解脲支原体感染对大鼠生精细胞线粒体核糖体蛋白S22的影响及知柏地黄汤的干预作用
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摘要
目的:观察解脲支原体(UU)感染对大鼠睾丸生精细胞线粒体核糖体蛋白S22(MRPS22)表达的影响及知柏地黄汤的干预作用。方法:UU感染SD雄性大鼠模型45只,随机分成模型组、知柏地黄汤组、西药组,并以健康大鼠为对照组,每组15只。造模成功后第10天起知柏地黄汤组予以知柏地黄汤灌胃[1 g/(k·d)],西药组予以阿奇霉素灌胃[0.105 g/(k·d)],对照组、模型组予以同体积生理盐水灌胃,连续灌胃21 d。采用TUNEL法检测生精细胞凋亡,电镜观察生精细胞线粒体结构,流式细胞仪检测线粒体膜电位(MMP),RT-PCR检测大鼠睾丸生精细胞MRPS22 m RNA表达,Western印迹检测大鼠睾丸生精细胞MRPS22蛋白表达。观察各组大鼠睾丸生精细胞凋亡指数(AI)、生精细胞线粒体结构、MMP、生精细胞MRPS22 m RNA和蛋白表达情况。结果:模型组大鼠睾丸生精细胞AI[(11.23±1.65)%]显著高于知柏地黄汤组[(6.62±0.49)%]和西药组[(7.82±0.81)%](P <0. 01),知柏地黄汤组显著低于西药组(P <0.05)。知柏地黄汤组及西药组大鼠睾丸线粒体结构较模型组显著改善。模型组大鼠睾丸生精细胞MMP水平[(8. 77±1. 73)%]显著低于对照组[(22. 33±1. 66)%](P <0. 01),知柏地黄汤组[(18. 26±1. 32)%]显著高于模型组(P <0.01)和西药组[(15.91±1.69)%],西药组MMP水平显著高于模型组(P <0. 01)。模型组MRPS22 m RNA表达(8.02±3.21)与对照组(15.43±2.54)、知柏地黄汤组(11. 26±3. 82)相比均具有显著性差异(P <0. 01),知柏地黄汤组MRPS22 m RNA表达显著高于西药组(8. 79±2. 03)(P <0. 01)。模型组(22. 65±5. 31) MRPS22蛋白表达与对照组(33.31±7.09)、知柏地黄汤组(33.35±3.96)和西药组(28.11±4.13)相比均具有显著性差异(P <0. 01),知柏地黄汤组显著高于西药组(P <0.01)。大鼠睾丸生精细胞MRPS22蛋白表达量与MMP呈显著正相关(r=0.639,P <0.01)。结论:UU感染可能通过抑制大鼠睾丸生精细胞MRPS 22 m RNA及蛋白表达,降低MMP,引起生精细胞的凋亡。知柏地黄汤通过改善UU感染大鼠睾丸生精细胞MRPS22 m RNA及蛋白表达,提高MMP,减轻大鼠睾丸生精细胞凋亡。
Objective:To investigate the impact of Ureaplasma urealyticum(UU) infection(UUI) on the expression of the mitochondrial ribosomal protein S22(MRPS22) in rat spermatogenic cells and the intervening effect of Zhibai Dihuang Decoction(ZBDH).Methods:Forty-five SD male rats were randomly divided into four groups of equal number:normal control,UUI model control,ZBDH and azithromycin,and the UUI model was made by bladder injection of the standard UU strain in the latter three groups.After modeling,the rats in the ZBDH and azithromycin groups were treated intragastrically with ZBDH at 1 g/kg/d and azithromycin at 0.105 g/kg/d respectively,while those in the normal and UUI model control groups with normal saline at 1 ml/kg/d.At 21 days after intervention,all the animals were sacrificed and their testes harvested for observation of the apoptosis and mitochondrial ultrastructure of the spermatogenic cells,measurement of the mitochondrial membrane potential(MMP) by flow cytometry,and determination of the m RNA and protein expressions of MRPS22 by RT-PCR and Western blot,respectively.Results:The apoptotic index of the rat spermatogenic cells was significantly higher in the UUI model control than in the ZBDH and azithromycin groups([11.23 ± 1.65]% vs [6.62 ±0.49]% and [7.82 ± 0.81]%,P < 0.01),but lower in the ZBDH than in the azithromycin group(P < 0.05).The mitochondrial ultrastructure of the spermatogenic cells was markedly improved in the ZBDH and azithromycin groups as compared with that in the model control.The MMP level was remarkably lower in the model control than in the normal control([8.77 ± 1.73]% vs [22.33 ± 1.66]%,P <0.01),but higher in the ZBDH([18.26 ± 1.32] %) than in the model control(P < 0.01) and the azithromycin group([15.91 ±1.69]%)(P < 0.01).The m RNA and protein expressions of MRPS22 were significantly lower in the model control(8.02 ± 3.21 and22.65 ± 5.31) than in the normal control(15.43 ± 2.54 and 33.31 ± 7.09),ZBDH(11.26 ± 3.82 and 33.35 ± 3.96),and azithromycin group(8.79 ± 2.03 and 28.11 ± 4.13)(all P < 0.01),but both higher in the ZBDH than in the azithromycin group(P < 0.01).There was a positive correlation between the MRPS22 protein expression and MMP(r = 0.639,P < 0.01).Conclusion:UU infection induces the apoptosis of rat spermatogenic cells by inhibiting the m RNA and protein expressions of MRPS22 and reducing the mitochondrial membrane potential,while ZBDH can decrease the apoptosis of spermatogenic cells by improving the m RNA and protein expressions of MRPS22 and enhancing the mitochondrial membrane potential.
Objective:To investigate the impact of Ureaplasma urealyticum(UU) infection(UUI) on the expression of the mitochondrial ribosomal protein S22(MRPS22) in rat spermatogenic cells and the intervening effect of Zhibai Dihuang Decoction(ZBDH).Methods:Forty-five SD male rats were randomly divided into four groups of equal number:normal control,UUI model control,ZBDH and azithromycin,and the UUI model was made by bladder injection of the standard UU strain in the latter three groups.After modeling,the rats in the ZBDH and azithromycin groups were treated intragastrically with ZBDH at 1 g/kg/d and azithromycin at 0.105 g/kg/d respectively,while those in the normal and UUI model control groups with normal saline at 1 ml/kg/d.At 21 days after intervention,all the animals were sacrificed and their testes harvested for observation of the apoptosis and mitochondrial ultrastructure of the spermatogenic cells,measurement of the mitochondrial membrane potential(MMP) by flow cytometry,and determination of the m RNA and protein expressions of MRPS22 by RT-PCR and Western blot,respectively.Results:The apoptotic index of the rat spermatogenic cells was significantly higher in the UUI model control than in the ZBDH and azithromycin groups([11.23 ± 1.65]% vs [6.62 ±0.49]% and [7.82 ± 0.81]%,P < 0.01),but lower in the ZBDH than in the azithromycin group(P < 0.05).The mitochondrial ultrastructure of the spermatogenic cells was markedly improved in the ZBDH and azithromycin groups as compared with that in the model control.The MMP level was remarkably lower in the model control than in the normal control([8.77 ± 1.73]% vs [22.33 ± 1.66]%,P <0.01),but higher in the ZBDH([18.26 ± 1.32] %) than in the model control(P < 0.01) and the azithromycin group([15.91 ±1.69]%)(P < 0.01).The m RNA and protein expressions of MRPS22 were significantly lower in the model control(8.02 ± 3.21 and22.65 ± 5.31) than in the normal control(15.43 ± 2.54 and 33.31 ± 7.09),ZBDH(11.26 ± 3.82 and 33.35 ± 3.96),and azithromycin group(8.79 ± 2.03 and 28.11 ± 4.13)(all P < 0.01),but both higher in the ZBDH than in the azithromycin group(P < 0.01).There was a positive correlation between the MRPS22 protein expression and MMP(r = 0.639,P < 0.01).Conclusion:UU infection induces the apoptosis of rat spermatogenic cells by inhibiting the m RNA and protein expressions of MRPS22 and reducing the mitochondrial membrane potential,while ZBDH can decrease the apoptosis of spermatogenic cells by improving the m RNA and protein expressions of MRPS22 and enhancing the mitochondrial membrane potential.
引文
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[11]Wang P,Luo C,Li Q,et al.Mitochondrion-mediated apoptosis isinvolved in reproductive damage caused by BPA in male rats.Environ Toxicol Pharmacol,2014,38(3):1025-1033.
[12]Guo M,Zhang P,Xi HJ.The expression of laminin in mouse developing kidney.Acta Anatomca Sinica,2005,36(6):688-691.
[13]Kondo S,Scheef EA,Sheibanei N,et al.PECAM-1 iso-formspecific regulation of kidney endothelial cell migration and capillary morphogenesis.Am J Physiol Cell Physiol,2007,292(6):2070-2083.
[14]Lai L,Chen J,Hao CM,et al.Aldosterone promotes fibronectin production through a Smad 2-dependent TGF-beta1 pathway in mesangial cells.Biochem Biophys Res Commun,2006,348(1):70-75.
[15]何清湖,宾东华,周青,等.知柏地黄汤对解脲脲原体感染模型大鼠精子线粒体呼吸链复合物的影响.中华男科学杂志,2016,22(11):1005-1010.
[16]刘朝圣,卢芳国,何清湖,等.知柏地黄汤对解脲脲原体感染大鼠生精细胞凋亡及Caspase-3,Gaspase-9表达的影响.中国中西医结合杂志,2011,31(9):1254-1258.
[2]Huang YF,Xiong CL,Shang XJ,et al.Ureaplasmaurealyticum infection and apoptosis of spermatogenic cells.Asian J Androl,1999,1(3):127-129.
[3]郭炫佐,何清湖,李迎秋.知柏地黄汤对UU感染大鼠生精细胞凋亡及凋亡因子Cyt-c、AIF表达的影响.湖南中医药大学学报,2016,36(1):15-18.
[4]Guan Y,Martin GB.Cellular and molecular responses of adult testis to changes in nutrition:Novel insights from the sheep model.Reproduction,2017,154(5):R133-R141.
[5]郭军华,李迎秋,郭炫佐,等.知柏地黄汤对解脲脲原体感染大鼠生精细胞线粒体细胞色素氧化酶的影响.中华男科学杂志,2017,23(8):722-727.
[6]孙鲁宁,宋晓宇,张静萍,等.线粒体核糖体蛋白S22对细胞凋亡的影响.中国现代医学杂志,2011,21(32):3984-3987.
[7]王喆,陆承荣,罗渊,等.解脲支原体大鼠感染模型的建立.实验动物科学,2011,28(2):74-75.
[8]Lech T,Styrna J,Kotarska K.The contribution of p53 and Ychromosome longarm genes to regulation of apoptosis in mouse testis.Reprod Fertil Dev,2018,30(3):469-476.
[9]Eskandari M,Jani S,Kazemi M,et al.Ameliorating effect of Ginseng on epididymo-orchitis inducing alterations in sperm quality and spermatogenic cells apoptosis following infection by uropathogenic Escherichia coli in rats.Cell J,2016,18(3):446-457.
[10]董煜,王为,陈国元.线粒体途径在生精细胞凋亡中的研究进展.国外医学:医学地理分册,2013,34(1):65-70.
[11]Wang P,Luo C,Li Q,et al.Mitochondrion-mediated apoptosis isinvolved in reproductive damage caused by BPA in male rats.Environ Toxicol Pharmacol,2014,38(3):1025-1033.
[12]Guo M,Zhang P,Xi HJ.The expression of laminin in mouse developing kidney.Acta Anatomca Sinica,2005,36(6):688-691.
[13]Kondo S,Scheef EA,Sheibanei N,et al.PECAM-1 iso-formspecific regulation of kidney endothelial cell migration and capillary morphogenesis.Am J Physiol Cell Physiol,2007,292(6):2070-2083.
[14]Lai L,Chen J,Hao CM,et al.Aldosterone promotes fibronectin production through a Smad 2-dependent TGF-beta1 pathway in mesangial cells.Biochem Biophys Res Commun,2006,348(1):70-75.
[15]何清湖,宾东华,周青,等.知柏地黄汤对解脲脲原体感染模型大鼠精子线粒体呼吸链复合物的影响.中华男科学杂志,2016,22(11):1005-1010.
[16]刘朝圣,卢芳国,何清湖,等.知柏地黄汤对解脲脲原体感染大鼠生精细胞凋亡及Caspase-3,Gaspase-9表达的影响.中国中西医结合杂志,2011,31(9):1254-1258.