Baohuoside I Inhibits the Proliferation of Hepatocellular Carcinoma Cells via Apoptosis Signaling and NF-kB Pathway

Baohuoside I is a flavonoid isolated from Epimedium koreanum Nakai and has many pharmacological activities. However, its role in liver cancer remains unclear. This study aimed to investigate the inhibitory effect of Baohuoside I on the Human Hepatocellular Carcinoma (HCC) cell lines QGY7703, and underlying mechanisms. QGY7703 cells were used as the model to assess the function of Baohuoside I in vitro. The effects of Baohuoside I on QGY7703 cells’ growth, proliferation, and invasiveness were confirmed by CCK-8, lactate dehydrogenase release, and invasion assays. Cell apoptosis was analyzed by flow cytometry, and the levels of cleaved Caspase-3, Bax, and Bcl-2 were quantified by western blot. Western blot analysis, nuclear translocation of NF-κB, and Q-PCR were used to measure the expression of affected molecules. In QGY7703 cells, Baohuoside I induced the expression of molecules related to NF-κB pathway. The toxicity of Baohuoside I on QGY7703 cells was also confirmed in vivo, in a tumor model. Baohuoside I had a significant toxic effect on QGY7703 cells from a concentration of 10 μM. This compound significantly inhibited the proliferation of QGY7703 cells by inducing apoptosis and downregulating NF-κB signaling pathway. Thus, Baohuoside I is a novel candidate drug and opens new possibilities of clinical strategies for HCC treatment.     

枸杞多糖的提取纯化及组成分析

采用水提法从枸杞中提取分离枸杞多糖(LBP)用DEAE纤维素柱色谱和凝胶柱色谱进行分离纯化,采用GPC-LLS法、红外光谱和气相色谱等方法对其组成进行研究。结果表明LBP含有3个级分,LBP的分子质量约为1.497×105,由阿拉伯糖(Ara),鼠李糖(Rha),木糖(Xyl),甘露糖(Man),半乳糖(Gal)和葡萄糖(Glc)6种中性单糖组成。     

Lycium barbarum polysaccharide protects against LPS-induced ARDS by inhibiting apoptosis,oxidative stress,and inflammation in pulmonary endothelial cells

Acute respiratory distress syndrome (ARDS) is a heterogenous syndrome characterised by diffuse alveolar damage, with an increase in lung endothelial and epithelial permeability. Lycium barbarum polysaccharide (LBP), the most biologically active fraction of wolfberry, possesses antiapoptotic and antioxidative effects in distinct situations. In the present study, the protective effects and potential molecular mechanisms of LBP against lipopolysaccharide (LPS)-induced ARDS were investigated in the mice and in the human pulmonary microvascular endothelial cells (HPMECs). The data indicated that pretreatment with LBP significantly attenuated LPS-induced lung inflammation and pulmonary oedema in vivo. LBP significantly reversed LPS-induced decrease in cell viability, increase in apoptosis and oxidative stress via inhibiting caspase-3 activation and intracellular reactive oxygen species (ROS) production in vitro. Moreover, the scratch assay verified that LBP restored the dysfunction of endothelial cells (ECs) migration induced by LPS stimulation. Furthermore, LBP also significantly suppressed LPS-induced NF-κB activation, and subsequently reversed the release of cytochrome c. These results showed the antiapoptosis and antioxidant LBP could partially protect against LPS-induced ARDS through promoting the ECs survival and scavenging ROS via inhibition of NF-κB signalling pathway. Thus, LBP could be potentially used for ARDS against pulmonary inflammation and pulmonary oedema.     

Taxol induced Bcl-2 protein phosphorylation in human hepatocellular carcinoma QGY-7703 cell line

Bcl-2 family proteins play a critical role in the regulation of apoptosis. Treatment of a human hepatocellular carcinoma cell line, QGY-7703, with Taxol induced apoptosis and Bcl-2 protein phosphorylation. Microscopic observation indicated that apoptotic bodies (0-15%) of Taxol-treated QGY cells appeared after 12 h of treatment, and apoptotic QGY cells gradually increased to 40% after 24 h and 70% after 48 h. A DNA fragmentation assay showed that Taxol induced genomic DNA cleavage into 200 bp DNA fragments. Bcl-2 protein was phosphorylated in Taxol-treated QGY cells within 3 h of treatment, and continued gradually up to 24 h. By 48 h, the protein was unphosphorylated. Other Bcl-2 family proteins, including Bax (a heterodimerization partner of Bcl-2), Bcl-XL, Bak and Bad, were expressed, but at constant levels. The results show a close correlation between Bcl-2 phosphorylation and apoptosis in QGY cells. The inactivation of Bcl-2 protein phosphorylation could be one of the key mechanisms needed for the induction of apoptosis in Taxol-treated QGY cells.     

Down-regulation of adenine nucleotide translocase 3 and its role in camptothecin-induced apoptosis in human hepatoma QGY7703 cells

Adenine nucleotide translocase (ANT) is known as a core component of the mitochondrial permeability transition pore (MPTP) and a key player in cell death. However, its role in camptothecin (CPT)-induced apoptosis has not been examined. We showed that CPT-induced apoptosis in QGY7703 cells and down-regulated the expression of ANT3. Using ANT3 knock-out and overexpression experiments, we provide further evidence that ANT3 plays a contributive role in CPT-induced apoptosis through induction of MPTP. We speculate that the down-regulation of ANT3 upon stimulation with CPT may be part of the molecular basis underlying the mechanism of acquired resistance to CPT.     

Lycium barbarum polysaccharides reduce neuronal damage, blood-retinal barrier disruption and oxidative stress in retinal ischemia/reperfusion injury

Neuronal cell death, glial cell activation, retinal swelling and oxidative injury are complications in retinal ischemia/reperfusion (I/R) injuries. Lycium barbarum polysaccharides (LBP), extracts from the wolfberries, are good for "eye health" according to Chinese medicine. The aim of our present study is to explore the use of LBP in retinal I/R injury. Retinal I/R injury was induced by surgical occlusion of the internal carotid artery. Prior to induction of ischemia, mice were treated orally with either vehicle (PBS) or LBP (1 mg/kg) once a day for 1 week. Paraffin-embedded retinal sections were prepared. Viable cells were counted; apoptosis was assessed using TUNEL assay. Expression levels of glial fibrillary acidic protein (GFAP), aquaporin-4 (AQP4), poly(ADP-ribose) (PAR) and nitrotyrosine (NT) were investigated by immunohistochemistry. The integrity of blood-retinal barrier (BRB) was examined by IgG extravasations. Apoptosis and decreased viable cell count were found in the ganglion cell layer (GCL) and the inner nuclear layer (INL) of the vehicle-treated I/R retina. Additionally, increased retinal thickness, GFAP activation, AQP4 up-regulation, IgG extravasations and PAR expression levels were observed in the vehicle-treated I/R retina. Many of these changes were diminished or abolished in the LBP-treated I/R retina. Pre-treatment with LBP for 1 week effectively protected the retina from neuronal death, apoptosis, glial cell activation, aquaporin water channel up-regulation, disruption of BRB and oxidative stress. The present study suggests that LBP may have a neuroprotective role to play in ocular diseases for which I/R is a feature.     

Hypoglycemic and hypolipidemic effects and antioxidant activity of fruit extracts from Lycium barbarum

The hypoglycemic and hypolipidemic effects of Lycium barbarum fruit water decoction, crude polysaccharide extracts (crude LBP), and purified polysaccharide fractions (LBP-X) in alloxan-induced diabetic or hyperlipidemic rabbits were investigated through designed sequential trials and by measuring blood glucose and serum lipid parameters. Total antioxidant capacity was also assessed using trolox equivalent antioxidant capacity (TEAC) and oxygen radical absorbance capacity (ORAC) assay. It was found that the three Lycium barbarum fruit extracts/fractions could significantly reduce blood glucose levels and serum total cholesterol (TC) and triglyceride (TG) concentrations and at same time markedly increase high density lipoprotein cholesterol (HDL-c) levels after 10 days treatment in tested rabbits, indicating that there were substantial hypoglycemic and hypolipidemic effects. Hypoglycemic effect of LBP-X was more significant than those of water decoction and crude LBP, but its hypolipidemic effect seemed to be weaker. Total antioxidant capacity assay showed that all three Lycium barbarum extracts/fractions possessed antioxidant activity. However, water and methanolc fruit extracts and crude polysaccharide extracts exhibited stronger antioxidant activity than purified polysaccharide fractions because crude extracts were identified to be rich in antioxidants (e.g., carotenoids, riboflavin, ascorbic acid, thiamine, nicotinic acid). Lycium barbarum polysaccharides (glycocojugates), containing several monosaccharides and 17 amino acids, were major bioactive constituents of hypoglycemic effect. Both polysaccharides and vitamin antioxidants from Lycium barbarum fruits were possible active principles of hypolipidemic effect.     

Lycium barbarum polysaccharides: Protective effects against heat-induced damage of rat testes and H2O2-induced DNA damage in mouse testicular cells and beneficial effect on sexual behavior and reproductive function of hemicastrated rats

Lycium barbarum, a famous Chinese medicinal herb, has a long history of use as a traditional remedy for male infertility. Polysaccharides are the most important functional constituent in L. barbarum fruits. We systematically investigated the effect of L. barbarum polysaccharides (LBP) on rat testis damage induced by a physical factor (43 degrees C heat exposure), on DNA damage of mouse testicular cells induced by a chemical factor (H2O2), and on sexual behavior and reproductive function of hemicastrated male rats. The results showed that LBP provided a protective effect against the testicular tissue damage induced by heat exposure. When compared with negative control, a suitable concentration of LBP significantly increased testis and epididymis weights, improved superoxide dismutase (SOD) activity, and raised sexual hormone levels in the damaged rat testes. LBP had a dose-dependent protective effect against DNA oxidative damage of mouse testicular cells induced by H2O2. LBP improved the copulatory performance and reproductive function of hemicastrated male rats, such as shortened penis erection latency and mount latency, regulated secretion of sexual hormones and increased hormone levels, raised accessory sexual organ weights, and improved sperm quantity and quality. The present findings support the folk reputation of L. barbarum fruits as an aphrodisiac and fertility-facilitating agent, and provide scientific evidence for a basis for the extensive use of L. barbarum fruits as a traditional remedy for male infertility in China.     

枸杞多糖与黄芪多糖抑菌活性的研究*

目的:研究黄芪多糖和枸杞多糖的抑菌活性并探讨不同pH值对其抑菌活性的影响。方法:采用滤纸片扩散法,分析不同浓度黄芪多糖和枸杞多糖在不同pH值下对几种常见细茵和霉菌(大肠杆菌、沙门氏菌、金黄色葡萄球菌、黑曲霉、产黄青霉)的抑制效果。结果:对于细菌,枸杞多糖8mg/mL时出现抑菌圈,而黄芪多糖0.02 mg/mL时效果最佳;对于霉菌,随着枸杞多糖浓度的增大,抑菌圈的直径增大,而黄芪多糖0.02 mg/mL时效果最佳;当枸杞多糖和黄芪多糖在pH6的条件下,二者抑菌活性均最强。结论:枸杞多糖和黄芪多糖对细菌、霉菌都有一定的抑制效果,pH值可影响枸杞多糖和黄芪多糖的抑菌效果。     

Activation of the Nrf2/HO-1 Antioxidant Pathway Contributes to the Protective Effects of Lycium Barbarum Polysaccharides in the Rodent Retina after Ischemia-Reperfusion-Induced Damage

Lycium barbarum polysaccharides (LBP), extracts from the wolfberries, are protective to retina after ischemia-reperfusion (I/R). The antioxidant response element (ARE)–mediated antioxidant pathway plays an important role in maintaining the redox status of the retina. Heme oxygenase-1 (HO-1), combined with potent AREs in its promoter, is a highly effective therapeutic target for the protection against neurodegenerative diseases, including I/R-induced retinal damage. The aim of our present study was to investigate whether the protective effect of LBP after I/R damage was mediated via activation of the Nrf2/HO-1-antioxidant pathway in the retina. Retinal I/R was induced by an increase in intraocular pressure to 130 mm Hg for 60 minutes. Prior to the induction of ischemia, rats were orally treated with either vehicle (PBS) or LBP (1 mg/kg) once a day for 1 week. For specific experiments, zinc protoporphyrin (ZnPP, 20 mg/kg), an HO-1 inhibitor, was intraperitoneally administered at 24 h prior to ischemia. The protective effects of LBP were evaluated by quantifying ganglion cell and amacrine cell survival, and by measuring cell apoptosis in the retinal layers. In addition, HO-1 expression was examined using Western blotting and immunofluorescence analyses. Cytosolic and nuclear Nrf2 was measured using immunofluorescent staining. LBP treatment significantly increased Nrf2 nuclear accumulation and HO-1 expression in the retina after I/R injury. Increased apoptosis and a decrease in the number of viable cells were observed in the ganglion cell layer (GCL) and inner nuclear layer (INL) in the I/R retina, which were reversed by LBP treatment. The HO-1 inhibitor, ZnPP, diminished the LBP treatment-induced protective effects in the retina after I/R. Taken together, these results suggested that LBP partially exerted its beneficial neuroprotective effects via the activation of Nrf2 and an increase in HO-1 protein expression.     

枸杞多糖对H5N1流感全病毒灭活疫苗的体液免疫增强作用

探讨枸杞多糖(Lycium barbarum polysaccharide,LBP)作为佐剂对H5亚型流感病毒全病毒灭活疫苗的体液免疫增强效果。将流感病毒A/Vietnam/1194/2004(H5N1)灭活疫苗与不同剂量的枸杞多糖混合后以腹腔注射的方式共同免疫小鼠,免疫后三周收集血清用于特异性抗体检测。实验中设立氢氧化铝佐剂组作对照共同评价LBP作为佐剂的免疫增强效果。结果显示,小鼠血清中针对H5灭活疫苗的特异性抗体水平在一定范围内随着LBP剂量的增加而提高。LBP在800μg剂量时血清特异性抗体水平较无佐剂组显著增强,并与氢氧化铝佐剂组大致相当。因而,LBP有可能成为一种有效的流感灭活疫苗免疫佐剂。     

枸杞多糖诱导人脐血间充质干细胞向神经元样细胞分化的实验研究

目的:探讨枸杞多糖诱导人脐血间充质干细胞(MSCs)向神经元样细胞分化的可行性及其机制。方法:无菌条件下收集正常足月儿的脐带血,经肝素抗凝,用相对密度1.077的淋巴细胞分离液分离脐血的单个核细胞,用低糖DMEM培养基进行培养和纯化扩增。选取第3代细胞进行诱导实验,当传代细胞长满瓶底的80%以上时,先用含15?S和10ng/ml bFGF的DMEM完全培养基预诱导24小时,然后用不含血清含1g/L枸杞多糖的DMEM培养基诱导,光镜下观察细胞形态,用免疫组化技术检测细胞Nestin和NF的表达。结果:预诱导后MSCs没有变化,而经枸杞多糖诱导4h后细胞即出现形态学上的改变,细胞变成不规则形,立体感增强,从胞体伸出突起。免疫组化检测显示,细胞Nestin、NF呈阳性。结论:人脐血间充质干细胞经枸杞多糖诱导可转化为神经元样细胞,其诱导机制可能与枸杞多糖的抗氧化作用有关。     

Polysaccharides from wolfberry prevents corticosterone-induced inhibition of sexual behavior and increases neurogenesis

Lycium barbarum, commonly known as wolfberry, has been used as a traditional Chinese medicine for the treatment of infertility and sexual dysfunction. However, there is still a scarcity of experimental evidence to support the pro-sexual effect of wolfberry. The aim of this study is to determine the effect of Lycium barbarum polysaccharides (LBP) on male sexual behavior of rats. Here we report that oral feeding of LBP for 21 days significantly improved the male copulatory performance including increase of copulatory efficiency, increase of ejaculation frequency and shortening of ejaculation latency. Furthermore, sexual inhibition caused by chronic corticosterone was prevented by LBP. Simultaneously, corticosterone suppressed neurogenesis in subventricular zone and hippocampus in adult rats, which could be reversed by LBP. The neurogenic effect of LBP was also shown in vitro. Significant correlation was found between neurogenesis and sexual performance, suggesting that the newborn neurons are associated with reproductive successfulness. Blocking neurogenesis in male rats abolished the pro-sexual effect of LBP. Taken together, these results demonstrate the pro-sexual effect of LBP on normal and sexually-inhibited rats, and LBP may modulate sexual behavior by regulating neurogenesis.     

The role of reactive oxygen species (ROS) production on diallyl disulfide (DADS) induced apoptosis and cell cycle arrest in human A549 lung carcinoma cells

Diallyl disulfide (DADS), an oil soluble constituent of garlic (Allium sativum), has been reported to cause antimutagentic and anticarcinogenic effects in vitro and in vivo by modulating phases I and II enzyme activities. In recent years, several studies suggested that the chemopreventive effects of DADS can also be attributed to induction of cell cycle arrest and apoptosis in cancer cells. In the present study, we reported that DADS-induced cell cycle arrest at G2/M and apoptosis in human A549 lung cancer cells in a time- and dose-dependent manner. Additionally, a significant increase of intracellular reactive oxygen species (ROS) was induced in A549 cells less than 0.5 h after DADS treatment, indicating that ROS may be an early event in DADS-modulated apoptosis. Treatment of A549 cells with N-acetyl cysteine (NAC) completely abrogated DADS-induced cell cycle arrest and apoptosis. The result indicated that oxidative stress modulates cell proliferation and cell death induced by DADS.     

Protective effect of Lycium barbarum polysaccharides on oxidative damage in skeletal muscle of exhaustive exercise rats

The aim of this study was to determine the modulatory effect of Lycium barbarum polysaccharides (LBP) on the oxidative stress induced by an exhaustive exercise. 32 male Wistar rats were taken in the study. The experiment was a 30-day exhaustive exercise program. We determined the lipid peroxidation, glycogen levels, and anti-oxidant enzyme activities in skeletal muscle. The results demonstrated that L. barbarum polysaccharides administration significantly increases glycogen level and anti-oxidant enzyme activities, and decreased malondialdehyde (MDA) level and creatine kinase activities. In conclusion, L. barbarum polysaccharides administration can significantly decrease the oxidative stress induced by the exhaustive exercise.     

枸杞多糖的抗炎镇痛作用研究

为研究枸杞多糖的抗炎镇痛效应及其潜在分子机制,采用小鼠热板实验、小鼠福尔马林炎症疼痛模型和原代培养的背根神经节神经元细胞对疼痛指标和炎症相关分子进行了检测。实验结果显示枸杞多糖能显著缓解后足注射福尔马林引起的动物II相痛表现(P <0. 05),减少动物两后足重量差值(P <0. 001)和血液(P <0. 001)、脊髓(P <0. 05)组织中白细胞介素6的含量,而对小鼠热板潜伏期无明显影响;枸杞多糖降低原代培养的DRG细胞膜上TRPV1介导的钙信号(P <0. 05)。结果证明枸杞多糖具有良好的抗炎镇痛作用,其镇痛机制可能与降低炎症因子白细胞介素6表达及其引起的TRPV1活性增加有关。     

无花果果浆对肿瘤细胞增殖抑制和诱导凋亡作用

为了探讨无花果果浆(Fig fruit latex,FFL)对人肿瘤细胞的生长抑制作用及其机制,用无花果果浆处理体外培养的人肿瘤细胞,细胞增殖试验(MTT法)、克隆形成试验研究FFL对人肿瘤细胞的增殖抑制作用,Brdu掺人试验、吖啶橙/溴乙啶(AO/EB)染色、流式细胞术检测FFL对肿瘤细胞DNA合成、凋亡和细胞周期的影响.结果,用FFL处理后,肿瘤细胞增殖活性降低(P＜0.05),克隆形成下降(P＜0.05),Brdu标记指数降低(P＜0.05),吖啶橙染色凋亡细胞增多(P＜0.05);细胞周期分布改变,凋亡指数升高(P＜0.01),G0/G1期细胞数增加(P＜0.01),S期细胞数减少(P＜0.01),在一定剂量内对正常细胞无明显影响.试验结果提示,FFL对所试肿瘤细胞的增殖有显著地抑制作用,其作用机理可能与抑制肿瘤细胞DNA合成,诱导肿瘤细胞凋亡及细胞周期阻滞有关.     

枸杞果实ATP酶超微细胞化学定位研究

运用常规ATP酶超微细胞化学定位技术,对宁夏枸杞果实发育不同阶段的韧皮部和果肉库薄壁细胞ATP酶分布进行了观察研究.结果显示,在果实发育过程中SE/CC复合体与周围韧皮薄壁细胞间存在共质体隔离,韧皮薄壁细胞及果肉库薄壁细胞的胞间连丝较少,但是与果肉库薄壁细胞相邻的韧皮薄壁细胞的胞间连丝较多.囊泡和膜泡在筛管、韧皮薄壁细胞和库薄壁细胞中很丰富,并且质膜、囊泡膜、液泡膜上ATP酶沉淀物在韧皮部各细胞分布较少,在果肉库薄壁细胞分布较多,特别是在果实第二次快速生长期,果肉库薄壁细胞膜系统、细胞壁和胞间隙的ATP酶活性剧烈增强.此外,果肉库薄壁细胞的质膜ATP酶具极性分布特点.由此得出,枸杞果实韧皮部卸载是一种需要能量驱动的过程,其卸载途径主要以质外体途径为主,在从韧皮部向果肉库薄壁细胞卸出时可能为共质体和质外体途径共存.膜泡运输是枸杞果实同化物卸出和转运的重要方式,而韧皮薄壁细胞在同化物卸出和转运过程中承担了主要转运角色;果肉库薄壁细胞进行主动和定向卸载、积累同化物的能力很强.     

外源甜菜碱对盐胁迫下枸杞光合功能的改善

研究了外源甜菜碱对盐胁迫下枸杞扦插苗叶片光合功能的影响。结果表明,外源甜菜碱能使盐胁迫下的枸杞叶片叶绿素荧光动力学参数Fo、Fm、Fv、Fv／Fm、Fm／Fo和Fv／Fo增高,使光合色素叶绿素a(Chla)、叶绿素b(Chlb)和类胡萝卜素(Car)含量明显增加,叶绿素a与b的比值(Chla／Chlb)升高,类胡萝卜素与叶绿素的比值(Car／Chl)降低,说明外源甜菜碱有利于植物对光能的捕获、吸收、传递和转换,提高叶片的光合活性,降低盐胁迫对植物的抑制作用。     

Human immunodeficiency virus type 1 Vpr induces cell cycle arrest at the G(1) phase and apoptosis via disruption of mitochondrial function in rodent cells

Vpr of human immunodeficiency virus type 1 causes cell cycle arrest at the G(2)/M phase and induces apoptosis after G(2)/M arrest in primate cells. We have reported previously that Vpr also induces apoptosis independently of G(2)/M arrest in human HeLa cells. By contrast, Vpr does not induce G(2)/M arrest in rodent cells, but it retards cell growth. To clarify the relationship between cell cycle arrest and apoptosis, we expressed Vpr endogenously in rodent cells and investigated cell cycle profiles and apoptosis. We show here that Vpr induces cell cycle arrest at the G(1) phase and apoptosis in rodent cells. Vpr increased the activity of caspase-3 and caspase-9, but not of caspase-8. Moreover, Vpr-induced apoptosis could be inhibited by inhibitors of caspase-3 and caspase-9, but not by inhibitor of caspase-8. We also showed that Vpr induces the release of cytochrome c from mitochondria into the cytosol and disrupts the mitochondrial transmembrane potential. Finally, we showed that apoptosis occurred in HeLa cells through an identical pathway. These results suggest that disruption of mitochondrial functions by Vpr induces apoptosis via cell cycle arrest at G(1), but that apoptosis is independent of G(2)/M arrest. Furthermore, it appears that Vpr acts species-specifically with respect to induction of cell cycle arrest but not of apoptosis.