金花茶提取物对MMTV-PyMT转基因小鼠自发乳腺癌的预防作用研究

目的:考察金花茶二氯甲烷乙酸乙酯提取物对MMTV-Py MT转基因小鼠乳腺癌发生的实验预防作用。方法:MTT法检测金花茶二氯甲烷乙酸乙酯提取物对人乳腺癌细胞SKBR-3的体外增殖抑制作用;采用MMTV-Py MT转基因自发乳腺癌小鼠模型观察金花茶二氯甲烷乙酸乙酯提取物对小鼠体内肿瘤发生的预防作用。结果:金花茶二氯甲烷乙酸乙酯提取物对人乳腺癌细胞SKBR-3有显著地体外增殖抑制作用,其IC50为419.0±12.87μg/m L;转基因小鼠动物模型中,金花茶二氯甲烷乙酸乙酯提取物对小鼠肿瘤的发生和发展均有显著地抑制作用;模型组小鼠肿瘤平均发生数量为5.25个/只,给药组为3.75个/只;模型组组小鼠平均瘤重为0.47 g/只,给药组0.24 g/只。结论:金花茶二氯甲烷乙酸乙酯提取物对MMTV-Py MT转基因小鼠乳腺癌的发生有一定的预防作用。     

D-半乳糖诱导衰老小鼠睾丸结构与功能变化及其氧化应激相关机制

目的建立D-半乳糖(D-gal)致小鼠衰老模型,探讨睾丸结构与功能变化及相关机制。方法 C57BL/6J小鼠随机分为衰老组和对照组。衰老组小鼠皮下注射D-gal;对照组等时等量注射生理盐水。模型复制完成第2d,采集小鼠内眦静脉血测定血清睾酮水平;取小鼠睾丸测定睾丸指数;石蜡切片HE染色观察睾丸组织形态学;衰老相关β-半乳糖苷酶(SA-β-Gal)染色检测生精细胞衰老;TUNEL法检测生精细胞凋亡;制备睾丸组织匀浆上清,ELISA检测TNF-α、IL-1β、和IL-6的水平;硫代巴比妥酸法检测丙二醛(MDA)含量;酶学法检测超氧化物歧化酶(SOD)活性;ABTS法检测总抗氧化能力(TAC);Western blotting检测衰老相关蛋白p21与p53表达水平。结果衰老模型组小鼠血清睾酮水平显著下降,睾丸脏器指数无明显差异,生精小管结构紊乱,生精细胞病理损伤明显,SA-β-Gal染色阳性的生精细胞百分率上升,细胞凋亡指数上升,睾丸匀浆上清中TNF-α、IL-1β、IL-6水平显著上升,SOD活性、TAC能力显著降低,MDA含量上升,睾丸组织中衰老相关蛋白:p21与p53表达水平显著提高。结论 D-gal建立的衰老动物模型可见睾丸形态结构与功能出现衰老的生物学表现,其机制可能与氧化应激损伤及p21/p53通路激活有关。     

罗仙子提取物对肝癌的体内外抑制作用研究

本文观察中药罗仙子提取物在离体细胞水平以及整体动物模型中对肝癌的治疗作用。在罗仙子提取物干预人肝癌SMMC-7721细胞24 h后,采用MTT法检测细胞活率;采用光学显微镜、荧光显微镜、透射电镜观察细胞形态学改变,流式细胞仪检测细胞凋亡率;以Heps肝癌小鼠模型观察罗仙子提取物体内对肿瘤生长的抑制作用。研究发现不同浓度罗仙子提取物可抑制SMMC-7721细胞增殖同时诱导典型的细胞凋亡形态学变化,与对照组相比细胞凋亡率显著升高,且具有浓度依赖性;不同浓度罗仙子提取物可显著抑制Heps肝癌小鼠体内肿瘤生长。实验证明罗仙子提取物可显著抑制Heps肝癌小鼠体内肿瘤生长,该作用可能与罗仙子提取物抑制肝癌细胞增殖、诱导细胞凋亡有关。     

中风膏对氧糖剥夺/再复氧损伤大鼠海马神经干细胞增殖的影响

目的：研究中风膏对体外氧糖剥夺/再复氧损伤大鼠神经干细胞增殖的影响。方法：采用悬浮培养法分离纯化新生SD大鼠大脑海马神经干细胞,免疫荧光法鉴定细胞,第3代贴壁培养神经干细胞氧糖剥夺2 h后,复氧培养24 h造模。实验分为正常对照组、模型组、中风膏5%含药血清组、中风膏10%含药血清组、中风膏20%含药血清组,每组6个复孔,分1 d、3 d、5 d、7 d 4个时间点。CCK8法检测细胞活力,流式细胞仪测定细胞凋亡。结果：悬浮培养细胞均高表达巢蛋白（nestin）。在增殖试验中,正常组与模型对照组比较,模型对照组的细胞增殖能力明显降低（P<0.01）;与模型对照组相比,第3日,中风膏20%治疗组的细胞增殖能力明显增强（P<0.01）;实验第5日、7日,中风膏5%、10%、20%组细胞增殖能力明显增强（P<0.01）。在凋亡实验中,与正常组比较,模型对照组的细胞凋亡明显升高（P<0.01）;与模型对照组相比,中风膏20%组细胞凋亡显著下降（P<0.01）。结论：中风膏含药血清对海马神经干细胞OGD/R损伤后的增殖能力有显著的修复作用,20%中风膏含药血清对干细胞损伤有治疗作用,可降低细胞凋亡。     

不同剂量炔雌醚对雄性小鼠繁殖生理的作用

为探究不同剂量炔雌醚作用于雄性昆明小鼠时,对小鼠的繁殖生理指标的影响,现将18只雄性昆明小鼠分为3组:对照组(C组)、10 mg/kg给药组(E10组)、20 mg/kg给药组(E20组)进行实验。在给药后第6天进行剖检,测量其繁殖器官重量及血清睾酮浓度,观察其睾丸组织形态切片。结果发现,两给药组繁殖器官重量与对照组相比无明显差异;(E10组)血清睾酮浓度有所上升,(E20组)无明显变化;(E20组)曲细精管受到一定程度的损伤,而(E10组)的损伤不明显。因此,高浓度的炔雌醚可能对雄性昆明小鼠有更好的抗生育作用。本研究为进一步明确炔雌醚对鼠类不育控制的最佳浓度提供了一定的理论依据。     

Roxadustat对肾缺血再灌注损伤的保护作用及机制研究

目的:探讨一种新型PHD抑制剂Roxadustat对小鼠肾缺血再灌注损伤的保护作用及其可能的作用机制。方法:将雄性C57BL/6小鼠随机分为4组:假手术组(sham)、损伤组(IR)、损伤+低剂量给药组(IR+Rox10 mg/kg)以及损伤+高剂量组(IR+Rox25 mg/kg)。除假手术组外,其余各组分别于造模前1h、6h、12h给药,并于造模后6h、12h、24h、48h采血检测血肌酐(Scr)、尿素氮(BUN),1d、2d、5d取材肾脏进行病理检测。此外,利用HK-2细胞建立缺氧模型,测定给药后细胞活力和细胞凋亡情况的变化及凋亡通路蛋白和HIF-1α的表达情况。结果:与sham组和IR组相比,给药组Scr和BUN水平均明显降低,且高剂量组Scr和BUN水平显著低于低剂量组,且给药组形态学损伤更轻,细胞凋亡明显减少。细胞学实验显示,Roxadustat能提高低氧条件下HK-2细胞的活力,降低细胞凋亡,并抑制低氧导致的Bax升高,提高Bcl-2的表达,而用HIF-1α抑制剂2-MeOE2,可消除Roxadustat对凋亡的抑制作用。结论:Roxadustat能够通过上调HIF-1α表达,抑制线粒体途径凋亡通路相关蛋白表达,减少细胞凋亡,对小鼠肾脏缺血再灌注损伤产生保护作用。     

齐墩果酸对自然衰老大鼠睾丸DNA损伤保护作用及机制研究

探讨齐墩果酸(Oleanolic Acid,OA)对自然衰老大鼠睾丸DNA损伤的保护作用及其可能机制。将30只SPF级18月龄SD大鼠随机分为三组:自然衰老组、OA低、高剂量组,每组10只,另取10只3月龄大鼠作为青年对照组。OA低、高剂量组分别灌胃给予OA 5、25 mg/kg,自然衰老组和青年对照组给予生理盐水,每周灌胃6天,周日不给药,连续给药6个月。末次给药后禁食不禁水12 h,称重,麻醉后处死大鼠,快速取出睾丸组织。HE染色观察睾丸组织形态变化,免疫组织化学法检测DNA损伤相关蛋白53BP1、ATR的表达,Western Blot检测睾丸组织中衰老相关蛋白p21和DNA损伤相关蛋白γ-H2AX、Chk1、p-p53的表达水平。HE结果表明,OA能显著改善增龄所致的睾丸组织形态变化;与青年对照组相比,自然衰老组睾丸组织DNA损伤标志蛋白γ-H2AX表达水平明显升高,而给予OA干预后其表达显著降低;此外,与青年对照组比较,自然衰老组睾丸组织中衰老相关蛋白p21的表达显著增高,DNA损伤相关蛋白53BP1、ATR的阳性灶点数及其下游Chk1、p-p53的蛋白表达明显升高,OA低、高剂量组均能显著降低上述蛋白的表达。以上结果表明OA对自然衰老大鼠睾丸组织DNA损伤有较好的保护作用,机制与调节ATR/Chk1/P53信号通路有关。     

红芪胶囊对低剂量辐射损伤小鼠的保护作用

目的:采用低剂量辐射损伤小鼠模型,观察红芪胶囊的药理作用。方法:二级昆明种小鼠216只,除正常组36只外,其余180只采用多次低剂量累积辐射。将照射的180只小鼠按体重随机分为5组,每组12只,分别为:模型组,阳性对照组(1.20 g/kg),红芪胶囊小剂量组(0.175 g/kg),红芪胶囊中剂量组(0.35 g/kg),红芪胶囊大剂量组(0.70 g/kg);将未照射的36只小鼠设为正常组,每组又分为给药6、12、24 d三个时间点,每时间点12只动物。各组在末次照射当天分别灌胃上述药物,正常对照组及模型对照组灌胃等量蒸馏水,每日一次,连续给药24 d,观察红芪胶囊的药理作用。结果:1红芪胶囊给药12 d、24 d对辐射损伤小鼠的外周血象红细胞(RBC)、白细胞(WBC)、血小板(PLT)和血红蛋白含量(HGB)有明显的升高作用(P0.01或P0.05);2明显增加辐射损伤小鼠的肝脏、脾脏、大脑和睾丸组织的湿质量(P0.01或P0.05);3镜下观察可见红芪胶囊能明显改善损伤小鼠骨髓细胞活性。结论:红芪胶囊对低剂量辐射损伤具有一定保护作用。     

复方辣木颗粒剂影响雄性小鼠性功能的实验研究

目的观察复方辣木颗粒剂对雄性小鼠性功能的影响。方法建立醋酸铅致雄性小鼠生精障碍模型。将60只实验小鼠分为正常组、模型组、阳性对照组和复方辣木颗粒剂高、中、低剂量组,每组10只。给药后,观察小鼠体重、脏器指数、附睾精子活力、精子活率及睾丸的病理形态等。结果复方辣木颗粒剂对醋酸铅致少弱精子症小鼠体重无明显影响,各给药组小鼠附睾系数、睾丸系数、精子活力、精子活率均有所上升,生精细胞和间质细胞数量增多、排列更紧密。各剂量间比较,高剂量效果更显著(均P0.05)。结论复方辣木颗粒剂可显著改善小鼠性功能,为临床合理应用提供有利参考。     

氟化钠对小鼠睾丸组织的损伤及细胞凋亡的影响

目的研究氟中毒对小鼠睾丸组织的影响。方法给小鼠饮用含0 mg/L、10 mg/L、25 mg/L、50 mg/L氟化钠水,HE染色观察睾丸组织形态病理学变化,用TUNEL法检测小鼠睾丸组织细胞凋亡。结果氟中毒能诱导小鼠睾丸TUNEL阳性细胞,且凋亡细胞率较对照组明显增高。结论氟中毒可诱导小鼠睾丸组织细胞凋亡明显,凋亡发生部位与病理改变明显的部位相吻合,氟中毒小鼠睾丸组织细胞凋亡机制参与睾丸损害过程。     

镉对小鼠精母细胞凋亡及附睾内精子质量的影响

采用人工水质染毒的方法,利用透射电镜技术及流式细胞术（FCM）,探讨重金属镉对小鼠精巢内生殖细胞凋亡及附睾内成熟精子质量的影响.结果表明：各试验组小鼠生殖细胞处于凋亡时期的数量显著高于对照组,凋亡时期的生殖细胞超微结构呈现出线粒体空泡、核膜内陷、染色质周缘化及核固缩等形态特征,表明镉容易引起小鼠生殖细胞凋亡;各试验组精子早期凋亡的比例显著高于对照组,而活性精子的比例显著低于对照组（P<0.05）,其中高剂量组（0.10 mmol·L^-1）精子成活率（75.1%）显著低于对照组和其他试验组,而早期凋亡率（22.6%）则显著高于对照组;高剂量组睾丸生殖细胞DNA断裂率（18.2%）及附睾精子断裂率（26.5%）均显著高于对照组（3.3%、5.6 %）（P<0.05）.各试验组小鼠睾丸内DNA断裂的生殖细胞数量低于附睾内DNA断裂的精子数量.随着添加剂量的增加,小鼠睾丸内生殖细胞及附睾内精子凋亡率逐渐升高.表明小鼠生殖细胞凋亡及DNA损伤数量与镉剂量具有一定相关性.     

蒺藜中甾体皂苷对新生隐球菌生物膜形成的抑制作用

目的 观察蒺藜甾体皂苷类化合物TTS-12对新生隐球菌生物膜形成的影响,探讨其可能的作用机制.方法 光镜观察TTS-12对新生隐球菌生物膜生长形态的影响;MTT法观察TTS-12对新生隐球菌生物膜形成的影响;实时定量RTPCR观察不同浓度TTS-12对新生隐球菌细胞生物膜关键基因PMT4表达的影响.结果 经TTS-12处理的新生隐球菌生物膜结构更疏松,TTS-12可剂量依赖性地降低新生隐球菌生物膜生长动力学指标及PMT4基因表达水平(P＜0.01).结论 TTS-12可抑制新生隐球菌生物膜的形成.通过降低新生隐球菌PMT4基因表达可能是其抑制新生隐球菌生物膜的形成作用机制之一.     

The effect of Tribulus terrestris extract on motility and viability of human sperms after cryopreservation

BackgroundSemen cryopreservation produces significant amounts of reactive oxygen species (ROS), which may lead to impairment of sperm morphology, function, and ultimately, male fertility. Since Tribulus terrestris has antioxidant and free-radical-scavenging properties, this study aims to reveal the effect of the Tribulus terrestris extract on motility and vitality of human sperms after cryopreservation.Materials and methodsSemen specimens from 80 healthy volunteers were divided into eight groups: fresh control (group I), freeze control (group II), groups III, IV, and V, which had 20, 40, and 50 μg/mL doses of Tribulus terrestris extract added before cryopreservation, and groups VI, VII, and VIII, which were supplemented by these extract doses after the freeze-thaw process. To evaluate the effects of the Tribulus terrestris extract, the semen samples were incubated with the extract and evaluated with a light microscope for motility and viability.ResultsAfter cryopreservation, a significant improvement in spermatozoa viability was observed in group VII. In groups VII and VIII, motility, according to World Health Organization (WHO) criteria, increased considerably (p < 0.001). There was no significant difference among groups III, IV, and V.ConclusionThe present study demonstrated that the protective effects of Tribulus terrestris, which improves human sperm motility and viability, may be due to its antioxidant properties. On the basis of the results, the researchers concluded that Tribulus terrestris can be used as a safe therapeutic alternative to current modalities for the management of motility dysfunction in males.     

Histopathological changes in Poecilia latipinna male gonad due to Tribulus terrestris administration

Tribulus terrestris is a traditionally known non-toxic aphrodisiac herb for maleness. It was experimented recently to understand the effect and mechanism on mono sex production in Poecilia latipinna. It would help to develop a new eco-friendly way to masculinize P. latipinna, since males have higher commercial value than females. The different concentration (100, 150, 200, 250 and 300 mg/L) of T. terrestris extract and a control were tested for their effect on sex transformation/reversal in P. latipinna by immersing the newly born young ones in the respective concentrations for 2 mo. The obtained results indicate that a dose dependant masculinization is obtained due to T. terrestris administration, which improved the male proportion. Histological results revealed that the testes of fish treated with T. terrestris extract contained all stages of spermatogenesis, clearly demonstrating that the administration of T. terrestris extract to P. latipinna stimulated spermatogenesis. Thus, it is discernible that 0-d-old hatchlings of P. latipinna exposed to T. terrestris extract orient/reverse their sex more towards maleness besides yielding better growth and spermatogenesis which is a mandate for fancy fish industry.     

骨痹消对兔骨关节炎软骨细胞凋亡的影响

目的探讨中药早期治疗对兔实验性膝关节骨性关节炎(KOA)软骨细胞凋亡的影响。方法参照Okazaki等[1]伸膝制动OA动物模型法造模,随机分为正常组(A组)、模型组(B组)、骨痹消组(C组),于治疗6周、10周后测各组兔血清NO水平变化;软骨电镜结构及TUNEL法软骨细胞凋亡指数(AI)。结果 B组血清NO水平逐渐升高,C组有不同程度的降低;B组电镜下6周时可见凋亡细胞,10周可见凋亡小体;TUNEL法细胞凋亡原位检测:B组软骨细胞AI明显增高。治疗后C组下降明显,10周时与B组相比具极显著差异(P0.01)。结论骨痹消在一定程度上阻断软骨细胞凋亡,减少细胞凋亡数量,促进软骨的修复。     

应激对机体脑体功能的影响及其生物学机制初探

目的：探讨应激对机体脑体功能的损伤效应及其生物学机制,为应激损伤防护措施的制订提供科学依据。方法：采用束缚应激模型,观察模型动物认知功能、体能、海马LTP、血浆糖皮质激素、心电图、心肌组织结构等指标的变化。结果：应激动物学习记忆能力和运动耐力明显下降,血浆糖皮质激素水平显著升高,海马LTP诱发受到抑制,心电图异常改变,心功能紊乱,心肌组织结构出现病理损伤,心肌细胞凋亡率增加,心肌组织Hsp70表达水平随应激强度增加而逐渐降低。结论：应激诱导机体神经一内分泌功能紊乱,进而导致机体脑体功能损伤。     

麻黄素对仔鼠睾丸组织结构和Bax、EGF蛋白的影响

为探讨麻黄素对各发育期昆明小鼠(Mus musculus)仔鼠睾丸组织结构及Bax蛋白和表皮生长因子(epidermal growth factor,EGF)阳性表达的影响,选取雄性仔鼠采用递增剂量腹腔连续注射麻黄素,应用生物显微技术观察睾丸组织结构的变化,用免疫组织化学和体视学半定量方法检测Bax和EGF蛋白在睾丸组织中的表达.结果表明,麻黄素组仔鼠各发育阶段生精小管直径及上皮厚度明显小于对照组(P＜0.01或P＜0.05),初级精母细胞直径小于对照组(P＜0.01或P＜0.05),生精小管上皮各级生精细胞的发生较晚,且排列疏松而凌乱,细胞间空隙多,发育成熟期管腔内成熟精子较少;麻黄素组各发育期仔鼠睾丸中Bax和EGF蛋白的阳性表达与对照组相比显著增强(P＜0.01或P＜0.05).上述结果表明,麻黄素影响仔鼠睾丸组织的发育,麻黄素对各发育期仔鼠睾丸组织的损伤可能与Bax和EGF蛋白表达的增强有关.     

蝙蝠蛾拟青霉菌Cs-4菌株对腺嘌呤造成肾脏和睾丸损伤的恢复作用

本文用腺嘌呤建立肾脏和睾丸损伤的小鼠模型,探索蝙蝠蛾拟青霉菌Cs-4菌株对腺嘌呤造成的肾脏和睾丸损伤的恢复作用。将小鼠随机分为空白组、模型组和蝙蝠蛾拟青霉菌Cs-4菌粉的5个剂量组（350mg/kg、550mg/kg、750mg/kg、1 500mg/kg、3 000mg/kg）,连续给予相应药物21d,对各组小鼠的肾脏、睾丸通过HE染色、Masson染色,进行组织病理学检查,测定各组小鼠血清中尿素氮（BUN）、肌酐（Cr）、促卵泡刺激素（FSH）、黄体生成素（LH）含量,测定睾丸组织匀浆中的酸性磷酸酶（ACP）、琥珀酸脱氢酶（SDH）和乳酸脱氢酶（LDH）水平。结果表明,蝙蝠蛾拟青霉菌Cs-4菌株具有使小鼠体重逐渐恢复上升趋势、使受损的肾脏和睾丸组织结构恢复的作用,能使BUN、Cr、FSH、LH、ACP、SDH和LDH水平趋于正常化。     

p53 is involved in inducing testicular apoptosis in mice by the altered redox status following tertiary butyl hydroperoxide treatment

In testis, seminiferous epithelium is one of the most productive self-renewing systems in which apoptosis is an important phenomenon. Alteration in the cellular redox status has several detrimental effects on the cells, one of which is increased rate of apoptotic signals disturbing the natural balance. Since apoptotic responses to various therapeutic agents and toxicants follow diverse molecular mechanisms, therefore, the present study was designed to explore apoptosis in testes under the effect of oxidative stress. Tertiary butyl hydroperoxide (tBHP) was used to induce oxidative stress in mice. It was found that ROS production in testes by tBHP resulted in increased apoptosis. The apoptosis was evident from TUNEL staining in Zenker-fixed paraffin-embedded testicular sections of tBHP treated mice testis and DNA fragmentation analysis. Increased mRNA and protein expression of p53 in testis were observed by using RT-PCR and ELISA techniques, respectively. This indicates that p53 expression is linked to ROS generation in mice testes. The functional status of p53 was also assessed by upregulation of cyclin dependent kinase inhibitor, p21. Thus tBHP induced oxidative stress subject testicular cells to apoptosis which seems to involve p53.     

Activation of Bcl-2-Caspase-9 Apoptosis Pathway in the Testis of Asthmatic Mice

Background

Apoptosis plays a critical role in controlling the proliferation and differentiation of germ cells during spermatogenesis. Dysregulation of the fine-tuned balance may lead to the onset of testicular diseases. In this study, we investigated the activation status of apoptosis pathways in the testicular tissues under the background of an asthmatic mouse model.

Methods

Ten BALB/c mice were divided into two groups: the acute asthma group and the control group. In the acute asthma group, ovalbumin (OVA)-sensitized mice were challenged with aerosolized OVA for 7 days, while the control group was treated with physiological saline. After that, both epididymis and testis were collected to determine the sperm count and motility. Apoptosis in the testis was evaluated by DNA ladder, immunochemistry and further by PCR array of apoptosis-related genes. Finally, the cleavage of caspase-3 and poly ADP-ribose polymerase (PARP) was determined by western blot and the enzymatic activities of caspase-9 and 3/7 were assessed using Caspase-Glo kits.

Results

Compared with control mice, significant decreases in the body weight, testis weight, sperm count and motility were seen in the experimental group. DNA ladder and immunochemistry showed significant increase in apoptotic index of the asthmatic testis, whereas a decrease in mRNA expression of Bcl-2 and increases in Bax, BNIP3, caspase-9, and AIF were observed in the asthma group. Furthermore, protein levels of AIF were significantly upregulated, while the translational expression of Bcl-2 was downregulated markedly. Consistently, caspase-9 activity in the testis of asthma mice was significantly higher than that of the control group.

Conclusion

Collectively, these results showed that Bcl-2-caspase-9 apoptosis pathway was clearly activated in the testis of asthmatic mice with the increased expression of apoptosis-related genes and proteins. To our knowledge, this is the first report demonstrating that asthma could lead to the activation of the mitochondrial apoptosis signaling pathway in the mouse testis.