齐墩果酸对自然衰老大鼠睾丸DNA损伤保护作用及机制研究
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摘要
探讨齐墩果酸(Oleanolic Acid,OA)对自然衰老大鼠睾丸DNA损伤的保护作用及其可能机制。将30只SPF级18月龄SD大鼠随机分为三组:自然衰老组、OA低、高剂量组,每组10只,另取10只3月龄大鼠作为青年对照组。OA低、高剂量组分别灌胃给予OA 5、25 mg/kg,自然衰老组和青年对照组给予生理盐水,每周灌胃6天,周日不给药,连续给药6个月。末次给药后禁食不禁水12 h,称重,麻醉后处死大鼠,快速取出睾丸组织。HE染色观察睾丸组织形态变化,免疫组织化学法检测DNA损伤相关蛋白53BP1、ATR的表达,Western Blot检测睾丸组织中衰老相关蛋白p21和DNA损伤相关蛋白γ-H2AX、Chk1、p-p53的表达水平。HE结果表明,OA能显著改善增龄所致的睾丸组织形态变化;与青年对照组相比,自然衰老组睾丸组织DNA损伤标志蛋白γ-H2AX表达水平明显升高,而给予OA干预后其表达显著降低;此外,与青年对照组比较,自然衰老组睾丸组织中衰老相关蛋白p21的表达显著增高,DNA损伤相关蛋白53BP1、ATR的阳性灶点数及其下游Chk1、p-p53的蛋白表达明显升高,OA低、高剂量组均能显著降低上述蛋白的表达。以上结果表明OA对自然衰老大鼠睾丸组织DNA损伤有较好的保护作用,机制与调节ATR/Chk1/P53信号通路有关。
To study the protective effects of Oleanolic Acid on Testicular DNA Damage in natural aging rats,and to explore the possible mechanism. The 18-month old male SD rats were randomly divided into natural aging group,OA low and high dose group,with 10 rats in each group. Another 10 3-month old rats were used as young control group. OA low and high dose groups were given gastric gavage of OA 5 and 25 mg/kg,respectively,while the young control group and the natural aging group were given saline six times per week,lasting for 6 months. After fasting for 12 hours,the rats were sacrificed. Then,the testes were immediately removed and weighed. The change of testicular tissue morphology was observed by HE staining. The expression and localization of DNA damage-associated protein 53 BP1 and ATR were observed by Immunohistochemistry. The relative expression levels of senescence associated protein p21 and DNA damagerelated proteins γ-H2 AX,Chk1 and p-p53 in testes were detected by Western Blot. HE showed that OA significantly improved the testicular tissue structure of natural aging rats. Compared with young control group,the levels of γ-H2 AX protein in testis were significantly upregulated in natural aging group. However,the expression of γ-H2 AX significantly decreased in OA-treated groups. In addition,the numbers of ATR-and 53 BP1-positive cells,and the protein expression levels of p21,Chk1,and p-p53 were significantly upregulated in testis of rats in natural aging group when compared with young control group. As expected,OA significantly restored these changes. Taken together,OA protected against testicu-lar DNA damage of natural aging rats,which may be associated with ATR/Chk1/P53 signaling.
To study the protective effects of Oleanolic Acid on Testicular DNA Damage in natural aging rats,and to explore the possible mechanism. The 18-month old male SD rats were randomly divided into natural aging group,OA low and high dose group,with 10 rats in each group. Another 10 3-month old rats were used as young control group. OA low and high dose groups were given gastric gavage of OA 5 and 25 mg/kg,respectively,while the young control group and the natural aging group were given saline six times per week,lasting for 6 months. After fasting for 12 hours,the rats were sacrificed. Then,the testes were immediately removed and weighed. The change of testicular tissue morphology was observed by HE staining. The expression and localization of DNA damage-associated protein 53 BP1 and ATR were observed by Immunohistochemistry. The relative expression levels of senescence associated protein p21 and DNA damagerelated proteins γ-H2 AX,Chk1 and p-p53 in testes were detected by Western Blot. HE showed that OA significantly improved the testicular tissue structure of natural aging rats. Compared with young control group,the levels of γ-H2 AX protein in testis were significantly upregulated in natural aging group. However,the expression of γ-H2 AX significantly decreased in OA-treated groups. In addition,the numbers of ATR-and 53 BP1-positive cells,and the protein expression levels of p21,Chk1,and p-p53 were significantly upregulated in testis of rats in natural aging group when compared with young control group. As expected,OA significantly restored these changes. Taken together,OA protected against testicu-lar DNA damage of natural aging rats,which may be associated with ATR/Chk1/P53 signaling.
引文
1 Belloc S,Hazout A,Zini A,et al.How to overcome male infertility after 40:Influence of paternal age on fertility[J].Maturitas,2014,78(1):22-29.
2 Sibert L,Lacarrière E,Safsaf A,et al.Aging of the human testis[J].Presse Med,2014,43:171-177.
3 Jiang H,Zhu WJ,Li J,et al.Quantitative histological analysis and ultrastructure of the aging human testis[J].Int Urol Nephrol,2014,46:879-885.
4 Paul C,Robaire B.Ageing of the male germ line[J].Nat Rev Urolog,2013,10:227-234.
5 Grégoire M C,Massonneau J,Simard O,et al.Male-driven de novo mutations in haploid germ cells[J].Mol Hum Reprod,2013,19:495-499.
6 Goriely A,Wilkie A OM.Paternal age effect mutations and selfish spermatogonial selection:causes and consequences for human disease[J].Am J Hum Genet,2012,90:175-200.
7 State Pharmacopoeia Committee(国家药典委员会).Chinese pharmacopoeia(中华人民共和典)[J].Chin Trad Patent Med(中国传统医学专利),2015;1:45-46.
8 Pollier J,Goossens A.Oleanolic acid[J].Phytochemistry,2012,77(5):10-15.
9 Liu J.Oleanolic acid and ursolic acid:research perspectives[J].J Ethnopharmacol,2005,100(1-2):92-94.
10 Zhang J,Lu L,Zhou L.Oleanolic acid activates daf-16,to increase lifespan in Caenorhabditis elegans[J].Biochem Biophys Res Commun,2015,468:843-849.
11 Lee W,Yang EJ,Ku SK,et al.Anti-inflammatory effects of oleanolic acid on LPS-induced inflammation in vitro and in vivo[J].Inflammation,2013,36:94-102.
12 Liu L(刘玲),Zhao JL(赵建龙),Wang JG(王建刚).Oleanolic acid induces G2/M phase arrest and apoptosis in human hepatocellular carcinoma Bel-7402 cells[J].Chin J Chin Mater Med(中国中药杂志),2015,0:4897-4902.
13 Dong J(董静),Wu BY(吴勃岩),Liu YQ(刘艳秋),et al.Impact of oleanolic acid on apoptosis-related protein expression in mice bearing H22 tumor tissue[J].Chin J Exp Tradit Med Form(中国实验方剂学杂志),2015,16:129-131.
14 Cui D H(崔德华).Reaearch progress of brain aging and cognitive impairment[J].Pract Geriatr(实用老年学),2010,24(1):19-23.
15 Hua Y,Robinson TJ,Cao Y,et al.Cathepsin K knockout alleviates aging-induced cardiac dysfunction[J].Aging Cell,2015,14:345-351.
16 Gao ZQ(高志清),Hai CX(海春旭),Zhang XD(张晓迪),et al.Expression and significance of RAGE and p21 in the D-galactose induced aging model[J].Chin J Gerontol(中国老年学),2007,27:1661-1663.
17 Sharma A,Singh K,Almasan A.Histone H2AX phosphorylation:a marker for DNA damage[J].Methods Mol Biol,2012,920:613-626.
18 Gutierrezmartinez P,Rossi DJ,Beerman I.DNA damage and aging around the clock[J].Trends Mol Med,2016,22:635-637.
19 Panda S,Isbatan A,Adami GR.Modification of the ATM/ATR directed DNA damage response state with aging and long after hepatocyte senescence induction in vivo[J].Mech Ageing Dev,2008,129:332-340.
20 Manic G,Obrist F,Sistigu A,et al.Trial Watch:Targeting ATM-CHK2 and ATR-CHK1 pathways for anticancer therapy[J].Cell Mol Life Sci,2015,2(4):e10129761-17.
21 Shieh SY,Ahn J,Tamai K,et al.The human homologs of checkpoint kinases Chk1 and Cds1(Chk2)phosphorylate P53 at multiple DNA damage-inducible sites[J].Genes Dev,2000,14:289-300.
22 Zhao T,Tang X,Channakeshava sokke umeshappa,et al.Simulated microgravity promotes cell apoptosis through suppressing Uev1A/TICAM/TRAF/NF-κB-regulated anti-apoptosis and P53/PCNA-and ATM/ATR-Chk1/2-controlled DNA-damage response pathways[J].J Cell Iochem,2016,17:2138-2148.
23 Zilfou JT,Lowe SW.Tumor suppressive functions of P53[J].CSH Pespect Biol,2009,1(5):a001883.
24 Jiang YJ(江越菁),Zang Y(臧奕).The roles of 53BP1 in DNA double-strand breaks repair[J].Chin J Biochem Mol Biol(中国生物化学与分子生物学报),2016,32:1279-1285.
2 Sibert L,Lacarrière E,Safsaf A,et al.Aging of the human testis[J].Presse Med,2014,43:171-177.
3 Jiang H,Zhu WJ,Li J,et al.Quantitative histological analysis and ultrastructure of the aging human testis[J].Int Urol Nephrol,2014,46:879-885.
4 Paul C,Robaire B.Ageing of the male germ line[J].Nat Rev Urolog,2013,10:227-234.
5 Grégoire M C,Massonneau J,Simard O,et al.Male-driven de novo mutations in haploid germ cells[J].Mol Hum Reprod,2013,19:495-499.
6 Goriely A,Wilkie A OM.Paternal age effect mutations and selfish spermatogonial selection:causes and consequences for human disease[J].Am J Hum Genet,2012,90:175-200.
7 State Pharmacopoeia Committee(国家药典委员会).Chinese pharmacopoeia(中华人民共和典)[J].Chin Trad Patent Med(中国传统医学专利),2015;1:45-46.
8 Pollier J,Goossens A.Oleanolic acid[J].Phytochemistry,2012,77(5):10-15.
9 Liu J.Oleanolic acid and ursolic acid:research perspectives[J].J Ethnopharmacol,2005,100(1-2):92-94.
10 Zhang J,Lu L,Zhou L.Oleanolic acid activates daf-16,to increase lifespan in Caenorhabditis elegans[J].Biochem Biophys Res Commun,2015,468:843-849.
11 Lee W,Yang EJ,Ku SK,et al.Anti-inflammatory effects of oleanolic acid on LPS-induced inflammation in vitro and in vivo[J].Inflammation,2013,36:94-102.
12 Liu L(刘玲),Zhao JL(赵建龙),Wang JG(王建刚).Oleanolic acid induces G2/M phase arrest and apoptosis in human hepatocellular carcinoma Bel-7402 cells[J].Chin J Chin Mater Med(中国中药杂志),2015,0:4897-4902.
13 Dong J(董静),Wu BY(吴勃岩),Liu YQ(刘艳秋),et al.Impact of oleanolic acid on apoptosis-related protein expression in mice bearing H22 tumor tissue[J].Chin J Exp Tradit Med Form(中国实验方剂学杂志),2015,16:129-131.
14 Cui D H(崔德华).Reaearch progress of brain aging and cognitive impairment[J].Pract Geriatr(实用老年学),2010,24(1):19-23.
15 Hua Y,Robinson TJ,Cao Y,et al.Cathepsin K knockout alleviates aging-induced cardiac dysfunction[J].Aging Cell,2015,14:345-351.
16 Gao ZQ(高志清),Hai CX(海春旭),Zhang XD(张晓迪),et al.Expression and significance of RAGE and p21 in the D-galactose induced aging model[J].Chin J Gerontol(中国老年学),2007,27:1661-1663.
17 Sharma A,Singh K,Almasan A.Histone H2AX phosphorylation:a marker for DNA damage[J].Methods Mol Biol,2012,920:613-626.
18 Gutierrezmartinez P,Rossi DJ,Beerman I.DNA damage and aging around the clock[J].Trends Mol Med,2016,22:635-637.
19 Panda S,Isbatan A,Adami GR.Modification of the ATM/ATR directed DNA damage response state with aging and long after hepatocyte senescence induction in vivo[J].Mech Ageing Dev,2008,129:332-340.
20 Manic G,Obrist F,Sistigu A,et al.Trial Watch:Targeting ATM-CHK2 and ATR-CHK1 pathways for anticancer therapy[J].Cell Mol Life Sci,2015,2(4):e10129761-17.
21 Shieh SY,Ahn J,Tamai K,et al.The human homologs of checkpoint kinases Chk1 and Cds1(Chk2)phosphorylate P53 at multiple DNA damage-inducible sites[J].Genes Dev,2000,14:289-300.
22 Zhao T,Tang X,Channakeshava sokke umeshappa,et al.Simulated microgravity promotes cell apoptosis through suppressing Uev1A/TICAM/TRAF/NF-κB-regulated anti-apoptosis and P53/PCNA-and ATM/ATR-Chk1/2-controlled DNA-damage response pathways[J].J Cell Iochem,2016,17:2138-2148.
23 Zilfou JT,Lowe SW.Tumor suppressive functions of P53[J].CSH Pespect Biol,2009,1(5):a001883.
24 Jiang YJ(江越菁),Zang Y(臧奕).The roles of 53BP1 in DNA double-strand breaks repair[J].Chin J Biochem Mol Biol(中国生物化学与分子生物学报),2016,32:1279-1285.