吸烟对雄性大鼠生殖毒性的实验研究
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摘要
目的：研究吸烟对大鼠下丘脑-垂体-睾丸轴相关激素的影响及对睾丸细胞的毒性作用，通过对相关凋亡基因研究分析其可能的机制；研究香烟烟雾提取物对睾丸细胞的细胞毒性，分析其可能机制。方法：体内实验方法，选用5周SD大鼠80只，分为对照组与吸烟组，吸烟组采用静式染毒，每天1次，每次2h，连续8周；对照组采用空白对照。实验第2周、第4周、第6周与第8周分别处死对照组与吸烟组大鼠10只，测定各种指标。放射免疫法测定血清中睾酮、黄体生成激素、卵泡雌激素、胰岛素样生长因子-1及下丘脑促性腺激素释放激素；HE染色观察睾丸的结构形态；TUNEL法定性、定量测定睾丸的细胞凋亡；Western Blot检测各组大鼠睾丸组织中Fas/FasL与Bcl-2/Bax蛋白表达；RT-PCR检测各组大鼠睾丸组织中Fas/FasL与Bcl-2/BaxmRNA表达。用细胞培养研究香烟烟雾提取物对大鼠睾丸的支持细胞、间质细胞与生精细胞的影响。取对数生长期细胞用于试验，三种睾丸细胞培养，按照CSE干预浓度不同，将试验分为三组：0%CSE组、5%CSE组、10%CSE组。各组细胞培养12h后，采用MTT法检测CSE对细胞增殖的影响，流式细胞术检测细胞凋亡的情况，生物化学的方法检检测胞外乳酸脱氢酶的改变。结果：1）随着时间延长，吸烟组大鼠体重明显低于对照（P＜0.05）；2）大鼠血清中睾酮含量自第四周起吸烟组明显低于对照组（P＜0.05）；吸烟组大鼠血清T含量随染毒时间延长明显下降（P＜0.05）；3）大鼠血清中黄体生成素含量在对照组有随着时间延长逐渐增高，在吸烟组则随时间下降；第四周起吸烟组大鼠血清中黄体生成素含量明显低于对照组（P＜0.05）；4）大鼠血清中卵泡刺激素在对照组与吸烟组含量稳定，自第二周起吸烟组含量明显低于对照组（P＜0.05）；5）大鼠血清中胰岛素样生长因子-1含量随时间下降，吸烟组大鼠血清中胰岛素样生长因子-1在第八周含量明显低于第二周、第四周与第六周（P＜0.05），吸烟组第二周、第六周和第八周均明显低于对照组（P＜0.05）；6）大鼠下丘脑促性腺激素释放激素无论是对照组还是吸烟组随时间延长而升高，吸烟组与对照组比较差异无统计学意义（P＞0.05）；7）睾丸HE染色发现吸烟可以导致睾丸结构组织的破坏，随着时间的延长，组织改变明显加深。第八周组吸烟组睾丸组织出现明显的萎缩，大量的细胞裂解，细精管与间质组织出现明显的变性改变；8）TUNEL法测定睾丸细胞的凋亡率，吸烟组第四周、第六周、第八周细胞凋亡率明显增高（P＜0.05）；9）吸烟组大鼠睾丸组织中Fas/FasL与Bax蛋白上调，Bcl-2蛋白下调；Fas/FasL、Bax基因的mRNA表达水平有上调的趋势，结果与蛋白表达相同。吸烟第八周大鼠睾丸的Fas/FasL、Bax基因表达水平明显上升（P＜0.05），Bcl-2mRNA下调明显（P＜0.05）10）香烟烟雾提取物对支持细胞增值的影响没有显著性差异（P＞0.05）；对间质细胞、生精细胞增值随浓度的升高出现明显的下降（P＜0.05），且呈现一定的剂量反应关系；香烟烟雾提取物组的睾丸支持细胞、间质细胞和生精细胞组胞外LDH活力明显高于对照组（P＜0.05）；11）流式细胞仪测定结果表明CSE导致支持细胞、间质细胞和生精细胞组胞凋亡率上升。结论：吸烟是青春期大鼠生殖系统发育的有害因素，不仅下丘脑-垂体-睾丸轴中重要的激素有影响，而且对睾丸组织与细胞有不利影响，其可能的机制是吸烟激活了细胞凋亡调控基因，导致相应蛋白表达增加，同时抑制了保护性蛋白的表达；香烟烟雾提取物中含有导致睾丸支持细胞、间质细胞和生精细胞凋亡的化学物质，这提示吸烟可能导致青春期大鼠生殖功能发育迟缓，精子数量和功能的减少。
Objective: To research the effect of smoking on hypothalamic-pituitary-testicularaxis hormones and the toxic effects on testicular cells in rat, and analysis of the possiblemechanisms by related apoptosis gene. To study of cigarette smoke extract on testicularcell cytotoxicity and analysis of the possible mechanisms Methods:80SD rats wererandomly divided into8groups. Four groups were control group (C), and the others wereexposed groups, which was inhaled cigarette smoke for2h per day and continued for8weeks. The smoking group and the compared were respectively divided into4sub-groups,i.e. sub-group of2weeks inhalation, sub-group of4weeks inhalation, sub-group of6weeks inhalation, sub-group of8weeks inhalation and the reproduction group with10male mice in each group. By the end of2ed4th6th8thweek10male mice in the smokinggroup and the compared group were respectively killed and the index were observed. Thetestosterone (T), luteinizing hormone (LH), follicle stimulating hormone (FSH) andinsulin-like growth factor-1(IGF-1) in rat serum and gonadotropin-releasing hormone(GnRH) in rat hypothalamus were determined by radioimmunoassay kit. The testicularorganization structure were observed with HE staining, The apoptosis of testicular cellsof different groups was located and quantitated in morphologic and cellular levels bymethod of Methyl green-pyronine staining and temimal dexynucleotidyl tranferase-mdiated dUTP biotin nick end labeling (TUNEL). The Bcl-2、Bax、Fas、FasL expressionsin testis of different groups were located and quantitated by method of Western blotting.Each group the level of Fas, Fas-L, Bcl-2mRNA expression were measured by RT-PCR.It was studied that the effect of cigarette smoke extract (CSE) on the proliferation andgrowth of pdmarily cultured Sertoli cells, Leyding cell and Spermatogenic cells in vitro.The three cells were exposed to different concentrations of CSE for12hours, which weredivided into three groups according to its concentration, including0%CSE group、 5%CSEgroup10%CSE group. Lactate dehydrogenase (LDH) was detected bybiochemistry, cell proliferation by MTT assay and CSE induced cell apoptosis wasobserved by Flow Cytometry. Results:1) With the extension of exposed time of smoking,the increase of weight in the smoke group were apparently lower than those of thecompared group especially in the group of4th,6thand8thweeks inhalation (P＜0.05).2)The testosterone in the smoking group significantly lower than the control group after the4thweeks (P＜0.05) and it was reduced with the extension of exposed time (P＜0.05).3)LH in the control rat serum was obviously raised with time elapsed especially in thegroup of6thand8thweeks, while it in the smoking group with time extended declined.The difference of LH between the smoking group and the control group was alsosignificant (P＜0.05).4) FSH was stable in the rat serum of the control group and thesmoking group, and it was distinctly lowered since the second week of the smokinggroup than the control group (P＜0.05);5) IGF-1levels decreased with time. IGF-1insmoking group of the8thweeks was significantly lower than the2edweeks, the4thweekand the6thweek (P＜0.05), while the2edweek, the6thweek and the8thweek of thesmoking group were significantly lower than those in the control group (P＜0.05)6)Gonadotropin-releasing hormone of rat hypothalamus either the control group or thesmoking group increased with time. GnRH in the smoking group was not statisticallysignificant than the control group (P＞0.05);7) The result of HE stain was that smokingcan lead to the testis tissue destruction, and organizational change was deepened withtime. The testis in the8thweek of the smoking group smoking obvious atrophy, a largenumber of cell lysis significant degenerative changes in the seminiferous tubules andinterstitial tissue;8) The result of TUNEL was that apoptosis index of the cells in thetestis of the4th,6thand8thsmoking group significantly higher than the control (P＞0.05);9) Fas/FasL and Bax protein in rat testis of the smoking group was raised while the Bcl-2protein was decresed. The mRNA expression of Fas/FasL, Bax was increased, the sameresults with protein expression. The gene expression of Fas/FasL, Bax in the smoking8thweeks t Fas/FasL, Bax was increased significantly (P＜0.05) and Bcl-2mRNA waslowered significantly (P＜0.05);10) the cell proliferation of the Leyding cell andSpermatogenic cells with the increased CSEs’ concentration was decreased significantly(P＜0.05) and appeared a dose-response relationship; LDH of the Sertoli cells, theLeyding cells and the Spermatogenic cells in CSEs group was significantly higher thanthat in the control group (P＜0.05);11) The result measured by flow cytometry showedthat CSE leaded to the apoptosis rate of the Sertoli cells, the Leyding cells and the Spermatogenic was significantly increased (P＜0.05). Conclusion: Smoking is harmfulfactors on the development of pubertal rats reproductive system, and have an adverseeffect on not only the Hypothalamic-pituitary-Testicular axis hormones but also testiculartissue and cells. The activation apoptosis regulatory genes by smoking leaded to acorresponding increase in protein expression, while inhibition of the protective proteinexpression. Chemicals from cigarette smoke extract resulted in Sertoli cells, Leyding celland Spermatogenic cells apoptosis. It suggested that smoking may lead to growthretardation on development of reproductive system in pubertal rat, and reduction in spermcount and function.
Objective: To research the effect of smoking on hypothalamic-pituitary-testicularaxis hormones and the toxic effects on testicular cells in rat, and analysis of the possiblemechanisms by related apoptosis gene. To study of cigarette smoke extract on testicularcell cytotoxicity and analysis of the possible mechanisms Methods:80SD rats wererandomly divided into8groups. Four groups were control group (C), and the others wereexposed groups, which was inhaled cigarette smoke for2h per day and continued for8weeks. The smoking group and the compared were respectively divided into4sub-groups,i.e. sub-group of2weeks inhalation, sub-group of4weeks inhalation, sub-group of6weeks inhalation, sub-group of8weeks inhalation and the reproduction group with10male mice in each group. By the end of2ed4th6th8thweek10male mice in the smokinggroup and the compared group were respectively killed and the index were observed. Thetestosterone (T), luteinizing hormone (LH), follicle stimulating hormone (FSH) andinsulin-like growth factor-1(IGF-1) in rat serum and gonadotropin-releasing hormone(GnRH) in rat hypothalamus were determined by radioimmunoassay kit. The testicularorganization structure were observed with HE staining, The apoptosis of testicular cellsof different groups was located and quantitated in morphologic and cellular levels bymethod of Methyl green-pyronine staining and temimal dexynucleotidyl tranferase-mdiated dUTP biotin nick end labeling (TUNEL). The Bcl-2、Bax、Fas、FasL expressionsin testis of different groups were located and quantitated by method of Western blotting.Each group the level of Fas, Fas-L, Bcl-2mRNA expression were measured by RT-PCR.It was studied that the effect of cigarette smoke extract (CSE) on the proliferation andgrowth of pdmarily cultured Sertoli cells, Leyding cell and Spermatogenic cells in vitro.The three cells were exposed to different concentrations of CSE for12hours, which weredivided into three groups according to its concentration, including0%CSE group、 5%CSEgroup10%CSE group. Lactate dehydrogenase (LDH) was detected bybiochemistry, cell proliferation by MTT assay and CSE induced cell apoptosis wasobserved by Flow Cytometry. Results:1) With the extension of exposed time of smoking,the increase of weight in the smoke group were apparently lower than those of thecompared group especially in the group of4th,6thand8thweeks inhalation (P＜0.05).2)The testosterone in the smoking group significantly lower than the control group after the4thweeks (P＜0.05) and it was reduced with the extension of exposed time (P＜0.05).3)LH in the control rat serum was obviously raised with time elapsed especially in thegroup of6thand8thweeks, while it in the smoking group with time extended declined.The difference of LH between the smoking group and the control group was alsosignificant (P＜0.05).4) FSH was stable in the rat serum of the control group and thesmoking group, and it was distinctly lowered since the second week of the smokinggroup than the control group (P＜0.05);5) IGF-1levels decreased with time. IGF-1insmoking group of the8thweeks was significantly lower than the2edweeks, the4thweekand the6thweek (P＜0.05), while the2edweek, the6thweek and the8thweek of thesmoking group were significantly lower than those in the control group (P＜0.05)6)Gonadotropin-releasing hormone of rat hypothalamus either the control group or thesmoking group increased with time. GnRH in the smoking group was not statisticallysignificant than the control group (P＞0.05);7) The result of HE stain was that smokingcan lead to the testis tissue destruction, and organizational change was deepened withtime. The testis in the8thweek of the smoking group smoking obvious atrophy, a largenumber of cell lysis significant degenerative changes in the seminiferous tubules andinterstitial tissue;8) The result of TUNEL was that apoptosis index of the cells in thetestis of the4th,6thand8thsmoking group significantly higher than the control (P＞0.05);9) Fas/FasL and Bax protein in rat testis of the smoking group was raised while the Bcl-2protein was decresed. The mRNA expression of Fas/FasL, Bax was increased, the sameresults with protein expression. The gene expression of Fas/FasL, Bax in the smoking8thweeks t Fas/FasL, Bax was increased significantly (P＜0.05) and Bcl-2mRNA waslowered significantly (P＜0.05);10) the cell proliferation of the Leyding cell andSpermatogenic cells with the increased CSEs’ concentration was decreased significantly(P＜0.05) and appeared a dose-response relationship; LDH of the Sertoli cells, theLeyding cells and the Spermatogenic cells in CSEs group was significantly higher thanthat in the control group (P＜0.05);11) The result measured by flow cytometry showedthat CSE leaded to the apoptosis rate of the Sertoli cells, the Leyding cells and the Spermatogenic was significantly increased (P＜0.05). Conclusion: Smoking is harmfulfactors on the development of pubertal rats reproductive system, and have an adverseeffect on not only the Hypothalamic-pituitary-Testicular axis hormones but also testiculartissue and cells. The activation apoptosis regulatory genes by smoking leaded to acorresponding increase in protein expression, while inhibition of the protective proteinexpression. Chemicals from cigarette smoke extract resulted in Sertoli cells, Leyding celland Spermatogenic cells apoptosis. It suggested that smoking may lead to growthretardation on development of reproductive system in pubertal rat, and reduction in spermcount and function.
引文
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