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
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摘要
目的:探讨五子衍宗方(Wuzi Yanzong Fang,WYF)基于磷酯酰肌醇3-激酶(PI3K)/丝氨酸苏氨酸蛋白激酶(Akt)信号通路对老龄大鼠精原干细胞增殖的作用及其机制研究。方法:以自然衰老大鼠为研究对象,将40只18月龄SPF级SD雄性大鼠随机分为老龄模型组,WYF低、中、高剂量组,每组10只,另以10只2月龄大鼠作为青年组。WYF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。免疫荧光法检测Sertoli细胞数量,实时荧光定量聚合酶链式反应(Real-time PCR)检测胶质细胞源性神经营养因子(GDNF),前髓细胞锌指蛋白(PLZF),干细胞因子(SCF)和骨形态蛋白4(BMP4)mRNA表达水平,免疫荧光法检测精原干细胞数量及增殖细胞核抗原(PCNA)的表达,蛋白免疫印迹法(Western blot)和免疫荧光法检测磷酯酰肌醇3-激酶(PI3K)和磷酸化丝氨酸苏氨酸蛋白激酶(p-Akt)的表达与定位。结果:与青年组比较,老龄模型组Sertoli细胞数量未见明显差异,而GDNF,PLZF,SCF和BMP4 mRNA表达水平显著降低,精原干细胞数量减少,PCNA表达下降,PI3K,p-Akt蛋白表达降低(P<0.01);与老龄模型组比较,WYF各剂量组GDNF,PLZF,SCF和BMP4 mRNA表达水平显著升高,精原干细胞数量增多,PCNA表达上调,PI3K,p-Akt表达明显增多(P<0.05,P<0.01)。结论:WYF能够明显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PI3K/Akt信号通路有关。
Objective: To investigate effect of Wuzi Yanzong Fang( WYF) on proliferation of spermatogonial stem cells in aging rats through phosphoinositol 3-kinase( PI3 K)/protein kinase( Akt) signaling pathway. Method: The 18-month-old male SD rats were randomly divided into aging model group,and low,middle and high-dose WYF groups,with 10 rats in each group. Ten 2-month-old rats were used as young control group. low,middle and high-dose WYF groups were given WYF 0. 4,0. 8,1. 6 g·kg~(-1) by gavage for 4 months,respectively. Young control group and aging model group were given normal saline,and the administration was suspended for 2 days a week. The administration lasted for 4 months. The rats were put to death after the final administration of WYF,and then the testes were quickly collected. The numbers of Sertoli cells were detected by immunofluorescence. The relative mRNA expression levels of glial cell line-derived neurotrophicfactor( GDNF),promyelocyte Zinc finger protein( PLZF),bone morphogenetic protein4( BMP4) and stem cell factor( SCF) in testicular tissues were detected by Real-time quantitative PCR( Real-time PCR). The numbers of spermatogonial stem cells and the expression levels of PCNA were detected by immunofluorescence. The expressions and localizations of PI3 K,Akt,p-Akt were detected by Western blot and immunofluorescence. Result: Compared with young control group,there was no significant difference in the numbers of Sertoli cells in each group,the mRNA expression levels of GDNF,PLZF,BMP4 and SCF in testis of aging model group were significantly decreased. The number of spermatogonial stem cells and levels of PI3 K,p-Akt,PCNA expression decreased( P < 0. 01). After WYF treatment,the levels of GDNF, PLZF, BMP4 and SCF were significantly increased, the numbers of spermatogonial stem cells were increased,and PCNA,PI3 K,p-Akt expressions were up-regulated( P < 0. 05,P < 0. 01). Conclusion: WYF can effectively promote the proliferation of spermatogonial stem cells in aging rats through PI3 K/Akt signaling pathway.
Objective: To investigate effect of Wuzi Yanzong Fang( WYF) on proliferation of spermatogonial stem cells in aging rats through phosphoinositol 3-kinase( PI3 K)/protein kinase( Akt) signaling pathway. Method: The 18-month-old male SD rats were randomly divided into aging model group,and low,middle and high-dose WYF groups,with 10 rats in each group. Ten 2-month-old rats were used as young control group. low,middle and high-dose WYF groups were given WYF 0. 4,0. 8,1. 6 g·kg~(-1) by gavage for 4 months,respectively. Young control group and aging model group were given normal saline,and the administration was suspended for 2 days a week. The administration lasted for 4 months. The rats were put to death after the final administration of WYF,and then the testes were quickly collected. The numbers of Sertoli cells were detected by immunofluorescence. The relative mRNA expression levels of glial cell line-derived neurotrophicfactor( GDNF),promyelocyte Zinc finger protein( PLZF),bone morphogenetic protein4( BMP4) and stem cell factor( SCF) in testicular tissues were detected by Real-time quantitative PCR( Real-time PCR). The numbers of spermatogonial stem cells and the expression levels of PCNA were detected by immunofluorescence. The expressions and localizations of PI3 K,Akt,p-Akt were detected by Western blot and immunofluorescence. Result: Compared with young control group,there was no significant difference in the numbers of Sertoli cells in each group,the mRNA expression levels of GDNF,PLZF,BMP4 and SCF in testis of aging model group were significantly decreased. The number of spermatogonial stem cells and levels of PI3 K,p-Akt,PCNA expression decreased( P < 0. 01). After WYF treatment,the levels of GDNF, PLZF, BMP4 and SCF were significantly increased, the numbers of spermatogonial stem cells were increased,and PCNA,PI3 K,p-Akt expressions were up-regulated( P < 0. 05,P < 0. 01). Conclusion: WYF can effectively promote the proliferation of spermatogonial stem cells in aging rats through PI3 K/Akt signaling pathway.
引文
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[19]Hossini A M,Quast A S,Pl9tz M,et al.PI3K/AKT signaling pathway is essential for survival of Induced pluripotent stem cells[J].PLo S One,2016,11(5):e0154770.
[20]Takashima S,Kanatsushinohara M,Tanaka T,et al.Functional differences between GDNF-dependent and FGF2-dependent mouse spermatogonial stem cell selfrenewal[J].Stem Cell Reports,2015,4(3):489-502.
[2]Gunes S,Hekim G N T,Arslan M A,et al.Effects of aging on the male reproductive system[J].J Assist Reprod Genet,2016,33(4):441-454.
[3]Giassetti M I,Goissis M D,Moreira P V,et al.Effect of age on expression of spermatogonial markers in bovine testis and isolated cells[J].Anim Reprod Sci,2016,170:68-74.
[4]柴智,王协和,樊慧杰,等.五子衍宗丸对生殖系统的保护作用及其临床研究进展[J].中华中医药杂志,2016,31(9):3662-3664.
[5]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.
[6]刘珍财,赵海霞,马娜,等.五子衍宗方对自然衰老大鼠睾丸损伤的保护作用及其机制研究[J].中药材,2017,40(12):2923-2927.
[7]YANG H,LIU Y,HAI Y,et al.Efficient conversion of spermatogonial stem cells to phenotypic and functional dopaminergic neurons via the PI3K/Akt and P21/Smurf2/Nolz1 pathway[J].Mol Neurobiol,2015,52(3):1654-1669.
[8]Kimura T,Nakano T.Regulation of stem cell systems by PI3K/Akt signaling[M].New York:Regul Networks in Stem Cells,2009:309-318.
[9]Almeida S,Rato L,Sousa M,et al.Fertility and sperm quality in the aging male[J].Current Pharmaceutical Design,2017,23(30):4429-4437.
[10]Silván U,Moreno P,Aréchaga J.The male germinal stem cell niche in mammals[M].Ottawa Ontario:Tissue-Specific Stem Cell Niche.Springer International Publishing,2015:313-326.
[11]Oatley J M,Brinster R L.The germline stem cell niche unit in mammalian testes[J].Physiol Rev,2012,92(2):577-595.
[12]Takashima S,Kanatsushinohara M,Tanaka T,et al.Functional differences between GDNF-dependent and FGF2-dependent mouse spermatogonial stem cell selfrenewal[J].Stem Cell Reports,2015,4(3):489-502.
[13]YANG Y,FENG Y,FENG X,et al.BMP4 cooperates with retinoic acid to induce the expression of differentiation markers in cultured mouse spermatogonia[J].Stem Cells Int,2016,2016(2):e9536192.
[14]Fran9a L R,Hess R A,Dufour J M,et al.The Sertoli cell:one hundred fifty years of beauty and plasticity[J].Andrology,2016,4(2):189-212.
[15]Rebourcet D,WU J,Cruickshanks L,et al.Sertoli cells modulate testicular vascular network development,structure,and function to influence circulating testosterone concentrations in adult male mice[J].Endocrinology,2016,157(6):2479-2488.
[16]Burkovics P,Dome L,Juhasz S,et al.The PCNAassociated protein PARI negatively regulates homologous recombination via the inhibition of DNA repair synthesis[J].Nucleic Acids Res,2016,44(7):3176-3189.
[17]Schultz M B,Sinclair D A.When stem cells grow old:phenotypes and mechanisms of stem cell aging[J].Development,2016,143(1):3-14.
[18]NIU B,BO L,WU C,et al.Melatonin promotes goat spermatogonia stem cells(SSCs)proliferation by stimulating glial cell line-derived neurotrophic factor(GDNF)production in Sertoli cells[J].Oncotarget,2016,7(47):77532-77542.
[19]Hossini A M,Quast A S,Pl9tz M,et al.PI3K/AKT signaling pathway is essential for survival of Induced pluripotent stem cells[J].PLo S One,2016,11(5):e0154770.
[20]Takashima S,Kanatsushinohara M,Tanaka T,et al.Functional differences between GDNF-dependent and FGF2-dependent mouse spermatogonial stem cell selfrenewal[J].Stem Cell Reports,2015,4(3):489-502.