【作者】 孙国平；

【导师】 徐叔云；

【作者基本信息】 安徽医科大学 ， 药理学， 2003， 博士

【摘要】 丹皮酚（Paeonol，Pae）又称牡丹酚，是毛莨科植物牡丹Paeonia Suffruicosa Andr.根皮和萝摩科植物长徐长卿Pycnostelma Paniculatum（Bunge）K．Schum干燥根或全草的主要有效成分。Pae是一种小分子的酚类化合物，呈白色针状结晶，具有熔点低（mp51℃±1℃）、易挥发及水溶性差的特性。其分子量（Mr）为166.18，分子式为C₉H₁₀O₃，化学结构是2-羟基-4-甲氧基苯乙酮。有关Pae药理作用的研究可以追溯到1947年，当时学者报道牡丹皮水煎液在试管中能抑制伤寒杆菌的生长。此后，国内外诸多学者逐渐重视其药理学研究。研究结果表明，Pae具有镇静催眠、解热镇痛、抗菌消炎、免疫调节、抗氧化及保护心脑血管等广泛的药理活性。我们在前期研究中发现，Pae对体外培养的多种肿瘤细胞的增殖有较强的抑制作用。本文以体内、体外实验相结合，对Pae的抗肿瘤活性作了进一步的考察，并通过观察免疫指标、细胞凋亡及凋亡相关蛋白表达的变化，探讨Pae的抗肿瘤作用机制，现分述如下： 1．Pae的体内和体外抗肿瘤作用 1．1 Pae的体外抗肿瘤作用 采用MTT法检测Pae对体外培养的肿瘤细胞的增殖抑制作用。Pae在7.81～250mg·L^-1剂量下对体外培养的人红白血病细胞系K₅₆₂、乳腺癌基因细胞系T_6-17、肝癌细胞系BEL-7404及宫颈癌细胞系Hela的增殖均有抑制作用。药物浓度越高，抑制作用越强，有明显的剂量效应关系。对不同的细胞系抑制作用不同，安徽医科大学博士学位论文JU一eso值分别为:30.83一119吃.’（K562）、x3.44川g一J一‘（’l’。一，:）、69.89 1119·L‘’（BEL一7404）及93.91 mg·L‘’（llela）。以l，ael25 1119·l厂’及250，119·xJ一’分别作川J几以一上到}})J包，于24h、48h、72h及961:观察红!})j包I’I勺生长情况，发现Pae又寸红l})】包的J曾夕八JqJ制作川有明显的时间效应关系，作川时间越长，抑制作川越强。1，2 Pae的体内抗肿瘤作用 Pae灌胃给药川莫型小鼠HePA肿瘤的生长有明支谈抑;ljlJ作川。在50mg .kg一’心一’X 14d剂量下抑瘤率为40%左右，当剂量增至800mg·kg一’·d一‘X14d时抑瘤率.箭达70%左右。表明Pae具有良好的抗肿瘤作用。在总共二批试验158只小鼠中，死亡8只，其中50 mg·kg一，·d一，剂量组死亡l只，200和400 mg·kg‘’·d一，剂量组各死一亡2只，800 mg·kg一’·d一’剂量组死亡3只。各剂量组小鼠死亡率均低于20%，用药后小鼠体重均有增加，未见明显毒副作用，表明Pae在本实验条件下具有良好的安全性。2.Pae对荷瘤小鼠免疫功能的影响2.1 pae对荷瘤小鼠淋巴细胞增殖反应的影响 采用3H一护fdR掺入法测定淋巴细胞增殖反应。接种1 4d的模型小鼠脾淋巴细胞ConA及LPS诱导的增殖反应均减低，与正常对照组比较差异均有显著性。Pae在50、200 mg·kg一!·d一1 X 14d剂量下对conA诱导的增殖反应有增强作)}J;而在800mg·kg一’·d一!X 14d剂里下，conA诱导的增殖反应有一};降趋势。在50，200，800mg·kg一’·d’‘x14d剂量下对荷瘤小鼠LPs诱导的增殖反应均有抑制作用。结果提示，Pae对荷瘤小鼠的B淋巴细胞功能有抑制作用，但在一定剂量下可增强T淋巴细胞功能。2.2 Pae对IL一2生成的影响 采月」ltlA法检测IL一2含量，模型红L小鼠Jfl!.拿!‘示!L一2含员及肿细胞产生IL一2均低于一眨常对‘1狱组，l，ae在50，200，800，119·kg一’·d一’x 1 4d剂量’一}‘均不，J‘使模型小鼠脾细胞产生IL一2能力恢复至正常对1以组水平;在上述剂量一};Pae亦可使模型小鼠安徽医科大学博士学位论文血.清IL一2含量升高，其『1， 200、800mg·kg一’·d一’火14tlfrlJ员下与模型对!!砚纠比较差异有显著性。 体外试验中，Pae在6.25，12.5，25及50mg·L一’浓度卜川莫型小鼠脾细胞生成lL一2均有促进作川，其，}，在12.5和25 mg·L’‘浓2交卜诱生作川址强，具有统计学差异（p<0.05）。2.3 Pae对下N卜。生成的影响 TNF一u含最测定采川l{IA法。模型组小鼠l州青『rNF一。水平及PM中产生rrNF-。与正常对·1以红1比较无统不I学差异。I，ae在50，200，800:119·kg均，IJ‘使模型组小鼠!f一1.护!，了1’NF一。含量)1·高，l〕M中)c，二生‘l’Nl了一u一’·d一!x 14d剂量能力增强。体外试验，}，，l):，e了J56，3.]3，6.25，一2.5及25，119·l了一’浓度一J‘又寸模型小鼠l，M。卜生)戊1’NF一u具丫J‘统乙}·学j，’均有促进作用，其，}“在6.25及12.5 mg·L一’浓度下促进作用最强，异。 以_「结果提示，!’:Le的抗月中瘤作川可能与增强细胞免疫功能，促进比一2及‘fNI了-u的生成有关。3.Pae诱导肿瘤细胞凋亡3.1 Pae诱导肿瘤细胞凋亡的形态学观察 HE染色光镜及电子显微镜下均可见典型的肿瘤细胞凋亡改变。光镜下，凋l’:细胞体积缩小，核染色质浓缩深染，胞质深嗜伊红染，!阿坏死细胞形态不规则，胞质淡染，细胞膜溶解不完整，核查.戈碎片状。电镜下，.让见体积缩小的凋亡细胞，染色质浓缩致密，并沿核膜分布形成新月状体，凋亡晚期细胞，可见细胞分解为1哎包裹的凋亡小体。3.2 Pae?更多还原

【Abstract】 Paeonol(Pae), the main component of paeony bark, possesses multiple pharmacological effects such as sedation, hypnosis, anti-pyresis, analgesia, anti-inflammation, anti-oxidation and immuno-regulation. We have found in previous study that Pae inhibited the proliferation of different types of tumor cells in vitro. In the present study, we will further explore the anti-tumor activity of Pae and its mechanism by examining the immunological indexes, apoptosis and the protein expression related to apoptosis.1. Anti-tumor effect of Pae in vivo and in vitro 1.1 Anti-tumor effect of Pae in vitroThe inhibitory effect of Pae on tumor cells proliferation in vitro was assayed by MTT dye reduction. Pae at concentrations of 7.81-250 mg L-1 inhibited proliferation of four tumor cell lines in vitro (human erythroleukemic cell line K562, breast cancer gene cell lineT6-17, hepatocarcinoma cell line BEL-7404 and cervical cancer cell line Hela) with different 1C50 (30.83, 13.44, 69.89 and 93.91 mg L-1 respectively). It was observedthat higher concentration of Pae, more inhibitory effect, suggesting obvious concentration-effect relationship of Pae. The inhibitory effects were augmented with the prolongation of culture time (24h, 48h, 72h to 96h), suggesting time-effect relationship of Pae.1.2 Anti-tumor effect of Pae in vivoPae ig suppressed the tumor growth significantly in HepA bearing mice model. The tumor-inhibitory rates were 40% and 70% when the mice were administered with Pae at doses of 50 mg kg-1 d-1 and 800 mg kg-1 d-1, respectively, for 14 days. There were 8 mice deceased in 4 treated groups with 158 mice (1 in the group with 50 mg kg-1 d-1 ,2 in the group with 200mg kg-1 d-1 , 2 in the group with 400 mg kg-1 d-1 and 3 in the group with 800 mg kg-1 d-1). The deceased rate in each group was lower than 20%. The body weight of mice after Pae administration increased significantly, and no obvious toxic and side effects were observed, suggesting Pae is safe in the present experimental condition.2. Effects of Pae on immune function with tumor-bearing mice 2.1 Effect of Pae on lymphocyte proliferation in tumor-bearing mice3H-TdR incorporation was used to detect lymphocyte proliferation. Con A- and LPS-induced splenic lymphocyte proliferation were decreased on day 14 after inoculation in tumor-bearing mice. Pae could augment decreased Con A-induced lymphocyte proliferation at doses 50 and 200 mg kg-1 d-1 and further decrease this reaction at doses 800 mg kg-1 d-1 for 14 days. However, Pae at all above doses suppress LPS-induced lymphocyte proliferation in tumor bearing mice. The results suggest thatPae inhibited B lymphocyte function and increased T lymphocyte function at certain doses in tumor bearing mice.2.2 Effect of Pae on IL-2 productionIL-2 content was assayed by RTA. Serum IL-2 content and IL-2 production from splenocytes in tumor bearing mice was lower than that in control group.Pae at doses of 50, 200, and 800 mg kg-1 d-1 increased the IL-2 production by splenocytes in tumor bearing mice, which became higher than that in no-treated tumor bearing mice and normal mice. Pae could also increase serum IL-2 level in this model, and the significant effects were seen in the groups treated with 200 and 800 mg kg-1 d-1 Pae.In vitro experiments showed that Pae at concentrations of 6.25, 12.5, 25 and 50 mg L-1 promoted IL-2 production by splenocytes from the above model. The most pronounced effect was seen in the group treated with 12.5 and 25 mg L-1 Pae.2.3 Effect of Pae on TNF- a productionTNF- a content was assayed by RTA. Serum TNF- a level and TNF- a production by peripheral macrophages (PMO) in tumor bearing mice model were higher than that in normal group, but no significant differences were observed. PAE at doses of 50, 200, and 800 mg kg d-1 could increase serum TNF- a content and promote TNF- a production by PMO in the tumor bearing mice model.In vitro experiments showed that Pae at concentrations of 6.25, 12.5, 25 and 50 mg L-1 promoted TNF-a更多还原