冬凌草甲素和冬凌草多糖的制备及其抗膀胱肿瘤作用的实验研究
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摘要
在我国，膀胱癌是泌尿系统最常见的恶性肿瘤。90％的膀胱癌是移行细胞癌，其中70-75％是浅表性膀胱癌。在浅表性膀胱癌中，95％为高危险性浅表性癌。膀胱癌复发率较高，单纯手术治疗约有40％在两年内复发，远期复发率几乎100％，其中浅表性膀胱癌有10％进展为浸润性膀胱癌。高危浅表性肿瘤术后若不给予任何药物治疗，其近期复发率为60-90％。根据膀胱癌易复发的特点，实行术后药物灌注可以达到清除残余瘤体，预防复发和延缓进展的目的。
BCG是一种有效的膀胱腔内灌注药，可以预防膀胱肿瘤复发，治疗膀胱原位癌，防治膀胱肿瘤进展及降低死亡率。在众多浅表性膀胱癌的免疫治疗方法中，BCG作为预防性免疫治疗方法是最有价值的；但仍有30-45％的病人对腔内BCG灌注治疗无反应。而且随着BCG治疗的病例数的增加，腔内BCG灌注所致的膀胱局部、全身反应和毒副作用逐渐增加。其中5％患者可出现严重并发症，0.5％甚至可以危及生命。并发症的发生严重影响了患者的依从性，使患者不能完成整个治疗过程。在Pasadoro报道的治疗组中，只有19％的患者完成了全部治疗，其余病例均因并发症或不良反应而放弃维持治疗。因此，BCG腔内灌注用于预防膀胱肿瘤复发和治疗浅表膀胱肿瘤受到一定的限制。有鉴于此，寻找与研究一种患者依从性好、并发症低、具有抗膀胱肿瘤作用的、拥有我国独立知识产权的膀胱灌注治疗方法。
冬凌草是近年来发现的具有广泛抗肿瘤作用的纯天然植物。但作为膀胱腔内灌注药物预防与治疗浅表性膀胱肿瘤的作用机制尚不清楚。
为了提取与制备适合膀胱腔内灌注的天然药物制剂，本课题采用乙醇回流法从冬凌草中分离得到了高纯度的ORI，水提取法得到了RRP；细胞实验分别研究了ORI和RRP抗膀胱肿瘤细胞系MB49细胞、EJ细胞和T24细胞的作用及其机制；在我们建立的C57BL／6小鼠原位膀胱肿瘤模型上，初步研究了ORI和RRP膀胱灌注治疗小鼠膀胱肿瘤的作用及其机制，为冬凌草提取物膀胱灌注预防和治疗浅表膀胱癌天然药物的开发提供科学的实验依据。
第一部分
冬凌草甲素和冬凌草多糖的制备
目的：提取分离冬凌草甲素和冬凌草多糖，为抗膀胱肿瘤细胞实验和动物实验提供样品。方法：乙醇回流法提取总二萜类，采用硅胶柱层析法分离纯化冬凌草甲素，以高效液相层析法(HPLC)鉴定和测定冬凌草甲素的含量。冬凌草多糖采用水提取、乙醇沉淀法进行提取分离，用有机溶剂纯化。结果：1000g冬凌草叶共提取得到冬凌草甲素2.2g，纯度高达98％，提取率为0.22％。200g冬凌草叶共提取得到冬凌草多糖约8.1g，提取率为4.05％。结论：乙醇回流法是较理想的提取冬凌草甲素的方法，水提取、乙醇沉淀法是较理想的提取冬凌草多糖的方法。
第二部分
冬凌草甲素和冬凌草多糖抗膀胱肿瘤细胞系的实验研究
目的：研究ORI和RRP对膀胱肿瘤细胞系MB49细胞、EJ细胞和T24细胞的作用及机制。方法：用MTT法检测ORI和RRP对MB49、EJ、T24的生长抑制作用；用光学显微镜和电子显微镜、Hoechst33258荧光染色法观察细胞的形态学变化；凝胶电泳观察细胞的DNA片断化；FCM Annexin V-PI双染色法检测细胞凋亡率；Caspase分光光度法检测Caspase-3、Caspase-8和Caspase-9活性变化；Western blot检测Bcl-2和Bax表达的变化；PCR-TRAP-银染法检测端粒酶活性变化。结果：ORI、RRP对MB49细胞、EJ细胞和T24细胞均有明显的生长抑制作用，呈明显的时间-效应关系和浓度-效应关系。作用24h时，ORI对MB49细胞、EJ细胞和T24细胞的IC50分别为16μmol／L、25.6μmol／L和27.2μmol／L。联合应用ORI和RRP对MB49细胞有相加作用，对EJ细胞和T24细胞有协同作用。形态学观察，凋亡细胞数随ORI作用时间延长而增加，见凋亡小体出现。DNA电泳看到DNA ladder。Annexin V-FITC-PI双染色FCM检查发现，随着ORI作用浓度增加，凋亡细胞数和坏死细胞数均增加，浓度越高，坏死细胞的比率增加越多。Caspase分光光度法检测结果发现，随着ORI作用时间延长，MB49细胞、EJ细胞和T24细胞的Caspase-3和Caspase-9活性均升高，EJ细胞和T24的Caspase-8活性升高，但MB49细胞的Caspase-8未见明显变化。Western blot显示，随着ORI作用时间延长，三种细胞Bcl-2的表达均逐渐减弱，Bax的表达均逐渐增强。PCR-TRAP-银染法检测显示，MB49细胞的端粒酶活性随着ORI作用时间的延长而逐渐减弱。结论：冬凌草提取物ORI和RRP对膀胱肿瘤细胞系有明显的抗肿瘤作用。ORI低浓度时主要引起膀胱肿瘤系凋亡，高浓度时有直接的细胞毒作用。在EJ细胞和T24细胞，ORI可能主要通过Fas／FasL-线粒体途径和Apo2L／TRAIL-Caspase-8激活Caspase家族，导致凋亡的发生；在MB49细胞，ORI可能主要通过Fas／FasL-线粒体途径导致凋亡的发生。
第三部分
冬凌草甲素和冬凌草多糖膀胱灌注治疗小鼠膀胱肿瘤的实验研究
目的：建立小鼠原位膀胱肿瘤模型；探讨ORI和RRP膀胱灌注后的疗效及其机制；观察膀胱灌注用药对膀胱及全身重要器官的影响。方法：采用硝酸银灼伤、MB49细胞膀胱灌注的方法建立C57BL／6小鼠原位膀胱肿瘤模型，常规病理检查和电子显微镜检查诊断膀胱肿瘤，计算肿瘤接种成功率；建立模型后10d，四组小鼠各15只，分别灌注PBS、ORI、RRP、ORI和RRP，每5d灌注一次，灌注四次后1w处死小鼠，FCM检查血液淋巴细胞亚群变化，常规病理检查膀胱和心、肝、脾、肺、肾，了解灌注后对膀胱和重要器官的影响，并计算各自的治愈率。TUNEL检测残存肿瘤凋亡。结果：C57BL／6小鼠原位膀胱肿瘤模型的成瘤率为96.7％。ORI组治愈率为80％，RRP为60％，联合用药组为93.3％，均明显高于对照组的13.3％；ORI组和联合用药组明显高于RRP组。TUNEL检测显示，ORI组、RRP组和联合用药组残存肿瘤细胞出现凋亡，越近肿瘤表面，凋亡越明显。灌注后，血液淋巴细胞亚群各组无差别，膀胱除ORI组和联合用药组的移行上皮明显变薄外，无其它异常改变。各组的肝、脾、肺、心、肾未见明显损害。结论：硝酸银灼伤、MB49膀胱灌注建立起来的C57BL／6小鼠原位膀胱肿瘤模型是一种较为理想的原位肿瘤模型。ORI、RRP具有明显的抗小鼠膀胱肿瘤作用，其作用机制可能为诱发肿瘤细胞凋亡。ORI、RRP膀胱灌注治疗小鼠膀胱肿瘤对膀胱及全身重要器官是安全的。
Bladder cancer is the most common malignancy of urinary system in China.Ninety percent of bladder cancer is transitional cell carcinoma (BTCC), of whichseventy to seventy five percent is superficial bladder cancer. Of superficial bladdercancer, ninety five percent is high-grade BTCC. After surgery, forty percent tends torecur in two years.with a long-term recurrence of almost 100%. Of the superficialBTCC, ten percent will progress to be invasive. Of the high-grade superficial BTCC,the short-term recurrence is up to sixty to ninety percent if not interfered. Because ofthat, intravesical instillation of anticancer agents should aim at clearing tumor,preventing recurrence and delaying progression of tumor.
Although intravesical administration of Bacillus Calmette-Guerin (BCG) has beenaccepted as the most effective therapy for superficial bladder cancer and carcinoma insitu of the bladder, thirty to forty five percent of patients never respond to BCGtherapy. Furthermore, long-term remission (more than five years) is only achieved infifty percent of patients. Toxicity associated with the BCG therapy is frequent withoccurrence of severe adverse effects in five percent of patients and life-threateningcomplications in 0.5 percent of patients. Patients hardly accept the regular instillationperiod because of severe cystitis and complications. It's reported that the percentageof patients who kept accepting regular instillation is not more than nineteen percent.So, it's necessary to explore a new agent acceptable by patients for intravesicalinstillation with better effects and fewer complications.
Nowadays, research on antineoplastic agents has been highlighted by naturalproducts. Rabdosia rubescens is a traditional Chinese herb with a variety of pharma-cological effects, especially anticancer effects. Oridonin and rabdosia rubescenspolysaccharide (RRP) are the two important ingredients which exhibit remarkable inhibitory effects on many tumor cell lines. Clinical reports have demonstrated thatcoarse extract ofrabdosia rubescens can prevent recurrence of bladder cancer.
In this dissertation, oridonin and RRP extraction was studied on at first. Afteralcohol back-streaming and separation by silica gel column chromatography, oridoninof which the purity is ninety eight percent was obtained. After water-extraction withalcohol-precipitation, RRP was obtained. Second, we studied the antineoplasticeffects of oridonin and RRP on bladder cancer cell lines, MB49 cells, EJ cells andT24 cells. After establishing an orthotopic murine BTCC model successfully, wefinally studied the effects of oridonin and RRP on murine BTCC and its mechanism.
SectionⅠ
Extraction and Separationof Oridonin and Polysaccharide from Rabdosia Rubescens
Objective: To prepare oridonin and polysaccharide from the traditional Chineseherb, Rabdosia rubescens, for the next experiments. Methods: Total diterpenoidswere obtained by alcohol back-streaming. Oridonin was separated by silica gelcolumn chromatography, assayed by high performance liquid chromatography.Polysaccharide was obtained by water-extraction, precipitated by alcohol, andpurified by organic solvents. Results: Extraction rate of oridonin was 0.22 percent(2.2/1000) and the purity of oridonin was 98 percent. Extraction rate ofPolysaccharide was 4.05 percent (8.1/200). Conclusions: The alcohol back-streamingis a better method for extraction of oridonin. The water extraction withalcohol-precipitation is a practical method for preparation of RRP.
SectionⅡ
Studyon Antineoplastie Effects of Oridonin and Rabdosia Rubescens Polysaccharide on Bladder Cancer Cell Lines
Objective: To study the antineoplastic effects of oridonin and rabdosia rubescenspolysaccharide (RRP) on bladder cancer cell lines, MB49 cells, EJ cells and T24 cellsin vitro. Methods: The inhibitory rate of the cells was measured by MTT assay. Cellapoptotic rate was detected by flow cytometry (FCM) using Annexin V-FITC andpropidium iodide (PI) detection kit. Morphology of cell apoptosis was observed byHoechst 33258 fluorescence staining and electron/light microscopy. DNA fragmen-tation was assayed by agarose gel electrophoresis. Caspase-3, Caspase-8 andCaspase-9 were assayed with colorimetric assay kits. Bcl-2 and bax expressions weredetected by Western Blot. Telomerase was detected by PCR-TRAP-Silver staining.
Results: Oridonin and RRP could inhibit the growth of MB49 cells, EJ cells and T24cells and cause apoptosis significantly. The suppression was both in a time-anddose-dependent manner. IC50 at 24 hours is 16μmol/L in MB49 cells, 25.6μmol/L inEJ cells and 27.2μmol/L in T24 cells respectively. Combined administration oforidonin and RRP could be synergetic in EJ cells and T24 cells. Marked morpholo-gical changes of cell apoptosis including condensation of chromatin and apoptosisbody were observed clearly. Oridonin could down-regulate the Bcl-2 expression andup-regulate the Bax expression of MB49 cells, EJ cells and T24 cells. Oridonin coulddecrease the telomerase activity of MB49 Cells. Oridonin could increase the activityof Caspase-3 and Caspase-9 of the abovementioned 3 cells, and increase the activityof Caspase-8 of EJ cells and T24 cells, but not of MB49 cells. Conclusions: Oridoninand RRP, especially oridonin, have apparent inhibition and apoptosis-inducing effectson MB49 cells, EJ cells and T24 cells. Low concentration of oridonin mainly inducesapoptosis, while high concentration of oridonin may induce necrosis in bladdercancer cell lines involved. Oridonin may induce apoptosis by activating Caspase-3through both Fas/FasL-mitochondria and Apo 2L/TRAIL-Caspase-8 pathways in EJcells and T24 cells, but only via Fas/FasL-mitochondria pathway in MB49 cells.
SectionⅢ
Study on Intravesical Instillation of Oridonin andRabdosia Rubescens Polysaccharide for Murine Bladder Cancer
Objective: To establish a model of orthotopic murine bladder cancer, study theeffects of intravesical instillation of oridonin and RRP on murine bladder cancer andobserve the morphological changes in bladder, heart, liver, spleen, kidneys and lungs.
Methods: Orthotopic C57BL/6 murine bladder cancer model was established byintravesical instillation of MB49 cells after argentine nitrate cautery. The diagnosis ofbladder cancer was made by routine pathology and electron microscopy. Ten daysafter instillation of MB49 cells, mice were divided into 4 groups, 15 mice in eachgroup. 100 microliters of PBS, oridonin, RRP, combined administration of oridoninand RRP were instilled intravesically in different group respectively. Afterwards,instillation was given in each group every 5 days, 4 times in all. One week after that,mice were all sacrificed. Blood subtypes of lymphocyte were detected by FCM.Bladder, heart, liver, spleen, lungs and kidneys were anatomized out for pathology tocalculate the cure rate and observe morphological changes. TUNEL detection wasmade in tumor-loaded mice. Results: Tumor-taking rate was 96.7 percent in ouranimal model. There were no significant differences in lymphocyte subtypes betweencontrol group and experiment groups. The cure rate in the combined administrationgroup was 93.3 percent, which was higher than those both in oridonin group (80percent) and in RRP group (60 percent), of which differences were significant. Therewere no apparent degeneration, necrosis, inflammatory leukocyte infiltration andfibrosis in the abovementioned viscera. TUNEL detection showed that there wereapparent apoptosis cells in each group. The closer the tumor surface approaches, themore apparent the apoptosis is. Conclusions: The orthotopic C57BL/6 murinebladder cancer model established by instillation of MB49 cells after argentine nitrate cautery is practical for animal experiment of instillation therapy for bladder cancer.Oridonin and RRP have apparent antineoplastic effects, of which mechanism may beinduction of apoptosis. After instillation of oridonin and RRP, heart, liver, kidneys,lungs and spleen are safe without damages.
BCG是一种有效的膀胱腔内灌注药，可以预防膀胱肿瘤复发，治疗膀胱原位癌，防治膀胱肿瘤进展及降低死亡率。在众多浅表性膀胱癌的免疫治疗方法中，BCG作为预防性免疫治疗方法是最有价值的；但仍有30-45％的病人对腔内BCG灌注治疗无反应。而且随着BCG治疗的病例数的增加，腔内BCG灌注所致的膀胱局部、全身反应和毒副作用逐渐增加。其中5％患者可出现严重并发症，0.5％甚至可以危及生命。并发症的发生严重影响了患者的依从性，使患者不能完成整个治疗过程。在Pasadoro报道的治疗组中，只有19％的患者完成了全部治疗，其余病例均因并发症或不良反应而放弃维持治疗。因此，BCG腔内灌注用于预防膀胱肿瘤复发和治疗浅表膀胱肿瘤受到一定的限制。有鉴于此，寻找与研究一种患者依从性好、并发症低、具有抗膀胱肿瘤作用的、拥有我国独立知识产权的膀胱灌注治疗方法。
冬凌草是近年来发现的具有广泛抗肿瘤作用的纯天然植物。但作为膀胱腔内灌注药物预防与治疗浅表性膀胱肿瘤的作用机制尚不清楚。
为了提取与制备适合膀胱腔内灌注的天然药物制剂，本课题采用乙醇回流法从冬凌草中分离得到了高纯度的ORI，水提取法得到了RRP；细胞实验分别研究了ORI和RRP抗膀胱肿瘤细胞系MB49细胞、EJ细胞和T24细胞的作用及其机制；在我们建立的C57BL／6小鼠原位膀胱肿瘤模型上，初步研究了ORI和RRP膀胱灌注治疗小鼠膀胱肿瘤的作用及其机制，为冬凌草提取物膀胱灌注预防和治疗浅表膀胱癌天然药物的开发提供科学的实验依据。
第一部分
冬凌草甲素和冬凌草多糖的制备
目的：提取分离冬凌草甲素和冬凌草多糖，为抗膀胱肿瘤细胞实验和动物实验提供样品。方法：乙醇回流法提取总二萜类，采用硅胶柱层析法分离纯化冬凌草甲素，以高效液相层析法(HPLC)鉴定和测定冬凌草甲素的含量。冬凌草多糖采用水提取、乙醇沉淀法进行提取分离，用有机溶剂纯化。结果：1000g冬凌草叶共提取得到冬凌草甲素2.2g，纯度高达98％，提取率为0.22％。200g冬凌草叶共提取得到冬凌草多糖约8.1g，提取率为4.05％。结论：乙醇回流法是较理想的提取冬凌草甲素的方法，水提取、乙醇沉淀法是较理想的提取冬凌草多糖的方法。
第二部分
冬凌草甲素和冬凌草多糖抗膀胱肿瘤细胞系的实验研究
目的：研究ORI和RRP对膀胱肿瘤细胞系MB49细胞、EJ细胞和T24细胞的作用及机制。方法：用MTT法检测ORI和RRP对MB49、EJ、T24的生长抑制作用；用光学显微镜和电子显微镜、Hoechst33258荧光染色法观察细胞的形态学变化；凝胶电泳观察细胞的DNA片断化；FCM Annexin V-PI双染色法检测细胞凋亡率；Caspase分光光度法检测Caspase-3、Caspase-8和Caspase-9活性变化；Western blot检测Bcl-2和Bax表达的变化；PCR-TRAP-银染法检测端粒酶活性变化。结果：ORI、RRP对MB49细胞、EJ细胞和T24细胞均有明显的生长抑制作用，呈明显的时间-效应关系和浓度-效应关系。作用24h时，ORI对MB49细胞、EJ细胞和T24细胞的IC50分别为16μmol／L、25.6μmol／L和27.2μmol／L。联合应用ORI和RRP对MB49细胞有相加作用，对EJ细胞和T24细胞有协同作用。形态学观察，凋亡细胞数随ORI作用时间延长而增加，见凋亡小体出现。DNA电泳看到DNA ladder。Annexin V-FITC-PI双染色FCM检查发现，随着ORI作用浓度增加，凋亡细胞数和坏死细胞数均增加，浓度越高，坏死细胞的比率增加越多。Caspase分光光度法检测结果发现，随着ORI作用时间延长，MB49细胞、EJ细胞和T24细胞的Caspase-3和Caspase-9活性均升高，EJ细胞和T24的Caspase-8活性升高，但MB49细胞的Caspase-8未见明显变化。Western blot显示，随着ORI作用时间延长，三种细胞Bcl-2的表达均逐渐减弱，Bax的表达均逐渐增强。PCR-TRAP-银染法检测显示，MB49细胞的端粒酶活性随着ORI作用时间的延长而逐渐减弱。结论：冬凌草提取物ORI和RRP对膀胱肿瘤细胞系有明显的抗肿瘤作用。ORI低浓度时主要引起膀胱肿瘤系凋亡，高浓度时有直接的细胞毒作用。在EJ细胞和T24细胞，ORI可能主要通过Fas／FasL-线粒体途径和Apo2L／TRAIL-Caspase-8激活Caspase家族，导致凋亡的发生；在MB49细胞，ORI可能主要通过Fas／FasL-线粒体途径导致凋亡的发生。
第三部分
冬凌草甲素和冬凌草多糖膀胱灌注治疗小鼠膀胱肿瘤的实验研究
目的：建立小鼠原位膀胱肿瘤模型；探讨ORI和RRP膀胱灌注后的疗效及其机制；观察膀胱灌注用药对膀胱及全身重要器官的影响。方法：采用硝酸银灼伤、MB49细胞膀胱灌注的方法建立C57BL／6小鼠原位膀胱肿瘤模型，常规病理检查和电子显微镜检查诊断膀胱肿瘤，计算肿瘤接种成功率；建立模型后10d，四组小鼠各15只，分别灌注PBS、ORI、RRP、ORI和RRP，每5d灌注一次，灌注四次后1w处死小鼠，FCM检查血液淋巴细胞亚群变化，常规病理检查膀胱和心、肝、脾、肺、肾，了解灌注后对膀胱和重要器官的影响，并计算各自的治愈率。TUNEL检测残存肿瘤凋亡。结果：C57BL／6小鼠原位膀胱肿瘤模型的成瘤率为96.7％。ORI组治愈率为80％，RRP为60％，联合用药组为93.3％，均明显高于对照组的13.3％；ORI组和联合用药组明显高于RRP组。TUNEL检测显示，ORI组、RRP组和联合用药组残存肿瘤细胞出现凋亡，越近肿瘤表面，凋亡越明显。灌注后，血液淋巴细胞亚群各组无差别，膀胱除ORI组和联合用药组的移行上皮明显变薄外，无其它异常改变。各组的肝、脾、肺、心、肾未见明显损害。结论：硝酸银灼伤、MB49膀胱灌注建立起来的C57BL／6小鼠原位膀胱肿瘤模型是一种较为理想的原位肿瘤模型。ORI、RRP具有明显的抗小鼠膀胱肿瘤作用，其作用机制可能为诱发肿瘤细胞凋亡。ORI、RRP膀胱灌注治疗小鼠膀胱肿瘤对膀胱及全身重要器官是安全的。
Bladder cancer is the most common malignancy of urinary system in China.Ninety percent of bladder cancer is transitional cell carcinoma (BTCC), of whichseventy to seventy five percent is superficial bladder cancer. Of superficial bladdercancer, ninety five percent is high-grade BTCC. After surgery, forty percent tends torecur in two years.with a long-term recurrence of almost 100%. Of the superficialBTCC, ten percent will progress to be invasive. Of the high-grade superficial BTCC,the short-term recurrence is up to sixty to ninety percent if not interfered. Because ofthat, intravesical instillation of anticancer agents should aim at clearing tumor,preventing recurrence and delaying progression of tumor.
Although intravesical administration of Bacillus Calmette-Guerin (BCG) has beenaccepted as the most effective therapy for superficial bladder cancer and carcinoma insitu of the bladder, thirty to forty five percent of patients never respond to BCGtherapy. Furthermore, long-term remission (more than five years) is only achieved infifty percent of patients. Toxicity associated with the BCG therapy is frequent withoccurrence of severe adverse effects in five percent of patients and life-threateningcomplications in 0.5 percent of patients. Patients hardly accept the regular instillationperiod because of severe cystitis and complications. It's reported that the percentageof patients who kept accepting regular instillation is not more than nineteen percent.So, it's necessary to explore a new agent acceptable by patients for intravesicalinstillation with better effects and fewer complications.
Nowadays, research on antineoplastic agents has been highlighted by naturalproducts. Rabdosia rubescens is a traditional Chinese herb with a variety of pharma-cological effects, especially anticancer effects. Oridonin and rabdosia rubescenspolysaccharide (RRP) are the two important ingredients which exhibit remarkable inhibitory effects on many tumor cell lines. Clinical reports have demonstrated thatcoarse extract ofrabdosia rubescens can prevent recurrence of bladder cancer.
In this dissertation, oridonin and RRP extraction was studied on at first. Afteralcohol back-streaming and separation by silica gel column chromatography, oridoninof which the purity is ninety eight percent was obtained. After water-extraction withalcohol-precipitation, RRP was obtained. Second, we studied the antineoplasticeffects of oridonin and RRP on bladder cancer cell lines, MB49 cells, EJ cells andT24 cells. After establishing an orthotopic murine BTCC model successfully, wefinally studied the effects of oridonin and RRP on murine BTCC and its mechanism.
SectionⅠ
Extraction and Separationof Oridonin and Polysaccharide from Rabdosia Rubescens
Objective: To prepare oridonin and polysaccharide from the traditional Chineseherb, Rabdosia rubescens, for the next experiments. Methods: Total diterpenoidswere obtained by alcohol back-streaming. Oridonin was separated by silica gelcolumn chromatography, assayed by high performance liquid chromatography.Polysaccharide was obtained by water-extraction, precipitated by alcohol, andpurified by organic solvents. Results: Extraction rate of oridonin was 0.22 percent(2.2/1000) and the purity of oridonin was 98 percent. Extraction rate ofPolysaccharide was 4.05 percent (8.1/200). Conclusions: The alcohol back-streamingis a better method for extraction of oridonin. The water extraction withalcohol-precipitation is a practical method for preparation of RRP.
SectionⅡ
Studyon Antineoplastie Effects of Oridonin and Rabdosia Rubescens Polysaccharide on Bladder Cancer Cell Lines
Objective: To study the antineoplastic effects of oridonin and rabdosia rubescenspolysaccharide (RRP) on bladder cancer cell lines, MB49 cells, EJ cells and T24 cellsin vitro. Methods: The inhibitory rate of the cells was measured by MTT assay. Cellapoptotic rate was detected by flow cytometry (FCM) using Annexin V-FITC andpropidium iodide (PI) detection kit. Morphology of cell apoptosis was observed byHoechst 33258 fluorescence staining and electron/light microscopy. DNA fragmen-tation was assayed by agarose gel electrophoresis. Caspase-3, Caspase-8 andCaspase-9 were assayed with colorimetric assay kits. Bcl-2 and bax expressions weredetected by Western Blot. Telomerase was detected by PCR-TRAP-Silver staining.
Results: Oridonin and RRP could inhibit the growth of MB49 cells, EJ cells and T24cells and cause apoptosis significantly. The suppression was both in a time-anddose-dependent manner. IC50 at 24 hours is 16μmol/L in MB49 cells, 25.6μmol/L inEJ cells and 27.2μmol/L in T24 cells respectively. Combined administration oforidonin and RRP could be synergetic in EJ cells and T24 cells. Marked morpholo-gical changes of cell apoptosis including condensation of chromatin and apoptosisbody were observed clearly. Oridonin could down-regulate the Bcl-2 expression andup-regulate the Bax expression of MB49 cells, EJ cells and T24 cells. Oridonin coulddecrease the telomerase activity of MB49 Cells. Oridonin could increase the activityof Caspase-3 and Caspase-9 of the abovementioned 3 cells, and increase the activityof Caspase-8 of EJ cells and T24 cells, but not of MB49 cells. Conclusions: Oridoninand RRP, especially oridonin, have apparent inhibition and apoptosis-inducing effectson MB49 cells, EJ cells and T24 cells. Low concentration of oridonin mainly inducesapoptosis, while high concentration of oridonin may induce necrosis in bladdercancer cell lines involved. Oridonin may induce apoptosis by activating Caspase-3through both Fas/FasL-mitochondria and Apo 2L/TRAIL-Caspase-8 pathways in EJcells and T24 cells, but only via Fas/FasL-mitochondria pathway in MB49 cells.
SectionⅢ
Study on Intravesical Instillation of Oridonin andRabdosia Rubescens Polysaccharide for Murine Bladder Cancer
Objective: To establish a model of orthotopic murine bladder cancer, study theeffects of intravesical instillation of oridonin and RRP on murine bladder cancer andobserve the morphological changes in bladder, heart, liver, spleen, kidneys and lungs.
Methods: Orthotopic C57BL/6 murine bladder cancer model was established byintravesical instillation of MB49 cells after argentine nitrate cautery. The diagnosis ofbladder cancer was made by routine pathology and electron microscopy. Ten daysafter instillation of MB49 cells, mice were divided into 4 groups, 15 mice in eachgroup. 100 microliters of PBS, oridonin, RRP, combined administration of oridoninand RRP were instilled intravesically in different group respectively. Afterwards,instillation was given in each group every 5 days, 4 times in all. One week after that,mice were all sacrificed. Blood subtypes of lymphocyte were detected by FCM.Bladder, heart, liver, spleen, lungs and kidneys were anatomized out for pathology tocalculate the cure rate and observe morphological changes. TUNEL detection wasmade in tumor-loaded mice. Results: Tumor-taking rate was 96.7 percent in ouranimal model. There were no significant differences in lymphocyte subtypes betweencontrol group and experiment groups. The cure rate in the combined administrationgroup was 93.3 percent, which was higher than those both in oridonin group (80percent) and in RRP group (60 percent), of which differences were significant. Therewere no apparent degeneration, necrosis, inflammatory leukocyte infiltration andfibrosis in the abovementioned viscera. TUNEL detection showed that there wereapparent apoptosis cells in each group. The closer the tumor surface approaches, themore apparent the apoptosis is. Conclusions: The orthotopic C57BL/6 murinebladder cancer model established by instillation of MB49 cells after argentine nitrate cautery is practical for animal experiment of instillation therapy for bladder cancer.Oridonin and RRP have apparent antineoplastic effects, of which mechanism may beinduction of apoptosis. After instillation of oridonin and RRP, heart, liver, kidneys,lungs and spleen are safe without damages.
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