Protective Effect of Quercetin on Cadmium‐Induced Oxidative Toxicity on Germ Cells in Male Mice

Cadmium is a toxic heavy metal that is widely distributed in the environment. As a critical process, oxidative toxicity mediates the morphological and functional damages in germ cells after cadmium exposure. In this study, the protective effect of quercetin on cadmium‐induced oxidative toxicity was investigated in mouse testicular germ cells. After oral administration of cadmium chloride at 4 mg/kg body weight for 2 weeks, damages in spermatozoa occurred in the early stage of spermatogenesis. Cadmium treatment significantly decreased the testicular antioxidant system, including decreases in the glutathione (GSH) level, superoxide dismutase (SOD), and GSH peroxidase (GSH‐Px) activities. Moreover, exposure to cadmium resulted in an increase of hydrogen peroxide production and lipid peroxidation in testes. In addition, cadmium provoked germ cell apoptosis by upregulating expression of the proapoptotic proteins Bax and caspase‐3 and downregulating expression of the antiapoptotic protein Bcl‐XL. However, combined administration of a common flavonoid quercetin at 75 mg/kg body weight significantly attenuated cadmium‐induced germ cell apoptosis by suppressing the hydrogen peroxide production and lipid peroxidation in testicular tissue. Simultaneous supplementation of quercetin markedly restored the decrease in GSH level and SOD and GSH‐Px activities elicited by cadmium treatment. Additionally, quercetin protected germ cells from cadmium‐induced apoptosis by downregulating the expression of Bax and caspase‐3 and upregulating Bcl‐XL expression. These results indicate that quercetin, due to its antioxidative and antiapoptotic characters, may manifest effective protective action against cadmium‐induced oxidative toxicity in mouse testicular germ cells. Anat Rec, 2011. © 2010 Wiley‐Liss, Inc.     

Effect of Melatonin, Ascorbic Acid, and Succinic Acid on the Cumulative Toxic Effect of Repeated Treatment with Gammafos (Amifostine)

Daily treatment of outbred albino mice with gammafos in radioprotective doses of 300 and 500 mg/kg for 4 days produced a cumulative toxic effect. This effect was not observed after decreasing the dose of gammafos to 100 mg/kg. Repeated peroral administration of melatonin and ascorbic acid in a dose of 200 mg/kg 30 min before treatment with gammafos reduced its cumulative toxic effect. Succinic acid in a dose of 100 mg/kg was ineffective under these conditions. The cumulative death time for 50% animals receiving gammafos alone or in combination with melatonin, ascorbic acid, and succinic acid was 3.08, 4.29, 4.06, and 2.97 days, respectively.     

大鼠睾丸支持细胞的分离纯化与鉴定

目的　简化大鼠睾丸支持细胞的分离纯化方法 ,通过不同方法对Sertoli细胞进行形态学观察鉴定。方法　取鼠龄 16～ 2 2天的雄性Wistar大鼠睾丸 ,采用酶次第消化 ,培养过程中纯化Sertoli细胞 ,并用多种方法对Sertoli细胞进行鉴定。结果　大鼠睾丸支持细胞占培养细胞总数的 90 %以上。HE染色 ,Sertoli细胞突起很多 ,核仁清晰 ,在胞质中可见吞噬物和大小不等的空泡 ;甲基绿 派洛宁染色 ,Sertoli细胞富含RNA ;Feulgen染色和透射电镜 ,核仁周围可见卫星核小体。结论　本实验培养方法可获得更多、纯度高的Sertoli细胞 ,HE染色、甲基绿 派洛宁染色、Feulgen染色和透射电镜是鉴定Sertoli细胞的有效方法     

Morphological and Functional State of Rat Ovaries in Early and Late Periods after Administration of Platinum Cytostatics

Experiments on Wistar rats showed that cisplatin and carboplatin induced similar morphological alterations in the ovaries. Both agents reduced the number of structural and functional elements, but the effect of cisplatin was more pronounced. Morphological changes observed in the early period after injection of the preparations were accompanied by prolongation of the estrous cycle, which was longer in rats treated with carboplatin. Partial and reversible sterility was observed in females at the early terms after cisplatin treatment.     

Different Localization of ATP Sensitive K+ Channel Subunits in Rat Testis

ATP sensitive K⁺ (K_ATP) channels are important linkage of cell membrane excitability to its cellular bioenergetic state. These channels are composed of pore‐forming subunits and regulatory subunits. The present study focused on the cellular expressions and localizations of these subunits in rat testis. RT‐PCR analysis showed that rat testis contained five K_ATP channel subunits, Kir6.1, Kir6.2, SUR1, SUR2A and SUR2B. Immunoblot assay showed that proteins of Kir6.1, Kir6.2, SUR2A and SUR2B were expressed in rat testis. Immunohistochemistry revealed these K_ATP channel subunits were positive in different localizations of spermatogenic cells, Sertoli cells and Leydig cells, which implies these subunits playing important roles in spermatogenesis. Co‐localization of Kir6.2 with SUR2B was determined in acrosome or head cap of spermatids by double immunofluorescence analysis by indicating K_ATP channel might be formed by Kir6.2 and SUR2B in acrosome of spermatids. Different localizations of the K_ATP channel subunits in the cell membrane and membranous organelles of spermatogenic cells and Sertoli cells indicated the complex and multiple functions of K_ATP channels in rat testis. Anat Rec, 2011. © 2011 Wiley‐Liss, Inc.     

5‐Nitro‐2‐(3‐Phenylpropylamino) Benzoic Acid Induced Drug Resistance to Cisplatin in Human Erythroleukemia Cell Lines

Mechanisms of cisplatin resistance in cancer cells are not fully understood. Here, we showed a critical role for the chloride channel‐3 (ClC‐3) in cisplatin resistance in human erythroleukemia K562 and RK562 cells. We found that a chloride channel blocker 5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid (NPPB) could protect cells from cisplatin‐induced apoptosis. NPPB treatment decreased the mRNA and the protein expression of Bax/Bcl‐2, decreased the protein expressions of cytochrome C and caspase‐3, and increased the mRNA expressions of cyclin D1 and ClC‐3 in cells treated with cisplatin. The caspase‐3 activity was decreased significantly and the rate of cell apoptosis was decreased. NPPB treatment increased CIC‐3 expression, which could increase acidification of intracellular compartments, and increased sequestration of cisplatin, inducing decreased effective drug concentrations, and subsequently cell death. Collectively, our data indicate that NPPB can induce drug resistance to cisplatin by upregulating the expression of CIC‐3. NPPB‐induced CIC‐3 expression facilitates acidification of sequestrated cisplatin, and plays an important role in preventing cisplatin‐induced apoptosis in human erythroleukemia K562 and RK562 cells. Anat Rec,, 2011. © 2011 Wiley‐Liss, Inc.     

褪黑激素对小鼠睾丸生精细胞凋亡和睾丸间质细胞nNOS表达的影响

目的：观察外源性褪黑激素(MT)对睾丸生精细胞凋亡和nNOS表达的影响,探讨MT诱导生精细胞凋亡的可能机制。方法：20d和30d小鼠,应用化学发光法检测血清睾酮浓度,TUNEL测定生精细胞凋亡,免疫组化,SABC法观察nNOS在睾丸中的表达。结果：20d和30d实验组血清中睾酮水平明显下降,TUNEL阳性生精细胞数显著增多,TUNEL阳性生精细胞数与睾酮浓度呈负相关;20d实验小鼠中睾丸间质nNOS阳性细胞数与对照组相比明显减少,且与睾酮浓度呈正相关,而与TUNEL阳性生精细胞数呈负相关。结论：MT具有促进生精细胞凋亡的作用,与睾酮分泌受抑制有关;青春期前MT引起生精细胞凋亡可能还与nNOS表达有关。     

Effect of Pollack Liver Oil on Lipid Metabolism in Rat Testes

Rat testes were examined by biochemical and histochemical methods after 1, 3, 6, and 12-month pollack liver fat-supplemented diet (0.1 g/kg). Phospholipid concentration in the testicular tissue increased by 16% and cholesterol concentrations decreased by almost 20% after feeding fish oil for 1-3 months, while after 6-12-month diet further increase in cholesterol level in the testes and degeneration of some gonadal cells were noted. The most pronounced changes were seen in more differentiated elements of the spermatogenic epithelium in convoluted tubules.     

Distribution of Zonula Occludens‐1 and Occludin and alterations of testicular morphology after in utero radiation and postnatal hyperthermia in rats

In utero irradiation (IR) and postnatal hyperthermia (HT) exposure cause infertility by decreasing spermatogenic colony growth and the number of sperm in rats. Four groups were used: (i) Control group, (ii) HT group (rats exposed to hyperthermia on the 10th postnatal day), (iii) IR group (rats exposed to IR on the 17th gestational day) and (iv) IR + HT group. Three and six months after the procedures testes were examined by light and electron microscopy. Some degenerated tubules in the HT group, many vacuoles in spermatogenic cells and degenerated tight junctions in the IR group, atrophic tubules and severe degeneration of tight junctions in the IR + HT group were observed. ZO‐1 and occludin immunoreactivity were decreased and disorganized in the HT and IR groups and absent in the IR + HT group. The increase in the number of apoptotic cells was accompanied by a time‐dependent decrease in haploid, diploid and tetraploid cells in all groups. Degenerative findings were severe after 6 months in all groups. The double‐hit model may represent a Sertoli cell only model of infertility due to a decrease in spermatogenic cell and alterated blood‐testis barrier proteins in rat.     

The Protective Effect of Curcumin on Hepatotoxicity and Ultrastructural Damage Induced by Cisplatin

Abstract

We investigate the protective effect of curcumin (CU) on the hepatic ultrastructural damage induced by cisplatin in mice. 18 adult Kunming mice were randomly divided into normal saline (NS) group, cisplatin treatment group (CP) and CU?+?CP group (n?=?6 for each group). Mice in control group and CP group were administered with NS (20?mL/kg/day) and CU?+?CP group were i.p injected with CU (200?mg/kg/day) for 10 days. Then cisplatin (50?mg/kg/day) was injected in mice of CP group and CU?+?CP group, while those in control group were given the same volume of NS. Five days after injection all mice were killed and liver dissected. The hepatic morphological structures were observed under light microscope and transmission electron microscope. The results indicated that CU alleviated the hepatic histopathological damages induced by cisplatin, which included declined body weight, vacuolated cytoplasm and blurred liver trabecular structure. Moreover, no hepatic ultrastructural damages were observed in the CU protective group with condensed and marginated nuclear chromatin, bile canaliculi outstreched and bile deposited.     

db/db自发性糖尿病小鼠睾丸生殖细胞凋亡及相关基因Bcl-2和Bax表达

目的　研究糖尿病时生殖细胞凋亡及相关基因Bcl 2和Bax表达变化。方法　以脱氧核糖核酸末端转移酶介导的dUTP缺口末端标记法 (TUNEL)检测生殖细胞的凋亡 ,免疫组化ABC法检测凋亡相关基因Bcl 2和Bax的表达变化。结果　凋亡管数及凋亡阳性率糖尿病组均较正常高 ,而且随糖尿病病程进展呈明显增加趋势 ,对照组中随加龄二者也有增加。Bcl 2蛋白以散在的颗粒状大量分布于除精原细胞的各级生精细胞中 ,且以精子细胞质为最多。随糖尿病病变加重 ,Bcl 2表达减少 ,而且 ,糖尿病组比对照组Bcl 2表达率低。Bax蛋白比Bcl 2蛋白表达量少 ,以散在的颗粒状分布于曲细精管基底部的精原细胞和支持细胞中。随糖尿病病程进展和年龄增加 ,表达均呈增加趋势 ,而且 ,糖尿病组比对照组Bax表达率高。结论　在db/db自发性糖尿病小鼠睾丸 ,伴随生殖细胞凋亡增加 ,Bcl 2表达减少 ,Bax表达增加 ,凋亡及相关基因在糖尿病生殖功能障碍中起重要作用     

Sequential testicular atrophy involves changes in cellular proliferation and apoptosis associated with variations in aromatase P450 expression levels in Irs‐2‐deficient mice

Insulin receptor substrate 2 (Irs‐2) is an intracellular protein susceptible to phosphorylation after activation of the insulin receptor. Its suppression affects testis development and its absence induces peripheral resistance to insulin. The aim of this study was to identify changes induced by the deletion of Irs‐2 in the testicular structure and by the altered expression of cytochrome P450 aromatase, a protein necessary for the development and maturation of germ cells. Adult knockout (KO) mice (Irs‐2^?/?, 6 and 12 weeks old) and age‐matched wild‐type (WT) mice were used in this study. Immunohistochemistry and Western blot analyses were performed to study proliferation (PCNA), apoptosis (active caspase‐3) and P450 aromatase expression in testicular histological sections. Deletion of Irs‐2 decreased the number of epithelial cells in the seminiferous tubule and rete testis. Aberrant cells were frequently detected in the epithelia of Irs‐2^?/? mice, accompanied by variations in spermatogonia, which were shown to exhibit small hyperchromatic nuclei as well as polynuclear and anuclear structures. The amount of cell proliferation was significantly lower in Irs‐2^?/? mice than in WT mice, whereas apoptotic processes were more common in Irs‐2^?/? mice. Aromatase P450 reactivity was higher in 6‐week‐old KO mice than in WT mice of the same age and was even higher at 12 weeks. Our results suggest that Irs‐2 is a key element in spermatogenesis because silencing Irs‐2 induces the sequential development of testicular atrophy. The effects are observed mainly in germ cells present in the seminiferous tubule, which may be due to changes in cytochrome P450 aromatase expression.     

氯化血红素预处理对全脑缺血/再灌注大鼠海马神经元的保护作用

目的 研究氯化血红素对大鼠全脑缺血/再灌注后海马神经元的保护作用。方法 84只雄性Wistar大鼠采用4血管闭塞法制造大鼠全脑缺血模型,观察不同剂量氯化血红素对大鼠全脑缺血/再灌注后海马CA1区神经元形态学的影响,并应用流式细胞仪检测海马神经元凋亡率。结果 氯化血红素预处理组与缺血/再灌组相比海马神经元凋亡率下降,海马CA1区锥体细胞组织学分级明显降低,神经元密度明显升高(p<0.05)。而氯化血红素治疗组与缺血/再灌组相比无统计学意义(p>0.05)。结论 氯化血红素预处理对大鼠全脑缺血/再灌注后海马CA1区锥体神经元具有明显的保护作用。     

Effects of Exogenous Thyroid Hormone on the Postnatal Morphogenesis of the Rat Parotid Gland

Administration of thyroid hormone has been shown to accelerate the early postnatal development of the rat parotid gland, but these studies have dwelt almost entirely on biochemical changes. The objective of this study was to describe the effects of exogenous thyroid hormone on morphologic aspects of the developing parotid gland, in particular the transient appearance of scattered mucous cells in this otherwise serous gland. Pups were given a daily subcutaneous injection of thyroxine (T₄) of 0.1, 0.5, or 5.0 μg/g body weight, vehicle only (injection control), or no injection (normal control) beginning at 4 days, and killed for the collection of blood and parotid glands at intervals through 15 days. The serum was analyzed for T₄ and the glands were examined by light and electron microscopy. The results indicated that both serum T₄ and the pace of gland development were proportional to the dose of T₄. In particular, T₄ accelerated decreases in acinar size and gland area occupied by stroma and translocation of a subset of cells with small secretory granules, deeply stained with periodic acid–Schiff, from acini to intercalated ducts. However, the chronology of mucous cell disappearance was indifferent to treatment. In addition, signs of toxicity, including slower gain in body weight and greatly increased apoptosis and vacuoles in the glands, occurred with the higher doses of T₄. Anat Rec, 291:94–104, 2007. © 2007 Wiley‐Liss, Inc.     

噪声对大鼠睾丸影响的组织学和组织化学观察

本实验观察了Wistar大鼠在80dB(A)和100dB(A)中频、连续噪声作用下,睾丸的组织学、SDH 和3β-HSD 的改变。两组噪声作用后,各级生精细胞和支持细胞均有不同程度的退变,主要表现为线粒体肿胀变性,内质网扩张,胞质内出现较多空泡及顶体系统的改变等。曲细精管周组织发生皱折,局部增厚和纤维化,但间质细胞形态未见明显改变。生精上皮和间质细胞的SDH 活性和间质细胞的3β-HSD 活性明显减弱。提示噪声对大鼠生精过程有明显损害,并对睾丸的能量代谢和甾体合成代谢有抑制作用。噪声对睾丸损害的可能机理:①对睾丸细胞特别是细胞膜的直接损伤;②通过下丘脑--垂体系统对睾丸产生间接影响。     

Effect of long‐term vasectomy on seminiferous tubules in the guinea pig

The little previous work on the influence of vasectomy on the guinea pig testis has given controversial results. One group reports that the guinea pig suffers autoimmune orchitis while others claim damage may be mechanical. To clarify the issue, this study compares the morphology of seminiferous tubules 3 years after left unilateral vasectomy (8 guinea pigs) and control sham operation (6 animals). Grossly, left and right testes following left‐sided vasectomy were similar to controls and not significantly different in weight. On histology, left and right experimental testes and the control material showed various degrees of seminiferous tubular degeneration, including intraepithelial vesicle formation, loss of germ cells and intraluminal macrophages. Although vesicle formation was striking in most testes, quantitative analysis indicated that it was more frequent in the ipsilateral testis following unilateral vasectomy. It seems that vasectomy had exacerbated an age‐related phenomenon. Lymphocytic infiltration was seen in five of the left testes following vasectomy, in two of the corresponding right testes, but in none of the controls. Two vasectomized left testes, however, showed atrophic changes but no lymphocytic invasion. The results suggest that autoimmune orchitis follows vasectomy but that it may not be the primary cause of degeneration. Attempts to gain positive evidence for mechanical damage, however, were inconclusive. Clin. Anat. 12:250–263, 1999. © 1999 Wiley‐Liss, Inc.     

褪黑素对大鼠肝脏缺血再灌注损伤的保护作用

目的 探讨褪黑素对大鼠肝缺血再灌注损伤的保护作用及其机制。方法 将72只6—7周龄的健康雄性Wistar大鼠,随机分为3组,即褪黑素处理组（Mel组）、酒精溶媒对照组（Ale组）、生理盐水对照组（NS组）。建立肝缺血再灌注损伤模型,于不同时点用TBA法和黄嘌呤氧化酶法分别测定肝组织丙二醛（MDA）及超氧化物歧化酶（SOD）含量,免疫组织化学方法检测分析核因子-kB（NF-kB）的表达情况,结果MDA含量在再灌注后各时点的Mel组均显著低于Ale及NS对照组（P〈0．05）,SOD含量在再灌注后3h、12h、24h时点的Mel组显著高于Ale及NS对照组（P〈0．05）;Mel组再灌注后各时点的NF-kB蛋白表达阳性率均显著低于Ale组和NS组（P〈0．05）。以上各项指标在各时点Alc组与NS组相比无显著性差异。结论 Mel抗氧化及抑制肝细胞NF—kB的表扶,可能是大鼠肝缺血再灌注损伤的保护作用机制之一。     

Effect of Nicotine on Rat Gingival Fibroblasts In Vitro

Tobacco smoking is considered a major risk factor for the development and progression of periodontal diseases (Haber, J. and Wattles, J. (1994). J. Periodontol. 64, 16-23). The purpose of this study was to determine the effects of nicotine on rat gingival fibroblasts (RGF) cultured in vitro. After ether anesthesia, rat gingival tissues were obtained from the attached gingiva of a Wistar rat. Small fragments of gingiva were maintained in culture in Petri dishes. Fibroblasts developing from these explants were collected to obtain monolayer cultures. After the fourth passage (T4), cells were supplemented with nicotine at various concentrations. Control and treated cells were examined under phase contrast or transmission electron microscopy. They were compared as regards their DNA content, mitochondrial activity, collagen and protein synthesis, and cell death by apoptosis or necrosis. Nicotine from 0.05 uM to 1 miVl did not affect the DNA content or protein and collagen synthesis. At concentrations between 3 and 5 niM. growth was significantly diminished and the survival rate reduced. Ultrastructural analysis revealed dilated mitochondria and vacuolization in treated cells, suggestive of necrosis, but increased apoptosis was also revealed by cytometry. On the basis of this in vitro study, it appears that tobacco, through its component nicotine, may directly affect various functions of RGF.