复方中药体外保护肝细胞的机制

目的探讨复方中药制剂体外保护肝细胞的机制.方法用MTT法、荧光染色、透射电镜、DNA琼脂糖凝胶电泳及流式细胞术等方法观察不同浓度中药复方制剂(包括黄芩、黄柏、板蓝根、山豆根等)抑制TNF-α+Act-D诱导正常大鼠肝细胞凋亡的作用,并检测各组清蛋白的分泌结果自组中药复方制剂能明显改善肝细胞的凋亡状况,出现了典型的形态学和生化学改变,但存在药物浓度上的差异.中药组(0.2,2 mg.L-1)的凋亡率(10.4%±1.2%,13.9±0.4%)低于凋亡模型组(vs17.4%±1.9%,n=3,P＜0.05),中药组(20mg.L-1)的凋亡率(18.30%±1.15%)与凋亡模型组相比没有显著性差异(P＞0.05).除此之外,自组中药复方制剂还促进肝细胞清蛋白的分泌,但存在作用时间和药物浓度的交互作用.结论复方中药制剂能够抑制正常肝细胞凋亡并促进其清蛋白分泌.     

舒林酸抑制人胃腺癌细胞增殖及诱导凋亡

目的在体外对舒林酸抗人胃腺癌细胞增殖和凋亡诱导作用及其机制进行初步研究.方法采用人胃腺癌细胞株MKN45,MKN28作为研究对象.体外药物敏感试验(MTT)检测不同浓度和作用时间的舒林酸对细胞的增殖抑制效应;分别用Hoechst33258细胞核荧光染色、透射电镜和DNA琼脂糖凝胶电泳观察舒林酸诱导的细胞凋亡改变;应用Western斑点免疫印迹法观察经舒林酸2mmol·L-1和4mmol·L-1作用24 h后细胞内环氧化酶-2(COX-2)和凋亡抑制蛋白Bcl-2表达程度的变化.结果舒林酸对人胃腺癌细胞MKN45,MKN28的杀伤率随着剂量的增大和作用时间的延长而增加,舒林酸对不同类型的细胞杀伤率不同.舒林酸能够诱导人胃腺癌细胞MKN45,MKN28产生凋亡,凋亡诱导作用的强弱同样具有时间和剂量依赖性,而且对不同类型胃癌细胞的凋亡诱导效应有显著差异.经舒林酸2 mmol·L-1和4 mmol·L-1作用24 h后,MKN45细胞内COX-2和Bcl-2蛋白比未经舒林酸作用的细胞表达明显减少,而MKN28细胞内COX-2和Bcl-2蛋白的表达未出现明显变化.结论舒林酸在体外对人胃腺癌细胞株MKN45和MKN28有良好的增殖抑制作用,诱导凋亡是其抑制胃癌细胞增殖的重要作用机制.舒林酸对人胃腺癌细胞的抑制和凋亡诱导作用与其抑制细胞内环氧化酶-2,并进而抑制Bcl-2的表达有关,并可能与胃癌细胞的分化状态有关.     

肝症口服液含药血清对TGFα诱导SMMC-7721细胞增殖和ERK蛋白的影响

目的观察肝症口服液含药血清对TGFα诱导人肝癌细胞增殖和信号传导因子ERK影响.方法采用血清药理学方法,以病理鼠血清作对照,应用MTT比色法观察中药鼠血清对TGFα诱导人肝癌细胞SMMC-7721增殖的抑制作用.用免疫组化方法检测中药血清对信号传导因子ERK蛋白表达影响.结果中药血清作用24 h对SMMC-7721细胞抑制率达25%,P＜0.05;作用48 h抑制率达30%,P＜0.001;中药血清作用24 h对1μg·L-1 TGFα诱导的SMMC-7721细胞增殖抑制率为12.3%,P＞0.05;作用48 h抑制率为16%,P＜0.05;中药血清作用24 h对5μg·L-1TGFα诱导的SMMC-7721细胞增殖抑制率为8.9%,P＞0.05,作用48 h抑制率为17.2%,P＜0.001.中药血清对TGFα诱导的肝癌细胞SMMC-7721增殖有明显的抑制作用,抑制效应呈时间依赖性.中药血清能抑制ERK蛋白在细胞核中的表达.结论肝症口服液含药血清能够抑制TGFα诱导的人肝癌细胞增殖,阻断TGFα在细胞内的信号传导.     

藤梨根提取物对大肠癌LoVo细胞增殖的抑制作用及诱导凋亡的影响

目的:研究藤梨根提取物(ethanol extract from radix of actinidia chinensis,EERAC)对人大肠癌LoVo细胞增殖和凋亡的影响.方法:提取藤梨根抗癌有效活性成分(EERAC),按浓度分为4处理组(10、40、160、320mg/L)和空白对照组(0mg/L).各实验组经作用24、48、72h后,进行一般形态学和AO/EB荧光染色观察;MMT法检测细胞增殖的抑制情况;免疫组织化学(immunohistochemistry,IHC)法测定LoVo细胞中凋亡相关基因Bcl-2、Bax、Caspase-3的蛋白表达变化.结果:与空白对照组比较,一般形态学显示EERAC处理组能使细胞密度减低,增殖变慢;细胞逐渐变大,细胞间接触变松,胞浆中颗粒增多,细胞脱壁现象和周围碎片增多;荧光染色观察可见处理组细胞呈橙红色荧光,细胞核出现碎片状或固缩状的凋亡特征学形态改变,凋亡现象与EERAC的浓度呈正相关性;MTT法检测显示,EERAC处理组对LoVo细胞的最佳作用时间为72h,最大抑制率为79.48%,具有浓度和时间的依赖性(P<0.01);IHC检测结果显示EERAC作用LoVo细胞24h后,Bcl-2表达明显减弱,Bax、Caspase-3表达水平明显增高,Bcl-2/Bax比值下降,差异具有统计学意义(P<0.05),其效应与浓度相关.结论:EERAC具有明显抑制LoVo细胞增殖的作用,其机制可能与降低Bcl-2表达,上调Bax、Caspase-3的表达水平,激活线粒体凋亡途径有关.     

All teas are not created equal: the Chinese green tea and cardiovascular health

Tea is one of the most widely consumed beverages in the world, next only to water. It can be categorized into three types, depending on the level of fermentation, i.e., green (unfermented), oolong (partially fermented) and black (fermented) tea. In general, green tea has been found to be superior to black tea in terms of antioxidant activity owing to the higher content of (-)-epigallocatechin gallate. The processes used in the manufacture of black tea are known to decrease levels of the monometric catechins to a much greater extent than the less severe conditions applied to other teas. The cardioprotective effect of flavonoids from green tea can be attributed to not only antioxidant, antithrombogenic and anti-inflammatory properties but also improvement of coronary flow velocity reserve. In this article, I will discuss the effects of green tea on atherosclerosis, coronary heart disease, hypertension, diabetes, metabolic syndrome and obesity, and, finally, its comparison with black tea.     

Extraction and purification of TGFβ and its effect on the induction of apoptosis of hepatocytes

AIM To extract and purify the transforming growth factor β (TGF β), and to demonstrate its biological activity in vivo and induction of apoptosis of hepatocytes in vitro. METHODS TGF β was isolated from fresh bovine platelets by acid/ethanol extraction method and purified with ion exchange and gel chromatography. The extracted TGF β was injected subcutaneously to mice, and its biological activity in vivo was observed 72 hfs post-injection by HE staining. The morphological changes were observed by HE staining and the occurrence of apoptosis was detected by TUNEL method after the human normal hepatic cell line QZG was treated with 8 μg@L 1 TGFβ for 12 hrs in vitro. RESULTS The molecular mass 25 ku TGF β protein was successfully extracted. It was able to induce localized granulation tissue formation in vivo. TGF β-treated hepatocytes showed obvious apoptotic morphological changes, including the pyknosis and dense-stained nuclei and cytoplasm, the fragmentary, annular or crescent nuclei, and the "bubbling" cytoplasm. Moreover, its apoptotic rate was significantly higher than that of the control group (P＜0.05). CONCLUSION Biological active TGF β protein is extracted and purified successfully from bovine platelets, and it is able to induce the apoptosis of hepatocytes.     

腺病毒介导肝癌抑制基因-1联合5-氟尿嘧啶增强结直肠癌细胞杀伤作用及机制研究

目的 通过化学治疗药物5-氟尿嘧啶(5-FU)联合重组腺病毒(Ad)肝癌抑制基因-1(HCCS1)感染结直肠癌细胞,观察两者对结直肠癌细胞的联合杀伤作用.并初步探讨相关分子机制.方法 RT-PCR和Western印迹法检测Ad-HCCS1感染结直肠癌LoVo细胞后HCCS1的表达.活细胞计数试剂盒(CCK)-8法测定Ad-HCCS1联合5-FU处理时的细胞存活率.流式细胞仪检测各种处理后细胞凋亡的情况.Western印迹法检测各种处理后相关凋亡蛋白的变化.结果 ①Ad-HCCS1可成功介导HCCS1基因在LoVo细胞中的表达.②5-FU+Ad-HCCS1联合作用96 h时,LoVo细胞存活率为(11.23±4.61)%,与对照组比较差异有统计学意义[(92.23±3.77)%,P<0.01].③处理72 h后流式细胞仪分析结果 显示,5-FU+Ad-HCCS1联合组细胞凋亡率为(27.57±1.78)%,高于单用5-Fu组[(8.64±0.94)%]、单用Ad-HCCS1组[(13.19±1.32)%]和5-Fu+空载病毒组[(12.16±1.28)%],差异均有统计学意义(P值均<0.01).④在Ad-HCCS1感染LoVo细胞高表达HCCS1时,胞质蛋白中检测到组织蛋白酶(cathepsin)D;当5-FU加入后,胞质中的抗凋亡蛋白酶前体(procaspase)-8有所减少,而活性BH3结构域凋亡诱导蛋白(Bid)明显增加.在5-FU+Ad-HCCS1处理组,胞质中的Bax水平降低最为明显,而细胞色素C和活性凋亡蛋白酶(caspase)-3表达最多.结论 在5-FU基础上联合Ad-HCCS1可显著增强LoVo细胞的生长抑制和凋亡诱导,两种处理的联合作用点为Bax蛋白,为结直肠癌治疗提供了一种新策略.     

Extraction and purification of TGFbeta and its effect on the induction of apoptosis of hepatocytes

AIM: To extract and purify the transforming growth factor beta (TGF beta), and to demonstrate its biological activity in vivo and induction of apoptosis of hepatocytes in vitro. METHODS: TGF beta was isolated from fresh bovine platelets by acid/ethanol extraction method and purified with ion exchange and gel chromatography. The extracted TGF beta was injected subcutaneously to mice, and its biological activity in vitro was observed 72 hrs post-injection by HE staining. The morphological changes were observed by HE staining and the occurrence of apoptosis was detected by TUNEL method after the human normal hepatic cell line QZG was treated with 8 microg.(-1)TGFbeta for 12 hrs in vitro. RESULTS: The molecular mass 25 ku TGFbeta protein was successfully extracted. It was able to induce localized granulation tissue formation in vitro. TGF beta-treated hepatocytes showed obvious apoptotic morphological changes, including the pyknosis and dense-stained nuclei and cytoplasm, the fragmentary, annular or crescent nuclei, and the "bubbling" cytoplasm. Moreover, its apoptotic rate was significantly higher than that of the control group (P<0.05). CONCLUSION: Biological active TGF beta protein is extracted and purified successfully from bovine platelets, and it is able to induce the apoptosis of hepatocytes.     

The effect of an extract of green and black tea on glucose control in adults with type 2 diabetes mellitus: double-blind randomized study

Recent evidence suggests that tea from Camellia sinensis (eg, green, oolong, and black tea) may have a hypoglycemic effect. We evaluated the ability of an extract of green and black tea to improve glucose control over a 3-month period. A double-blind, placebo-controlled, randomized multiple-dose (0, 375, or 750 mg per day for 3 months) study in adults with type 2 diabetes mellitus not taking insulin was performed. The primary end point was change in glycosylated hemoglobin at 3 months. The 49 subjects who completed this study were predominantly white with an average age of 65 years and a median duration of diabetes of 6 years, and 80% of them reported using hypoglycemic medication. After 3 months, the mean changes in glycosylated hemoglobin were +0.4 (95% confidence interval, 0.2-0.6), +0.3 (0.1-0.5), and +0.5 (0.1-0.9) in the placebo, 375-mg, and 750-mg arms, respectively. The changes were not significantly different between study arms. We did not find a hypoglycemic effect of extract of green and black tea in adults with type 2 diabetes mellitus.     

中药960诱导人肝癌细胞凋亡的研究

目的：观察９６０抗癌合剂对人肝癌细胞的凋亡作用,揭示其原发性肝癌作用机理。方法：丫啶橙（ＡＯ）染色,琼脂糖凝胶电经丙锭染色细胞周期分析（ＰＩ－ＦＣＭ）和末端转移酶ＤＮＡ断裂点原位标记（ＴＵＮＥＬＦＣＭ）。结果：经９６０药和血清处理后。细胞皱缩,深染,ＡＯ染色,细胞呈致密浓染的黄绿药荧光;ＤＮＡ电位见典型“梯形”带;ＤＮＡ直方图上见典型凋亡峰（０．９５％～２７．７％）,ＴＵＮＥＬ标记率６４．１２％～     

Green and black tea are equally potent stimuli of NO production and vasodilation: new insights into tea ingredients involved

Epidemiological studies suggest that consumption of tea is associated with beneficial cardiovascular effects. Since different types of tea are consumed throughout the world, a question of much interest is whether green tea is superior to black tea in terms of cardiovascular protection. We therefore compared the effects of green and black tea on nitric oxide (NO) production and vasodilation and elucidated the tea compounds involved. We chose a highly fermented black tea and determined concentrations of individual tea compounds in both green and black tea of the same type (Assam). The fermented black tea was almost devoid of catechins. However, both teas stimulated eNOS activity and phosphorylation in bovine aortic endothelial cells (BAEC) as well as vasorelaxation in rat aortic rings to a similar extent. In green tea, only epigallocatechin-3-gallate (EGCG) resulted in pronounced NO production and NO-dependent vasorelaxation in aortic rings. During tea processing to produce black tea, the catechins are converted to theaflavins and thearubigins. Individual black tea theaflavins showed a higher potency than EGCG in NO production and vasorelaxation. The thearubigins in black tea are highly efficient stimulators of vasodilation and NO production. Green and black tea compounds induced comparable phosphorylation of eNOS and upstream signalling kinases. Whereas stimulation of eNOS activity by EGCG was only slightly affected by pretreatment of cells with various ROS scavengers, TF3(theaflavin-3′,3-digallate)-induced eNOS activity was partially inhibited by PEG-catalase. These results implicate that highly fermented black tea is equally potent as green tea in promoting beneficial endothelial effects. Theaflavins and thearubigins predominantly counterbalance the lack of catechins in black tea. The findings may underline the contribution of black tea consumption in prevention of cardiovascular diseases. Returned for 1. Revision: 7 January 2008 1. Revision received: 3 July 2008 Returned for 2. Revision: 1 August 2008 2. Revision received: 13 August 2008 Returned for 3. Revision: 26 September 2008 3. Revision received: 13 October 2008