紫草素诱导Raji细胞凋亡的机制研究

目的：研究紫草素（Shikonin）对Burkitt＇s淋巴瘤细胞凋亡及坏死的影响及Necrostatin-1（Nec-1）对紫草素诱导的细胞凋亡的作用。方法：对体外培养的Raji细胞进行荧光显微镜观察,Alarma Blue法检测细胞增殖率,流式细胞仪分析细胞凋亡及坏死,免疫印迹检测相关蛋白表达。结果：紫草素对Raji细胞生长有抑制作用,呈时间和浓度依糯。紫草素可诱导Raji细胞发生程序性坏死及凋亡。小分子化合物Nec-1不仅可以抑制紫草素诱导的程序性坏死,同时对细胞凋亡也有抑制作用。结论：紫草素可以抑制Raji细胞生长增殖,而小分子Nec-1可以抑制这一过程。     

Shikonin Isolated from Lithospermum erythrorhizon Downregulates Proinflammatory Mediators in Lipopolysaccharide-Stimulated BV2 Microglial Cells by Suppressing Crosstalk between Reactive Oxygen Species and NF-κB

According to the expansion of lifespan, neuronal disorder based on inflammation has been social problem. Therefore, we isolated shikonin from Lithospermum erythrorhizon and evaluated anti-inflammatory effects of shikonin in lipopolysaccharide (LSP)-stimulated BV2 microglial cells. Shikonin dose-dependently inhibits the expression of the proinflammatory mediators, nitric oxide (NO), prostaglandin E₂ (PGE₂), and tumor necrosis factor-α (TNF-α) as well as their main regulatory genes and products such as inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and TNF-α in LPS-stimulated BV2 microglial cells. Additionally, shikonin suppressed the LPS-induced DNA-binding activity of nuclear factor-κB (NF-κB) to regulate the key regulatory genes of the proinflammatory mediators, such as iNOS, COX-2, and TNF-α, accompanied with downregulation of reactive oxygen species (ROS) generation. The results indicate that shikonin may downregulate the expression of proinflammatory genes involved in the synthesis of NO, PGE₂, and TNF-α in LPS-treated BV2 microglial cells by suppressing ROS and NF-κB. Taken together, our results revealed that shikonin exerts downregulation of proinflammatory mediators by interference the ROS and NF-κB signaling pathway.     

紫草素对银屑病T淋巴细胞增殖、活化的影响

目的:研究具有凉血、解毒作用的中药单体紫草素对银屑病病理状态下异常活化的T淋巴细胞功能及相关信号转导通路的影响。方法:选取Jurkat E6-1 T淋巴细胞,以佛波醇酯和离子霉素活化Jurkat E6-1 T淋巴细胞,同时将0. 5～2μg/m L浓度的紫草素作用于细胞,采用CCK-8法检测T淋巴细胞增殖能力;流式细胞术检测细胞膜CD69的表达及细胞内游离钙离子浓度([Ca2+]i)的变化;用酶联免疫吸附试验(ELISA)检测T淋巴细胞释放白细胞介素-2(IL-2)、干扰素-γ(IFN-γ)、肿瘤坏死因子-α(TNF-α)的水平;用Realtime-PCR和Western blotting法分别观察核转录因子mRNA及蛋白的表达影响。结果:不同浓度的紫草素均可明显抑制细胞增殖、CD69表达及Th1类细胞因子分泌;均可有效降低[Ca2+]i和蛋白激酶C(PKC)磷酸化蛋白的水平;显著降低核转录因子NF-AT mRNA的表达,下调c-Jun的mRNA及蛋白表达并可抑制核因子-κB蛋白的表达。以上各项指标均有一定量效关系。结论:紫草素可以通过抑制过度活化的T淋巴细胞功能从而发挥免疫调控作用,为紫草素治疗银屑病的作用机制及应用前景提供实验依据。     

紫草素对大鼠重症急性胰腺炎ZO - 1与HSP70的影响

目的 观察紫草素对大鼠重症急性胰腺炎（SAP）胰腺HSP70、ZO - 1的表达及血清TNF - α和IL - 6的影响。方法 将大鼠随机分为模型组、低分子量肝素组（肝素组）;紫草素1 mg/kg和2 mg/kg组（紫草素1和2组）,采用牛磺胆酸钠制备SAP模型,分别检测术后12、24和72 h胰腺HSP70和ZO - 1表达、血清TNF - α和IL - 6水平及胰腺形态学变化。结果 术后达峰时间和顺序:ZO - 1表达为12 h,依次为肝素组（81.17±9.20）、紫草素2组（70.83±14.73）和1组（54.50±13.31）、模型组（31.67±6.06）,与模型组比较,差异有统计学意义（P＜0.01）;HSP70为24 h,紫草素2组（86.50±8.50）和1组（79.00±16.67）、肝素组（78.17±15.84）、模型组（40.17±16.92）,与模型组比较,差异有统计学意义（P＜0.01）;血清TNF - α和IL - 6均为24 h,模型组［（325.32±69.82）、（336.52±43.72） pg/ml］、紫草素1组［（175.23±43.66）、（227.66±62.66） pg/ml］、紫草素2组［（156.50±19.60）、（189.39±22.99） pg/ml］肝素组［（140.13±17.83,167.83±8.50）pg/ml］,与模型组比较,差异有统计学意义（P＜0.01）。胰腺病理变化及评分:模型组有不同程度胰腺水肿、出血、坏死和炎细胞浸润,小叶间隙增宽,评分均最高;紫草素1、2组和肝素组病变程度明显减轻,评分低于模型组,差异有统计学意义（P＜0.01）。结论 紫草素可减轻胰腺损伤,对胰腺具有保护作用。     

紫草素促进大鼠皮瓣成活的实验研究

目的 观察紫草素对大鼠狭长窄蒂皮瓣成活的影响及作用机制。方法 取Wistar大鼠在其背部设计2个类似“乒乓球拍”的皮瓣,每个狭长窄蒂（蒂的长宽比为2 cm∶1 cm）携带3 cm×3 cm皮瓣。48只大鼠随机分为对照组和紫草素组,各组相应处理:紫草素组（2 mg/kg）,对照组（给予等剂量溶剂,2 ml/ kg）,1次/d腹腔注射,连续7 d。免疫荧光组织化学染色分析术后皮瓣组织血管内皮生长因子（vascular endothelial growth factor,VEGF）、内皮型一氧化氮合酶（endothelial nitric oxide synthase,eNOS）和热休克蛋白70（Heat Shock Protein 70,HSP70）的表达;HE染色观察大鼠皮瓣形态学变化,并计算皮瓣成活面积百分比;ELISA 法检测动物血清一氧化氮（nitric oxide,NO）、超氧化物歧化酶（superoxide Dismutase,SOD）和丙二醛（malondialdehyde,MDA）的含量。结果 紫草素增加了术后大鼠皮瓣VEGF、eNOS和HSP70的表达,并促进新生毛细血管形成,提高了大鼠皮瓣的成活率;紫草素显著降低了大鼠血清MDA的水平,升高了NO和SOD的水平。结论 紫草素可促进狭长窄蒂皮瓣成活,机制与调节VEGF/eNOS/ NO信号分子表达和抗氧化作用有关。     

紫草素诱导生殖系肿瘤细胞凋亡的研究

目的:观察新疆紫草素对生殖系肿瘤细胞的体外增殖抑制和诱导凋亡作用。方法:由新疆紫草提取萘醌色素,制备微乳液,并加至人宫颈癌细胞系HeLas3和卵巢肿瘤细胞系OVCAR-3的体外培养中,共育24～72h。人胚肾HEK293和成纤维细胞系NIH3T3作为对照。MTT法测定细胞增殖。流式细胞术和TUNEL法分析细胞凋亡。结果:紫草素对HEK293和3T3的体外72h增殖无影响;但明显抑制宫颈癌以及卵巢肿瘤细胞系的生长;这种抑制作用呈现共育时间和药物浓度的依赖性。半数抑制浓度为2.58～0.19μg/ml。药物与卵巢肿瘤细胞系共育24h,肿瘤细胞凋亡明显增加,超过20%。结论:新疆紫草素诱导生殖系肿瘤细胞凋亡,从而抑制其体外增殖。     

Shikonin regulates HeLa cell death via caspase-3 activation and blockage of DNA synthesis

Shikonin, isolated from the plant Lithospermum erythrorhizon Sieb. Et Zucc, inhibited tumor cell growth and induced cell death in various tumor cells, with 50% growth inhibition of human cervical cancer cells, HeLa, at 18.9±1.1 μmol L^-1. Treated with 40 μmol L^-1 shikonin, HeLa cells underwent marked apoptotic morphological changes such as a round shape, membrane blebbing and apoptotic bodies derived from the fragmented nuclei. Another hallmark of apoptosis, DNA fragmentation, was observed by gel electrophoresis. Shikonin (10 μmol L^-1) significantly blocked the transition from G1 to S phase in the HeLa cell cycle. Pan-caspase inhibitor (Z-VAD-FMK), caspase-3 inhibitor (Z-DEVD-FMK) or caspase-8 inhibitor (Z-IETD-FMK) effectively inhibited shikonin-induced cell death, while caspase-1 inhibitor (Ac-YVAD-CMK) and caspase-9 inhibitor (Z-LEHD-FMK) failed to affect cell death. Caspase-3 activity significantly increased within 12 h after shikonin treatment. Reduced expression of inhibitor of caspase-activated deoxyribonuclease (ICAD) after exposure to shikonin for 12 h suggests the resultant activation of caspase-activated deoxyribonuclease (CAD), leading to apoptosis.     

紫草素对膀胱癌细胞T24增殖和凋亡的影响及其机制研究

目的:探讨紫草素抑制T24细胞生长的作用及其机制。方法:MTT法测定紫草素对T24细胞的生长抑制实验;用RT-PCR、westen-blot法检测不同浓度的紫草素作用于T24细胞时,对COX-2 mRNA和蛋白表达的影响;流式细胞仪检测细胞凋亡。结果:25、50和75μmol/L紫草素均能抑制T24细胞生长,诱导凋亡,并呈明显的量效关系和时间依赖性,紫草素能够抑制COX-2 mRNA和蛋白的表达。结论:紫草素能明显抑制T24细胞增殖,促其凋亡,其机制可能与COX-2表达下调有关。     

Shikonin modulates cell proliferation by inhibiting epidermal growth factor receptor signaling in human epidermoid carcinoma cells

Shikonin isolated from the roots of the Chinese herb Lithospermum erythrorhizon has been associated with anti-inflammatory properties. We evaluated shikonin's chemotherapeutic potential and investigated its possible mechanism of action in a human cutaneous neoplasm in tissue culture. Shikonin preferentially inhibits the growth of human epidermoid carcinoma cells concentration- and time-dependently compared to SV-40 transfected keratinocytes, demonstrating its anti-proliferative effects against this cancer cell line. Additionally, shikonin decreased phosphorylated levels of EGFR, ERK1/2 and protein tyrosine kinases, while increasing phosphorylated JNK1/2 levels. Overall, shikonin treatment was associated with increased intracellular levels of phosphorylated apoptosis-related proteins, and decreased levels of proteins associated with proliferation in human epidermoid carcinoma cells.     

一些紫草素衍生物的园二色谱研究

从四川密花滇紫草(Onosma confertum W.W.Smith)和新疆软紫草[Arnebia euchroma (Royle) Johust]的根中分到九个紫草素衍生物,其中七个为手性化合物。测定了它们的CD谱和UV谱,结果得到三对几乎完全对称的CD谱图和完全相同的UV谱图、确定了四川密花滇紫草成分均为R构型;新疆软紫草成分均为S构型。