淫羊藿苷激活Nrf2/HO-1信号通路减轻自然衰老大鼠睾丸生殖细胞DNA损伤研究
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摘要
目的探究淫羊藿苷对自然衰老大鼠睾丸生殖细胞DNA损伤的保护作用及其分子机制。方法将SPF级SD雄性大鼠随机分为4组,分别为青年对照组(2月龄)、衰老模型组(16月龄)和淫羊藿苷低、高剂量组(2、6 mg/kg,16月龄),每组8只,给予普通或含药饲料饲养4个月,禁食12 h后处死大鼠。HE染色法观察睾丸组织形态变化;黄嘌呤氧化酶法和硫代巴比妥酸法检测睾丸组织中超氧化物歧化酶(SOD)活力和丙二醛(MDA)水平;免疫荧光法检测睾丸组织中核因子E2相关因子2(Nrf2)及DNA损伤相关蛋白γ-H2AX的表达和定位;Western blotting法检测睾丸组织中Nrf2及其下游相关蛋白血红素氧合酶-1(HO-1)、醌氧化还原酶1(NQO1)、γ-H2AX和DNA损伤修复相关蛋白p-p53和p21表达水平。结果HE染色结果表明,淫羊藿苷能明显改善自然衰老大鼠睾丸组织形态学变化,显著提高衰老大鼠睾丸组织SOD活力,降低MDA水平;免疫荧光结果显示,淫羊藿苷能明显上调衰老大鼠睾丸组织中各级生精细胞内Nrf2蛋白表达水平,明显下调初级精母细胞和精原细胞中γ-H2AX蛋白表达水平;Western blotting结果显示,淫羊藿苷能显著上调衰老大鼠睾丸组织中Nrf2、HO-1和NQO1蛋白表达水平,下调γ-H2AX、p-p53和p21蛋白表达水平。结论淫羊藿苷可减轻自然衰老大鼠睾丸生殖细胞DNA损伤,其机制与激活Nrf2/HO-1信号通路有关。
Objective To study the protective effects of icariin on DNA damage of testicular germ cells in natural aging rats and explore the possible mechanism. Methods In the study, the SD rats were separated into four groups at random, with eight rats in each group: adult control group(2 months old), aging model group(16 months old), low and high doses of icariin-treated groups(2 mg/kg and 6 mg/kg, 16 months old). The adult control group and the aging model group were fed with normal diet for four months.Icariin-treated groups were given medicated feed for four months. After fasting for 12 h, all the rats were put to death. The testicular morphology was observed using HE staining. The activity of superoxide dismutase(SOD) and the content of malondialdehyde(MDA)in testicular tissue were detected by xanthine oxidase method and thiobarbituric acid method, respectively. The protein expression of Nrf2 and γ-H2 AX were detected by immunofluorescence. The relative protein expression levels of Nrf2, HO-1, NQO1, γ-H2 AX,p-p53, and p21 were detected by Western blotting. Results HE staining results showed that icariin significantly improved the structure of testicular tissue of natural aging rats. Compared with the aging model group, icariin significantly increased the SOD activity and decreased the MDA content in testis. In addition, immunofluorescence results showed that icariin significantly decreased the expression of DNA damage response protein γ-H2 AX and increased the protein expression of Nrf2 in testicular germ cells of aging rats.Moreover, Western blotting results showed that icariin significantly increased the relative protein expression levels of Nrf2, HO-1 and NQO1 in testis, when compared with aging model group. In parallel, the relative protein expression levels of γ-H2 AX, p-p53, and p21 were significantly decreased. Conclusion Icariin attenuates DNA damage in testicular germ cells of natural aging rats, which may be associated with the activation of Nrf2/HO-1 signaling pathway.
Objective To study the protective effects of icariin on DNA damage of testicular germ cells in natural aging rats and explore the possible mechanism. Methods In the study, the SD rats were separated into four groups at random, with eight rats in each group: adult control group(2 months old), aging model group(16 months old), low and high doses of icariin-treated groups(2 mg/kg and 6 mg/kg, 16 months old). The adult control group and the aging model group were fed with normal diet for four months.Icariin-treated groups were given medicated feed for four months. After fasting for 12 h, all the rats were put to death. The testicular morphology was observed using HE staining. The activity of superoxide dismutase(SOD) and the content of malondialdehyde(MDA)in testicular tissue were detected by xanthine oxidase method and thiobarbituric acid method, respectively. The protein expression of Nrf2 and γ-H2 AX were detected by immunofluorescence. The relative protein expression levels of Nrf2, HO-1, NQO1, γ-H2 AX,p-p53, and p21 were detected by Western blotting. Results HE staining results showed that icariin significantly improved the structure of testicular tissue of natural aging rats. Compared with the aging model group, icariin significantly increased the SOD activity and decreased the MDA content in testis. In addition, immunofluorescence results showed that icariin significantly decreased the expression of DNA damage response protein γ-H2 AX and increased the protein expression of Nrf2 in testicular germ cells of aging rats.Moreover, Western blotting results showed that icariin significantly increased the relative protein expression levels of Nrf2, HO-1 and NQO1 in testis, when compared with aging model group. In parallel, the relative protein expression levels of γ-H2 AX, p-p53, and p21 were significantly decreased. Conclusion Icariin attenuates DNA damage in testicular germ cells of natural aging rats, which may be associated with the activation of Nrf2/HO-1 signaling pathway.
引文
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[11]陈晓亮,张素琴.淫羊藿苷延缓衰老过程中对ATM-Chk2-p53通路的调节作用[J].老年医学与保健,2016,22(6):341-344.
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[13]马娜,赵海霞,陈茜,等.五子衍宗方对自然衰老大鼠睾丸生殖细胞DNA氧化损伤的保护作用研究[J].中国中药杂志,2018,43(14):2985-2990.
[14]Khodir A E,Said E,Atif H,et al.Targeting Nrf2/HO-1signaling by crocin:Role in attenuation of AA-induced ulcerative colitis in rats[J].Biomed Pharmacother,2019,doi:10.1016/j.biopha.2018.11.133.
[15]Song Y,Huang L,Yu J.Effects of blueberry anthocyanins on retinal oxidative stress and inflammation in diabetes through Nrf2/HO-1 signaling[J].J Neuroimmunol,2016,doi:10.1016/j.jneuroim.2016.11.001.
[16]Li Z,Feng H,Wang Y,et al.Rosmarinic acid protects mice from lipopolysaccharide/d-galactosamine-induced acute liver injury by inhibiting MAPKs/NF-κB and activating Nrf2/HO-1 signaling pathways[J].Int Immunopharmacol,2019,67:465-472.
[17]Li L,Dong H,Song E,et al.Nrf2/ARE pathway activation,HO-1 and NQO1 induction by polychlorinated biphenyl quinone is associated with reactive oxygen species and PI3K/AKT signaling[J].Chem Biol Interact,2014,doi:10.1016/j.cbi.2013.12.005.
[18]Liu X,Lin X,Zhang S,et al.Lycopene ameliorates oxidative stress in the aging chicken ovary via activation of Nrf2/HO-1 pathway[J].Aging,2018,10(8):2016-2036.
[19]Zhang H,Davies K J A,Forman H J.Oxidative stress response and Nrf2 signaling in aging[J].Free Radic Biol Med,2015,doi:10.1016/j.freeradbiomed.2015.05.036.
[20]刘建,陈朝晖,李文杰,等.氧化应激反应与不育症患者精子DNA损伤的相关性研究[J].临床医学工程,2018,25(6):791-792.
[21]Sharma A,Singh K,Almasan A.Histone H2AXphosphorylation:A marker for DNA damage[J].Methods Mol Biol,2012,doi:10.1007/978-1-61779-998-3_40.
[22]An J,Huang Y C,Xu Q Z,et al.DNA-PKcs plays a dominant role in the regulation of H2AX phosphorylation in response to DNA damage and cell cycle progression[J].BMC Mol Biol,2010,doi:10.1186/1471-2199-11-18.
[23]马娜,赵海霞,陈茜,等.基于P53/P21和碱基切除修复通路的五子衍宗方对自然衰老大鼠睾丸DNA损伤的保护作用研究[J].中草药,2018,49(6):1379-1384.
[24]Selvaratnam J S,Robaire B.Effects of aging and oxidative stress on spermatozoa of superoxide-dismutase1-and catalase-null mice[J].Biol Reprod,2016,doi:10.1095/biolreprod.116.141671.
[25]Speidel D.The role of DNA damage responses in p53biology[J].Arch Toxicol,2015,89(4):501-517.
[26]Talukder K A,Azmi I J,Ahmed K A,et al.Activation of p53/ATM-dependent DNA damage signaling pathway by shiga toxin in mammalian cells[J].Microb Pathog,2012,52(6):311-317.
[27]Kawase T,Kamiya M,Hayama K,et al.X-ray and ultraviolet C irradiation-inducedγ-H2AX and p53formation in normal human periosteal cells in vitro:Markers for quality control in cell therapy[J].Cytotherapy,2015,17(1):112-123.
[28]Dolan D W P,Zupanic A,Nelson G,et al.Integrated stochastic model of DNA damage repair by non-homologous end joining and p53/p21-mediated early senescence signalling[J].PLoS Comput Biol,2015,11(5):e1004246.
[29]Zilfou J T,Lowe S W.Tumor suppressive functions of p53[J].Cold Spring Harb Perspect Biol,2009,doi:10.1101/cshperspect.a001883.
[2]Sartorius G A,Nieschlag E.Paternal age and reproduction[J].Hum Reprod Update,2010,16(1):65-79.
[3]樊华,李文.雄性生殖氧化应激损伤的研究进展[J].中国临床医学,2016,23(2):242-246.
[4]Paul C,Nagano M,Robaire B.Aging results in molecular changes in an enriched population of undifferentiated rat spermatogonia[J].Biol Reprod,2013,doi:10.1095/biolreprod.113.112995.
[5]秦欣然,薛春然,彭麒桦,等.氧化应激对男性(雄性)生殖系统的影响[J].国际生殖健康/计划生育杂志,2018,37(6):492-497.
[6]王焕珍,柴艺汇,陈云志,等.淫羊藿化学成分与药理作用研究进展[J].亚太传统医药,2016,12(7):63-65.
[7]李丹,魏莎莉,谢怡,等.淫羊藿苷对糖尿病大鼠睾丸中亚硝基谷胱甘肽还原酶及Bcl-2的影响[J].解剖学杂志,2013,36(1):25-28.
[8]高学勇,林珊,韩咪莎.淫羊藿苷调节雄性大鼠生殖功能[J].解剖学杂志,2013,36(4):740-742.
[9]谢高宇,陈凯,刘浩然,等.淫羊藿苷对D-半乳糖致衰老大鼠睾丸组织MMP-2及MMP-9表达的影响[J].中国老年学杂志,2012,32(16):3458-3460.
[10]Zhang S Q,Cai W J,Huang J H,et al.Icariin,a natural flavonol glycoside,extends healthspan in mice[J].Exp Gerontol,2015,doi:10.1016/j.exger.2015.06.020.
[11]陈晓亮,张素琴.淫羊藿苷延缓衰老过程中对ATM-Chk2-p53通路的调节作用[J].老年医学与保健,2016,22(6):341-344.
[12]Miranda E P,Lorenzini F,Neves B,et al.Stereological and morphological analysis of the effects of aging on spermatogenesis in rat testis 1[J].Acta Cir Bras,2018,33(10):904-913.
[13]马娜,赵海霞,陈茜,等.五子衍宗方对自然衰老大鼠睾丸生殖细胞DNA氧化损伤的保护作用研究[J].中国中药杂志,2018,43(14):2985-2990.
[14]Khodir A E,Said E,Atif H,et al.Targeting Nrf2/HO-1signaling by crocin:Role in attenuation of AA-induced ulcerative colitis in rats[J].Biomed Pharmacother,2019,doi:10.1016/j.biopha.2018.11.133.
[15]Song Y,Huang L,Yu J.Effects of blueberry anthocyanins on retinal oxidative stress and inflammation in diabetes through Nrf2/HO-1 signaling[J].J Neuroimmunol,2016,doi:10.1016/j.jneuroim.2016.11.001.
[16]Li Z,Feng H,Wang Y,et al.Rosmarinic acid protects mice from lipopolysaccharide/d-galactosamine-induced acute liver injury by inhibiting MAPKs/NF-κB and activating Nrf2/HO-1 signaling pathways[J].Int Immunopharmacol,2019,67:465-472.
[17]Li L,Dong H,Song E,et al.Nrf2/ARE pathway activation,HO-1 and NQO1 induction by polychlorinated biphenyl quinone is associated with reactive oxygen species and PI3K/AKT signaling[J].Chem Biol Interact,2014,doi:10.1016/j.cbi.2013.12.005.
[18]Liu X,Lin X,Zhang S,et al.Lycopene ameliorates oxidative stress in the aging chicken ovary via activation of Nrf2/HO-1 pathway[J].Aging,2018,10(8):2016-2036.
[19]Zhang H,Davies K J A,Forman H J.Oxidative stress response and Nrf2 signaling in aging[J].Free Radic Biol Med,2015,doi:10.1016/j.freeradbiomed.2015.05.036.
[20]刘建,陈朝晖,李文杰,等.氧化应激反应与不育症患者精子DNA损伤的相关性研究[J].临床医学工程,2018,25(6):791-792.
[21]Sharma A,Singh K,Almasan A.Histone H2AXphosphorylation:A marker for DNA damage[J].Methods Mol Biol,2012,doi:10.1007/978-1-61779-998-3_40.
[22]An J,Huang Y C,Xu Q Z,et al.DNA-PKcs plays a dominant role in the regulation of H2AX phosphorylation in response to DNA damage and cell cycle progression[J].BMC Mol Biol,2010,doi:10.1186/1471-2199-11-18.
[23]马娜,赵海霞,陈茜,等.基于P53/P21和碱基切除修复通路的五子衍宗方对自然衰老大鼠睾丸DNA损伤的保护作用研究[J].中草药,2018,49(6):1379-1384.
[24]Selvaratnam J S,Robaire B.Effects of aging and oxidative stress on spermatozoa of superoxide-dismutase1-and catalase-null mice[J].Biol Reprod,2016,doi:10.1095/biolreprod.116.141671.
[25]Speidel D.The role of DNA damage responses in p53biology[J].Arch Toxicol,2015,89(4):501-517.
[26]Talukder K A,Azmi I J,Ahmed K A,et al.Activation of p53/ATM-dependent DNA damage signaling pathway by shiga toxin in mammalian cells[J].Microb Pathog,2012,52(6):311-317.
[27]Kawase T,Kamiya M,Hayama K,et al.X-ray and ultraviolet C irradiation-inducedγ-H2AX and p53formation in normal human periosteal cells in vitro:Markers for quality control in cell therapy[J].Cytotherapy,2015,17(1):112-123.
[28]Dolan D W P,Zupanic A,Nelson G,et al.Integrated stochastic model of DNA damage repair by non-homologous end joining and p53/p21-mediated early senescence signalling[J].PLoS Comput Biol,2015,11(5):e1004246.
[29]Zilfou J T,Lowe S W.Tumor suppressive functions of p53[J].Cold Spring Harb Perspect Biol,2009,doi:10.1101/cshperspect.a001883.