细菌脂多糖急性暴露通过炎症反应诱导小鼠睾丸损伤
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摘要
旨在研究细菌脂多糖(LPS)急性暴露对小鼠睾丸及其功能的影响。将健康雄性小鼠随机分为4组:对照组(Control)、1.25 mg·kg~(-1) LPS组、2.5 mg·kg~(-1) LPS组和5.0 mg·kg~(-1) LPS组,对照组腹腔注射200μL生理盐水,其余3组注射等体积含不同剂量的LPS,作用12 h。收集睾丸组织制作石蜡切片,染色后观察其病理变化,记录体重、睾丸和附睾指数,并测定精子品质相关指标,用RT-qPCR法检测炎症相关基因TNF-α、IL-6、GAS6和TGF-β1的转录,同时用ELISA检测血清中睾酮(T)的含量。结果表明,与对照组相比,LPS急性暴露时,以剂量依赖性方式降低小鼠体重(P<0.05或P<0.01),同时显著降低睾丸指数(P<0.05或P<0.01)、精子密度(P<0.01)及精子活力(P<0.05或P<0.01),增加精子畸形率(P<0.05或P<0.01);使睾丸组织发生不同程度的病理学变化,表现在生精细胞凋亡,曲细精管管腔增大,精子数量减少等;导致血清中睾酮(T)的含量显著下降;另外,LPS还呈剂量依赖方式增加促炎因子TNF-α和IL-6及抑制抗炎因子GAS6和TGF-β1的转录。结果提示,细菌LPS急性暴露可通过破坏机体中炎症因子的平衡,使睾丸组织受到损伤从而导致生精障碍。
The present study was conducted to investigate the effects of acute exposure to bacterial lipopolysaccharide(LPS) on testis and its function in mice. Healthy male mice were randomly divided into 4 groups: Control group, 1.25 mg·kg~(-1) LPS group, 2.5 mg·kg~(-1) LPS group and 5.0 mg·kg~(-1) LPS. The control group was intraperitoneally injected with 200 μL saline, while the other three groups were injected the same volume saline containing different doses of LPS and treated for 12 hours. Testes were collected to check the pathological changes after staining; body weight, testicular and epididymal indexes were recorded and the related sperm quality indexes were determined; testosterone(T) level in serum was measured by ELISA; Transcription of inflammatory genes(TNF-α, IL-6, GAS6 and TGF-β1) were detected by RT-qPCR. The results showed that LPS acute exposure reduced the body weight of mice in a dose-dependent manner(P<0.05 or P<0.01), significantly decreased testicular index(P<0.05 or P<0.01), sperm density(P<0.01) and sperm motility(P<0.05 or P<0.01), and increased the rate of sperm malformation(P<0.05 or P<0.01). Meanwhile, LPS exposure resulted in testicular pathological changes to varying degrees, such as spermatogenic cell apoptosis, enlargement of seminiferous duct cavity and less sperms, and significantly reduced the levels of testosterone(T) in serum. In addition, LPS also increased the transcription of pro-inflammatory factors(TNF-α and IL-6) and inhibited anti-inflammatory factors(GAS6 and TGF-β1) in a dose-dependent manner. These results indicated that acute exposure to bacterial LPS impairs testicular tissue and leads to spermatogenesis disorder via destroying the balance of inflammatory factors in the body.
The present study was conducted to investigate the effects of acute exposure to bacterial lipopolysaccharide(LPS) on testis and its function in mice. Healthy male mice were randomly divided into 4 groups: Control group, 1.25 mg·kg~(-1) LPS group, 2.5 mg·kg~(-1) LPS group and 5.0 mg·kg~(-1) LPS. The control group was intraperitoneally injected with 200 μL saline, while the other three groups were injected the same volume saline containing different doses of LPS and treated for 12 hours. Testes were collected to check the pathological changes after staining; body weight, testicular and epididymal indexes were recorded and the related sperm quality indexes were determined; testosterone(T) level in serum was measured by ELISA; Transcription of inflammatory genes(TNF-α, IL-6, GAS6 and TGF-β1) were detected by RT-qPCR. The results showed that LPS acute exposure reduced the body weight of mice in a dose-dependent manner(P<0.05 or P<0.01), significantly decreased testicular index(P<0.05 or P<0.01), sperm density(P<0.01) and sperm motility(P<0.05 or P<0.01), and increased the rate of sperm malformation(P<0.05 or P<0.01). Meanwhile, LPS exposure resulted in testicular pathological changes to varying degrees, such as spermatogenic cell apoptosis, enlargement of seminiferous duct cavity and less sperms, and significantly reduced the levels of testosterone(T) in serum. In addition, LPS also increased the transcription of pro-inflammatory factors(TNF-α and IL-6) and inhibited anti-inflammatory factors(GAS6 and TGF-β1) in a dose-dependent manner. These results indicated that acute exposure to bacterial LPS impairs testicular tissue and leads to spermatogenesis disorder via destroying the balance of inflammatory factors in the body.
引文
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[15] 何自标,兰瑞霞,陈晨,等.脂多糖对母兔下丘脑、卵巢、输卵管中TLR4及其相关细胞因子表达的影响[J].畜牧兽医学报,2014,45(6):910-917.HE Z B,LAN R X,CHEN C,et al.Effect of LPS on the expression of TLR4 and related cytokines in oviduct,ovary and hypothalamus of rabbits[J].Acta Veterinaria et Zootechnica Sinica,2014,45 (6):910-917.(in Chinese)
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[17] 杨露露,王华,姬艳丽,等.细菌脂多糖急性暴露对小鼠睾丸组织的损害作用[J].中华疾病控制杂志,2015,19(2):166-169.YANG L L,WANG H,JI Y L,et al.Acute exposure to lipopolysaccharide impairs testicular tissue in male mice[J].Chinese Journal of Disease Control & Prevention,2015,19(2):166-169.(in Chinese)
[18] 詹绪新,胡毓安,卿兴荣,等.脂多糖对雄性大鼠睾丸组织病理学和生殖内分泌功能的影响[J].中华男科学杂志,2014,20(4):304-308.ZHAN X X,HU Y A,QING X R,et al.Lipopolysaccharide affects testicular histology and reproductive endocrine function in male rats[J].National Journal of Andrology,2014,20(4):304-308.(in Chinese)
[19] IWASA T,MATSUZAKI T,TUNGALAGSUVD A,et al.Prenatal undernutrition results in greater lipopolysaccharide-induced changes in hypothalamic TNF-α expression,but does not affect the equivalent changes in the serum levels of luteinizing hormone and testosterone,in adult male rats[J].Int J Dev Neurosci,2016,48:80-83.
[20] TYNAN R J,WEIDENHOFER J,HINWOOD M,et al.A comparative examination of the anti-inflammatory effects of SSRI and SNRI antidepressants on LPS stimulated microglia[J].Brain Behav Immun,2012,26(3):469-479.
[21] HONG C Y,PARK J H,AHN R S,et al.Molecular mechanism of suppression of testicular steroidogenesis by proinflammatory cytokine tumor necrosis factor alpha[J].Mol Cell Biol,2004,24(7):2593-2604.
[22] WANG F,LIU W H,JIANG Q,et al.Lipopolysaccharide-induced testicular dysfunction and epididymitis in mice:a critical role of tumor necrosis factor alpha[J/OL].Biol Reprod,2018:doi:10.1093/biolre/ioy235[2019-03-05].https://doi.org/10.1093/biolre/ioy235.
[2] SAVI,et al.The effect of breed,boar and season on some properties of sperm[J].Biotechnol Anim Husb,2013,29(2):299-310.
[3] CHEN Q Y,DENG T T,HAN D S.Testicular immunoregulation and spermatogenesis[J].Semin Cell Dev Biol,2016,59:157-165.
[4] NI H J,LU L,DENG J P,et al.Effects of glutamate and aspartate on serum antioxidative enzyme,sex hormones,and genital inflammation in boars challenged with hydrogen peroxide[J].Mediat Inflamm,2016,2016:4394695.
[5] AZENABOR A,EKUN A O,AKINLOYE O.Impact of inflammation on male reproductive tract[J].J Reprod Infertil,2015,16(3):123-129.
[6] SHALOM-PAZ E,WEILL S,GINZBERG Y,et al.IUGR induced by maternal chronic inflammation:long-term effect on offspring's ovaries in rat model-a preliminary report[J].J Endocrinol Invest,2017,40(10):1125-1131.
[7] 果双双,李辉,施振旦,等.脂多糖对体外培养猪颗粒细胞增殖、凋亡和雌二醇分泌的影响[J].畜牧兽医学报,2016,47(4):693-699.GUO S S,LI H,SHI Z D,et al.Effects of lipopolysaccharide on proliferation,apoptosis and estradiol secretion of in vitro cultured porcine granulosa cells[J].Acta Veterinaria et Zootechnica Sinica,2016,47(4):693-699.(in Chinese)
[8] SHIMIZU T,ECHIZENYA R,MIYAMOTO A.Effect of lipopolysaccharide on progesterone production during luteinization of granulosa and theca cells in vitro[J].J Biochem Mol Toxicol,2016,30(4):206-211.
[9] SHIMIZU T,ISHIZAWA S,MAGATA F,et al.Involvement of lipopolysaccharide in ovarian cystic follicles in dairy cow:expressions of LPS receptors and steroidogenesis-related genes in follicular cells of cystic follicles[J].Anim Reprod Sci,2018,195:89-95.
[10] MAGATA F,ISHIDA Y,MIYAMOTO A,et al.Comparison of bacterial endotoxin lipopolysaccharide concentrations in the blood,ovarian follicular fluid and uterine fluid:a clinical case of bovine metritis[J].J Vet Med Sci,2015,77(1):81-84.
[11] FULLER E A,SOMINSKY L,SUTHERLAND J M,et al.Neonatal immune activation depletes the ovarian follicle reserve and alters ovarian acute inflammatory mediators in neonatal rats[J].Biol Reprod,2017,97(5):719-730.
[12] PELLATI D,MYLONAKIS I,BERTOLONI G,et al.Genital tract infections and infertility[J].Eur J Obstet Gynecol Reprod Biol,2008,140(1):3-11.
[13] WANG H,YANG L L,HU Y F,et al.Maternal LPS exposure during pregnancy impairs testicular development,steroidogenesis and spermatogenesis in male offspring[J].PLoS One,2014,9(9):e106786.
[14] VATANEN T,KOSTIC A D,D’HENNEZEL E,et al.Variation in microbiome LPS immunogenicity contributes to autoimmunity in humans[J].Cell,2016,165(4):842-853.
[15] 何自标,兰瑞霞,陈晨,等.脂多糖对母兔下丘脑、卵巢、输卵管中TLR4及其相关细胞因子表达的影响[J].畜牧兽医学报,2014,45(6):910-917.HE Z B,LAN R X,CHEN C,et al.Effect of LPS on the expression of TLR4 and related cytokines in oviduct,ovary and hypothalamus of rabbits[J].Acta Veterinaria et Zootechnica Sinica,2014,45 (6):910-917.(in Chinese)
[16] REDDY M M,MAHIPAL S V K,SUBHASHINI J,et al.Bacterial lipopolysaccharide-induced oxidative stress in the impairment of steroidogenesis and spermatogenesis in rats[J].Reprod Toxicol,2006,22(3):493-500.
[17] 杨露露,王华,姬艳丽,等.细菌脂多糖急性暴露对小鼠睾丸组织的损害作用[J].中华疾病控制杂志,2015,19(2):166-169.YANG L L,WANG H,JI Y L,et al.Acute exposure to lipopolysaccharide impairs testicular tissue in male mice[J].Chinese Journal of Disease Control & Prevention,2015,19(2):166-169.(in Chinese)
[18] 詹绪新,胡毓安,卿兴荣,等.脂多糖对雄性大鼠睾丸组织病理学和生殖内分泌功能的影响[J].中华男科学杂志,2014,20(4):304-308.ZHAN X X,HU Y A,QING X R,et al.Lipopolysaccharide affects testicular histology and reproductive endocrine function in male rats[J].National Journal of Andrology,2014,20(4):304-308.(in Chinese)
[19] IWASA T,MATSUZAKI T,TUNGALAGSUVD A,et al.Prenatal undernutrition results in greater lipopolysaccharide-induced changes in hypothalamic TNF-α expression,but does not affect the equivalent changes in the serum levels of luteinizing hormone and testosterone,in adult male rats[J].Int J Dev Neurosci,2016,48:80-83.
[20] TYNAN R J,WEIDENHOFER J,HINWOOD M,et al.A comparative examination of the anti-inflammatory effects of SSRI and SNRI antidepressants on LPS stimulated microglia[J].Brain Behav Immun,2012,26(3):469-479.
[21] HONG C Y,PARK J H,AHN R S,et al.Molecular mechanism of suppression of testicular steroidogenesis by proinflammatory cytokine tumor necrosis factor alpha[J].Mol Cell Biol,2004,24(7):2593-2604.
[22] WANG F,LIU W H,JIANG Q,et al.Lipopolysaccharide-induced testicular dysfunction and epididymitis in mice:a critical role of tumor necrosis factor alpha[J/OL].Biol Reprod,2018:doi:10.1093/biolre/ioy235[2019-03-05].https://doi.org/10.1093/biolre/ioy235.