Preliminary Study on Chinese Drug-Induced Apoptosis of Thyrocytes in Graves' Disease

Objective: To investigate the cellular and molecular mechanisms of some Chinese drugs on apoptosis of thyrocytes in Graves' disease. Methods: Two to ten weeks before and after using Chinese drugs on 13 Graves' disease patients with anti-thyroid drugs, patients' thyroid tissue was aspired with percutaneous puncturing needle. The effects of some Chinese drugs on thyrocytes was studied by applying cell morphology (stained with nuclear fast red, NFR), flow cytometry (stained with propidium iodide, PI), and terminal uridine deoxynucleotidyl end-labelling (TUNEL) technique (alkaline phosphatase and CBIP/NBT, NFR). The size of thyroid gland was measured by color Doppler ultrasonography. Results: After Chinese drug treatment, compared with single anti-thyroid drug group, the size of thyroid was significantly reduced (P<0.01). The apoptosis of thyrocytes can be observed by cell morphology such as chromatin condensation and nuclear fragmentation. The apoptosis rate is 18.66%±20.01% (n=13) in anti-thyroid drugs plus Chinese drugs group, 2.11%±1.78% (n=13) in single anti-thyroid drug group (P<0.01). For up to 2-10 weeks, more TUNEL-positive cells were found after treatment with Chinese drugs than those treated with anti-thyroid drug alone. Conclusion: Some Chinese drugs when used in combination with anti-thyroid drugs in treating Graves' disease could induce apoptosis of thyrocytes.

How do Chinese medicines that tonify the kidney inhibit dopaminergic neuron apoptosis?

Wistar rats were intragastrical y perfused with Chinese medicines used for tonifying the kidney. These included 0.180 g/mL of Herba Epimedi （Epimedium）, Semen Cuscutae （Dodder Seed）, or Herba Cistanches （Desertliving Cistanche）, 0.04 mg/mL monoamine oxidase-B inhibitor selegiline, or distil ed water for 14 consecutive days to prepare drug-containing serum or blank serum. MES23.5 cells in the logarithmic phase were cultured in media supplemented with 15%drug-containing serum for 24 hours, fol owed by incubation in culture solution containing 100μmol/L H2O2 for 3 hours. 3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） and flow tometry results showed that al drug-containing serums improved the survival rate of H 2 O 2-injured MES23.5 cells, inhibited pro-apoptotic FasL and caspase-3 expression, promoted anti-apoptotic Bcl-2 expression. However, drug-containing serums had little influence on Fas expression in H 2 O 2-injured MES23.5 cells. Enzyme-linked immunosorbent assay results showed that serum containing Herba Cistanches or Herba Epimedi increased the expression of nerve growth factor, brain-derived neurotrophic factor, and glial cellline-derived neurotrophic factor in injured MES23.5 cells;serum containing Semen Cuscutae only increased brain-derived neurotrophic factor expres-sion; while expression of the above neurotrophic factors remained the same in cells treated with serum containing selegiline. These findings indicate that Chinese medicines used to tonify the kid-ney can protect nerve cells by regulating the expression of apoptosis-related factors and neuro-trophic factors in MES23.5 cells.

Protective effects of components of the Chinese herb grassleaf sweetflag rhizome on PC12 cells incubated with amyloid-beta42

The major ingredients of grassleaf sweetflag rhizome are β-asarone and eugenol, which can cross the blood-brain barrier and protect neurons. This study aimed to observe the neuroprotective effects and mechanisms of β-asarone and eugenol, components of the Chinese herb grassleaf sweetflag rhizome, on PC12 cells. First, PC12 cells were cultured with different concentrations（between 1 × 10–10 M and 1 × 10–5 M） of β-asarone and eugenol. Survival rates of PC12 cells were not significantly affected. Second, PC12 cells incubated with amyloid-beta42, which reduced cell survival, were cultured under the same conditions（1 × 10–6 M β-asarone and eugenol）. The survival rates of PC12 cells significantly increased, while expression levels of the m RNAs for the pro-apoptotic protein Bax decreased, and those for the anti-apoptotic protein Bcl m RNA increased. In addition, the combination of β-asarone with eugenol achieved better results than either component alone. Our experimental findings indicate that both β-asarone and eugenol protect PC12 cells through inhibiting apoptosis, and that the combination of the two is better than either alone.

中药复方更年春含药血清及其主要单体对β淀粉样蛋白所致细胞损伤的保护作用

目的:观察中药复方更年春含药血清及其主要单体成分对β淀粉样蛋白(amyloidβ-protein,Aβ)所致肾上腺嗜铬细胞瘤细胞系PC12细胞损伤的保护作用。方法:采用Aβ25-35作用PC12细胞构建阿尔茨海默病(Alzheimers disease,AD)细胞模型,相差显微镜观察细胞形态改变。运用中药血清药理学方法制备大鼠更年春含药血清,以四甲基偶氮唑盐法观察更年春含药血清及其主要单体芍药苷、黄连素、知母皂苷A-Ⅲ、淫羊藿苷对体外培养的PC12细胞活力的影响及其拮抗Aβ损伤的作用,采用流式细胞仪观察更年春复方含药血清及复方主要单体对Aβ诱导的PC12细胞凋亡的影响。结果:PC12细胞在Aβ25-35作用后,有活力细胞数目减少,细胞间连接较松,胞浆较暗淡,细胞碎片较多,贴壁细胞欠透明,部分发生皱缩,胞浆中有较多颗粒。Aβ25-35剂量、时间依赖性地抑制PC12细胞的活力。更年春含药血清可以增强PC12细胞活力,不同浓度更年春含药血清培养细胞24、48、72h后,其增强PC12细胞活力均在20%浓度作用最强。更年春复方含药血清及复方主要单体之一的黄连素及主要单体混合液(包括芍药苷、黄连素、知母皂苷A-Ⅲ、淫羊藿苷)均有拮抗Aβ对PC12细胞损伤的作用,且均能抑制Aβ诱导的PC12细胞早期凋亡,其中更年春含药血清作用最强,中药单体混合液作用其次,而单体黄连素作用最弱。结论:更年春对AD细胞模型有保护作用,其含药血清作用强于主要中药单体及主要单体的混合液。

银杏平颤方对帕金森病模型小鼠多巴胺神经元丢失及其凋亡的影响

背景:现代药理研究证明银杏叶及其提取物生物具有抗氧化作用以及刺激神经生长因子产生等保护多巴胺神经元的存活,减少其凋亡。目的:验证银杏平颤方对帕金森病小鼠多巴胺神经元丢失及其凋亡的影响以及的神经母细胞瘤衍生细胞株SH-SY5Y的增殖情况的影响。方法:(1)C57BL小鼠随机分成3组。正常组10只不处理。其他2组小鼠每天腹腔注射MPTP建立小鼠帕金森病的模型,连续6周;每次腹腔注射前30 min进行灌胃处理,模型组10只灌胃生理盐水;治疗组10只灌胃中药银杏平颤方。分别在造模开始后的第15天和第30天取脑组织,免疫组织化学观察中脑黑质多巴胺神经元丢失和细胞凋亡情况;(2)体外培养神经母细胞瘤衍生细胞株SH-SY5Y并用银杏平颤方处理细胞,MTT法检测人SH-SY5Y的增殖,流式细胞术检测细胞凋亡情况;Real-time PCR和Western blotting检测细胞PARP、PTEN mR NA和蛋白质表达。结果与结论:(1)动物实验结果:正常组小鼠的中脑黑质致密部酪氨酸羟化酶阳性神经元的数目多于模型组,并存在时间依赖关系;银杏平颤方中药治疗组小鼠中脑黑质致密部酪氨酸羟化酶阳性神经元的数目多于模型组(P<0.05);(2)体外细胞实验结果:银杏平颤方处理的细胞凋亡率低于模型组;银杏平颤方处理细胞的PARP、PTEN的mR NA及蛋白水平均低于模型组。(3)结果说明,银杏平颤方治疗可在一定程度上抑制中脑神经元细胞的凋亡;其分子机制可能是银杏平颤方通过抑制PTEN表达进而降低细胞凋亡水平,保护多巴胺神经元,阻止其丢失,起到防治帕金森病的作用。