摘要
第一部分洁泽1号治疗解脲脲原体宫颈炎的试验研究
目的:观察洁泽1号治疗解脲脲原体宫颈炎的临床疗效。
方法:310例符合诊断标准无合并症的解脲脲原体宫颈炎患者,随机分为三组:A组:阿奇霉素联合洁泽Ⅰ号阴道灌洗治疗组102例;B组:洁泽Ⅰ号阴道灌洗治疗组87例;C组:口服阿奇霉素治疗组121例。三组治疗期间安全套避孕,均治疗满一疗程,停药21d后复查解脲脲原体并行妇科检查,观察三组临床疗效。
结果:洁泽Ⅰ号对解脲原体临床疗效,总有效率A组85.3%;B组:67.8%,C组:60.3%,A组与B组、C组相比统计学有极显著性差异(P＜0.01,P＜0.01),B组与C组相比差异无显著性意义(P＞0.05)。宫颈分泌物Uu转阴率A组78.4%,B组60.9%,C组47.9%;A组与B组、C组相比统计学有极显著性差异(P＜0.01,P＜0.01),B组与C组相比差异无显著性意义(P＞0.05),对耐药菌株Uu转阴率,A组48.1%,B组42.1%,C组16.1%,A组与B组相比无显著性差异(P＞0.05),A组与C组相比有极显著性差异(P＜0.01),B组与C组相比有显著性差异(P＜0.05),
结论:洁泽1号对解脲脲原体宫颈炎有一定的疗效,联合阿奇霉素有较好的治疗效果,特别是对耐药菌株疗效有显著优势,值得进一步研究。
第二部分洁泽1号体外对阴道常见抗病原微生物药效的实验研究
目的:观察洁泽1号体外对生殖道常见病原体金黄色葡萄球菌、大肠杆菌、白色念珠菌、乙型链球菌、解脲脲原体、阴道毛滴虫及脆弱拟杆菌等的MIC50、MIC90、MBC50、MBC90,大肠杆菌的杀菌曲线KC_s;洁泽1号对单纯性疱疹病毒2型(HSV-2)的体外抗病毒作用,为寻找具有消炎、预防生殖道感染作用的外用药具提供实验依据。
方法:采用琼脂稀释法检测的最小抑菌浓度(MIC)和最小杀菌浓度(MBC),并与洁尔阴对照;阴道毛滴虫及脆弱拟杆菌与甲硝唑对比;微量肉汤稀释法检测支原体MIC和MBC,并与红霉素对照;运用CPE法和MTT法检测洁泽1号对HSV-2的体外抗病毒作用,
结果:洁泽1号对大肠杆菌和金黄色葡萄球菌的抑制作用优于洁尔阴,对乙型溶血性链球菌的作用与洁尔阴相当,而对白色念珠菌的作用弱于洁尔阴。洁泽1号对Uu临床分离株的MIC_(50)为31.25mg·ml~(-1),MIC_(90)为62.5mg·ml~(-1),并能对UU红霉素耐药株有一定的抑制作用。JZ2作用2h滴虫全部死亡所需的JZ1浓度为0.25g.ml~(-1),所需的N-9浓度为0.625mg.ml~(-1),而单用JZ1作用2小时滴虫全部死亡的浓度为0.5g.ml~(-1),N-9浓度为1mg.ml~(-1),JZ1和N-9表现出较好的协同性;JZ2 0.25g.ml~(-1)和0.125g.ml~(-1)浓度组与同浓度JZ1组、JZ2 0.0625mg.ml~(-1)和0.03125mg.ml~(-1)浓度组与同浓度N-9组比较,滴虫死亡率在2h-8h差异有显著性意义(P＜0.05或P＜0.01),洁泽1号对阴道毛滴虫及脆弱拟杆菌有较好的杀菌作用,与壬苯醇醚有协同作用。JZ2的作用明显强于同浓度的JZ1和N-9;洁泽1号对阴道毛滴虫及脆弱拟杆菌有较好的杀菌作用,与壬苯醇醚有协同作用。洁泽1号对Vero细胞的半数无毒浓度TC_(50)为6.3mg·ml~(-1)(CPE法),8mg·ml~(-1)(MTT法),最低无毒浓度TC_0为1.6 mg·ml~(-1)(CPE法和MTT法)。抑制复制实验中洁泽1号半数抑制浓度IC_(50)为0.89mg·ml~(-1)(CPE法),0.98mg·ml~(-1)(MTT法),治疗指数TI为7.07(CPE法)和8.16(MTT法);洁泽1号抑制吸附的IC_(50)为0.15 mg·ml~(-1),TI为42(与CPE测得的TC_(50)比),53(与MTT法测得的TC_(50)比)。
结论:洁泽1号对受试菌的活性依次为:阴道毛滴虫、大肠杆菌、金葡菌、乙型链球菌、脆弱拟杆菌和白色念球菌。并且对Uu临床分离株有抑制作用。洁泽1号具有一定的抑制HSV-2吸附和复制作用,值得进一步研究。
第三部分洁泽1号对壬苯醇醚体外杀精增效作用的实验研究
目的:观察洁泽1号、壬苯醇醚(N-9),以及洁泽1号与不同浓度N-9混合液体外杀精作用。
方法:观察药物作用20s和3min的最低杀精浓度和精子活力恢复实验。并观察药物作用20s、3min和30min时精子前向运动率、活动率和活率。
结果:洁泽1号3min体外最低杀精浓度为1g·ml~(-1)。N-9 20s、3min最低杀精浓度均为0.25mg·ml~(-1)。混合液中N-9 20s、3min最低杀精浓度分别为0.125mg·ml~(-1)、0.0625mg·ml~(-1)。精子活力恢复实验显示失活精子不能恢复活力。洁泽1号浓度大于0.25g·ml~(-1)时,20s、3min和30min精子前向运动率、活动率和存活率与生理盐水对照组比较,统计学有极显著性差异(P＜0.01)。混合液中N-9浓度为0.0625mg·ml~(-1)时,精子在20s、3min和30min的前向运动率与相同浓度N-9单独使用比较,统计学有极显著性差异(P＜0.01)。混合液中N-9浓度为0.125mg·ml~(-1)、0.0625mg·ml~(-1)时,精子在20s和3min的活动率和存活率分别与相应浓度N-9单独使用比较,统计学均有极显著性差异(P＜0.01)。
结论:洁泽1号对壬苯醇醚体外杀精有增效作用,联合使用可降低N-9用量。
第四部分洁泽1号对小鼠滴虫性阴道炎预防作用的实验研究
目的:观察中药复方洁泽1号(JZ1)与有效避孕剂量的外用杀精剂壬苯醇醚(N-9)的复合制剂洁泽2号(JZ2)对小鼠滴虫性阴道炎的预防作用。
方法:将112只小鼠随机分为JZ2组(高、中、低)、JZ1组(高、中、低)、壬苯醇醚(N-9)组、甲硝唑组、空白凝胶组及模型组,造模前,每只小鼠阴道给予相应的药物凝胶或生理盐水20ul,注药2min后每只小鼠接种滴虫液20ul,在接种后的2、4、7、10、12、14天取小鼠阴道冲洗液涂片镜检及培养,观察小鼠感染情况,并计算各组小鼠不同时间的感染率。
结果:JZ2组、N-9组小鼠感染率都为0,与相同时间模型组比较差异有显著性意义(P＜0.01);甲硝唑组小鼠感染率很低,前4天仅为10%,之后为0,前10天与模型组比较有显著差异性(P＜0.01);JZ1组小鼠滴虫感染率比模型组低,2g.ml~(-1)浓度组在第2天与模型组比较差异有显著性意义(P＜0.05);空白凝胶组小鼠在不同时间的滴虫感染率与模型组比较差异均无显著性意义(P＞0.05);JZ2组、N-9组、JZ1 2g.ml~(-1)和1g.ml~(-1)。剂量组小鼠滴虫感染率与甲硝唑组比较差异无显著性意义(P＞0.05);JZ2与JZ1组比较,0.5g.ml~(-1)组在第2天感染率差异有显著性(P＜0.05)。
结论:预防性给药后,JZ2高、中、低剂量组和N-9组都能完全预防小鼠滴虫性阴道炎的感染,其作用强于同浓度的JZ1。
第五部分洁泽1号对小鼠白色念珠菌阴道炎预防作用的实验研究
目的:观察洁泽1号(JZ1)、壬苯醇醚(N-9)以及洁泽2号(JZ2)[中药复方洁泽1号(JZ1)与外用杀精剂壬苯醇醚(N-9)混合制剂]对小鼠白色念珠菌性阴道炎的预防作用。
方法:造模前阴道内按分组分别注入各药物凝胶20ul,1min后阴道接种白色念珠菌液5×10~4个/只,分别于造模后第2、4、7、14d观察小鼠阴道灌洗液菌丝生成及菌落计数结果,阴道、子宫组织PAS染色,各组阴道组织的炎症分值及阴道炎预防率。
结果:随着JZ2凝胶中JZ1浓度的降低,小鼠白色念珠菌性阴道炎预防率逐渐下降,呈现一定的量效关系。JZ2高剂量凝胶组预防率可达65.0%,与模型组预防率0.0%、空白凝胶组预防率20.0%比较差异有显著性意义(P＜0.01),与西药对照组(联苯苄唑凝胶)作用相当,但强于N-9组,且差异有显著性意义(P＜0.05)。JZ2中、低剂量凝胶组预防率较低,但与模型组之间的差异统计学有显著性意义(P＜0.05)。
结论:JZ2凝胶对白色念珠菌性阴道炎具有较好的预防作用。
第六部分洁泽1号对小鼠生殖器疱疹预防作用的实验研究
目的:建立小鼠生殖器疱疹动物模型,并观察洁泽1号对小鼠生殖器疱疹的预防作用。
方法:生殖器疱疹模型建立实验:小鼠32只,按体重随机分为4组,分别为高、中、低三个病毒滴度组和生理盐水对照组,每只小鼠皮下注射孕激素,5天后戊巴比妥钠麻醉,经阴道接种20ul相应滴度的2型单纯疱疹病毒(HSV-2)或给予生理盐水,建立小鼠生殖器疱疹模型。并通过阴道外阴症状评分、阴道分泌物毒力检测和间接酶联免疫吸附法(ELISA)检测小鼠血清HSV-2 IgG判断模型成功与否和模型病变程度。预防实验:72只小鼠按体重随机分为预防组(洁泽1号高、中、低剂量组)、模型对照组、阳性药物对照组(无环鸟苷组)和空白对照组(生理盐水对照组)。每组12只,预防组在造模的同时分别给予1g·ml~(-1),0.5g·ml~(-1)和0.25g·ml~(-1)三个不同剂量的洁泽1号凝胶,阳性药物对照组和模型组造模同时分别给予12%无环鸟苷或生理盐水,空白对照组仅给予生理盐水。每日对小鼠进行症状评分;给药造模后第1、4、7、9、12天测量小鼠阴道分泌物毒力;第12天处死小鼠,摘眼球取血用ELISA法检测小鼠血清抗HSV-21gG抗体阳性率;阴道组织一部分做病理切片,一部分用于定量PCR方法检测阴道组织HSV-2DNA拷贝量。
结果:模型情况:高滴度病毒模型组小鼠死亡率达50.0%,中滴度组达12.5%,低滴度组未出现小鼠死亡,外阴阴道症状评分提示高滴度组症状最严重,其次为中滴度组和低滴度组。高、中滴度组小鼠血清抗体阳性率达100.0%,低滴度组小鼠阳性率达50.0%。预防实验:(1)洁泽1号高、中剂量组都能很好的降低小鼠外阴阴道症状评分,阴道分泌物病毒毒力,血清抗体阳性率和阴道组织病毒DNA含量,并且这些指标与模型组比较均有显著性差异;(2)洁泽1号高、中剂量与无环鸟苷组比较,外阴阴道症状评分,分泌物毒力和阴道组织病毒DNA含量均无显著性差异(P＞0.05);(3)中剂量组小鼠外阴阴道症状评分、阴道分泌物毒力、血清抗体阳性率和阴道组织病毒DNA拷贝量低于高剂量组,但无显著性差异(P＞0.05)。(4)与生理盐水对照组比较,洁泽1号高、中剂量组和无环鸟苷组小鼠阴道每日症状评分、阴道分泌物毒力和小鼠阴道组织HSV-2DNA含量有显著性差异(P＜0.05)。(5)洁泽1号高、中剂量组、无环鸟苷组和模型组血清抗体阳性率分别为:75.0%、58.0%、41.6%和91.6%;洁泽1号高、中剂量组、无环鸟苷组和模型组细胞培养法阳性率分别为:58.3%、41.6%、50.0%和100.0%。(6)肉眼观察模型组小鼠可见阴道黏膜下有点状充血,并且阴道较其它组体积大,而洁泽1号高、中、低剂量组和无环鸟苷组阴道均无充血现象,组织形态良好;(7)组织学切片HE染色模型组可见黏膜不完整,大量淋巴细胞浸润,并可见增生的毛细血管。洁泽1号高、中剂量组和无环鸟苷组组织学切片可见黏膜完整,无淋巴细胞浸润,炎症较模型组明显减轻,但是低剂量组有轻微的淋巴细胞浸润。
结论:模型建立时高滴度组小鼠死亡率较高,中滴度组不仅模型成功率高,而且小鼠死亡率也低,低滴度组小鼠模型成功率仅达50%,所以选择中滴度组小鼠作为后续预防实验的动物模型。预防性给药后,洁泽1号高、中剂量组和无环鸟苷组小鼠病情明显轻于模型组,且中剂量组效果优于高剂量组,其预防效果与12%无环鸟苷相当。说明洁泽1号能降低小鼠生殖器疱疹的感染率或减轻生殖器疱疹的病情。
第七部分洁泽1号抗生育作用的实验研究
目的:观察中药复方洁泽1号(JZ1)凝胶对外用杀精剂壬苯醇醚(N-9)的体内杀精及抗生育的作用效果。
方法:采用改良的Sander—cramer方法观察JZ1、N-9、JZ1与N-9的混合制剂(JZ2)凝胶不同剂量组对大鼠、家兔体内杀精作用,大鼠、家兔阴道内给予相应组药物凝胶后雌雄合笼,分别于交配后1min、3min、5min、10min观察阴道内精子的活动率、死亡率及精子全部死亡的时间;在交配后的第20天剖腹观察雌鼠、雌兔黄体数、胚泡数、计算受精率及妊娠率以评估抗生育作用。
结果:大鼠实验表明:JZ1凝胶组(1g/ml)交配后1min内有少量原地活动精子,3min时未见活动精子,N-9 20s、3min最低杀精浓度均为0.25mg/ml,与洁泽1号混合(即JZ2)后,N-9最低杀精浓度分别为0.125mg/ml、0.0625mg/ml。交配后1min,各单纯N-9及JZ2凝胶组大鼠阴道内均未见活动精子。N-9(12%及以上浓度)、JZ2(含10%及以上浓度N-9)各组均有较高的避孕率,与自然对照组及空白凝胶组之间的差异均有极显著意义(P＜0.01)。JZ2(含14%、12%N-9)药物凝胶组抗生育率均可达100%,与14%N-9作用相当。JZ2(含10%N-9及8%N-9组)抗生育率分别为90%、40%,与自然对照组之间的有显著性差异。JZ2(含12%N-9和10%N-9)凝胶组对大鼠的避孕作用强于相同浓度的N-9组,且比较差别有显著性意义(P＜0.05)。家兔实验表明:同一时间下,JZ2高、中、低剂量组精子活动率和死亡率与生理盐水对照组比较差异均有显著性意义(P＜0.01),与JZ1比较,在1min差异有显著性意义(P＜0.01),低剂量组与同浓度的N-9组比较差异在1min有显著性意义(P＜0.05);JZ2高、中、低剂量组精子全部死亡时间分别为0.625±1.77min,4.00±12.25min,4.50±9.26min,三组与生理盐水对照组比较差异均有显著性意义(P＜0.01),JZ2低剂量组与同浓度的N-9组比较差异有显著性意义(P＜0.05),JZ2高剂量组与JZ1组比较差异有显著性意义(P＜0.01);交配后家兔黄体数在各组间差异均无显著性意义(P＞0.05);JZ2组家兔胚泡数量、受精率及妊娠率低于生理盐水组和JZ1组(P＜0.01),JZ2低剂量组与同浓度的N-9组比较差异有显著性意义(P＜0.05)
结论:由剂量-反应关系曲线可以看出JZ1对N-9的杀精作用具有协同作用,两药混合后(即JZ2)可以减少N-9的剂量;JZ2凝胶大鼠、家兔阴道内用药具有较好的体内杀精及抗生育作用,在相同避孕效果下可减少N-9的用量。其作用优于单用JZ1或N-9。
第八部分洁泽复方对小鼠阴道乳杆菌、糖原及乳铁蛋白影响的实验研究
目的:观察中药复方洁泽1号凝胶、洁泽2号凝胶对小鼠阴道乳杆菌、糖原及乳铁蛋白的作用,评价洁泽1号凝胶、洁泽2号凝胶对小鼠阴道内环境的影响。
方法:取雌性昆明小鼠180只,随机分为空白对照组、空白凝胶组(基质对照组)、JieZeNO.1凝胶高(2g/ml)、中(1g/ml),低(0.5g/ml)剂量组,JieZeNO.2号凝胶高(2g/ml)、中(1g/ml),低(0.5g/ml)剂量组,10%N-9组,分别于用药24h或72h观察乳杆菌、糖原、乳铁蛋白,共18组,每组10只。每只小鼠皮下注射生理剂量的苯甲酸雌二醇(E_2)2ug,72h后,小鼠阴道分别注入上述各药物凝胶20ul,并于注射药物凝胶后24h或72h阴道灌洗液活菌计数及培养,检测阴道组织糖原含量及乳铁蛋白表达。
结果:各浓度洁泽药物组与空白对照组在乳杆菌计数、糖原含量方面比较均无显著差异,与10%N-9组有显著差异(P＜0.01),将各浓度洁泽药物24h组与72h组相比亦无显著差异;乳铁蛋白表达结果表明任意两组比较均无显著差异。
结论:一次性使用JieZeNO.1凝胶、JieZeNO.2凝胶对小鼠阴道内乳杆菌、糖原及乳铁蛋白无明显影响,与单用N-9相比,对小鼠正常阴道内环境影响较小。
PartⅠClinical Research of JZNo.1 on Cervicitis Caused by Ureaplasma Urealyticum
Objectives To observe the clinical therapeutic effect on cervicitis caused by ureaplasma urealyticumand.
Methods 310 patients who suffered from cervicitis caused by ureaplasma urealyticum were randomly divided into 3 groups:Group A:the treated group integrated with traditional Chinese herbs Jieze No.1 30rains in vaginal once a day for 10days and Azithromycin orally 1.0g once 72hours for three times;Group B:the group treated with Jieze No.1 30mins in vaginal once a day for 10days;Group C:the group treated with Azithromycin orally 1.0g once 72hours for three times.
Results The clinical observation showed that the overall clinical crue rate of group A reached 85.3%;group B reached 67.8%,group C reached 60.3%,The overall clinical crue rate of group A showed significant differences compared with group B and group C (P＜0.01,P＜0.01).The clearing rate of Uu in group A is 78.4%,group is 60.9%,group C is 47.9%;Group A also showed great differences in clearing rate of Uu compared with group B and group C(P＜0.01,P＜0.01).Especially to drug-resistant strain,the clearing rate of Uu in A group reached 48.1%,group B reached 42.1%,and group C reached 16.1%.The clearing rate of Uu in group A and group B has significant differences compared with group C(P＜0.01,P＜0.05).while group A and group B has no significant differences(P＞0.05).
Conclusion JZ No.1 combined with azithromycin can effectively treat cervicitis caused by ureaplasma urealyticum,Eespecially it takes a remarkable effect on drug-resistant strains,It deserves further research.
PartⅡThe Effect of Jieze No.1 on Vaginal Microorganism in Vitro
Objectives:To observe antibacterial effect of Jieze NO.1(JZ1) on vaginal microorganism in vitro.
Methods:Minimum Inhibitory Concentration(MIC) and Minimum Bactericidal Concentration(MBC) of staphylococcus aureus(SA),Escherichia coli(EC),candida albicans(CA),and streptococcu s(ST),trichomonas vaginalis and B.fragilis were detected by an agar dilution method.We then try to obtain killing curves which relate the antibacterial effect of JZ NO.1 to EC.MIC and MBC of UU were detected by a microdilution broth method.And the Kirby-Bauer method was used to measure the bacterial inhibiting diameters of Jieze NO.1 decoction on lactobacillus.CPE,MTT and plaque assay are used to estimate JZ1 antiviral activity on herpes simplex virus type 2 (HSV-2).
Results:JZ NO.1 decoction produces better results on EC and SA,equivalent on ST, and not better on CA.KCS test indicate that 7.8mg·ml~(-1) JZ NO.1 decoction can inhibit the growth of EC weakly,62.5mg·ml~(-1) JZ NO.1 decoction can reduce the amount of EC greatly, 125 mg·ml~(-1) JZ NO.1 decoction can expel EC completely.JZ NO.1 decoction has strong inhibitory effect on clinical strain Uu.After treated with Jieze NO.2 decoction of 0.25g.ml~(-1) +0.0625mg.ml~(-1) concentration for 2h or 0.125g.ml~(-1)+0.03125mg.ml~(-1) concentration for 48h,the mortality of trichomonas vaginalis was all reached 100%,which was higher than that of N-9 and Jieze NO.1 decoction at the same concentration and Metronidazole of 1/100 concentration;Jieze NO.2 decoction,Jieze NO.1 decoction and Metronidazole all showed some antibacterial effect on B.fragilis,and their MIC were 0.125g.ml~(-1)+0.25mg.ml~(-1)、0.5mg.ml~(-1),0.625ug.ml~(-1) respectively;N-9 showed little antibacterial activation on B. fragilis,and its MIC was more than 20mg.ml~(-1).50%Inhibitory Concentration(IC_(50)) of JZ NO.1 on HSV-2 is 0.89mg·ml~(-1) by CPE,0.98 mg·ml~(-1) by MTT.Therapeutic Index(TI) is 7.07 by CPE and 8.16 by MTT.
Conclusions:The antibacterial effect of JZ NO.1 decoction on trichomonas vaginalis is better than that on EC,SA,ST,B.fragilis and CA.JZ NO.1 decoction also can inhibit the growth of Uu,and possesses antiactivity of HSV-2 in vitro,
PartⅢThe Effect of Jieze No.1 on Spermicidal Action with Nonoxynol-9 in Vitro
Objective:Spermicidal effect of JiezeNO.1(JZ1) decoction and Nonoxynol-9(N-9) on human sperm was examined in vitro.
Methods:The minimum spermicidal concentration of JZ1 decoction,N-9 and their mixture solution in 20s and 3min were examined by improved spermicidal test of Sander-cramer in vitro.The percentage of progressively moving spermatozoa,moving spermatozoa and viable spermatozoa were also observed in 20s、3min and 30min after the liquid medicine.
Results:(1) The minimal spermicidal concentration of JiezeNO.1 decoction in 3 min is 1g·ml~(-1),and minimal spermicidal concentration of N-9 in 20s is 0.25mg·ml~(-1).The minimal spermicidal concentration of N-9 with 1g·ml~(-1) JiezeNO.1 decoction in 20s and 3 min is 0.125mg·ml~(-1) and 0.0625mg·ml~(-1),and it is lower than concentration of N-9 (0.25mg·ml~(-1)) in 20s and 3min.(2) Sperms did not recover their activitys in a revival test.when the minimum spermicidal concentration of either JZ1 decoction,or N-9,or the mixed solution of the two agents,was used.(3)N-9(JZ1 in mix group) showed significant differences in the percentages of progressively moving spermatozoa,moving spermatozoa, and visible spermatozoa in 20s,3min,and 30min,when compared with N-9 alone(P＜0.01).
Conclusions:JiezeNO.1 decoction can improve N-9 spermicidal action in vitro,and also reduce the dose of N-9.when combined with N-9,it has proved of synergic effects.
PartⅣThe Preventing Effect of Jieze NO.1 Gel on richomonas Vaginitis in Mice
Objective To observe the preventing effect of Jieze NO.1 gel on trichomonas vaginitis in mice.
Methods 112 mice were separated into Jieze NO.1 group(high titer、median titer and lower titer)、Nonoxynol-9 group,Jieze NO.2 group(high titer、median titer and lower titer)、Nonoxynol-9 group、Metronidazole group、gel control group and model group. Every mouse was inoculated by 20ul gel of different drugs or saline intravaginally before modeling composition,2min later,all mice were inoculated by 20ul trophozoites.We collected the lotion to test under microscope and culture in the medium at 2、4、7、10、12、14 day after inoculation,checking active trichomond and comparing infection rate between each group.
Results The infection rate of Jieze NO.2 and N-9 group was 0.0%,it had significant differences(P＜0.01)compared with model group at the same time(P＜0.01);The infection rate of metronidazole group was 10.0%in 4 days,it was lower than that of control group in 10 days(P＜0.01);The infection rate of Jieze NO.1 2g.ml~(-1) group was lower than that of control group before 2day(P＜0.05);The infection rate had no difference between jel control group and model group;When compared with metronidazole group,the infection rate of Jieze NO.2、N-9、2g.ml~(-1) and 1g.ml~(-1) of Jieze NO.1 group had no difference(P＞0.05); The infection rate of 0.5g.ml~(-1) of Jieze NO.2 was lower than Jieze NO.1 at 2days(P＜0.05).
Conclusion The high titer group、the median titer group and the lower titer group of Jieze NO.2 all can effectively prevent the occurrence of trichomonas vaginitis.
PartⅤThe Preventive Studies of Jieze NO.1 on Candida Albicans
Objective:To evaluate prevention effect of Jieze NO.1(JZ1) gel,the combination of JZ1 and Nonoxynol-9(JZ2)gel on Candida Albicans Vaginitis in mice.
Methods:Mice were infunded intravaginally with 20ul gel of blank and drug,1min later inoculated intravaginally with 5×10~4 stationary-phase C.albicans blastoconidia (ATCC90028) in 20μl of phosphate-buffered saline(PBS).On day 2,4,7 and 14 after inoculation,the pseudohyphae in vaginal lavage fluid,the vaginal fungal burden(CFU),the scores of inflammation in vaginal tissue,PAS of vaginal and uterus tissues were observed. And the prevention rate of vaginitis was calculated.
Results:There was dosage-dependent effect of the prevention rate on JZ1 in JZ2 gel. Compared with groups of model and placebo of gel,there were significant difference on the prevention rate(65%) in group of high concentration of JZ2(P＜0.01),which could equal to the effect of group of Bifonazole Gel,and better than the group of 10%N-9(P＜0.05). There were low prevention rate(P＜0.05) in other groups of JZ2.But they were significant difference when compare with the group of model.
Conclusion:JZ2 had better prevention effect on Candida Albicans Vaginitis.
PartⅥExperimental Research on Preventing Effect of Jieze NO.1 Gel on Genital Herpes in Mice
Objective:To set up genital herpes model in mice and observe the preventive effect of Jieze NO.1 gel on this model.
Methods:We separated 32 mice into four groups,i.e.,the high titer group(HT),the median titer group(MT),the low titer group(LT) and saline control group(NS),All mice were injected subcutaneously with progesterone.On the fifth day,mice were anesthetized by 3%pentobarbital.The mice in virus groups are inoculated by 20ul different titers of HSV-2 invaginally,2.5×10~6 PFU·ml~(-1)(HT),2.5×10~5 PFU·ml~(-1)(MT),and 2.5×10~4 PFU·ml~(-1)(LT),respectively;The mice in NS were given 20ul saline.Then we evaluated the success of genital herpes model in mice through genital pathology scores,viral titers in vaginal washes and ELISA for anti-HSV-2 IgG respectively.Secondly,we separated 72 mice into 6 preventive groups,i.e.,Jieze NO.1 high dose group(JZH),the median dose group(JZM),the low dose group(JZL),the ACV group(ACVR):the model group(MOD) and the saline control group(NS).At the same time of virus challenging,the mice were pretreated intravaginally with 1g·ml~(-1)(JZH),0.5g·ml~(-1)(JZM),and 0.25g·ml~(-1)(JZL), respectively,the mice in NS and MOD with saline intravaginally,and the mice in ACV with 12%ACV intravaginally.Genital pathology was monitored daily and scored daily,and vaginal washes were analyzed for viral titers by plaque assays.Once the experiment was finished,all mice were killed and the following steps are implemented:(1) The serum was detected for anti-HSV-2 IgG through ELISA,(2)part of the vaginal tissue was analyzed histopathologically,and(3) the other part the vaginal tissue was analyzed through FQ-PCR for HSV-2 DNA.
Results:The death rate of HT in model is 50.0%,that of MT is 12.5%,while no mice in LT are dead.The pathology score of HT is higher than MT and LT.The positive rates of anti-HSV-2 IgG in both HT and MT is 100.0%and 50.0%in LT.In preventive experiment, (1) the genital pathology scores,the viral titers in vaginal washes,and HSV DNA in vaginal tissues in JZH,JZM and ACVR have significant difference in comparison to MOD(P＜0.05,P＜0.01);(2) the genital pathology scores,the viral titers in vaginal washes,and HSV DNA in vaginal tissues in both JZH and JZM are lower than those of ACVR,and have no significant difference in comparison to ACVR(P＞0.05);(3) the genital pathology scores and the viral titers in vaginal washes in JZH are higher than those JZM,although the difference are not statistically significant(P＞0.05).(4) the genital pathology scores,the viral titers in vaginal washes,and HSV DNA in vaginal tissues in JZH and JZL have significant difference in comparison to NS(P＜0.05);(5) The positive rate through ELISA of JZH,JZM,JZL,ACV and MOD is 75.0%、58.0%、41.6%and 91.6%, respectively;For comparison,the positive rate of JZH,JZM,JZL,ACV and MOD is 58.3%、41.6%、50%and 100%respectively by cell culture.(6) The mice in MOD appear dots of conjestion under their membrane,and the volume of their vaginal becomes significantly bigger.These features,however,are not observed in the mice in JZH,JZM, and JZL.(7) Lots of lymphocyte infiltrations occur and epithelium is histopathologically damaged in the mice in MOD,while few lymphocyte infiltrations in JZH,JZM,and ACV, and some lymphocyte infiltrations in JZL.
Conclusions:The death rate of mice in HT is highest,but the mice in MT have low death rate and high positive rate,and the mice in LT have low positive rate,and thus,we choose the MT as the animal model for subsequent preventive experiment.JZH,JZM and ACV can relieve the vaginal pathology,reduce the viral titers in vaginal washes and HSV-2 DNA in vaginal tissues and raise the anti-HSV-2 IgG positive rate.Although JZL can not reduce the antibody positive rate and the amount of the HSV-2 DNA,but can relieve vaginal pathology,reduce the viral titers in vaginal washes slightly.Moreover,1g·ml~(-1) JZ NO.1,0.5g·ml~(-1) JZ NO.1 and 12%ACV can effectively prevent the occurrence of genital herpes in mice,and if genital herpes do occur,JZ1 can relieve the degree of illness.
PartⅦSpermicidal and Intravaginal Contraception Effects of Jieze NO.1 gel
Objective:To evaluate the spermicidal and contraceptive effects of Jieze NO.1 gel, nonoxynol(N-9),the combination of Jieze NO.1 and N-9(JZ2) gel in rats and rabbits.
Methods:The drug was applied in female rats or rabbits intraviginally before coition, then we collected the vaginal washes in 1min、3min、5min、10min、15min and 30min to observe the percentage of moving spermatozoa、the percentage of death spermatozoa and the death time of all sperms under microscope after mating;The corpora luteum numbers and embryos were counted after 20 days,calculating the fertility rate and fertilized eggs rate.
Results:The minimal spermicidal concentration of N-9 in 20s and 3min were 0.25mg/ml.The minimal spermicidal concentration of N-9 in JZ2 was 0.125mg/ml in 20s and 0.0625mg/ml in 3 min.There were not any mobile sperms in the rats vagina within 1 minute after copulation in all groups of gel of simple N-9 and JZ2.There were higher antifertility rates in groups of JZ2 gel with 10%N-9 and greater and had significant difference when compared with natural control gel and placebo gel.The antifertility rate could reach 100%in JZ2 gel with 14%N-9 and 12%N-9,equaled to the group of 14%N-9, 90%and 40%in JZ2 gel with 10%N-9 and 8%N-9,all showing significant decrease(P＜0.01),when compared with group of natural control gel(0%).The groups of JZ2 gel with 12%N-9 and 10%N-9 showed significant difference when compared with N-9 alone in the same concentration(P＜0.05) in rats.In rabbit,The percentage of moving spermatozoa and death spermatozo in group of the high、median and the low titer of Jieze NO.2 had significant difference(P＜0.01) when compared with the control group at the same time and Jieze NO.1 group in 1min,and between the low titer of Jieze NO.2 and N-9 group;The death time of all sperms in groups of the high,median and the low titer of Jieze NO.2 were 0.625±1.77min,4.00±12.25min,4.50±9.26min separately,which had significant difference(P＜0.01) when compared with the control group and between the the high titer group of Jieze NO.2 and Jieze NO.1 group、the low titer of Jieze NO.2 and N-9 group;The corpora luteum numbers had no variability between each groups;Embryos、fertility rate and fertilized eggs rate of the high titer、the median titer and the low titer of Jieze NO.2 group were lower than that of Jieze NO.1 and control group(P＜0.01),which had variability when compared between the low titer of Jieze NO.2 and N-9 group.
Conclusion:JZ1 could improve N-9 spermicidal action,Jieze NO.2 gel had strong spermicidal and contraceptive effects in female rats and rabbits,of which the effect was better than that of using Jieze NO.1 gel or N-9 gel alone.
PartⅧEffect of Jieze Prescription on Lactobacillus,Glycogen and Lactoferrin in Mice Vagina
Objective:To study the effect of Jieze NO.1 gel,jieze No.2 gel on Lactobacillus、glycogen and lactoferrin in mice vagina,and evaluate its side effect.
Methods:180 female Kunming mice were divided into the groups of blank control, blank gel(matrix control)、high(2g/ml)、middle(1g / ml)、low(0.5g/ml) does of JieZe NO.1 gel、high(2g/ml)、middle(1g/ml)、low(0.5g/ml) does of JieZe NO.2 gel and 10% Nonoxynol-9,each group observed follow indexs on 24h and 72h after injected gel intravaginally.Each mouse was subcutaneously injected physiological estradiol benzoate (E_2) 2ug,72h later infunded intravaginally with 20ul Drug gel.On 24h and 72h after injected gel intravaginally,respectively,colons-forming units(CFU) of Lactobacillus in vaginal lavage fluid from each mice was counted,glycogen content was detected by vaginal pathological section and expression of Lactoferrin in vaginal tissue by Western Blotting.
Results:The results of CFU of Lactobacillus and conteat of glycogen showed there were no significant differences between groups injected drug gel intravaginally and the group of blank control, the results of groups of high,middle and low does of JieZe NO.1 gel and JieZe NO.2 gel which detected on 24h and the groups of 72h showed no significant difference too,but there were significant differences between groups injected drug gel intravaginally compared with 10%Nonoxynol-9(P＜0.01).The results of expression lactoferrin showed there were no significant differences between each two groups.
Conclusion:One-time using Jieze NO.1 or Jieze NO.2 gel could have no effect on mouse vaginal lactobacillus、glycogen、lactoferrin.
引文
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