当归贝母苦参煎剂治疗实验性慢性细菌性前列腺炎的研究

【作者】 何丽清；

【导师】 傅延龄；

【作者基本信息】 北京中医药大学 ， 中医临床基础， 2009， 博士

【摘要】 本论文分为文献综述、理论研究和实验研究三部分。1文献综述全面查阅整理了近十余年来国内外有关慢性细菌性前列腺炎(CBP)的研究文献资料,掌握了目前西医、中医对慢性细菌性前列腺炎的流行病学、发病机理、治疗用药及治疗方法等方面的研究进展,对当归贝母苦参丸方中药物的研究和临床应用进行了系统的总结和综述,为本课题的研究提供了前期工作基础。慢性细菌性前列腺炎的发病率不断上升,发病年龄趋向年轻化,而抗生素治疗难以取得满意疗效,容易复发。中医药既可调节机体整体状况,又针对前列腺局部的病理变化,有独特的优势。当归贝母苦参丸主要用于治疗泌尿和生殖系统感染,且治疗效果很好,但对本方的实验研究和全方的药理研究未见文献报道,至今仍是空白,值得我们深入探讨和研究。2理论研究在中医理论指导下,结合中西医的研究现状,探讨了CBP与湿热和血瘀的关系。CBP的发生与湿热、血瘀血虚密切相关,湿热兼瘀证是其常见证型,其治则当是清利湿热,活血化瘀,兼以温补。通过参考历代文献对当归贝母苦参丸全方及方中药物的论述,分析了该方的组方特点为苦辛同用,清宣兼施,利湿之中有润燥之品相兼,治血之中有理气之味匡助,气血并调,上下兼顾,药味简炼,配伍精当。阐明了治疗慢性细菌性前列腺炎用该方的科学性和可行性,为本课题的研究提供了理论依据。3实验研究目的:(1)对当归贝母苦参煎剂治疗CBP的效果进行观察、评价;(2)对当归贝母苦参煎剂治疗CBP的作用机理进行探讨。方法:将体重250±20g的雄性SD大鼠,随机分为空白对照组、模型组、西药对照组、当归贝母苦参煎剂治疗高、中、低剂量组,共6组,每组10只。适应性喂养3天后,利用微创技术开腹在直视下前列腺局部注射大肠埃希菌制备慢性细菌性前列腺炎动物模型(空白对照组注射等量的生理盐水),造模2周后,各组大鼠灌胃用药。当归贝母苦参丸由传统的丸剂改为适合用于实验的煎剂,按成人临床每日服用剂量当归15g、浙贝母20g、苦参15g,高、中、低剂量组分别为成人用药剂量的14倍、7倍和3.5倍,含生药浓度为1.1667g/ml、0.5833g/ml和0.2917g/ml。西药对照组用诺氟沙星悬浮液,按成人每日用量0.8g的7倍,药物浓度为0.0093g/ml。模型组和空白对照组用等容量蒸馏水灌胃。各组大鼠按1ml/100g体重/d,每日1次,用药4周后取材,观测指标。(1)肉眼观察各组大鼠前列腺大小、硬度、颜色及与周围组织的粘连情况;(2)光镜下观察各组大鼠前列腺组织的病理变化;(3)各组大鼠前列腺白细胞(WBC)及卵磷脂小体(SPL)计数;(4)测各组大鼠全血粘度;(5)测各组大鼠前列腺组织匀浆液中超氧化歧化酶(SOD)、丙二醛(MDA)的含量;(6)免疫组化染色法观察各组大鼠前列腺组织肿瘤坏死因子-α(TNF-α)、粘附分子-1(ICAM-1)的表达情况;(7)放免法测各组大鼠前列腺组织匀浆液中双氢睾酮(DHT)、白介素-1β(IL-1β)、白介素-2(IL-2)、白介素-6(IL-6)、白介素-8(IL-8)、分泌型免疫球蛋白A(SIgA)含量的改变;(8)逆转录聚合酶链反应法(RT-PCR)从基因水平测各组大鼠前列腺组织IL-1βmRNA、TNF-αmRNA的基因表达。结果:(1)当归贝母苦参煎剂能显著减小CBP大鼠病变范围,减轻前列腺与周围组织的粘连;能显著降低炎性细胞浸润程度和间质纤维增生程度,增加腺腔内分泌物;能显著减少CBP大鼠前列腺组织中的白细胞数,增加其中的卵磷脂小体数。实验数据表明,当归贝母苦参高剂量煎剂治疗CBP的效果为最好。(2)当归贝母苦参煎剂能显著降低CBP模型大鼠的全血粘度;对于CBP模型大鼠的前列腺组织,当归贝母苦参煎剂可以显著升高其中的SOD含量和降低MDA含量;可显著降低其中DHT的含量;对其中的SIgA含量变化无统计学差异;能显著下调其中ICAM-1的表达;能显著升高其中的IL-2含量和降低IL-8含量,而对IL-6含量有轻微降低,但无统计学意义;能显著减少其中的炎症细胞因子TNF-α、IL-1β的产生,这不仅是在细胞水平基础上,而且在基因转录水平上得到印证。(3)当归贝母苦参高剂量煎剂在降低CBP模型大鼠的全血粘度,升高其前列腺组织中的SOD、IL-2的含量,降低其中ICAM-1、TNF-α的免疫组化阳性表达,降低其中的IL-1β、IL-8、DHT含量,降低其前列腺组织中TNF-αmRNA、IL-1βmRNA的基因表达等方面优于阳性对照药。结论:(1)本课题利用现代科学手段,验证了当归贝母苦参煎剂治疗实验性CBP具有良好的效果,证实了当归贝母苦参丸治疗CBP疗效的确切性。(2)从前列腺组织的整体水平、细胞水平和基因水平阐述了当归贝母苦参煎剂治疗慢性细菌性前列腺炎的作用机理。当归贝母苦参煎剂主要作用机理可能在于:①具有较好的抗炎、抗纤维增生、杀菌作用;②可恢复前列腺的分泌功能,对腺体有促分泌作用;能促进局部组织的血液循环,改善药物在前列腺中的渗透;③具有抗氧化、清除自由基作用;④能降低前列腺组织中的DHT;⑤能提高机体的免疫功能,对机体内的免疫分子有调节作用,降低ICAM-1的阳性表达,增强抗炎性细胞因子表达,抑制促炎性细胞因子表达。这为临床上使用本方提供了确切证据,表明本研究具有临床实用意义和创新性。但本实验对当归贝母苦参煎剂调节前列腺中IL-6和SIgA水平的机制尚需进一步研究阐明。更多还原

【Abstract】 There are three parts in this thesis:Literature Reviews,Theoretical Research and Experimental Research.1 Literature ReviewsThrough the comprehensive literature of the chronic bacterial prostatitis (CBP) at home and abroad in latest over ten years,the progress in clinical epidemiology,pathogeny,medicine and methods of treatment were grasped.The pharmacology of herbs in Dangguibeimukushen pill(DGBMKS pill) and the clinical application of DGBMKS pill also were summarized.Based on these researches,the foundation of this project was laid.The morbility of CBP is increasing worldwide and the disease age is getting younger,but the satisfactory effects can’t be gained through antibiotics and CBP easily has a relapse.Chinese medicine can not only adjust the whole condition of the organism,but also cure the regional pathological changes of prostate,so it has the distinct advantages.DGBMKS pill is mainly applied to treat the infection of urinary and reproductive systoms and tbe therapeutic efficacy is well.But there aren’t the literature of the experimental and pharmacological researches on DGBMKS pill,so these researches should be carried out.2 Theoretical ResearchBased on the guiding of Chinese medical theories and the literature reviews, the relationships between the dampness-heat,blood stasis and CBP were explored.The occurance of CBP is closely related to dampness-heat,blood stasis and blood deficiency.The Zheng of dampness-heat with blood stasis is the common Zheng style of CBP.The principle of treatment is clearing away dampness -heat,activating blood circulation to dissipate blood stasis,warming and tonifying.According to the literature on the herbs of DGBMKS pill,its characteristics were analyzed.These are the herbs pungent in flavour,warm in nature and having the functions of clearing and dispersing are comprised;there are the herbs of clearing away dampness in company with moistening dryness and treating blood in company with regulating Qi in this prescription;this prescription can adjust blood and Qi and give consideration to the upper and the lower of body;it is brief and refined.The part showed the project was scientific and feasible and gave the theoretical support to the project.3 Experimental Research Objective:(1)To observe and estimate the effect of Dangguibeimukushen decoction(DGBMKS decoction) on CBP;(2)To explore the mechanisms of DGBMKS decoction treating CBP through the experimental research.Methods:60 male,250±20g,Sprague-Dawley rats were randomly divided into 6 groups(10 in each):the control group(A),the model group(B),the western medicine group(C),the high(D),middle(E) and low(F)-dose groups of DGBMKS decoction.After 3 days of adaptively feeding,the CBP models were established in the latter 5 groups by injecting E.coli into the dorsal lobes of the prostate while the control group being injected 0.9%NS instead of E.coli.Two weeks Iater,DGBMKS decoction was perfused into the rat stomachs of D,E and F groups.The density of high-dose was 1.1667g/ml,the middle was 0.5833g/ml and the low was 0.2917g/ml.These were 14 times,7 times and 3.5 times of the adult dose which was DANGGUI 15g,ZHEBEIMU 20g and KUSHEN 15g in clinic respectively.Norfloxacin suspension was administrated to C group.Its density was 0.0093g/ml,whichwas 7 times of the adult dose—0.8g.While the same volume distilled water was administrated to the control group and the model group.The intragastric administration was 1ml/100g/d,once daily and for four weeks.Items detected:(1)the size,hardness,color and adhesion of the prostates; (2) the histopathological changes of the prostates;(3) White blood cell(WBC) count and the density of Small particle of lecithin(SPL) in the prostates;(4)the whole blood viscosity;(5)the contents of Superoxide dismutase(SOD) and malondialdehyde(MDA) in the prostates;(6)the expression of Tumor Necrosis Factor-α(TNF-α) and Intercellular adhesion molecule-1(ICAM-1) by immunohistochemistry; (7)the contents of Dihydrotestosterone(DHT)、Interleukin-1β(IL-1β)、Interleukin-2(IL-2)、Interleukin-6(IL-6)、Interleukin-8(IL-8)、Secreted Immunoglobulin A(SIgA) in the prostates by radioimmunoassay (RIA);(8)the mRNA gene expression of IL-1β、TNF-αin the prostates through Reverse transcription polymerase chain reaction(RT-PCR).Results:(1)The extent of disease and adhesion of the prostates were significantly decreased;The inflammatory cell inflatration and the hyperplasia of fibrous tissue were significantly diminished and the secretion in prostate was significantly increased;the numbers of WBC was significantly decreased while the density of SPL was significantly increased by DGBMKS decoction.According to the data of experiments,the high-dose DGBMKS decoction had the best effect on CBP. (2)The whole blood viscosity was significantly decreased;the contents of SOD were significantly increased and MDA were significantly decreased;the content of DHT was significantly decreased;the content of SIgA was not statistically significant;the expression of ICAM-1 was significantly decreased;the content of IL-2 was significantly increased and that of IL-8 was significantly decreased;the content of IL-6 was decreased,but it was not statistically significant;the mRNA gene expression of TNF-αand IL-1βwere significantly inhibited by DGBMKS decoction.(3)The high-dose DGBMKS decoction was better effective in decreasing the whole blood viscosity,increasing the contents of SOD and IL-2,decreasing the immunohistochemistry expressiones of ICAM-1 and TNF-α,decreasing the contents of IL-1β、IL-8 and DHT and inhibiting mRNA gene expressiones of TNF-αand IL-1βthan the positive medicine(Norfloxacin).Conclusion:(1)Through the modern and scientific methods;the favourable effect of DGBMKS decoction on CBP was verified.This illustrated the certainty of DGBMKS decoction curing CBP.(2)The mechanisms of DGBMKS decoction on treating CBP were expounded on the levels of the whole,cell and gene.The mechanisms lay on:①reducing inflammation, depressing the hyperplasia of fibrous tissue and sterilizing;②recovering the secretion of prostate,improving microcirculation and permeation of medicine;③antioxidating and removing free radical;④decreasing DHT;⑤promoting immune function and adjusting the cytokine.The project provided the precise evidences to administrate DGBMKS decoction.The practical significance in clinical and the creativeness were demonstrated.But the mechanisms of DGBMKS decoction adjusting IL-6 and SIgA need to be studied further.更多还原