脂多糖诱导的炎症对雄性大鼠生殖功能的影响及其机制研究

发布时间：2018-05-20 05:32

本文选题：脂多糖 + 生殖功能　；参考：《南方医科大学》2014年硕士论文

【摘要】：研究背景近年来,随着社会的发展,环境恶化和人们不良生活习惯影响,男性生育力呈下降趋势,主要表现在人类的精液质量包括精子浓度和精子活力等指标在逐渐下降。不育症患病人群不断增长,也越来越受到人们的关注。不育症是一种影响人类生存、繁衍的疾病,根据有关部门统计,我国约有10%育龄夫妇存在不育问题,其中一半是由于男方因素引起。男性不育症成为男科学领域重要的研究课题。导致男性不育的因素众多,如先天性生殖器官发育异常、遗传疾病、内分泌疾病、生殖系统感染和炎症以及理化因素等,其中生殖道感染和炎症与男性不育密切相关,是男性不育的重要致病因素之一,也是当前研究热点之一。男性生殖道炎症能够影响睾丸、附睾等部位,从而在精子生成和发育阶段对精子造成一定的影响。尽管已有大量关于生殖道炎症与男性不育相关研究,然而炎症如何发展并影响男性生育及其可能的机制尚不完全明了。脂多糖(Lipopolysaccharide,LPS)是革兰阴性菌细胞壁的主要组成成分,也是革兰阴性菌的主要致病因子,已被广泛用于制造动物炎症模型,研究炎症对机体的作用。体内注射LPS可产生类似感染的炎症反应,抑制动物睾丸类固醇激素生成,影响精子发生,也可导致血-睾屏障损伤,引起抗精子抗体产生,并诱导精子和生精细胞凋亡等,从而损害生育。我们给予雄性大鼠腹膜内注射LPS诱导炎症反应,观察了LPS作用后大鼠睾丸组织病理学改变和附睾精子质量变化,并在此基础上调查了LPS诱导的炎症对大鼠附睾氧化应激状态、睾丸生精细胞凋亡率和血清内分泌激素水平的影响。有机阳离子/肉碱转运蛋白2(Organic cation/carnitine transporter2,OCTN2)是左卡尼汀(L-carnitine, LC)的主要转运载体。附睾上皮细胞基底膜上的OCTN2将血液中的LC主动转运到附睾腔内,使得LC在附睾液中的浓度比血浆中浓度高出2000倍以上。LC又称左旋肉碱,是广泛存在于体内的一种类维生素物质,作为脂肪酸的运输载体,将长链脂肪酸从线粒体膜外转移到膜内,使其在线粒体基质中进行β-氧化,为人体提供所需要的能量-ATP,促进脂肪酸的代谢。在附睾中,LC运送长链脂肪酸进入精子,氧化供能,为精子提供动力,可启动精子运动、促进精子成熟和提高精子受精能力。精浆LC水平与精子浓度、活动率、正常形态精子百分率等精液参数之间存在相关性。鉴于LC在男性生殖系统中的重要作用,那么若OCTN2表达改变,将会导致LC转运障碍,精浆LC水平降低,影响精子成熟。因此,我们调查了LPS作用后大鼠附睾组织OCTN2表达改变。本课题利用LPS诱导雄性大鼠炎症反应,探索了LPS诱导的炎症对雄性大鼠生殖功能的影响并初步在分子水平探索其可能的机制。目的 1.给予雄性SD大鼠腹膜内注射LPS,诱导炎症反应,观察大鼠睾丸组织病理学改变和附睾精子质量变化,探讨LPS诱导炎症对雄性大鼠生殖功能的影响； 2.在1的基础上,调查大鼠附睾氧化应激状态、睾丸生精细胞凋亡率和血清T、LH、FSH水平,并进一步研究大鼠附睾组织OCTN2表达改变,探讨LPS诱导的炎症影响雄性大鼠生殖功能的可能机制； 3.通过以上研究,探讨临床生殖道感染和炎症损害男性生育的相关机制,以期最终实现将研究成果进行转换,为临床不育症诊疗提供实验和理论依据。方法 36只雄性SD大鼠(400～450g)随机均分为4组：A组(对照组)腹膜内注射无菌生理盐水；其余3组腹膜内注射LPS (5mg/kg,溶于无菌生理盐水中),分别于给药12h(B组)、24h(C组)和72h(D组)后麻醉处死。取左侧附睾称重后,采用WLJY-9000型伟力数码彩色精子质量检测系统检测附睾头、尾精子活动率,以精子总活动率计算精子活动率；用血细胞计数板按常规方法计算附睾尾精子数目,并计算精子相对计数值(106个/100mg附睾重)；取大鼠左侧睾丸,制备睾丸组织切片,HE染色后,镜下观察睾丸组织病理学变化；用硫代巴比妥酸比色法检测附睾组织匀浆中丙二醛(MDA)含量,黄嘌呤氧化酶比色法测定附睾组织匀浆中超氧化物歧化酶(SOD)活性来反映附睾氧化应激状态；流式细胞术检测睾丸生精细胞凋亡率；酶联免疫分析法(ELISA)检测血清睾酮(T)、黄体生成素(LH)和卵泡刺激素(FSH)水平反映生殖内分泌激素改变；提取大鼠附睾组织总RNA, Real-time RT-PCR技术检测大鼠附睾OCTN2mRNA表达水平变化；提取大鼠附睾组织总蛋白,Western blot技术检测大鼠附睾OCTN2蛋白表达水平变化。结果 1.一般情况给药前,大鼠饮食正常,状态良好。给予LPS作用后约2h开始,大鼠出现寒战、身体扭曲、波状缘毛、口鼻分泌物增多和嗜睡等现象,其炎症反应明显,4h后状态逐渐恢复。 2.大鼠附睾精子计数和精子活动率变化与A组大鼠附睾头、尾精子活动率[分别为(62.65±3.61)%和(68.01±1.80)%]相比,B组大鼠附睾头、尾精子活动率[分别为(56.84±2.65)%和(62.28±4.06)%]明显降低(P0.05),而C组大鼠附睾头、尾精子活动率[分别为(37.28±4.97)%和(45.35±3.39)%]以及D组大鼠附睾头、尾精子活动率[分别为(24.64±4.78)%和(34.85±4.42)%]极显著降低(P0.01)；与A组大鼠附睾尾精子相对计数[(38.94±4.08)x106个/100mg]相比,B组大鼠附睾尾精子相对计数[(37.15±2.54)×106个/100mg]减少,但无统计学差异(P0.05),而C组[(31.97±2.81)×106个/100mg]和D组[(28.60±4.03)×106个/1OOmg]大鼠附睾尾精子相对计数极显著减少(P0.01)。 3.大鼠睾丸组织HE染色结果 HE染色镜下观察显示,A组大鼠睾丸生精小管结构清晰,各级生精细胞排列整齐,生精小管管腔内精子数目较多,无脱落的生精细胞；B组大鼠睾丸生精小管结构清晰,各级生精细胞排列基本整齐,部分生精小管管腔内精子数目有所减少,并可见脱落的生精细胞；C组大鼠睾丸生精上皮变薄,生精小管结构较紊乱、不规则,各级生精细胞减少且排列不整齐,生精小管管腔内精子数目减少,并可见脱落的生精细胞；D组大鼠睾丸生精上皮变薄,生精小管结构较紊乱、不规则,呈明显肿胀状态,各级生精细胞层次紊乱,生精小管管腔内精子数目明显减少,可见较多脱落生精细胞阻塞管腔。 4.大鼠附睾组织氧化应激状态改变硫代巴比妥酸比色法检测结果显示,与A组大鼠附睾组织匀浆中MDA含量[(4.66±1.49)nmol/mgprot]相比,B组[(15.95±3.26)nmol/mgprot]和C组[(12.93±2.54)nmol/mgprot]大鼠附睾MDA含量极显著升高(P0.01),而D组大鼠附睾MDA含量[(9.67±1.68)nmol/mgprot]明显升高(P0.05)；黄嘌呤氧化酶比色法测定结果显示,与A组大鼠附睾组织匀浆中SOD活性[(879.335±105.089)U/mgprot]相比,B组大鼠附睾组织匀浆中SOD活性[(729.265±93.783)U/mgprot]降低,但无统计学差异(P0.05),C组[(694.126±58.530)U/mgprot]和D组[(655.352±115.215)U/mgprot]大鼠附睾组织匀浆中SOD活性显著降低(P0.05)。 5.大鼠睾丸生精细胞凋亡率变化流式细胞术结果显示,与A组大鼠睾丸生精细胞凋亡率(4.21±1.67)%相比,B组大鼠睾丸生精细胞凋亡率(11.01±3.30)%明显增加(P0.05),而C组(23.88±4.58)%和D组(41.28+2.28)%大鼠生精细胞凋亡率极显著增加(P0.01)。 6.大鼠血清T、LH和FSH水平改变 ELISA检测结果显示,与A组大鼠血清T水平[(0.490±0.028)ng/ml]相比,B组大鼠血清T水平[(0.460±0.024)ng/ml]降低,但无统计学差异(P0.05),而C组[(0.417±0.021)ng/ml]和D组[(0.378±0.021)ng/ml]大鼠血清T水平极显著降低(P0.01)；与A组大鼠血清LH水平[(6.290±0.515)ng/L]相比,B组大鼠血清LH水平[(5.881±0.124)ng/L]降低,但无统计学差异(P-0.05),而C组[(5.123±0.271)ng/L]和D组[(4.504±0.279)ng/L]大鼠血清LH水平极显著降低(P0.01);与A组大鼠血清FSH水平[(1.837±0.127)IU/L]相比,B组大鼠血清FSH水平[(1.707±0.098)IU/L]降低,但无统计学差异(P0.05),而C组[(1.620±0.115)IU/L]和D组[(1.562±0.216)IU/L]大鼠血清FSH水平显著降低(P0.05)。 7.大鼠附睾组织OCTN2表达改变 Real-time RT-PCR检测结果和Western blot检测结果相符：与A组大鼠附睾组织OCTN2mlRNA表达量(1.00±0.00)相比,B组大鼠附睾OCTN2mRNA表达量(0.962±0.016)降低,但无统计学差异(P0.05),而C组(0.706±0.026)和D组(0.474±0.037)大鼠附睾OCTN2mRNA表达量极显著降低(P0.01)；与A组大鼠附睾组织OCTN2蛋白表达量(1.707±0.092)相比,B组大鼠附睾OCTN2蛋白表达量(1.458±0.165)降低,但无统计学差异(P0.05),而C组(1.197±0.118)和D组(1.023±0.136)大鼠附睾OCTN2蛋白表达量极显著降低(P0.01)。结论 1. LPS (5mg/kg)腹膜内注射诱导大鼠炎症反应,导致大鼠附睾精子计数和精子活动率降低以及睾丸组织病理学改变,影响大鼠生殖功能； 2.LPS诱导的炎症反应破坏大鼠附睾氧化应激状态、增加大鼠睾丸生精细胞凋亡率并降低血清T、LH、FSH水平,这些指标改变可能是LPS诱导炎症损害生殖功能的相关因素； 3.LPS诱导的炎症反应抑制大鼠附睾组织OCTN2表达,可能是LPS诱导炎症损害雄性大鼠生殖功能的机制之一。
[Abstract]:Background of the study

In recent years , with the development of society , the deterioration of environment and people ' s bad habits , the fertility of male is decreasing . The quality of human semen includes such indexes as sperm concentration and sperm motility .

Male infertility has many factors , such as abnormal reproductive organs development , genetic diseases , endocrine diseases , reproductive system infection , inflammation and physical and chemical factors , among which reproductive tract infection and inflammation are closely related to male infertility , which is one of the important pathogenic factors of male infertility .

lipopolysaccharides ( LPS ) is the main component of the cell wall of Gram - negative bacteria , and is the main pathogenic factor of Gram - negative bacteria , and has been widely used in the manufacture of animal inflammation models .

This study investigated the effects of LPS on the reproductive function of male rats and explored the possible mechanism of LPS - induced inflammation in male rats .

Purpose

1 . LPS was injected intraperitoneally to male SD rats , inflammatory reaction was induced , pathological changes of rat testis were observed , and the changes of sperm quality were observed , and the effects of LPS - induced inflammation on the reproductive function of male rats were investigated .

2 . On the basis of 1 , the oxidative stress state , apoptosis rate and serum T , LH and FSH levels were investigated in rats , and the expression of OCTN2 in the testis of rats was further studied , and the possible mechanism of LPS - induced inflammation on the reproductive function of male rats was discussed .

3 . Through the above research , the relative mechanism of reproductive tract infection and inflammation is discussed in order to realize the transformation of the research results and provide the experimental and theoretical basis for the diagnosis and treatment of clinical infertility .

method

36 male SD rats ( 400 - 450 g ) were randomly divided into 4 groups : group A ( control group ) intraperitoneal injection of sterile physiological saline ;
In the remaining three groups , LPS ( 5 mg / kg , dissolved in sterile physiological saline ) was injected intravenously for 12 h ( group B ) , 24 h ( group C ) and 72 h ( group D ) .
The number of sperm count was calculated by routine method by blood cell counting plate , and the relative count value of sperm was calculated ( 106 / 100 mg / 100 mg ) ;
The testis of the left side of the rat was obtained , the testis tissue sections were prepared , and the pathological changes of the testis were observed under the microscope after HE staining ;
The content of malondialdehyde ( MDA ) in the homogenate of the tissue homogenate was determined by thiobarbituric acid colorimetry , and the activity of superoxide dismutase ( SOD ) in the homogenate of the tissue homogenate was determined by xanthine oxidase colorimetric method .
The apoptosis rate of spermatogenic cells was detected by flow cytometry .
Serum testosterone ( T ) , LH and FSH levels were measured by enzyme linked immunosorbent assay ( ELISA ) .
The expression level of OCTN mRNA was detected by real - time RT - PCR in rats with total RNA and Real - time RT - PCR .
The expression level of OCTN2 protein was detected by Western blot .

Results

1 . General situation

Before administration , the rats had normal diet and good condition . After administration of LPS for about 2 hours , the rats appeared cold war , body torsion , undulate margin , mouth - nasal discharge increased and drowsiness , etc . , their inflammatory responses were obvious , and the state gradually recovered after 4h .

2 . Changes of sperm count and sperm motility in rats

Compared with group A , the activity of sperm motility was ( 56.84 卤 2.65 ) % and ( 62.28 卤 4.06 ) % , respectively ( 56.84 卤 4.97 ) % and ( 45.35 卤 3.39 ) % and ( 34.85 卤 4.42 ) % , respectively ( P < 0.01 ) .
Compared with group A , the relative counts of sperm in tail sperm of rats ( 37.15 卤 2.54 ) 脳 106 / 100 mg / kg were significantly decreased ( P < 0.01 ) , but there was no statistical difference ( P < 0.01 ) .

3 . HE staining results of rat testis

Under HE staining , the structure of spermatogenic tubules was clear in group A , and the spermatogenic cells at all levels were arranged in order , and the number of spermatogenic cells in the tubules of spermatogenic tubules was more than that of spermatogenic cells .
In group B , the structure of spermatogenic tubules was clear , the arrangement of spermatogenic cells at all levels was basically regular , the number of sperm in the tubules of partially spermatogenic tubules decreased , and the spermatogenic cells shed were observed .
In group C , the spermatogenic epithelium of the testis was thinner , the structure of spermatogenic tubules was disordered , irregular , the spermatogenic cells at all levels decreased and the arrangement was irregular , the number of spermatogenic cells in the tubules of spermatogenic tubules decreased , and the detached spermatogenic cells were observed ;
In group D , the spermatogenic epithelium of the testis was thinned , the structure of spermatogenic tubules was disordered , irregular , and the level of spermatogenic cells at all levels was disordered , and the number of sperm cells in the tubules of spermatogenic tubules was significantly decreased .

4 . Changes of oxidative stress state in the rat testis

Compared with group A , the content of MDA was significantly higher in group B than that in group A ( 15.95 卤 3.26 ) nmol / mg in group B ( 12.93 卤 2.54 ) nmol / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg / mg , respectively , compared with that in group A ( P < 0.01 ) .
Compared with group A , the activity of SOD decreased ( 729,265 卤 93.783 ) U / mgT lymphocyte decreased , but there was no statistical difference ( P0.05 ) .

5 . Apoptosis rate of spermatogenic cells in rat testis

Compared with group A , the apoptosis rate of spermatogenic cells ( 11.01 卤 3.30 ) % increased significantly ( P < 0.01 ) , while the apoptosis rate of spermatogenic cells in group C ( 23.88 卤 4.58 ) % and group D ( 41.28 + 2.28 ) % increased significantly ( P0.01 ) .

6 . Changes of serum T , LH and FSH levels in rats

The results of ELISA showed that the serum T level in group B was significantly lower than that in group A ( 0.490 卤 0.028 ) ng / ml , but there was no statistical difference ( P < 0.05 ) , but the serum T level was significantly lower in group C ( 0.417 卤 0.021 ) ng / ml and D group ( 0.378 卤 0.021 ) ng / ml ( P0.01 ) .
Compared with group A , the serum LH levels were significantly lower in group B ( 5.881 卤 0.124 ) ng / L , but there was no statistical difference ( P - 0.05 ) , but there was no statistical difference ( P - 0.05 ) . The serum FSH levels in group B were significantly lower than those in group A ( 1.620 卤 0.115 ) IU / L and D group ( 1.562 卤 0.216 ) IU / L , respectively ( P0.05 ) .

7 . Changes of OCTN2 Expression in the Testis of Rats

The results of real - time RT - PCR and Western blot analysis showed that the expression of OCTN mRNA was decreased in group B rats ( 0 . 962 卤 0 . 016 ) , but there was no statistical difference ( P 0 . 05 ) , while that of C group ( 0 . 706 卤 0.026 ) and D group ( 0 . 474 卤 0 . 37 ) decreased significantly ( P0.01 ) .
The OCTN2 protein expression ( 1.458 卤 0.165 ) was lower in group B than in group A ( 1.707 卤 0.092 ) , but there was no statistical difference ( P < 0.05 ) , while the expression of OCTN2 protein in group C ( 1.197 卤 0.118 ) and group D ( 1.023 卤 0.136 ) decreased significantly ( P0.01 ) .

Conclusion

1 . Intraperitoneal injection of LPS ( 5mg / kg ) induced inflammatory reaction in rats , which resulted in the decrease of sperm count and sperm motility and pathological changes of testis in rats , which could affect the reproductive function of rats .

2 . LPS - induced inflammatory reaction destroys the oxidative stress state of rat testis , increases the apoptosis rate of rat testis spermatogenic cells and decreases serum T , LH and FSH levels .

3 . LPS - induced inflammatory response can inhibit the expression of OCTN2 in rats , which may be one of the mechanisms of LPS - induced inflammation to damage the reproductive function of male rats .
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R698.2

【参考文献】