柚皮素对心肌梗死大鼠血管新生的促进作用及机制研究

[目的]观察柚皮素对心肌梗死大鼠血管新生的促进作用及相关机制。[方法]将50只无特定病原体(specific pathogen free,SPF)级SD大鼠随机分为假手术组、模型组、阿司匹林组、柚皮素高剂量组和柚皮素低剂量组,每组10只。采用冠脉动脉前降支结扎法建立大鼠心肌梗死模型,造模第2天开始给药,柚皮素高剂量组和柚皮素低剂量组给予100mg·kg-1和50mg·kg-1柚皮素混悬液灌胃,阿司匹林组予100mg·kg~(-1)阿司匹林水溶液灌胃,假手术组和模型组予0.9%氯化钠溶液20mL·kg-1灌胃,1次/d,连续4周。末次给药结束后检测大鼠心功能指标,苏木素-伊红(hematoxylin-eosin,HE)染色和Masson染色观察心肌组织病理学变化,免疫组化检测心肌组织血小板内皮细胞黏附分子-1 (platelet endothelial cell adhesion molecule-1,PECAM-1/CD31)表达,计算微血管密度(microvascular density,MVD),Western blot检测心肌组织中血管内皮生长因子(vascular endothelial growth factor,VEGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)蛋白表达情况。[结果]与假手术组比较,模型组左室射血分数(left ventricular ejection fractions,LVEF)和左室短轴缩短率(left ventricular factional shortening,LVFS)降低,左室收缩末期内径(left ventricular end-systolic dimension,LVESD)和左室舒张末期内径(left ventricular end-diastolic dimension,LVEDD)增加;与模型组比较,阿司匹林组、柚皮素高剂量组和柚皮素低剂量组LVEF和LVFS增加,LVESD和LVEDD降低。HE染色和Masson染色结果显示,与假手术组比较,模型组大鼠心肌组织排列紊乱,纤维化明显;与模型组比较,柚皮素高剂量组心肌组织排列较为整齐,胶原纤维沉积减少,但柚皮素低剂量组心肌组织分布相对较紊乱,仍有明显胶原纤维沉积。免疫组化检测结果显示,模型组CD31表达水平和MVD高于假手术组;与模型组比较,柚皮素高剂量组和柚皮素低剂量组CD31表达水平和MVD增加,其中柚皮素高剂量组CD31表达水平和MVD高于阿司匹林组和柚皮素低剂量组。Western blot检测结果显示,与假手术组比较,模型组VEGF、bFGF蛋白表达水平增加;与模型组比较,阿司匹林组、柚皮素高剂量组和柚皮素低剂量组VEGF、b FGF蛋白表达水平增加,其中柚皮素高剂量组VEGF、b FGF蛋白表达水平高于阿司匹林组和柚皮素低剂量组。上述指标差异均有统计学意义(P0.05)。[结论]柚皮素能够促进心肌梗死大鼠血管新生,其作用机制可能与促进VEGF、bFGF蛋白表达有关。     

核桃花化学成分的研究

目的 研究核桃Juglans regia花的化学成分。方法 利用正和反相硅胶、Sephadex LH-20及制备液相等柱色谱方法进行分离纯化,根据谱学数据及理化性质鉴定化合物结构,并采用MTT法检测化合物的体外细胞毒活性。结果 从核桃花95％乙醇水提取物中分离得到12个黄酮类化合物和7个生物碱类化合物,分别鉴定为金合欢素（1）、柚皮素（2）、5,7-二羟基-6,8,4''-三甲氧基黄酮（3）、木犀草素（4）、草质素（5）、白杨素（6）、异野樱素（7）、3,5-二羟基-4'',7-二甲氧基黄酮醇（8）、槲皮苷（9）、异槲皮苷（10）、山柰酚（11）、槲皮素（12）、2-（4-羟基苯乙基）异吲哚啉-1,3-二酮（13）、吲哚-3-甲酸乙酯（14）、1,2,3,4-四氢去甲哈尔满-1-酮（15）、3-醛基吲哚（16）、马齿苋酰胺E（17）、青兰素C（18）、N-苯甲酰-L-苯丙氨酸（19）。结论 除化合物4和9～12外,其余化合物均为首次从胡桃属中分离得到,其中化合物13为1个新的天然产物。化合物2、3和5具有抑制人结肠癌HCT-116细胞、人肝癌HepG2细胞、人胃癌BGC-823细胞、人肺支气管癌NCI-H1650细胞、人卵巢癌A2780细胞的增殖作用。     

Naringenin modulates circulatory lipid peroxidation, anti-oxidant status and hepatic alcohol metabolizing enzymes in rats with ethanol induced liver injury

We have investigated the modulatory efficacy of naringenin on circulatory lipid peroxidation and anti-oxidant status, hepatic alcohol metabolizing enzymes in rats with ethanol induced hepatotoxicity. Rats were divided into four groups: groups 1 and 2 received isocaloric glucose and 0.5% carboxymethyl cellulose; groups 3 and 4 received 20% ethanol equivalent to 6 g/kg body weight everyday for the total experimental period of 60 days. In addition, groups 2 and 4 were given naringenin (50 mg/kg) everyday for the last 30 days of the experiment. The results showed significantly elevated levels/activities of bilirubin, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides (LOOH), conjugated dienes (CD) and phase I enzymes, and significantly lowered the activities of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), superoxide dismutase (SOD), catalase (CAT) and phase II enzymes in ethanol-fed rats as compared to those of the control. Supplementation with naringenin for the last 30 days of the experiment to ethanol-fed rats, significantly decreased the levels/activities of bilirubin, ALP, LDH, TBARS, LOOH, CD and phase I enzymes, and significantly elevated the activities of ADH, ALDH, SOD, CAT and phase II enzymes as compared to control rats. These findings suggest that naringenin can effectively modulate the hepatic alcohol metabolizing enzymes in rats with ethanol induced liver injury.     

离体人肠道菌群对柚皮苷的代谢动力学分析

目的:研究人肠道菌群对柚皮苷的代谢程度与速度,为该成分的新药开发提供参考。方法:采集健康志愿者新鲜粪便制备肠道菌群混悬液,与柚皮苷在厌氧环境下孵育,运用HPLC监测柚皮苷浓度变化,HPLC-MS鉴定代谢产物结构。柚皮苷HPLC色谱条件为检测波长288 nm,流动相甲醇-0.1%磷酸溶液梯度洗脱。结果:柚皮苷与人肠道菌群共同孵育15 min后,柚皮苷色谱峰后面可见1个代谢产物;孵育至120 min时,柚皮苷代谢率100%。经HPLC-MS鉴定代谢产物为柚皮素。柚皮苷代谢过程符合非线性动力学过程。HPLC监测发现柚皮素可被进一步代谢,导致柚皮素峰面积降低。结论:柚皮苷可被人肠道菌群代谢,代谢速度快,代谢产物主要为柚皮素,可能存在柚皮苷的二级代谢产物。     

柚皮素纳米结构脂质载体的处方优化和初步评价

目的制备一种具有缓释作用的柚皮素(NG)新型纳米结构脂质载体(NG-NLC),并对其理化性质进行初步考察。方法以乳化蒸发-低温固化法制备NG-NLC。采用星点设计-效应面法考察柚皮素-脂质材料比、单硬脂酸甘油酯-辛癸酸甘油酯比,以及乳化剂用量对包封率和载药量的影响。通过包封率、载药量、粒径、DSC分析以及体外释放度来评价NG-NLC的特性。结果经过处方优化,确定NG-NLC最佳工艺条件为柚皮素-脂质材料比为20.77,单硬脂酸甘油酯-辛癸酸甘油酯比为1.85,乳化剂用量为58.45 mg,制备的NG-NLC包封率为(80.13±1.45)%,载药量为(3.59±0.06)%,平均粒径为(134.1±9.1)nm,多分散系数(PDI)为0.152±0.044;体外释放实验表明,NG-NLC在p H 7.4的缓冲溶液中前期有突释现象,后期则有缓释特征。结论采用乳化蒸发-低温固化法成功制备了NG-NLC,为柚皮素的临床应用奠定了基础。     

Identification of potential therapeutic target of naringenin in breast cancer stem cells inhibition by bioinformatics and in vitro studies

Cancer therapy is a strategic measure in inhibiting breast cancer stem cell (BCSC) pathways. Naringenin, a citrus flavonoid, was found to increase breast cancer cells’ sensitivity to chemotherapeutic agents. Bioinformatics study and 3D tumorsphere in vitro modeling in breast cancer (mammosphere) were used in this study, which aims to explore the potential therapeutic targets of naringenin (PTTNs) in inhibiting BCSCs. Bioinformatic analyses identified direct target proteins (DTPs), indirect target proteins (ITPs), naringenin-mediated proteins (NMPs), BCSC regulatory genes, and PTTNs. The PTTNs were further analyzed for gene ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, protein–protein interaction (PPI) networks, and hub protein selection. Mammospheres were cultured in serum-free media. The effects of naringenin were measured by MTT-based cytotoxicity, mammosphere forming potential (MFP), colony formation, scratch wound-healing assay, and flow cytometry-based cell cycle analyses and apoptosis assays. Gene expression analysis was performed using real-time quantitative polymerase chain reaction (q-RT PCR). Bioinformatics analysis revealed p53 and estrogen receptor alpha (ERα) as PTTNs, and KEGG pathway enrichment analysis revealed that TGF-ß and Wnt/ß-catenin pathways are regulated by PTTNs. Naringenin demonstrated cytotoxicity and inhibited mammosphere and colony formation, migration, and epithelial to mesenchymal transition in the mammosphere. The mRNA of tumor suppressors P53 and ERα were downregulated in the mammosphere, but were significantly upregulated upon naringenin treatment. By modulating the P53 and ERα mRNA, naringenin has the potential of inhibiting BCSCs. Further studies on the molecular mechanism and formulation of naringenin in BCSCs would be beneficial for its development as a BCSC-targeting drug.     

Impact of Naringenin on Oxytetracycline-Mediated Oxidative Damage in Kidney of Rats

The aim of this study was to investigate the effect of naringenin on oxytetracycline-induced nephrotoxicity in rats. Oxytetracycline (200 mg/kg body weight, ip) was administered in 0.5ml of sterile physiological saline for 15 days, resulting in a significant increase in serum urea and creatinine and reduction in creatinine clearance. A significant increase in lipid peroxidation markers (TBARS and lipid hydroperoxide) and decrease in antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) and low molecular weight antioxidants (vitamin C, vitamin E, and reduced glutathione) levels were also observed in oxytetracycline-treated rats. The oral administration of naringenin (50 mg/kg body weight) attenuated the oxytetracycline-induced nephrotoxicity by significantly decreased levels of serum urea and creatinine with the significant normalization of creatinine clearance. Upon the administration of naringenin, the depleted renal antioxidant defense system (enzymatic and non-enzymatic antioxidants) was significantly increased in rats treated with oxytetracycline. These biochemical observations were supplemented by histopathological examination of kidney section. The present results suggest that the supplementation of naringenin might be helpful to alleviate the oxytetracycline-induced oxidative injury in kidney.     

Naringenin inhibited migration and invasion of glioblastoma cells through multiple mechanisms

Glioblastoma (GBM) is the most mortality brain cancer in the world. Due to high invasion and drug resistance cause the poor prognosis of GBM. Naringenin, an ingredient of citrus, exhibits many cellular functions such as antioxidant, anti‐inflammation, and anticancer. Naringenin inhibits the migration of bladder and lung cancer via modulation of MMP‐2 and/or MMP‐9 activities, Naringenin inhibits migration and trigger apoptosis in gastric cancer cells through downregulation of AKT pathway. However, the effects of naringenin in GBM still remain to be elucidated. In this study, we reveal the molecular mechanisms of naringenin in the inhibition of migration and invasion in GBM. No overt alternation of cell proliferation was found in of GBM 8901 cells treated with different concentration of naringenin. Slight decreased cell viability was found in GBM 8401 cell treated with 200 and 300 μM naringenin. Significant reduction of migration and invasion as assayed by Boyden chamber analysis was found in of GBM cells treated with 100, 200, and 300 μM naringenin. Zymography analysis also revealed that the activities of MMP‐2 and MMP‐9 of GBM cells were significantly inhibited in response to 100, 200, or 300 μM naringenin treatment. Proteins of MMP‐2 and MMP‐9 were downregulated in naringenin treated GBM cells. In addition, naringenin also attenuated the activities of ERK and p38. Naringenin decreased mesenchymal markers (snail and slug) expression as revealed by Western blot analysis. Taken together, our findings indicated that naringenin eliminated the migration and invasion of GBM cells through multiple mechanisms including inhibition of MMPs, ERK, and p38 activities and modulation of EMT markers. Our results also suggested that naringenin may be a potential agent to prevent metastasis of GBM.     

Antidiabetic and toxicological evaluations of naringenin in normoglycaemic and NIDDM rat models and its implications on extra-pancreatic glucose regulation

Aim: The present investigation was designed to determine the in vivo antidiabetic effect of naringenin (NG) in normoglycaemic and diabetic rat models through blood glucose (GLU) measurements following acute and subchronic time periods. Possible modes of action of NG were investigated and its acute toxicity determined. Methods: Normoglycaemic and non‐insulin‐dependent diabetes mellitus (NIDDM) rat models were treated for acute and subchronic (5 days) time periods with 50 mg/kg/day of NG. Blood biochemical profiles were determined after 5 days of the treatment in normoglycaemic and NIDDM rats using commercial kits for GLU, triglycerides (TG), total cholesterol (CHOL) and high‐density lipoprotein (HDL). In order to elucidate its antidiabetic mode of action, NG was administered intragastrically and an oral glucose tolerance test performed using GLU and sucrose (2 g/kg) as substrates. The inhibitory effect of a single concentration of NG (10 μM) on 11β‐hydroxysteroid dehydrogenase type 1 (11β‐HSD1) activity in vitro was determined. Finally, the preclinical safety and tolerability of NG was determined by toxicological evaluation in mice and rats using Organization for Economic Cooperation and Development (OECD) protocols. Results: Intragastrically administered NG (50 mg/kg) induced a significant decrease in plasma GLU in normoglycaemic and NIDDM rat models (p < 0.05) following acute and subchronic time periods. After 5 days of administration, NG produced significant diminished blood GLU and TG levels in streptozotocin–nicotinamide–induced diabetic rats. The administration of NG to normal rats significantly increased the levels of TG, CHOL and HDL (p < 0.05). NG (5 and 50 mg/kg) induced a total suppression in the increase of plasma GLU levels after administration of substrates (p < 0.01), but NG did not produce inhibition of α‐glucosidase activity in vitro. However, NG (10 μM) was shown to inhibit 11β‐HSD1 activity by 39.49% in a cellular enzyme assay. Finally, NG showed a Medium Lethal Dose LD₅₀ > 5000 mg/kg and ranking at level five based on OECD protocols. Conclusion: Our findings suggest that NG may exert its antidiabetic effect by extra‐pancreatic action and by suppressing carbohydrate absorption from intestine, thereby reducing the postprandial increase in blood GLU levels.     

Naringenin Enhances the Anti-Tumor Effect of Doxorubicin Through Selectively Inhibiting the Activity of Multidrug Resistance-Associated Proteins but not P-glycoprotein

Purpose  Naringenin has shown paradoxical results to modulate the function of multidrug resistance-associated proteins (MRPs). The aim of this study is to interpret whether naringenin can reverse intrinsic and/or acquired resistance of cancer cells to chemotherapeutic agents. Methods  The effects of naringenin on the uptake, retention and cytotoxicity of doxorubicin were investigated in A549, MCF-7, HepG2 and MCF-7/DOX cells. Cellular efflux pathways modulated by naringenin were assessed with their specific substrates and inhibitors. The improved antitumor activity of doxorubicin in combination with naringenin was also investigated in vivo. Results  The IC₅₀ values of doxorubicin in combination with naringenin in A549 and MCF-7 cells were approximately 2-fold lower than that of doxorubicin alone. The increased sensitivity to doxorubicin by naringenin in HepG2 and MCF-7/DOX cells was not observed. Naringenin increased the cellular doxorubicin accumulation through inhibiting doxorubicin efflux in the cells expressing MRPs but not P-gp. In contrast to doxorubicin alone, doxorubicin in combination with naringenin enhanced antitumor activity in vivo with low systemic toxicity. Conclusion  Naringenin enhances antitumor effect of doxorubicin by selective modulating drug efflux pathways. Naringenin will be a useful adjunct to improve the effectiveness of chemotherapeutic agents in treatment of human cancers.