五子衍宗方通过调节miR-let-7-Imp轴对老龄大鼠精原干细胞增殖的影响
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摘要
目的:通过miR-let-7-Imp轴探讨五子衍宗方(WZ)对老龄大鼠精原干细胞增殖的作用及其机制。方法:将40只18月龄SPF级SD雄性大鼠随机分为老龄模型组,WZ低、中、高剂量组,每组10只,另以10只2月龄大鼠作为青年组。WZ低、中、高剂量组分别灌胃给药0. 4,0. 8,1. 6 g·kg~(-1),青年组、老龄模型组分别灌胃给予生理盐水,每周停药2 d,持续给药4个月。末次给药后麻醉处死大鼠,快速取出睾丸组织。实时荧光定量聚合酶链式反应(Real-time PCR)检测miR-let-7,Imp mRNA表达水平,蛋白免疫印迹法(Western blot)和免疫荧光检测睾丸精原干细胞增殖相关信号通路磷酸化(p)-两面神激酶2(JAK2)/JAK2,p-信号传导及转录激活因子3(STAT3)/STAT3蛋白的表达与定位,免疫荧光检测精原干细胞数量及增殖相关蛋白增殖细胞核抗原(PCNA)的表达,苏木素-伊红(HE)染色观察睾丸组织形态学变化。结果:Real-time PCR结果显示,与青年组比较,老龄模型组miR-let-7 mRNA表达水平显著升高(P <0. 01),Imp mRNA表达水平显著降低(P <0. 01),而给予WZ干预后,miR-let-7 mRNA水平明显降低(P <0. 05,P <0. 01),Imp mRNA表达水平明显升高(P <0. 05); Western blot和免疫荧光结果显示,精原干细胞增殖相关信号通路蛋白p-JAK2,p-STAT3表达降低(P <0. 01),WZ处理后,p-JAK2,p-STAT3表达明显增多(P <0. 05,P <0. 01);免疫荧光结果显示,与青年组比较,老龄模型组中精原干细胞数量减少,PCNA表达下降,WZ处理后,精原干细胞数量增多,PCNA表达上调; HE结果也显示,WZ能够明显改善自然衰老所致的睾丸组织形态结构变化。结论:WZ能够促进老龄大鼠精原干细胞的增殖,其作用机制可能与调节睾丸内miR-let-7-Imp轴有关。
Objective: To explore the effect and mechanism of Wuzi Yanzong Fang( WZ) on proliferation of spermatogonial stem cells in aging rats by regulating miR-let-7-Imp axis. Method: A total of 40 18-month-old male SD rats were randomly divided into aging model group,and low,middle and high-dose WZ groups,with 10 rats in each group. Ten 2-month old rats were used as young control group. Low,middle and high-dose WZ groups were given by gastric WZ 0. 4,0. 8,1. 6 g·kg~(-1) respectively. Young control group and aging model group were given normal saline for 4 months,which was suspended for 2 days every week. Rats were put to death after the final treatment with WZ,and then the testes were quickly removed from rats. The relative mRNA expression levels of miR-let-7 and Imp were detected by Real-time fluorescence quantitative polymerase chain reaction( Realtime PCR). The expressions and localization of p-JAK2/JAK2, phosphate( p)-signal transduction and transcriptional activators3( STAT3)/STAT3 signaling pathway proteins were detected by Western blot and immunofluorescence. The numbers of spermatogonial stem cells and the expression levels of proliferating cell nuclear antigen( PCNA) were detected by immunofluorescence. The testicular tissue morphology was observed using htoxylin eosin( HE) staining. Result: Compared with young control group,the expression levels of miR-let-7 mRNA were significantly increased,while the expression levels of Imp mRNA were decreased in the aging model group( P < 0. 01). Conversely,the expression levels of miR-let-7 mRNA were significantly decreased( P < 0. 05,P < 0. 01),while the mRNA expression levels of Imp mRNA were significantly increased after WZ treatment( P <0. 05). The results of Western blotting and immunofluorescence showed that the expression levels of p-JAK2,p-STAT3 in aging model group were decreased significantly( P < 0. 01),while WZ significantly increased the expression levels of p-JAK2,p-STAT3( P < 0. 05,P < 0. 01). Immunofluorescence results showed that compared with young control group,the number of spermatogonial stem cells and PCNA expression decreased in aging model group. After WZ treatment,the number of spermatogonial stem cells was increased,whereas PCNA expression was up-regulated. HE showed that WZ significantly improved the testicular tissue structure of aging rats. Conclusion:WZ effectively promote the proliferation of spermatogonial stem cells by regulating miR-let-7-imp axis in testis.
Objective: To explore the effect and mechanism of Wuzi Yanzong Fang( WZ) on proliferation of spermatogonial stem cells in aging rats by regulating miR-let-7-Imp axis. Method: A total of 40 18-month-old male SD rats were randomly divided into aging model group,and low,middle and high-dose WZ groups,with 10 rats in each group. Ten 2-month old rats were used as young control group. Low,middle and high-dose WZ groups were given by gastric WZ 0. 4,0. 8,1. 6 g·kg~(-1) respectively. Young control group and aging model group were given normal saline for 4 months,which was suspended for 2 days every week. Rats were put to death after the final treatment with WZ,and then the testes were quickly removed from rats. The relative mRNA expression levels of miR-let-7 and Imp were detected by Real-time fluorescence quantitative polymerase chain reaction( Realtime PCR). The expressions and localization of p-JAK2/JAK2, phosphate( p)-signal transduction and transcriptional activators3( STAT3)/STAT3 signaling pathway proteins were detected by Western blot and immunofluorescence. The numbers of spermatogonial stem cells and the expression levels of proliferating cell nuclear antigen( PCNA) were detected by immunofluorescence. The testicular tissue morphology was observed using htoxylin eosin( HE) staining. Result: Compared with young control group,the expression levels of miR-let-7 mRNA were significantly increased,while the expression levels of Imp mRNA were decreased in the aging model group( P < 0. 01). Conversely,the expression levels of miR-let-7 mRNA were significantly decreased( P < 0. 05,P < 0. 01),while the mRNA expression levels of Imp mRNA were significantly increased after WZ treatment( P <0. 05). The results of Western blotting and immunofluorescence showed that the expression levels of p-JAK2,p-STAT3 in aging model group were decreased significantly( P < 0. 01),while WZ significantly increased the expression levels of p-JAK2,p-STAT3( P < 0. 05,P < 0. 01). Immunofluorescence results showed that compared with young control group,the number of spermatogonial stem cells and PCNA expression decreased in aging model group. After WZ treatment,the number of spermatogonial stem cells was increased,whereas PCNA expression was up-regulated. HE showed that WZ significantly improved the testicular tissue structure of aging rats. Conclusion:WZ effectively promote the proliferation of spermatogonial stem cells by regulating miR-let-7-imp axis in testis.
引文
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[16] TONG M,Debra M,Ryan E, et al. Expression of Mirlet7 family MicroRNAs in response to retinoic acidinduced spermatogonial differentiation in mice[J]. Biol Reprod,2011,85:189-197.
[17] Yoon H J,Mukesh K G,JI Y S,et al. MicroRNA signature in testes-derived male germ-line stem cells[J]. Mol Hum Reprod,2010,16(11):804-810.
[18] Fabrizio J J,Hickey C A,Stabrawa C,et al. Imp(IGF-II mRNA-binding protein)is expressed during spermatogenesis in Drosophila melanogaster[J]. Fly(Austin),2008,2(1):47-52.
[19] Vainchenker W, Constantinescu S N. JAK/STAT signaling in hematological malignancies[J]. Oncogene,2013,32(21):2601-2613.
[20] Levy O,Ruvinov E,Reem T,et al. Highly efficient osteogenic differentiation of human mesenchymal stem cells by eradication of STAT3 signaling[J]. Int J Biochem Cell Biol,2010,42(11):1823-1830.
[21] Oatley J M,Kaucher A V,Avarbock M R,et al.Regulation of mouse spermatogonial stem cell differentiation by STAT3 signaling[J]. Biol Reprod,2010,83(3):427-433.
[22] Oatley J M,Brinster R L. Regulation of spermatogonial stem cell self-renewal in mammals[J]. Annu Rev Cell Dev Biol,2008,24(1):263-286.
[23] Abike F,Tapisiz O L,Zergeroglu S,et al. PCNA and Ki-67 in endometrial hyperplasias and evaluation of the potential of malignancy[J]. Eur J Gynaecol Oncol,2011,32(1):77-80.
[24] Ryu B Y,Orwig K E,Oatley J M,et al. Effects of aging and niche microenvironment on spermatogonial stem cell self-renewal[J]. Stem Cells,2006,24:1505-1511.
[2] Matzuk M, Lamb D J. The biology of infertility:research advances and clinical challenges[J]. Nature Med,2008,14(11):1197-1213.
[3] Oatley J M,Brinster R L. Regulation of spermatogonial stem cell self-renewal in mammals[J]. Annu Rev Cell Dev Biol,2008,24:263-286.
[4] ZHANG X S,Ebata K T,Robaire B,et al. Aging of male germ line stem cells in mice[J]. Biol Reprod,2006,74:119-124.
[5] Boyerinas B,Park S M,Hau A,et al. The role of let-7in cell differentiation and cancer[J]. Endocr Relat Cancer,2010,17(1):19-36.
[6]李娌,王学美.五子衍宗方的临床运用及现代研究[J].中国实验方剂学杂志,2007,13(4):63-67.
[7]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损伤和凋亡的保护作用研究[J].中国中药杂志,2018,43(8):1675-1681.
[8]曾庆琪,孙磊,聂超,等.五子衍宗丸的药理学研究进展[J].中华中医药学刊,2014,32(8):1935-1937.
[9]刘珍财,赵海霞,马娜,等.基于PI3K/Akt通路观察五子衍宗方对老龄大鼠精原干细胞增殖的影响[J].中国实验方剂学杂志,2018,24(23):119-125.
[10]黄威峰,张长城,刘静,等.五子衍宗方对环磷酰胺致成年雄性小鼠睾丸生殖细胞凋亡的保护作用[J].中药材,2016,39(5):1143-1147.
[11]王虹滕,鸿琦,施珏平,等. let-7microRNA调控动物器官发育的研究进展[J].生命科学,2011,23(4):364-368.
[12] Pasquinelli A E, Reinhart B J, Slack F, et al.Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA[J]. Nature,2000,408(6808):86-89.
[13] Toledanol H,D'Alteriol C,Czech B,et al. The let-7-Imp axis regulates ageing of the Drosophila testis stemcell niche[J]. Nature,2012,485(7400):605-610.
[14] Melton C, Judson R L, Blelloch R. Opposing microRNA families regulate self-renewal in mouse embryonic stem cells[J]. Nature,2010,463(7281):621-626.
[15] Ramachandran R,Fausett B V,Goldman D. Ascl1a regulates muller glia dedifferentiation and retinal regeneration through a Lin-28-dependent, let-7microRNA signalling pathway[J]. Nat Cell Biol,2010,12(11):1101-1107.
[16] TONG M,Debra M,Ryan E, et al. Expression of Mirlet7 family MicroRNAs in response to retinoic acidinduced spermatogonial differentiation in mice[J]. Biol Reprod,2011,85:189-197.
[17] Yoon H J,Mukesh K G,JI Y S,et al. MicroRNA signature in testes-derived male germ-line stem cells[J]. Mol Hum Reprod,2010,16(11):804-810.
[18] Fabrizio J J,Hickey C A,Stabrawa C,et al. Imp(IGF-II mRNA-binding protein)is expressed during spermatogenesis in Drosophila melanogaster[J]. Fly(Austin),2008,2(1):47-52.
[19] Vainchenker W, Constantinescu S N. JAK/STAT signaling in hematological malignancies[J]. Oncogene,2013,32(21):2601-2613.
[20] Levy O,Ruvinov E,Reem T,et al. Highly efficient osteogenic differentiation of human mesenchymal stem cells by eradication of STAT3 signaling[J]. Int J Biochem Cell Biol,2010,42(11):1823-1830.
[21] Oatley J M,Kaucher A V,Avarbock M R,et al.Regulation of mouse spermatogonial stem cell differentiation by STAT3 signaling[J]. Biol Reprod,2010,83(3):427-433.
[22] Oatley J M,Brinster R L. Regulation of spermatogonial stem cell self-renewal in mammals[J]. Annu Rev Cell Dev Biol,2008,24(1):263-286.
[23] Abike F,Tapisiz O L,Zergeroglu S,et al. PCNA and Ki-67 in endometrial hyperplasias and evaluation of the potential of malignancy[J]. Eur J Gynaecol Oncol,2011,32(1):77-80.
[24] Ryu B Y,Orwig K E,Oatley J M,et al. Effects of aging and niche microenvironment on spermatogonial stem cell self-renewal[J]. Stem Cells,2006,24:1505-1511.