当归多糖对D-半乳糖致衰老小鼠睾丸的保护作用

邱竹 姜蓉 汪子铃 陈粼波 陈雄斌 王璐 黄国宁 王亚平*

doi:10.16098/j.issn.0529-1356.2019.04.017

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解剖学报 ›› 2019, Vol. 50 ›› Issue (4) : 506-511. DOI: 10.16098/j.issn.0529-1356.2019.04.017

组织学胚胎学发育生物学

当归多糖对D-半乳糖致衰老小鼠睾丸的保护作用

邱竹¹姜蓉¹ 汪子铃¹陈粼波¹陈雄斌¹ 王璐¹ 黄国宁²王亚平^1*

作者信息 +

Protective effects of Angelica Sinensis polysacchairides on the testis ofaging model mice induced by D-galactose

QIU Zhu¹JIANG Rong¹ WANG Zi-ling¹ CHEN Lin-bo¹ CHEN Xiong-bin¹WANG Lu¹ HUANG Guo-ning² WANG Ya-ping^1*

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摘要

目的探讨当归多糖（ASP）对D-半乳糖（D-Gal）所致衰老小鼠睾丸的保护作用与机制。方法 C57BL/6 J雄性小鼠40只，随机分为对照组、ASP对照组、衰老模型组ASP衰老模型组，每组10只。衰老模型组：小鼠颈背部皮下注射D-Gal(120 mg/kg，qd×42)；对照组小鼠皮下注射等时与等量生理盐水；ASP对照组小鼠皮下注射等量生理盐水15 d，第16天开始腹腔注射ASP(140 mg/kg，qd×27)；ASP衰老模型组小鼠建立方法同衰老模型组小鼠，第16天开始腹腔注射ASP（同ASP对照组）。模型复制完成第2天，采集各组小鼠内眦静脉血测定血清睾酮水平；取睾丸测定脏器指数；石蜡切片，HE染色观察睾丸组织学形态；做冷冻睾丸组织切片，衰老相关β-半乳糖苷酶（SA-β-Gal）染色观察睾丸组织细胞衰老情况；制备睾丸组织匀浆上清液，酶学法检测超氧化物歧化酶(SOD)活性，2，2’-连氮基-双（3-乙基苯并二氢噻唑啉-6-磺酸）二铵盐（ABTS）法检测总抗氧化能力(T-AOC)，硫代巴比妥酸法检测丙二醛(MDA)含量；Western blotting检测衰老相关蛋白P53、P21表达水平。结果各组间睾丸湿重及睾丸器官指数无明显差异，但衰老模型组小鼠睾丸组织结构损伤明显，生精小管的细胞层数减少，生精细胞出现退变，睾丸间质细胞明显减少；血清睾酮水平显著降低；SA-β-Gal染色阳性细胞密度显著增加；睾丸组织匀浆中SOD活性及T-AOC显著降低，MDA含量显著升高；P53、P21蛋白表达显著升高。与衰老模型组比较，注射ASP干预衰老过程，睾丸组织学结构损伤明显减轻，生精细胞退变与睾丸间质细胞减少均不明显；血清睾酮水平降低不明显；SA-β-Gal染色阳性细胞密度减少；SOD活性和T-AOC增高，MDA含量降低；P53、P21蛋白表达显著降低。结论 ASP具有拮抗致衰剂D-gal对睾丸的损伤作用，其机制可能与ASP抑制氧化应激损伤及抑制衰老相关基因表达有关。

Abstract

Objective To investigate the protective effects of Angelica Sinensis polysacchairides (ASP) on the testis of aging mice induced by D-galactose(D-Gal) and its mechanism. Methods Forty male C57BL/6 J mice were randomly divided into the normal,ASP normal,aging model and ASP aging model groups,with 10 mice in each group.The aging model group mice were subcutaneously injected with D-Gal(120 mg/kg，qd×42);the normal group mice were subcutaneously injected with saline of the same dose at the same time;the ASP normal group mice were subcutaneously injected with the same amount of saline,qd×15,and following intraperitoneally injected with ASP(140 mg/kg，qd×27);the ASP aging model group were the same as the aging model group, from the 16th day of establishment of the aging model group on, the mice were subcutaneously injected with the same dose and the same time of ASP.The 2nd day after model establishment,peripheral blood was collected from the inner canthus vein and the content of serum testosterone was measured;the testes were sampled and testicular index determined; paraffin sections and HE staining of the testes were prepared,the testis histopathology was observed;frozen sections of the testes were prepared,the aging of testicular cells was detected by senescence associated-β-galactosidase(SA-β-Gal) staining;the tissue homogenate of testes was prepared,the activity of superoxide dismutase(SOD) was evaluated by enzymatic method ,the total antioxidant capacity(T-AOC) was detected by 2,2’-amino-di(3-ethyl-benzothiazoline sulphonic acid-6) ammonium salt(ABTS),the content of malondialdehyde(MDA) was measured by thiobarbituric acid method ;the expression of aging-related proteins P53 and P21 was detected by Western blotting. Results There is no significant difference in the wet weight of testis and the index of testicular organs in the groups,but the testicular tissue structure of the aging model group was obviously damaged.The layers of the spermatogenic cells in seminiferous epithelium,the testicular interstitial cells and the serum testosterone level significantly decreased;the positive cells detected by SA-β-Gal stain markedly increased;the activity of superoxide dismutase(SOD) and the total antioxidant capacity(T-AOC) obviously decreased,while the content of malondialdehyde(MDA) increased significantly;and the expression of P53 and P21 was evidently up-regulated.Compared the ASP aging model group with the aging model group,the testicular tissue structure was not obviously damaged.The number of the spermatogenic cells,the testicular interstitial cells and the serum testosterone level decreased less evidently,the SA-β-Gal stain positive cells decreased significantly;the activity of superoxide dismutase(SOD) and the total anti oxidant capacity(T-AOC) increased significantly,while the content of malondialdehyde(MDA) decreased significantly;and the expression of P53 and P21 was remarkably down-regulated. Conclusion ASP can antagonize testis aging induced by D-galactose in mice.Inhibition of oxidative stress damage and the expression of senescence-associated genes may be the underlying mechanism.

导出引用

邱竹姜蓉汪子铃陈粼波陈雄斌王璐黄国宁王亚平. 当归多糖对D-半乳糖致衰老小鼠睾丸的保护作用[J]. 解剖学报. 2019, 50(4): 506-511 https://doi.org/10.16098/j.issn.0529-1356.2019.04.017

QIU Zhu JIANG Rong WANG Zi-ling CHEN Lin-bo CHEN Xiong-bin WANG Lu HUANG Guo-ning WANG Ya-ping. Protective effects of Angelica Sinensis polysacchairides on the testis ofaging model mice induced by D-galactose[J]. Acta Anatomica Sinica. 2019, 50(4): 506-511 https://doi.org/10.16098/j.issn.0529-1356.2019.04.017

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