中药“四神丸”对小鼠结肠炎性病变后诱发结肠癌预防作用的研究

【作者】 曹阳；

【导师】 田振国；

【作者基本信息】 辽宁中医药大学 ， 中西医结合临床， 2013， 博士

【摘要】 目的：研究四神丸对结肠炎性病变后诱发结肠癌小鼠的干预和影响，观察四神丸对结肠炎性病变后诱发结肠癌小鼠一般状态、体重变化、便血和脱肛等症状以及胸腺、脾脏、结肠器官重量的影响，检验四神丸对结肠炎性病变后诱发结肠癌小鼠结肠组织成癌率和结肠组织病理改变结果的影响，进而明确四神丸对小鼠结肠炎性病变后诱发结肠癌的化学预防作用；研究四神丸对结肠炎性病变后诱发结肠癌小鼠结肠组织环氧化酶2（COX-2）蛋白表达的影响，从蛋白层面分析四神丸对肠炎性病变后诱发结肠癌的化学预防作用的可能机制；检测四神丸对结肠炎性病变后诱发结肠癌小鼠结肠组织环氧化酶2（COX-2）相关基因mRNA表达量的影响，从基因层面分析四神丸对肠炎性病变后诱发结肠癌的化学预防作用的可能机制；观察四神丸对结肠炎性病变后诱发结肠癌小鼠结肠组织NF-E2相关因子2∕抗氧化反应元件（Nrf2∕ARE）通路的影响，探讨四神丸对肠炎性病变后诱发结肠癌的化学预防作用的可能机制；综合分析各部分实验数据，明确四神丸口服与灌肠两种给药方式中的最佳给药方式。材料与方法：第一部分：四神丸对结肠炎性病变后诱发结肠癌小鼠器官、成癌率以及病理的影响。采用给予小鼠强致炎剂右旋葡聚糖苷钠（DSS）饮水两周，配合腹腔注射强致癌剂1，2-二甲肼（DMH）制造结肠炎性病变后发展为结肠癌的小鼠模型。四神丸严格按照原方组方、配比、剂量,经打粉、研磨、煎煮、提取、回流、浓缩、干燥、再打粉备用。取昆明种小鼠50只，随机分为5组,分为正常对照组、模型组、口服四神丸组、灌肠四神丸组、西药对照组，每组10只小鼠。对照组正常饲养，不作任何处理。模型组给予小鼠饮用含20g/L右旋葡聚糖苷钠(DSS)的饮用水,一周后给予小鼠腹腔注射含40mg/kg的1,2-二甲肼(DMH)的生理盐水,隔天一次,共三次，两周后停止饮用DSS饮用水，改为正常饮水，直到处死。除正常对照组外和模型组，其他各组，除与模型组相同处理外，从第二周开始,口服四神丸组给予灌胃0.2ml(按体表面积计算)的四神丸浸膏剂(生药含量2g/ml)，直到处死。从第二周开始,灌肠四神丸组每天给予0.2ml(按体表面积计算)的四神丸浸膏剂(生药含量2g/ml)灌肠，直到处死。从第二周开始,西药对照组每天给予柳氮磺胺吡啶蒸馏水溶液0.2ml（药物含量按照体表面积计算）灌胃，直到处死。在此期间密切观察各组小鼠一般状态和体重以及便血、脱肛等症状的变化。第20周后，摘取脾脏、胸腺、结肠组织，用滤纸吸干并称重。观察小鼠结肠组织肿瘤个数并计算成瘤率。并将各组小鼠结肠组织切片经HE染色，镜下观察，并进行比较。第二部分：基于Nrf2/ARE通路的结肠炎性病变后诱发结肠癌预防机制的研究。动物饲养、中药处理、动物分组、造模处置、造模时间、处理因素和取材时间与方法同第一部分。通过免疫印迹（Western-blot）法检测NF-E2相关因子2∕抗氧化反应元件（Nrf2∕ARE）通路中的NF-E2相关因子2（Nrf2）蛋白表达量，经结肠组织总蛋白的提取、SDS-PAGE凝胶电泳、蛋白质从凝胶转移至滤膜，最后分析NF-E2相关因子2（Nrf2）蛋白光密度值，并进行分析比较。第三部分：基于环氧合酶2(COX-2)的结肠炎性病变后诱发结肠癌预防机制的研究。动物饲养、中药处理、动物分组、造模处置、造模时间、处理因素和取材时间与方法同第一部分。首先，从基因层面通过反转录聚合酶链式反应（RT-PCR）检测环氧合酶2(COX-2)相关基因mRNA,并计算环氧合酶2(COX-2)相关基因mRNA相对表达量，进行统计分析。从蛋白层面，通过免疫组化方法检测环氧合酶2(COX-2)蛋白表达量，并作光密度测定，通过计算进行统计分析。结果：1.四神丸对结肠炎性病变后诱发结肠癌小鼠器官、成癌率以及病理的影响1.1体重的变化：模型组、口服四神丸组、灌肠四神丸组、西药组体重均比正常对照组明显下降（P<0.01），其中以模型组体重下降的最多。除正常对照组外，其他各组之间比较没有统计学意义。1.2便血、脱肛症状的比较：出现便血的小鼠中模型组最多，共4只，达到40%；其次是口服四神丸组和西药组，为1只，达到10%；最少的为正常对照组和灌肠四神丸组，为0只。出现脱肛小鼠最多的是模型组，有3只，达到33%。其他各组未见脱肛症状，均为0只。1.3胸腺、脾脏、结肠组织重量比较：DMH/DSS诱导的模型小鼠与对照组相比,脾脏、结肠质量明显升高(P<0.01),胸腺质量则明显降低(P<0.01)。口服四神丸组、灌肠四神丸组、西药对照组与模型组比较，胸腺只有灌肠四神丸组好于模型组，且有统计学意义（P<0.05）。脾脏上述三组数据均明显好于模型组(P<0.01)。结肠组织上述三组数据也均明显好于模型组(P<0.01)。口服四神丸组、灌肠四神丸组、西药对照组三组之间对比，从数据来看，灌肠四神丸组作用强于口服四神丸组，口服四神丸组强于西药组。胸腺数据灌肠四神丸组好于口服四神丸组和西药对照组，且有统计学意义（P<0.05）。脾脏和结肠数据灌肠四神丸组和口服四神丸组均好于西药对照组，有明显统计学意义(P<0.01)。1.4四神丸对结肠炎性病变后诱发结肠癌小鼠成瘤率影响只使用DMH+DSS的模型组小鼠，经过21周，在结肠炎的基础上大多形成了结肠癌，成癌率是90%，且肿瘤数目不止一个，小肠和其他器官未见肿瘤发生(表1-7)。肿瘤直径2-6mm，多见于粘膜表面，凸向肠腔，颜色暗红，质脆，表面时有溃疡。口服四神丸组和灌肠四神丸组的成瘤率均为20%，且肿瘤数目多为1个，且直径较小。未成瘤小鼠，结肠组织多稍有增厚，显示炎性浸润，停留在结肠炎状态，未向结肠癌发展(图1)。西药对照组小鼠成瘤率为40%，高于四神丸处理的两组，但低于模型组，成瘤的小鼠结肠肿瘤数量多于四神丸处理的两组。1.5结肠组织PH染色病理切片比较正常对照组为正常小鼠腺体结构，腺体结构排列有序，无炎细胞侵润，粘膜完整；模型组除有部分炎症细胞存在外，可见腺体结构已经紊乱，并呈侵润性生长，说明造模成功；口服四神丸组、灌肠四神丸组和西药对照组有一定量的炎症细胞侵润，腺体结构虽有增厚，但腺体结构基本保持正常，停留在结肠炎，未向结肠癌转变，没有出现向周围组织侵润的迹象。炎症程度灌肠四神丸组和西药对照组略小于口服四神丸组。2.基于Nrf2/ARE通路的结肠炎性病变后诱发结肠癌预防机制的研究模型组Nrf2蛋白表达量明显比正常对照组降低（P<0.01）；口服四神丸组和灌肠四神丸组Nrf2蛋白表达量明显高于模型组（P<0.01），也明显高于西药组（P<0.01）；而西药组与模型组对比则没有区别，不具统计学意义（P＞0.05）。3.基于环氧合酶2(COX-2)的结肠炎性病变后诱发结肠癌预防机制的研究3.1COX2相关基因mRNA RT-PCR检测与对照组相比，模型组COX-2相关基因mRNA表达量明显增高（P<0.01）；而口服四神丸组、灌肠四神丸组和西药组与模型组相比则明显抑制了COX-2相关基因mRNA的表达（P<0.01），但三者之间区别没有统计学意义（P＞0.05）。3.2小鼠结肠组织COX2蛋白免疫组化图中黄色颗粒即为阳性表达。从肉眼看，模型组黄色颗粒明显多于其他各组，正常对照组几乎没有阳性表达，而口服四神丸组、灌肠四神丸组和西药对照组则有不同程度的黄色颗粒分布。从光密度值检测来看，模型组COX-2蛋白免疫组化染色光密度值明显较正常对照组增高（P<0.01）；口服四神丸组、灌肠四神丸组和西药对照组光密度值均小于模型组光密度值，且有统计学意义（P<0.01），但口服四神丸组和灌肠四神丸组、西药对照组三组之间表光密度值比较，灌肠四神丸组小于小于其他两小组，有统计学意义（P<0.05）。结论：1．四神丸可降低结肠炎性病变后诱发结肠癌小鼠便血和脱肛等症状的发生。2．四神丸可抑制结肠炎性病变后诱发结肠癌小鼠脾脏增大、胸腺缩小、结肠增重。3．四神丸可降低结肠炎性病变后诱发结肠癌小鼠的成癌率。4．四神丸可阻止小鼠结肠炎性病变后向结肠癌转变，即对小鼠结肠炎性病变后诱发结肠癌具有化学预防作用。5．四神丸可以抑制结肠炎性病变后诱发结肠癌引起的Nrf2蛋白的下调。6．四神丸对结肠炎性病变后诱发结肠癌的化学预防作用可能与抑制结肠炎性病变后诱发结肠癌引起的Nrf2蛋白的下调有关。7.四神丸能够在基因层面抑制肠炎性病变后诱发结肠癌小鼠结肠组织COX-2蛋白相关基因mRNA的表达。8.四神丸能够在蛋白层面降低肠炎性病变后诱发结肠癌小鼠结肠组织COX-2蛋白的表达。9.四神丸对结肠炎性病变后诱发结肠癌的化学预防作用可能与抑制结肠组织COX-2蛋白相关基因mRNA的表达和降低结肠组织COX-2蛋白的表达有关。10.从给药方式来看，四神丸对结肠炎性病变后诱发结肠癌的化学预防作用，口服和灌肠没有明显差异。更多还原

【Abstract】 Objective：To study the intervention and influence of Si-Shen bolus on colitis associatedcancer in mice;to observe mice’ general status and organs of colitis associated cancer;To clearSi-Shen bolus’s chemical prevention for colitis associated cancer; to study Si-Shen bolusaffectting on colon cancer’s COX-2protein expression induced by colitis; to analysis Si-Shenbolus’ possible mechanisms of chemical prevention effect of colitis associated cancer;toobserve the effect of colon tissue’s Nrf2ARE signal pathway in mice;to discuss Si-Shenbolus’ possible mechanisms of chemical prevention effect of colitis associated cancer;toanalysis test data and clear the best intake way of Si-Shen bolus.Materials and Methods:Part Ⅰ:Si-Shen bolus’ influence to mice’ organs、cancer rate and pathology with colitisassociated cancer.We gave mice strong inflammatory agent dextran sodium sulfate(DSS)drinking water for two weeks, and gave strong carcinogenic agent1,2-dimethyl hydrazine(DMH) by intraperitoneal injection at the same time.We manufactured mouse model of colitisassociated cancer finally. Si-Shen bolus was accordanced in strict with the original formula,proportion and dosage, powder, grinding, decocting, extraction, circumfluence, concentration,drying and powder and set aside.50mice were randomly divided into5groups: normalcontrol group, model group, oral group, enema group, western medicine control group, eachgroup10mice. Control group does not make any processing. Model group mice was givendrink water containing20g/L dextran sodium sulfate (DSS).A week later, mice was givenabdominal cavity injection containing40mg/kg,1,2-dimethyl hydrazine (DMH) in salinesolution,the next day at a time, a total of three times. After two weeks stop drinking DSS,drinking water to drink water, until death. Other groups’early treatment was the same ofmodel group. Oral group was given0.2ml(calculated by surface area, with traditionalChinese medicine2g/ml) Si-Shen bolus extract. Enema group was given0.2ml (calculatedby surface area, with traditional Chinese medicine2g/ml)Si-Shen bolus extract by enemaevery day. Western medicine control group was given willow nitrogen sulphanilamidepyridine distilled water solution for a0.2ml(calculated by surface area) every day. Duringthis period closely observe general status in mice, the symptom such as prolapse and hemafecia. After20weeks,we gathered the spleen, thymus, colon, and weighed with filterpaper. We observe mice colon tissue and calculated tumor rate.We observe each group micecolon tissue’s section of HE staining under microscopic. PartⅡ:prevention mechanismresearch based on the Nrf2/ARE pathways induced colitis associated cancer. Animal feeding,Chinese medicine treatment, animal group, building disposal,processing factors, buildingtime,based method were same of the first part. By Western blot method,we detected theNrf2ARE signal pathways’ Nrf2protein expression.By extraction of total protein in thecolonic tissue,Polypropylene phthalic amide gel electrophoresis,protein from gel tomembrane,we analyse Nrf2protein’s optical density value. PartⅢ:prevention mechanismresearch based on COX-2protein induced colitis associated cancer. Animal feeding, Chinesemedicine treatment, animal group, building disposal,processing factors, building time,basedmethod were same of the first part. By reverse transcription polymerase chain reaction (rt-pcr)we detected of cox-2protein related genes mRNA, Calculated cox-2related genes mRNAexpression. By immunohistochemical we detected cox-2protein expression,and analysedoptical density.Result:1. Part Ⅰ:Si-Shen bolus’ influence to mice’ organs、cancer rate and pathology with colitisassociated cancer.1.1Changes in body weightModel group, oral group, enema group, western medicine control group was significantlylower than the normal control group (P <0.01), weight loss in model group was most. Inaddition to the normal control group, other is no statistical significance between groups.1.2Bloody and rectoceleMost bloody appeared in the model group, total4only, at40%. Followed by oral groupand western medicine group,1only, at10%. Normal control group and enema group was atleast,0only.Most rectocele appeared in model group,total3,up to33%.1.3The thymus, spleen and colon tissue weightCompared to control group, the spleen and colon quality of model group increased significantly (P <0.01), thymus quality is significantly lower (P <0.01).Oral group, enema group, western medicine control group compared with the modelgroup, only the data of enema group’s thymus was better than the model group, and havestatistical significance (P<0.05). The data of the three groups’s spleen was significantly betterthan the model group (P<0.01). The data of three groups’s colon tissue was significantlybetter than the model group (P<0.01) too.Oral group,enema group, western medicine control group compare between eachother,from the data, enema group effect is better than oral group, oral group effect is betterthan western medicine control group. The data of enema group’s thymus was better than oralgroup and western medicine control group, and have statistical significance (P<0.05). Thedata of enema group and oral group’s spleen and colon was better than the western medicinecontrol control group, and there was statistical significance (P<0.01).1.4The effect on tumor formation rateModel group mice using only DMH+DSS，after21weeks,most mice formated to coloncancer from colitis.It’s cancer rate is90%, and the number of tumor was more than one.Thesmall intestine and other organs showed no tumor. Tumor diameter was2-6mm, found in themucosal surface,convex to the intestinal lumen, color dark red, crisp, surface ulcers.Thetumor formation rate of Oral group and enema group was20%.The number of tumor was1,and the smaller diameter. The mice without colon tumor slightly thickened, showedinflammation sex embellish, stayed in the colitis state, not to the development of colon cancer.Control group ‘s tumor formation rate was40%, higher than the two group of Si-Shen bolustreatment, but lower than that in the model group. It’s colon tumor number was more than thetwo groups of Si-Shen bolus treatment.1.5The colon tissue PH dyeing pathological comparisonNormal control group had normal gland structure, glandular structures arranged orderly,no inflammatory cells invasion, Mucous membrane was complete. Model group glandsstructure was disorder and Invasive growth.Model building was successful. There was acertain amount of inflammatory cells invasion in oral group,enema group and westernmedicine control group. There was thickening of the gland structure,but glands keep normalstructure.It stayed in colitis,not to colon cancer.There was no signs of surrounding tissue invasion. enema group and western medicine control group’s Inflammation degree was lessthan oral group’s.2.PartⅡ:prevention mechanism research based on the Nrf2/ARE pathways induced colitisassociated cancer.Nrf2protein expression quantity of model group was obviously decreased than thenormal control group (P <0.01). Oral and enema group Nrf2protein expression issignificantly higher than model group (P <0.01). Oral and enema group Nrf2proteinexpression is significantly higher than western medicine control group (P <0.01) too.Therewas no difference and statistical significance compared with model group and westernmedicine control group（P＞0.05）.3.PartⅢ:prevention mechanism research based on COX-2protein induced colitis associatedcancer.3.1Cox-2related gene mRNA RT-PCR detectionCompared with the control group, model group expression of cox-2related gene mRNAwas significantly higher (P <0.01). Cox-2related gene mRNA of oral group, enema groupand western medicine control group expression was significantly inhibited (P <0.01)compared with model group. But the difference between the three has no statisticalsignificance (P>0.05).3.2Mice colon tissue COX2protein immunohistochemicalYellow particles are the positive expression. Look from the naked eye，the yellowparticles in model group was obviously more than other groups. There was almost no positiveexpression in normal control group.But Oral group, enema group and western medicinecontrol group have different level of yellow granular distribution in their pictures.Model group’s cox-2protein immunohistochemical staining optical density valueincreased significantly compared with normal control group (P <0.01). Oral group,enemagroup and western medicine control group optical density value were all less than modelgroup optical density value, and there was statistical significance (P <0.01).But comparisonbetween oral group,enema group and western medicine control group, enema group’s opticaldensity value was less than less than other two groups.It has statistically significant (P <0.05). Conclusion:1. Si-Shen bolus can reduce colitis associated cancer symptoms such as bloody and rectocelein mice.2. Si-Shen bolus can inhibit changes of spleen,thymus,colon in colitis associated cancer mice.3. Si-Shen bolus can reduce colon cancer rate in colitis associated cancer mice.4. Si-Shen bolus can prevent colitis to colon cancer in mice after lesions, and Si-Shen bolushave chemoprevention effect for colitis associated cancer in mice.5. Si-Shen bolus can inhibit the Nrf2protein decrease in colitis associated cancer.6. Si-Shen bolus’s chemoprevention effect for colitis associated cancer may be related toinhibition of Nrf2protein decrease.7. Si-Shen bolus can inhibit colon tissue’s Cox-2related gene mRNA expression in colitisassociated cancer mice at genetic level.8. Si-Shen bolus can reduce colon tissue’s Cox-2protein expression in colitis associatedcancer miceat protein level.9. Si-Shen bolus’s chemoprevention effect for colitis associated cancer may be related toinhibition of Cox-2related gene mRNA expression and reducing Cox-2protein expression incolon tissue.10.From the point of delivery way, oral and enema was no significant difference inSi-Shen bolus’s chemoprevention effect for colitis associated cancer。更多还原