组织,应用激光捕获显微技术获取肝细胞,对mt DNA ND1和ND4区克隆和测序。结果其余各组肝细胞的mt DNA ND4序列距离与参考序列的平均距离与对照组比较,差异均有高度统计学意义(P<0.01);AZT组肝细胞的mt DNA ND4平均d N与对照组比较,差异有统计学意义(P<0.05);肝细胞mt DNA ND1的序列距离与参考序列的平均距离,AZT组和3TC组序列距离与对照组比较,差异均有统计学意义(P<0.01);DDI组肝组织mt DNA ND1的平均d S与对照组比较,差异有统计学意义(P<0.05);AZT组和3TC组肝细胞mt DNA ND1的平均d S与对照组比较,差异均有统计学意义(P<0.05)。结论长期暴露于NAs可导致小鼠肝细胞mt DNA ND1和ND4区病变。
Objective To investigate whether long-term use of nucleoside analogues(NAs) can lead to damage of DNA(mt DNA) ND1 and ND4 regions in liver mitochondria of mice. Methods According to the simple random method, twenty-five 7-weeks-old female Balb/c mice were divided into 5 groups, with 5 cases in each group. The control group received double-distilled water by intraperitoneal injection 5 days per week for 3 months. The each test group was respectively given D4 T 50 mg/kg, AZT 100 mg/kg,3 TC 50 mg/kg,or DDI 50 mg/kg by intraperitoneal injection per day,5 days per week for 3 months. Single liver cell was captured from mouse liver tissues by laser capture microdissection. The products of mt DNA ND1 and ND4 region PCR amplicons were cloned and sequenced in mice liver cells. Results The mean distance to the reference sequence of mt DNA ND4 in the captured hepatocytes of AZT, D4 T and 3 TC groups were longer than that in the control group, the differences were statistically significant(P < 0.01). The mean mt DNA ND4 d N of the captured hepatocytes in the AZT group was higher than that in the control group, the difference was statistically significant(P < 0.05). The mean distance to the reference sequence of mt DNA ND1 in the captured hepatocytes of AZT and 3 TC groups were longer than that in the control group, the differences were statistically significant(P <0.01). The mean mt DNA ND1 d S of the liver tissue in the DDI group was higher than that in the control group, the difference was statistically significant(P < 0.05). The mean mt DNA ND1 d S of AZT and 3 TC groups were higher than that in the control group, the differences were statistically significant(P < 0.05). Conclusion Long-term exposure to nucleoside analogue can result in mt DNA ND1 and ND4 regions lesion in mouse hepatocytes.
Objective To investigate whether long-term use of nucleoside analogues(NAs) can lead to damage of DNA(mt DNA) ND1 and ND4 regions in liver mitochondria of mice. Methods According to the simple random method, twenty-five 7-weeks-old female Balb/c mice were divided into 5 groups, with 5 cases in each group. The control group received double-distilled water by intraperitoneal injection 5 days per week for 3 months. The each test group was respectively given D4 T 50 mg/kg, AZT 100 mg/kg,3 TC 50 mg/kg,or DDI 50 mg/kg by intraperitoneal injection per day,5 days per week for 3 months. Single liver cell was captured from mouse liver tissues by laser capture microdissection. The products of mt DNA ND1 and ND4 region PCR amplicons were cloned and sequenced in mice liver cells. Results The mean distance to the reference sequence of mt DNA ND4 in the captured hepatocytes of AZT, D4 T and 3 TC groups were longer than that in the control group, the differences were statistically significant(P < 0.01). The mean mt DNA ND4 d N of the captured hepatocytes in the AZT group was higher than that in the control group, the difference was statistically significant(P < 0.05). The mean distance to the reference sequence of mt DNA ND1 in the captured hepatocytes of AZT and 3 TC groups were longer than that in the control group, the differences were statistically significant(P <0.01). The mean mt DNA ND1 d S of the liver tissue in the DDI group was higher than that in the control group, the difference was statistically significant(P < 0.05). The mean mt DNA ND1 d S of AZT and 3 TC groups were higher than that in the control group, the differences were statistically significant(P < 0.05). Conclusion Long-term exposure to nucleoside analogue can result in mt DNA ND1 and ND4 regions lesion in mouse hepatocytes.
引文
[1]Seto WK,Liu K,Wong DK,et al.Patterns of hepatitis B surface antigen decline and HBV DNA suppression in Asian treatment-experienced chronic hepatitis B patients after three years of tenofovir treatment[J].J Hepatol,2013,59(4):709-716.
[2]Obiako OR,Abdu-Aguye I,Ogunniyi A.Effect of Stavudine-Based antiretroviral therapy on the severity of polyneuropathy in HIV/AIDS patients:a preliminary report from Zaria,Northern Nigeria[J].West Afr J Med,2011,30(5):354-358.
[3]Dragovic G,Jevtovic D.The role of nucleoside reverse transcriptase inhibitors usage in the incidence of hyperlactatemia and lactic acidosis in HIV/AIDS patients[J].Biomed Pharmacother,2012,66(4):308-311.
[4]胡倩倩,时丽丽,谭初兵.核苷类似物线粒体毒性机制及临床表现[J].中国药理学与毒理学杂志,2013,27(5):885-888.
[5]张维,乔录新,丁渭,等.核苷类似物对小鼠肝脏线粒体DNA D-loop区突变的影响[J].中国医药导报,2016,13(15):9-12.
[6]任林柱,张英.分子生物学实验原理与技术[M].北京:科学出版社,2015.
[7]Moyle G.Toxicity of antiretroviral nucleoside and nucleotide analogues:is mitochondrial toxicity the only mechanism?[J].Drug safety,2000,23(6),467-481.
[8]Dalakas MC.Peripheral neuropathy and antiretroviral drugs[J].J Peripher Nerv Syst,2001,6(1),14-20.
[9]Youle M.Acetyl-L-carnitine in HIV-associated antiretroviral toxic neuropathy[J].CNS Drugs,2007,21 Suppl 1:25-30,45-46.
[10]Antoniades C,Macdonald C,Knisely A,et al.Mitochondrial toxicity associated with HAART following liver transplantation in an HIV-infected recipient[J].Liver transpl,2004,10(5),699-702.
[11]Laguno M,Milinkovic A,de Lazzari E,et al.Incidence and risk factors for mitochondrial toxicity in treated HIV/HCV-coinfected patients[J].Antivir Ther,2005,10(3),423-429.
[12]Macias J,Castellano V,Merchante N,et al.Effect of antiretroviral drugs on liver fibrosis in HIV-infected patients with chronic hepatitis C:harmful impact of nevirapine[J].AIDS,2004,18(5),767-774.
[13]Antoniades C,Macdonald C,Knisely A,et al.Mitochondrial toxicity associated with HAART following liver transplantation in an HIV-infected recipient[J].Liver Transpl,2004,10,699-702.
[14]Gu F,Chauhan V,Kaur K,et al.Alterations in mitochondrial DNA copy number and the activities of electron transport chain complexes and pyruvate dehydrogenase in the frontal cortex from subjects with autism[J].Transl Psychiatry,2013,3,e299.doi:10.1038/tp.2013.68.
[15]Restrepo NA,Mitchell SL,Go odloe RJ,et al.Mitochondrial variation and the risk of age-related macular degeneration across diverse populations[J].Pac Symp Biocomput,2015,243-254.
[16]Gurses C,Azakli H,Alptekin A,et al.Mitochondrial DNA profiling via genomic analysis in mesial temporal lobe epilepsy patients with hippocampal sclerosis[J].Gene,2014,538(2),323-327.
[17]Zong NC,Li HM,Li H,et al.Integration of cardiac proteome biology and medicine by a specialized knowledgebase[J].Circ Res,2013,113(9),1043-1053.
[18]Garlick K.The potential of combination drug therapy for hepatitis B to cause mitochondrial damage[J].https://researchspace.auckland.ac.nz/handle/2292/22450.
[19]Zhang Y,Song F,Gao Z,et al.Long-term exposure of mice to nucleoside analogues disrupts mitochondrial DNA maintenance in cortical neurons[J].PLo S One,2014,9(1):e85637.
[2]Obiako OR,Abdu-Aguye I,Ogunniyi A.Effect of Stavudine-Based antiretroviral therapy on the severity of polyneuropathy in HIV/AIDS patients:a preliminary report from Zaria,Northern Nigeria[J].West Afr J Med,2011,30(5):354-358.
[3]Dragovic G,Jevtovic D.The role of nucleoside reverse transcriptase inhibitors usage in the incidence of hyperlactatemia and lactic acidosis in HIV/AIDS patients[J].Biomed Pharmacother,2012,66(4):308-311.
[4]胡倩倩,时丽丽,谭初兵.核苷类似物线粒体毒性机制及临床表现[J].中国药理学与毒理学杂志,2013,27(5):885-888.
[5]张维,乔录新,丁渭,等.核苷类似物对小鼠肝脏线粒体DNA D-loop区突变的影响[J].中国医药导报,2016,13(15):9-12.
[6]任林柱,张英.分子生物学实验原理与技术[M].北京:科学出版社,2015.
[7]Moyle G.Toxicity of antiretroviral nucleoside and nucleotide analogues:is mitochondrial toxicity the only mechanism?[J].Drug safety,2000,23(6),467-481.
[8]Dalakas MC.Peripheral neuropathy and antiretroviral drugs[J].J Peripher Nerv Syst,2001,6(1),14-20.
[9]Youle M.Acetyl-L-carnitine in HIV-associated antiretroviral toxic neuropathy[J].CNS Drugs,2007,21 Suppl 1:25-30,45-46.
[10]Antoniades C,Macdonald C,Knisely A,et al.Mitochondrial toxicity associated with HAART following liver transplantation in an HIV-infected recipient[J].Liver transpl,2004,10(5),699-702.
[11]Laguno M,Milinkovic A,de Lazzari E,et al.Incidence and risk factors for mitochondrial toxicity in treated HIV/HCV-coinfected patients[J].Antivir Ther,2005,10(3),423-429.
[12]Macias J,Castellano V,Merchante N,et al.Effect of antiretroviral drugs on liver fibrosis in HIV-infected patients with chronic hepatitis C:harmful impact of nevirapine[J].AIDS,2004,18(5),767-774.
[13]Antoniades C,Macdonald C,Knisely A,et al.Mitochondrial toxicity associated with HAART following liver transplantation in an HIV-infected recipient[J].Liver Transpl,2004,10,699-702.
[14]Gu F,Chauhan V,Kaur K,et al.Alterations in mitochondrial DNA copy number and the activities of electron transport chain complexes and pyruvate dehydrogenase in the frontal cortex from subjects with autism[J].Transl Psychiatry,2013,3,e299.doi:10.1038/tp.2013.68.
[15]Restrepo NA,Mitchell SL,Go odloe RJ,et al.Mitochondrial variation and the risk of age-related macular degeneration across diverse populations[J].Pac Symp Biocomput,2015,243-254.
[16]Gurses C,Azakli H,Alptekin A,et al.Mitochondrial DNA profiling via genomic analysis in mesial temporal lobe epilepsy patients with hippocampal sclerosis[J].Gene,2014,538(2),323-327.
[17]Zong NC,Li HM,Li H,et al.Integration of cardiac proteome biology and medicine by a specialized knowledgebase[J].Circ Res,2013,113(9),1043-1053.
[18]Garlick K.The potential of combination drug therapy for hepatitis B to cause mitochondrial damage[J].https://researchspace.auckland.ac.nz/handle/2292/22450.
[19]Zhang Y,Song F,Gao Z,et al.Long-term exposure of mice to nucleoside analogues disrupts mitochondrial DNA maintenance in cortical neurons[J].PLo S One,2014,9(1):e85637.