Super Antibiotics, Part II. Hyperforin, Mass Spectroscopy (MS) and Gas Chromatography-Mass Spectrometry (GC-MS), Evidence of Permeability of the Blood-Testis Barrier (BTB) and the Blood-Brain Barrier (BBB) to Hyperforin

In the first article of this series, we presented some evidence of hyperforin as an antibiotic, antiprotozoal, antiviral, anticancer, and immunomodulatory substance. In the present article, evidence of the permeability of the blood-testis barrier (BTB) and blood-brain barrier (BBB) to hyperforin and its distribution in other organs of the domestic pig (Sus scrofa domesticus) are revealed. Seven-month-old male boars with a body mass of 100 kg were fed a diet containing hyperforin. Organs were surgically removed under anesthesia. Organs were suitable prepared and extracted, and then analyzed using gas chromatography-mass spectrometry with supersonic molecular beams (GC-MS with SMB). The presence of hyperforin was recorded in all organs and body fluids. Special attention was paid to the evaluation of the presence of hyperforin in the brain and testes of experimental animals. The presence of hyperforin in the brain and testes of experimental animals was established by GC-MS with SMB. The results are of interest because penicillin and numerous other antibiotics cannot pass through the BTB or BBB if healthy or non-inflamed, which limits their use in patients with meningitis and gonorrhea. The findings are also of interest in cases of penicillin- and multi-antibiotic-resistant bacterial infections.

Blood-testis barrier and spermatogenesis： lessons From genetically-modified mice

The blood-testis barrier （BTB） is found between adjacent Sertoli cells in the testis where it creates a unique microenvironment for the development and maturation of meiotic and postmeiotic germ cells in seminiferous tubes. It is a compound proteinous structure, composed of several types of cell junctions including tight junctions （TJs）, adhesion junctions and gap junctions （GJs）. Some of the junctional proteins function as structural proteins of BTB and some have regulatory roles. The deletion or functional silencing of genes encoding these proteins may disrupt the BTB, which may cause immunological or other damages to meiotic and postmeiotic cells and ultimately lead to spermatogenic arrest and infertility. In this review, we will summarize the findings on the BTB structure and function from genetically-modified mouse models and discuss the future perspectives.

Effect of Electromagnetic Pulse Exposure on Permeability of Blood-testicle Barrier in Mice

Objective To study the effect of electromagnetic pulse （EMP） exposure on the permeability of blood-testicle barrier （BTB） in mice. Methods Adult male BALB/c mice were exposed to EMP at 200 kV/m for 200 pulses with 2 seconds interval. The mice were injected with 2% Evans Blue solution through caudal vein at different time points after exposure, and the permeability of BTB was monitored using a fluorescence microscope. The testis sample for the transmission electron microscopy was prepared at 2 h after EMP exposure. The permeability of BTB in mice was observed by using Evans Blue tracer and lanthanum nitrate tracer. Results After exposure, cloudy Evans Blue was found in the testicle convoluted seminiferous tubule of mice. Lanthanum nitrate was observed not only between testicle spermatogonia near seminiferous tubule wall and sertoli cells, but also between sertoli cells and primary spermatocyte or secondary spermatocyte. In contrast, lanthanum nitrate in control group was only found in the testicle sertoli cells between seminiferous tubule and near seminifdrous tubule wall. Conclusion EMP exposure could increase the permeability of BTB in the mice.

Ⅳ型胶原蛋白NC1片段在精子发生及血睾屏障中作用的研究进展

在哺乳动物睾丸中,位于生精上皮基底膜附近的血睾屏障(blood-testis barrier,BTB)对精子发生至关重要。BTB由相邻的支持细胞(Sertoli细胞)连接而成,将生精上皮分为靠近基底膜的“基底室”和靠近生精小管管腔的“近腔室”,为精子发生提供了免疫屏障。Ⅳ型胶原蛋白是基底膜的重要组成成分,啮齿类动物的睾丸中Ⅳ型胶原蛋白主要是由3条α3链组成的三螺旋结构。大鼠睾丸的Ⅳ型胶原蛋白α3链C端可以水解成大小为28 ku的蛋白片段,称为C端非胶原结构域(noncollagenous domain-peptide,NC1片段)。NC1片段作为一种基底膜源性多肽在参与精子发生与维系BTB功能方面发挥了重要作用。本文通过分析、总结近期研究结果,概述Ⅳ型胶原蛋白NC1片段在睾丸中的作用及其机制,尤其是其在精子发生与BTB中作用的研究进展。

Regulation of blood-testis barrier dynamics by the mT0RCl/rpS6 signaling complex:an in vitro study

During spermatogenesis, developi ng germ cells that lack the cellular ultrastructures of filopodia and lamellipodia gen erally found in migrating cells, such as macrophages and fibroblasts, rely on Sertoli cells to support their transport across the seminiferous epithelium. These in elude the transport of preleptote ne spermatocytes across the blood-testis barrier (BTB), but also the transport of germ cells, in particular developing haploid spermatids, across the seminiferous epithelium, that is to and away from the tubule lumen, depending on the stages of the epithelial cycle. On the other hand, cell junctions at the Sertoli cell-cell and Sertoli-germ cell in terface also un dergo rapid remodeli ng, invo Iving disassembly and reassembly of cell j un ctions, which, in turn, are supported by actin- and microtubule-based cytoskeletal remodeling. Interestingly, the underlying mechanism(s) and the invoIving biomolecule(s) that regulate or support cytoskeletal remodeling remain largely unknown. Herein, we used an in vitro model of primary Sertoli cell cultures that mimicked the Sertoli BTB in vivo overexpressed with the ribosomal protei n S6 (rpS6, the down stream signali ng protein of mammalian target of rapamycin complex 1 [mTORCl]) cloned into the mammalian expression vector pCI-neo, namely, quadruple phosphomimetic and constitutively active mutant of rpS6 (pCI-neo/p-rpS6-MT) versus pCI-neo/rpS6-WT (wild-type) and empty vector (pCI-neo/Ctrl) for studies. These findings provide compelling evidence that the mT0RCl/rpS6 signal pathway exerted its effects to promote Sertoli cell BTB remodeling. This was mediated through changes in the organization of actin- and microtubulebased cytoskeletons, involving changes in the distribution and/or spatial expression of actin- and microtubule-regulatory proteins.

胶质瘤血瘤屏障体外模型的形态学观察

目的 探讨胶质瘤血瘤屏障体外模型的形态学特征。 方法 利用电镜技术观察血管内皮细胞ECV30 4 和C6 胶质瘤细胞共培养 (混合共培养、Transwell共培养、Transwell膜两面共培养 )后的内皮细胞的孔窗、内皮细胞之间的连接及内皮细胞与肿瘤细胞的相互关系、瘤细胞的“血管周足”等的形态学特征 ;并与 4例人脑胶质瘤组织标本的血瘤屏障进行比较。 结果 电镜下发现ECV30 4 细胞经与C6 细胞按 3种不同方式共培养汇合后均为无孔窗型内皮细胞 ,细胞间出现紧密连接 ;Transwell共培养的膜上C6 细胞未见伸出伪足突向膜的微孔 ;经膜两面共培养的瘤细胞则可通过Transwell的微孔突向内皮细胞侧 ,但瘤细胞未伸出伪足包绕内皮细胞或突入内皮细胞间隙 ,瘤细胞的“血管周足”不完整 ,后两者与脑胶质瘤组织标本的血瘤屏障的形态特征相类似。 结论 在体外经Transwell膜两面共培养的ECV30 4 和C6 胶质瘤细胞的系统可在一定程度模拟体内的血瘤屏障的形态学特征。

睾酮通过抑制Rab13表达影响支持细胞屏障通透性

探讨Rab13 GTPase对血睾屏障(blood testis barrier,BTB)通透性的调节机制。体外分离培养20 d龄大鼠睾丸支持细胞,睾酮处理后测定跨上皮电阻(transepithelial electrical resistance,TER),FITC-鬼笔环肽染色分析F-actin分布,并采用Western印迹检测Rab13在睾酮处理前后的表达变化;si RNA干扰培养支持细胞中Rab13的表达,Western印迹检测沉默效果并检测连接蛋白表达变化;TER测定Rab13沉默前后上皮屏障功能改变,F-actin染色分析Rab13沉默对肌动蛋白丝分布的影响。结果显示,睾酮处理可使体外支持细胞屏障TER值升高,F-actin在膜下聚集增强,且Rab13在睾酮处理后表达量明显降低;Rab13 si RNA处理不影响连接蛋白质的表达,但可引起F-actin在细胞连接处的分布增强,导致体外BTB屏障功能增强。表明Rab13可通过调节F-actin的排布影响体外BTB通透性。

Expression of E-cadherin and α-catenin in a varicocele-induced infertility rat model

The roles of E-cadherin and α-catenin were evaluated in the development of varicocele-induced infertility. Analysis of the association between the expression of E-cadherinla-catenin and clinical/pathological parameters was performed. Thirty lO-week-old male rats （experimental group） were used for the experiments; the left renal vein was ligated to form a varicocele. The abdomen was incised in 30 rats （control group） and no procedure was performed on 10 rats （baseline group）. The weights of the left testis, serum reactive oxygen species （ROS）, testosterone, luteinizing hormone （LH）, follicle-stimulating hormone （FSH） and degenerative changes in the seminiferous tubules after 4 and 8 weeks were recorded. The expression of E-cadherin and α-catenin was evaluated by immunohistochemical （IHC） staining and Western blot analysis. The ROS increased in the 8-week experimental group, compared with the baseline and control groups （P〈0.001 for both）. Additionally, FSH significantly increased in the 4- and 8-week experimental group compared with the control groups （P= 0.013 and P= 0.032, respectively）. The ratio of degenerative changes in the seminiferous tubules of the experimental groups increased. The IHC staining showed that the expression of E-cadherin and a-catenin decreased in the 4- and 8-week experimental groups. Similar to the IHC staining, the experimental group had decreased reactivity on Western blot analysis. The expression of E-cadherin and a-catenin was significantly associated with the ROS and degenerative changes in the seminiferous tubules. The results of this study suggest that damage to the blood-testis barrier （BTB） is associated with varicocele-induced male infertility, and that ROS may cause damage to the BTB.

Evidence on Invasion of Blood,Adipose Tissues,Nervous System and Reproductive System of Mice After a Single Oral Exposure:Nanoplastics versus Microplastics

Objective This study was designed to provide the evidences on the toxicokinetics of microplastics(MPs)and nanoplastics(NPs)in the bodies of mammals.Methods 100 nm,3μm,and 10μm fluorescent polystyrene(PS)beads were administered to mice once by gavage at a dose of 200 mg/kg body weight.The levels and change of fluorescence intensity in samples of blood,subcutaneous fat,perirenal fat,peritesticular fat,cerebrum,cerebellum,testis,and epididymis were measured at 0.5,1,2,and 4 h after administration using an IVIS Spectrum small-animal imaging system.Histological examination,confocal laser scanning,and transmission electron microscope were performed to corroborate the findings.Results After confirming fluorescent dye leaching and impact of pH value,increased levels of fluorescence intensity in blood,all adipose tissues examined,cerebrum,cerebellum,and testis were measured in the 100 nm group,but not in the 3 and 10μm groups except in the cerebellum and testis at 4 h for the 3μm PS beads.The presence of PS beads was further corroborated.Conclusion After a single oral exposure,NPs are absorbed rapidly in the blood,accumulate in adipose tissues,and penetrate the blood-brain/testis barriers.As expected,the toxicokinetics of MPs is significantly size-dependent in mammals.

Rearrangement of Actin Cytoskeleton by Zika Virus Infection Facilitates Blood–Testis Barrier Hyperpermeability

In recent years,various serious diseases caused by Zika virus(ZIKV)have made it impossible to be ignored.Confirmed existence of ZIKV in semen and sexually transmission of ZIKV suggested that it can break the blood–testis barrier(BTB),or Sertoli cell barrier(SCB).However,little is known about the underlying mechanism.In this study,interaction between actin,an important component of the SCB,and ZIKV envelope(E)protein domainⅢ(EDⅢ)was inferred from coimmunoprecipitation(Co-IP)liquid chromatography–tandem mass spectrometry(LC–MS/MS)analysis.Confocal microscopy confirmed the role of actin filaments(F-actin)in ZIKV infection,during which part of the stress fibers,the bundles that constituted by paralleled actin filaments,were disrupted and presented in the cell periphery.Colocalization of E and reorganized actin filaments in the cell periphery of transfected Sertoli cells suggests a participation of ZIKV E protein in ZIKV-induced F-actin rearrangement.Perturbation of F-actin by cytochalasin D(CytoD)or Jasplakinolide(Jas)enhanced the infection of ZIKV.More importantly,the transepithelial electrical resistance(TEER)of an in vitro mouse SCB(mSCB)model declined with the progression of ZIKV infection or overexpression of E protein.Co-IP and confocal microscopy analyses revealed that the interaction between F-actin and tight junction protein ZO-1 was reduced after ZIKV infection or E protein overexpression,highlighting the role of E protein in ZIKV-induced disruption of the BTB.We conclude that the interaction between ZIKV E and F-actin leads to the reorganization of F-actin network,thereby compromising BTB integrity.

乙醇在HBV DNA转染中对大鼠血睾屏障的影响及其机制探讨(英文)

目的:探讨机体摄入大量乙醇后,血睾屏障(blood-testis barrier,BTB)能否有效阻止含乙型肝炎病毒(hepatitis B virus,HBV)DNA的质粒转染生精小管生精上皮细胞和影响BTB完整性的因素。方法:取20只Wistar成熟雄性大鼠随机分为实验组(A组)和对照组(B组),应用聚合酶链反应(polymerase chain reaction,PCR)和原位杂交(in situ hybridization,ISH)检测HBVDNA的存在和生精小管的转染情况,应用透射电子显微镜(transmission electron microscopy,TEM)技术检测BTB与生精内环境的超微结构形态学变化。结果:①PCR:A组样本可见特异的HBVDNA阳性条带;②原位杂交:A组发现阳性杂交信号弥散,可被广泛发现于生精上皮基底室和近腔室的生精细胞上;③TEM:A组大鼠睾的生精小管基膜厚薄不均,基膜组织疏松增厚,成波浪式皱褶,可见基膜断裂,精原细胞与支持细胞及生精小管的基膜之间出现较多空泡,生精小管、生精上皮、生精细胞及支持细胞与相邻细胞之间的间隙扩大。结论:BTB的完整性是其起保护功能的重要基础,乙醇可以破坏其完整性,会使全部生精细胞易受HBV的感染。

肌动蛋白结合蛋白细丝蛋白A参与肿瘤坏死因子α诱导的支持细胞屏障功能降低的研究

目的:探讨肌动蛋白结合蛋白细丝蛋白A(filamin A,FLNa)在肿瘤坏死因子α(tumor necrosis factorα,TNFα)调节支持(Sertoli)细胞屏障功能过程中发挥的作用。方法:体外分离培养20日龄大鼠睾丸支持细胞,用10μg/L TNFα处理后测定跨上皮电阻(trans-epithelial electrical resistance,TER),反映屏障功能改变;Western blotting分析TNFα处理后FLNa表达水平变化;细胞免疫荧光或鬼笔环肽染色分别检测FLNa及微丝(F-actin)的定位改变;FLNa si RNA干扰后检测支持细胞屏障功能。结果:TNFα处理后TER值较对照组显著降低(P<0.01);Western blotting结果显示TNFα处理组FLNa水平明显下降(P<0.01);细胞形态学检测亦表明TNFα处理后FLNa及F-actin在支持细胞的分布有显著改变;TER测定结果表明FLNa经si RNA沉默后可降低血睾屏障(BTB)功能(P<0.01)。结论:FLNa可通过调节F-actin在支持细胞中的排布参与TNFα引起的BTB功能降低。

血脑/血瘤屏障体外模型的构建、形态与功能特性

目的构建并观测血脑/血瘤屏障体外模型的形态与功能特性,为中枢神经系统药物跨血脑和(或)血瘤屏障研究提供体外实验模型。方法将分离的Balb/C小鼠脑微血管内皮细胞 (BMEC)在铺有明胶的微孔膜上单层培养或与大鼠胶质瘤细胞C6双室共培养,分别建立血脑屏障 (BBB)和血瘤屏障(BTB)模型,采用光镜和扫描电镜观察BMEC形态,采用Millicell-ERS系统检测屏障中的跨内皮电阻(TEERs),免疫细胞化学检测P-糖蛋白(P-gp)表达,并比较BBB和BTB中 BMEC的形态和功能特征。结果两组模型中的BMEC均形成单层生长和良好的细胞间紧密连接, 产生较高的TEERs值,并检测到P-gp表达。瘤细胞诱导使BMEC间出现间隙,同时相点TEERs值降低,P-gp表达减弱。结论两种体外模型可用于研究药物跨血脑和(或)血瘤屏障特性,其中BTB 模型可能更适合抗肿瘤药物的研究。

高功率微波对大鼠血睾屏障通透性的影响及防护研究

目的探讨高功率雷达微波辐照对大鼠血睾屏障通透性的影响及防护研究。方法将成年雄性SD大鼠随机分为两大组(分别进行单次和连续微波辐照实验研究)。每大组又随机分为对照组、微波辐照组、药物防护组和物理防护组4个小组,每组10只。药物防护组大鼠在辐照前用安多霖药物灌胃。对微波辐照组、药物防护组和物理防护组大鼠进行辐照,其中物理防护组辐照盒外面套有防电磁波辐射面料。对照组大鼠行假辐照。辐照后取大鼠睾丸组织用含有硝酸镧颗粒的戊二醛固定液固定,用透射电镜观察血睾屏障通透性的变化。结果与对照组相比,辐照组的睾丸组织生精细胞间隙明显增大,发生不同程度的变性、线粒体空泡化,血睾屏障紧密连接处可见镧颗粒沉积,而在药物防护组和物理防服组,硝酸镧颗粒未穿过血睾屏障紧密连接。结论高功率雷达微波辐照能够使大鼠血睾屏障通透性显著增加,对机体产生一定程度的生殖毒性,安多霖胶囊和防电磁波辐射面料表现出了较好的防护效果。

Molecular basis of cryptorchidism-induced infertility

Artificial cryptorchidism or local testicular heat treatment can induce reversible oligospermia or azoospermia in monkeys and rats via germ cell apoptosis. Local warming of monkey testes in water at 43°C for 2 consecutive days (30 min per day) decreased the number of sperm in the semen by up to 80% on d 28, and the effect was completely reversed on d 144. Germ cells rely heavily on Sertoli cells for structural and nutritional support. Specialized junctions that play a pivotal role in spermatogenesis occur at sites of Sertoli-Sertoli and Sertoli-germ cell contact in the seminiferous epithelium. We demonstrated that expression of tight junction (TJ)-associated molecules, such as occludin and zonula occludens-1 (ZO-1), were greatly reduced 24–48 h after heat treatment, while the permeability of the blood-testis barrier (BTB) was simultaneously increased, but recovered 10 d later. These results indicate a reversible disruption of the BTB associated with transient inductions of transforming growth factor (TGF) β2 and β3 expression, p38 mitogen-activated protein kinase and extracellular signal-regulated kinase activation, and concomitant loss of occludin and ZO-1. This suggests that expression of TJ-associated molecules and the BTB was reversibly perturbed by mild testicular hyperthermia, and that the heat-induced induction of TGF-β might be involved in downregulating TJ-associated proteins, leading to cell junction reduction. This review discusses the changes in total gene expression patterns after experimental cryptorchidism in adult mouse testes, and the cloning of several novel, physiologically significant spermatogenesis-specific genes.

Claudin-11 and occludin are major contributors to Sertoli cell tight junction function, in vitro

The Sertoli cell tight junction （T J） is the key component of the blood-testis barrier, where it sequesters developing germ cells undergoing spermatogenesis within the seminiferous tubules. Hormonally regulated claudin-11 is a critical transmembrane protein involved in barrier function and its murine knockout results in infertility. We aimed to assess quantitatively the significance of the contribution of claudin-11 to TJ function, in vitro, using siRNA-mediated gene silencing. We also conducted an analysis of the contribution of occludin, another intrinsic transmembrane protein of the TJ. Silencing of claudin-11 and/or occludin was conducted using siRNA in an immature rat Sertoli cell culture model. Transepithelial electrical resistance was used to assess quantitatively TJ function throughout the culture. Two days after siRNA treatment, cells were fixed for immunocytochemical localization of junction proteins or lyzed for RT-PCR assessment of mRNA expression. Silencing of claudin-11, occludin, or both resulted in significant decreases in TJ function of 55% （P 〈 0.01）, 51% （P 〈 0.01）, and 62% （P 〈 0.01）, respectively. Data were concomitant with significant decreases in mRNA expression and marked reductions in the localization of targeted proteins to the Sertoli cell TJ. We provide quantitative evidence that claudin-11 contributes significantly （P 〈 0.01） to Sertoli cell TJ function in vitro. Interestingly, occludin, which is hormonally regulated but not implicated in infertility until late adulthood, is also a significant （P 〈 0.01） contributor to barrier function. Our data are consistent with in vivo studies that clearly demonstrate a role for these proteins in maintaining normal TJ barrier structure and function.

Ezrin： a regulator of actin microfilaments in ce unctions of the rat testis

Ezrin, radixin, moesin and merlin （ERM） proteins are highly homologous actin-binding proteins that share extensive sequence similarity with each other. These proteins tether integral membrane proteins and their cytoplasmic peripheral proteins （e.g., adaptors, nonreceptor protein kinases and phosphatases） to the microfilaments of actin-based cytoskeleton. Thus, these proteins are crucial to confer integrity of the apical membrane domain and its associated junctional complex, namely the tight junction and the adherens junction. Since ectoplasmic specialization （ES） is an F-actin-rich testis-specific anchoring junction-a highly dynamic ultrastructure in the seminiferous epithelium due to continuous transport of germ cells, in particular spermatids, across the epithelium during the epithelial cycle-it is conceivable that ERM proteins are playing an active role in these events. Although these proteins were first reported almost 25 years and have since been extensively studied in multiple epithelia/endothelia, few reports are found in the literature to examine their role in the actin filament bundles at the ES. Studies have shown that ezrin is also a constituent protein of the actin-based tunneling nanotubes （TNT） also known as intercellular bridges, which are transient cytoplasmic tubular ultrastructures that transport signals, molecules and even organelles between adjacent and distant cells in an epithelium to coordinate cell events that occur across an epithelium. Herein, we critically evaluate recent data on ERM in light of recent findings in the field in particular ezrin regarding its role in actin dynamics at the ES in the testis, illustrating additional studies are warranted to examine its physiological significance in spermatogenesis.