中药二黄散对实验大鼠宫颈炎的治疗效果及药物作用机制的探讨
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摘要
研究背景:慢性宫颈炎是妇女常见病、多发病,发病率高达60%以上,其中以宫颈糜烂最为常见。由于其与宫颈癌的发生密切相关,因而对该病的防治应引起足够的重视。
目前,临床对于宫颈糜烂的防治尚无安全有效的方法,其治疗仍以物理疗法为主,主要通过冷、热等各种物理手段(激光、冷冻、红外线及微波等),将宫颈糜烂面单层柱状上皮破坏,使其坏死脱落后,为新生的复层鳞状上皮覆盖。物理疗法治疗宫颈糜烂的疗效是肯定的,但存在术后阴道长时间分泌物增多,甚至有大量水样排液及脱痂时出血,创面愈合时间长,且在创面尚未愈合期间(4～8周)需禁止性生活等缺点,许多患者不愿接受物理疗法。因此寻找一种能有效治疗宫颈糜烂,又能避免物理治疗的副反应,对于防止宫颈癌变具有非常积极的意义。
近年来的研究认为,炎症和创伤修复受多种细胞因子和生长因子的调节和影响,其中血小板活化因子(PAF)、前列腺素E(PGE2)和表皮生长因子(EGF)、血小板源性衍生生长因子(PDGF)是目前研究最多、最深入、应用最广泛的细胞因子。现代医学研究发现,PAF和PGE2在组织炎症反应中起着重要的作用,促使炎症的发生、发展,对组织的愈合起着不利的作用;而EGF和PDGF是体内一种正常微量的物质,在人体组织内分布极为广泛,起着调节细胞DNA合成、促进细胞分裂和刺激血管生成的作用,对受伤后组织的快速修复起着十分重要的作用。正常情况下,EGF、PDGF含量很低,因而组织的自身修复是一个缓慢的过程。
国内外学者在应用中草药防治组织器官炎症的大量研究中发现黄连、雄黄等中药对金黄色葡萄球菌、志贺氏痢疾杆菌等有较强的抗菌作用,对炎症介质的作用也得到了普遍认可。黄连水煎液提取物对组织的修复再生有很好的作用。临床上含有黄连或雄黄的中药复方治疗宫颈糜烂有较好的疗效,我们前期的临床工作也表明,中药二黄散对宫颈糜烂也有很好的治疗效果。但目前局部用中药治疗宫颈炎的研究仍停留在药物的临床研究上,对中药组方及其药理作用的研究尚未见报道。
本研究根据中医辨证施治理论,将黄连、雄黄等五味中药合理配伍,并按一定比例配方,制成了散剂,取名为“二黄散”。通过对动物实验、临床治疗效果评估,并运用病理形态学、分子免疫学、分子生物学的方法,探讨中药二黄散治疗宫颈炎的药效学,以及药物的分子作用机制,为研制和开发治疗宫颈炎实用有效的中药新方剂提供实验依据。
本研究分为以下三部分,现分述如下:
第一部分大鼠宫颈炎模型的建立与鉴定
目的用低浓度苯酚胶浆(20%)制备大鼠宫颈炎模型。
方法取苯酚4ml,阿拉伯树胶4ml,加蒸馏水至20ml,配成20%的苯酚胶浆。除正常对照组15人外,其余54只均用导尿管向阴道深部注入苯酚胶浆,每只动物注入1.5ml,2天1次,共5次。观察实验大鼠的症状和体征,光学显微镜观察大鼠宫颈组织病理形态学变化,对制作的大鼠宫颈炎模型进行鉴定。
结果我们运用20%苯酚胶浆隔日给药5次,可见大鼠阴道口红肿、有的有脓性分泌物流出;阴道粘膜变性或脱落,形成溃疡,宫颈充血水肿、炎细胞浸润、炎性肉芽组织或纤维增生;大鼠食欲减退、活动减少、体重减轻。
结论采用20%苯酚胶浆隔日给药5次,能成功复制大鼠阴道及宫颈炎动物模型,且可重复性好。
第二部分中药二黄散对实验大鼠宫颈炎的治疗效果与作用机制
目的运用中药二黄散对大鼠宫颈炎进行治疗,观察治疗效果,探讨其药物作用机制。
方法造模后第2天,开始阴道局部给药,正常对照组不处理,阳性对照组给消糜栓,阴性对照组给空白基质,二黄散Ⅰ组和二黄散Ⅱ组分别给予相当于人临床用量的30倍和45倍的二黄散。1次/天,隔日1次,共12次。治疗结束后次日处死动物,解剖子宫后取出观察,并记录肉眼所见。做常规石蜡切片,HE染色,镜下观察阴道至子宫组织形态学变化,评价组织愈合效果。采用放射免疫分析法测定PAF和PGE2含量,免疫组织化学技术,检测经二黄散治疗的实验大鼠宫颈炎组织中EGF和PDGF的蛋白表达,RT-PCR方法检测EGF和PDGFmRNA的表达,免疫印迹法检测EGFR蛋白的表达。
结果中药二黄散Ⅰ、Ⅱ组和消糜栓组(阳性对照组)与阴性对照组相应部位比较,病变及炎症程度明显减轻,炎症细胞浸润明显减少,未见出血和坏死,比较差异有显著意义(P<0.05)。中药二黄散Ⅰ、Ⅱ组与阳性对照组对宫颈组织形态学的影响无显著性差异(P>0.05)。
中药二黄散Ⅰ、Ⅱ组中PAF和PGE2含量减低,治疗前后的含量分别为: 745.22±153.10,624.55±70.21;614.65±128.53,594.52±143.46;治疗后PAF、PGE2接近正常对照组,与阴性对照组比较差异有显著意义(P<0.05)。
EGF、PDGF免疫组化学结果显示,中药二黄散Ⅰ、Ⅱ组蛋白表达均显著高于阴性对照组和正常对照组,差异有统计学意义(P<0.05),中药二黄散Ⅰ、Ⅱ组EGFmRNA平均灰度分别为(4.53±0.39),(4.88±0.28),显著高于阴性对照组和正常对照组(2.33±0.57、1.87±0.40)(P<0.05,P<0.01);二黄散Ⅰ、Ⅱ组EGFR蛋白表达均显著高于阴性对照组,差异有统计学意义(P<0.05)。而PDGFmRNA平均灰度分别为(2.94±0.42),(3.51±0.29),高于正常对照组和阴性对照组(1.55±0.52)(1.43±0.45),差异有统计学意义(P<0.05)。阴性对照组EGF和PDGFmRNA与正常对照组相比,差异无统计学意义(P>0.05)。
结论二黄散能有效地改善实验大鼠宫颈炎炎症程度,宫颈炎大鼠用药后,炎性细胞数量明显减少,症状显著减轻,二黄散对实验大鼠宫颈炎有很好的治疗作用。二黄散能降低宫颈炎组织中PAF、PGE2的含量,促进宫颈组织中生长因子EGF和PDGFmRNA转录和蛋白合成,提高EGF受体蛋白的表达,促使宫颈鳞状上皮细胞的增殖、分化,有利于糜烂组织愈合。使损伤与修复因素趋于平衡,这可能是二黄散治疗宫颈炎的机制之一。
第三部分中药二黄散治疗慢性宫颈炎临床疗效评估
目的观察二黄散对不同类型宫颈糜烂的治疗效果及药物作用机制。
方法用二黄散治疗宫颈糜烂患者60例,其中轻度糜烂18例、中度23例、重度19例;单纯型26例,颗粒型21例,乳突型13例。将二黄散药粉喷敷于宫颈糜烂面上,隔日1次,7次为一疗程,连用2～4个疗程。观察治疗前后糜烂面的大小、病变的深浅以及患者的主观症状等。ELISA检测治疗前后宫颈分泌物中EGF、PDGF的含量变化。
结果(1)轻度糜烂有效率为94.4%,中度糜烂有效率为86.9%,重度糜烂有效率为73.7%;单纯型糜烂有效率为96.1%,颗粒型糜烂有效率为90.4%,乳突型糜烂有效率为53.8%。总有效率为85%。(2)治疗前后患者EGF宫颈粘液中含量为(41.56±16.51、97.45±20.12);PDGF宫颈粘液中的平均含量分别为:(32.74±10.22、56.20±12.33)。治疗前后宫颈粘液中EGF和PDGF的含量明显增高,比较差异具有显著意义(P<0.05)。
结论中药二黄散能明显改善宫颈糜烂患者的临床症状,有效地治疗宫颈糜烂,促进宫颈上皮细胞的增殖和分化,有利于组织的修复,该药对宫颈糜烂治愈率高、操作安全、简便,无明显的毒副作用,患者乐于接受。
Background Chronic cervicitis is one of the commonest disease of gynecology,It is often occurred in childbearing women with the incidence of more than 60%. And cervical erosion as a common pathological change is closely related to cervical carcinogenesis. So we should pay more attention to the pathogeny, diagnosis and treatment for it.
At present, routine clinical treatment remains as basic treatment and locally administered corrosive medicine. The mainstay treatment for chronic cervicitis is physiotherapy. It includes: laser therapy, cryotherapy, infrared light condensation therapy, microwave therapy, and etc. A common mechanism they shared is to destroy the simple columnar epithelium of area of the cervical erosion, and make the necrotic tissue desquamated, and then the area is recovered by regenerative stratified squamous epithelium. All the above physiotherapies bring some complications: increment of distinguishing discharge in vagina, water sample apocenosis, bleeding of decrustation, and prohibited sexual life before the healing of raw surface of the cervix (about four to eight weeks), so patiants with chronic cervicitis would not like to accept physiotherapy. Because of these reason, to seek an effective therapy is our duty as an accoucheur.
Recovery of inflammatary tissue is a complex course. It includes a series of stages: inflammatory of the tissue, formation of granulation tissue movement, formation of epithelium cell. Antiinfection enhancing nutrition and protecting local wound are main traditional therapeutic methods. platelet activating factor(PAF), mitogen-activated protein kinase(PGE2) , epidermal growth facto(rEGF)and platelet-derived growth factor( PDGF) are the most widely distributed in human body. They widely exist in central nervous system and peripheral system. And it can nourish and diferentiate many kinds of cells which generated from mesoderm and nerve ectoderm. Current medical research shows that they play very important roles in the rapid repair of tissue. It can promote metabolism and mitosis of all kinds of cells normally. For the concentration of them in body is very low, the healing of inflammatary tissue is also a slow course.
Recently, herbal medicine plays an important role in the periodontal treatment, also the effect of inflammatory medium. Scholars found that Radix scutellariae and Realgar could inhibit many kinds of bacteria and virus. Researchers also found that Chinese medicine could promote proliferation and attachment to root surface of cultured. Nowadays, the research on the local use of Chinese herbal medicine still remains in medical effects of single medicine, whereas complex prescription and its pharmaceutical mechanisms are still unknown.
Based on the above backgrounds, we investigated Erhuansan of a new Chinese medicine. This study used diagnosis based on overall analysis of patients' condition, mixed Radix scutellariae and Realgar in the reasonable proportion, then made ustained release preparation.We evaluated Erhuansan sustained-release preparation's to eliminate inflammation and promote period on talregeneration in animal and in human body, and observed the activity of inhibiting inflammation animal experiment and clinical application. This research provided experimental evidence in developing effective and practical Chinese herbal preparation on cervical erosion treatment. It will contribute to the step research on effect and pharmaceutical mechanisms of Chinese drugs Erhuansan to the cervical erosion.and provide the theory instruction for pharmaceutical application.
The paper is divided into three parts as follows.
Part one Research on the Animal Model with Colpitis and Cervicitis in Wistar rats
Objective To build the model with colpitis and cervicitis in Wistar rats.
Methods 15 normal Wistar rats and 54 animal models were studied. Preparing animal models as following, 20%Phenol mucilage are made of Phenol 4ml, Arabia balbta 4ml and distilled water 12ml. In the pathological model of cervical inflammation in rats set up by deep injection of phenol mucilage 1.5ml into every rat vagina by catheter for every two days, total five times. The change of the pudendum is observed for ten days. Then the model rats were killed, the weight of the rat’s body and inflammatory cells of its cervix were taken and quantified, and its index examined and calculated by pathology.
Results In model groups, the rats lost their appetite, reduced movement and the weight of them. At the same time, we also found that perineum tissue swelled and excreted pusappeared in the vagina orifices. Putrescence or desquamation appeared in the vagina and cervical mucous membrane. Under the vagina and cervical mucous membrane, inflammation cells soakage and acestom a hyperplasia were discovered.
Conclusion We could successfully establish the wistar rat model with colpitis and cervicitis by deep injection of 20% phenol mucilage 1.5ml into every rat vagina by catheter, five times. It was very convenient and done repeatedly.
Part two Study on the curative effect and mechnism of Chinese medicine Erhuansan on the animal model with cervicitis
Objective To research on effect and pharmaceutical mechanisms of Chinese drugs Erhuansan to animal model with cervicitis.
Methods After injecting phenol slurry for five times, we operated to the rats. Five normal Wistar rats and six animal model rats were observed. The rats of the positive and negative control groups were treated by Xiaomishuan and blank matrix suppository. The rats of ErhuansanⅠand ErhuansanⅡgroups were treated by 30 and 45 times of normal clinical human dose. The expression of PAF and PGE2 were remarkably decreased by radiommunoassay. The protein levels and mRNA of EGF and PDGF were determined by immunohistochemistry and the reverse transcriptional polymerase chain reaction (RT-PCR). The expressions of EGFR were assessed by Western blot method.
Results The Pathomorphology of positive control group and ErhuansanⅠ, ErhuansanⅡgroups were all changed better than that of the negative ones (the model group). The inflammation cells were reduced significantly in the former. The configuration of squamous cells was normal. It also showed that Erhuansan could improve the degree of Wistar rat model with colpitis and cervicitis. It could alleviate the signs and symptoms of them, such as swelling, discharge in the vagina orifices and putrescence, desquamation appeared in the vagina and cervix mucous membrane.
After treatment, the contents of PAF and PGE2 were (745.22±153.10, 624.55±70.21; 614.65±128.53, 594.52±143.46). The level decreased significantly in the Erhuansan treated group in comparison with the blank treated group(P<0.05).The content of them in ErhuansanⅠand ErhuansanⅡg roups were close to those of the normal control group.
The expressions of EGF and PDGF were significantly increased in ErhuansanⅠandⅡgroup compared to the normal ones(P<0.05). There were no differences between the negative control group and normal one. The transcription levels of the EGF mRNA in ErhuansanⅠandⅡgroup were significantly higher than that of normal groups(4.53±0.39vs 1.87±0.40; 4.88±0.28 vs 1.87±0.40, P<0.01). However, the expression levels of PDGF mRNA were higher in cervix tissue compared to control as assessed by RT-PCR(2.94±0.42vs 1.55±0.52; 3.51±0.29 vs 1.55±0.52, P<0.05). In negative control groups EGF and PDGF in cervix tissues were all no significantly than those in normal one(2.33±0.57, 1.43±0.45, P>0.05). The concentrations of EGFR in Erhuansan groups were significantly higher than those in normal one and negative one.
Conclusion Erhuansan can promote the healing of cervical erosion on rats. After treatment, the content of PAF and PGE2 were lower and the expressions of EGF, EGFR and PDGF in cervititis tissue were increased.The balance of inflammation medium was setted up.This was probably the mechanisms of Erhuansan on the animal model with cervicitis.
Part three Clinical Study of Erhuansan of chines medicine preparation in cervical erosion
Objective To study the curative effect of Erhuansan in cervical erosion .
Methods 60 women with cervical erosion were investigated. Every one was treated by Erhuansan for every two days, seven times for each period. On the basis of pathological changes, it was given two to four periods of treatment. During the treatment, the changes of cervix surface were watched by naked eye and colposcopy, and examined the content of EGF and PDGF in cervix secretion by enzyme linked immunosorbent assay.
Results The curative rates of mild, moderate and severe cervical erosion cases were 94.4%, 86.9%, 73.7% respectively. And the ones of three types were 96.1%, 90.4%, 53.8%. The total efficient rate was 85%. After treatment the content of EGF was increased (97.45±20.12)in cervix mucus. There was no change in serum.The same thing was to the content of PDGF. In cervix mucus, the content of EGF and PDGF in cervix tissues were all significantly higher than before(P<0.05).
Conclusion The efficient rates of Erhuansan on cervical erosion cases were high. This method was safe, easy, efficient, Patients were taken pleasure in it. It can promote the re-epithelium.
目前,临床对于宫颈糜烂的防治尚无安全有效的方法,其治疗仍以物理疗法为主,主要通过冷、热等各种物理手段(激光、冷冻、红外线及微波等),将宫颈糜烂面单层柱状上皮破坏,使其坏死脱落后,为新生的复层鳞状上皮覆盖。物理疗法治疗宫颈糜烂的疗效是肯定的,但存在术后阴道长时间分泌物增多,甚至有大量水样排液及脱痂时出血,创面愈合时间长,且在创面尚未愈合期间(4～8周)需禁止性生活等缺点,许多患者不愿接受物理疗法。因此寻找一种能有效治疗宫颈糜烂,又能避免物理治疗的副反应,对于防止宫颈癌变具有非常积极的意义。
近年来的研究认为,炎症和创伤修复受多种细胞因子和生长因子的调节和影响,其中血小板活化因子(PAF)、前列腺素E(PGE2)和表皮生长因子(EGF)、血小板源性衍生生长因子(PDGF)是目前研究最多、最深入、应用最广泛的细胞因子。现代医学研究发现,PAF和PGE2在组织炎症反应中起着重要的作用,促使炎症的发生、发展,对组织的愈合起着不利的作用;而EGF和PDGF是体内一种正常微量的物质,在人体组织内分布极为广泛,起着调节细胞DNA合成、促进细胞分裂和刺激血管生成的作用,对受伤后组织的快速修复起着十分重要的作用。正常情况下,EGF、PDGF含量很低,因而组织的自身修复是一个缓慢的过程。
国内外学者在应用中草药防治组织器官炎症的大量研究中发现黄连、雄黄等中药对金黄色葡萄球菌、志贺氏痢疾杆菌等有较强的抗菌作用,对炎症介质的作用也得到了普遍认可。黄连水煎液提取物对组织的修复再生有很好的作用。临床上含有黄连或雄黄的中药复方治疗宫颈糜烂有较好的疗效,我们前期的临床工作也表明,中药二黄散对宫颈糜烂也有很好的治疗效果。但目前局部用中药治疗宫颈炎的研究仍停留在药物的临床研究上,对中药组方及其药理作用的研究尚未见报道。
本研究根据中医辨证施治理论,将黄连、雄黄等五味中药合理配伍,并按一定比例配方,制成了散剂,取名为“二黄散”。通过对动物实验、临床治疗效果评估,并运用病理形态学、分子免疫学、分子生物学的方法,探讨中药二黄散治疗宫颈炎的药效学,以及药物的分子作用机制,为研制和开发治疗宫颈炎实用有效的中药新方剂提供实验依据。
本研究分为以下三部分,现分述如下:
第一部分大鼠宫颈炎模型的建立与鉴定
目的用低浓度苯酚胶浆(20%)制备大鼠宫颈炎模型。
方法取苯酚4ml,阿拉伯树胶4ml,加蒸馏水至20ml,配成20%的苯酚胶浆。除正常对照组15人外,其余54只均用导尿管向阴道深部注入苯酚胶浆,每只动物注入1.5ml,2天1次,共5次。观察实验大鼠的症状和体征,光学显微镜观察大鼠宫颈组织病理形态学变化,对制作的大鼠宫颈炎模型进行鉴定。
结果我们运用20%苯酚胶浆隔日给药5次,可见大鼠阴道口红肿、有的有脓性分泌物流出;阴道粘膜变性或脱落,形成溃疡,宫颈充血水肿、炎细胞浸润、炎性肉芽组织或纤维增生;大鼠食欲减退、活动减少、体重减轻。
结论采用20%苯酚胶浆隔日给药5次,能成功复制大鼠阴道及宫颈炎动物模型,且可重复性好。
第二部分中药二黄散对实验大鼠宫颈炎的治疗效果与作用机制
目的运用中药二黄散对大鼠宫颈炎进行治疗,观察治疗效果,探讨其药物作用机制。
方法造模后第2天,开始阴道局部给药,正常对照组不处理,阳性对照组给消糜栓,阴性对照组给空白基质,二黄散Ⅰ组和二黄散Ⅱ组分别给予相当于人临床用量的30倍和45倍的二黄散。1次/天,隔日1次,共12次。治疗结束后次日处死动物,解剖子宫后取出观察,并记录肉眼所见。做常规石蜡切片,HE染色,镜下观察阴道至子宫组织形态学变化,评价组织愈合效果。采用放射免疫分析法测定PAF和PGE2含量,免疫组织化学技术,检测经二黄散治疗的实验大鼠宫颈炎组织中EGF和PDGF的蛋白表达,RT-PCR方法检测EGF和PDGFmRNA的表达,免疫印迹法检测EGFR蛋白的表达。
结果中药二黄散Ⅰ、Ⅱ组和消糜栓组(阳性对照组)与阴性对照组相应部位比较,病变及炎症程度明显减轻,炎症细胞浸润明显减少,未见出血和坏死,比较差异有显著意义(P<0.05)。中药二黄散Ⅰ、Ⅱ组与阳性对照组对宫颈组织形态学的影响无显著性差异(P>0.05)。
中药二黄散Ⅰ、Ⅱ组中PAF和PGE2含量减低,治疗前后的含量分别为: 745.22±153.10,624.55±70.21;614.65±128.53,594.52±143.46;治疗后PAF、PGE2接近正常对照组,与阴性对照组比较差异有显著意义(P<0.05)。
EGF、PDGF免疫组化学结果显示,中药二黄散Ⅰ、Ⅱ组蛋白表达均显著高于阴性对照组和正常对照组,差异有统计学意义(P<0.05),中药二黄散Ⅰ、Ⅱ组EGFmRNA平均灰度分别为(4.53±0.39),(4.88±0.28),显著高于阴性对照组和正常对照组(2.33±0.57、1.87±0.40)(P<0.05,P<0.01);二黄散Ⅰ、Ⅱ组EGFR蛋白表达均显著高于阴性对照组,差异有统计学意义(P<0.05)。而PDGFmRNA平均灰度分别为(2.94±0.42),(3.51±0.29),高于正常对照组和阴性对照组(1.55±0.52)(1.43±0.45),差异有统计学意义(P<0.05)。阴性对照组EGF和PDGFmRNA与正常对照组相比,差异无统计学意义(P>0.05)。
结论二黄散能有效地改善实验大鼠宫颈炎炎症程度,宫颈炎大鼠用药后,炎性细胞数量明显减少,症状显著减轻,二黄散对实验大鼠宫颈炎有很好的治疗作用。二黄散能降低宫颈炎组织中PAF、PGE2的含量,促进宫颈组织中生长因子EGF和PDGFmRNA转录和蛋白合成,提高EGF受体蛋白的表达,促使宫颈鳞状上皮细胞的增殖、分化,有利于糜烂组织愈合。使损伤与修复因素趋于平衡,这可能是二黄散治疗宫颈炎的机制之一。
第三部分中药二黄散治疗慢性宫颈炎临床疗效评估
目的观察二黄散对不同类型宫颈糜烂的治疗效果及药物作用机制。
方法用二黄散治疗宫颈糜烂患者60例,其中轻度糜烂18例、中度23例、重度19例;单纯型26例,颗粒型21例,乳突型13例。将二黄散药粉喷敷于宫颈糜烂面上,隔日1次,7次为一疗程,连用2～4个疗程。观察治疗前后糜烂面的大小、病变的深浅以及患者的主观症状等。ELISA检测治疗前后宫颈分泌物中EGF、PDGF的含量变化。
结果(1)轻度糜烂有效率为94.4%,中度糜烂有效率为86.9%,重度糜烂有效率为73.7%;单纯型糜烂有效率为96.1%,颗粒型糜烂有效率为90.4%,乳突型糜烂有效率为53.8%。总有效率为85%。(2)治疗前后患者EGF宫颈粘液中含量为(41.56±16.51、97.45±20.12);PDGF宫颈粘液中的平均含量分别为:(32.74±10.22、56.20±12.33)。治疗前后宫颈粘液中EGF和PDGF的含量明显增高,比较差异具有显著意义(P<0.05)。
结论中药二黄散能明显改善宫颈糜烂患者的临床症状,有效地治疗宫颈糜烂,促进宫颈上皮细胞的增殖和分化,有利于组织的修复,该药对宫颈糜烂治愈率高、操作安全、简便,无明显的毒副作用,患者乐于接受。
Background Chronic cervicitis is one of the commonest disease of gynecology,It is often occurred in childbearing women with the incidence of more than 60%. And cervical erosion as a common pathological change is closely related to cervical carcinogenesis. So we should pay more attention to the pathogeny, diagnosis and treatment for it.
At present, routine clinical treatment remains as basic treatment and locally administered corrosive medicine. The mainstay treatment for chronic cervicitis is physiotherapy. It includes: laser therapy, cryotherapy, infrared light condensation therapy, microwave therapy, and etc. A common mechanism they shared is to destroy the simple columnar epithelium of area of the cervical erosion, and make the necrotic tissue desquamated, and then the area is recovered by regenerative stratified squamous epithelium. All the above physiotherapies bring some complications: increment of distinguishing discharge in vagina, water sample apocenosis, bleeding of decrustation, and prohibited sexual life before the healing of raw surface of the cervix (about four to eight weeks), so patiants with chronic cervicitis would not like to accept physiotherapy. Because of these reason, to seek an effective therapy is our duty as an accoucheur.
Recovery of inflammatary tissue is a complex course. It includes a series of stages: inflammatory of the tissue, formation of granulation tissue movement, formation of epithelium cell. Antiinfection enhancing nutrition and protecting local wound are main traditional therapeutic methods. platelet activating factor(PAF), mitogen-activated protein kinase(PGE2) , epidermal growth facto(rEGF)and platelet-derived growth factor( PDGF) are the most widely distributed in human body. They widely exist in central nervous system and peripheral system. And it can nourish and diferentiate many kinds of cells which generated from mesoderm and nerve ectoderm. Current medical research shows that they play very important roles in the rapid repair of tissue. It can promote metabolism and mitosis of all kinds of cells normally. For the concentration of them in body is very low, the healing of inflammatary tissue is also a slow course.
Recently, herbal medicine plays an important role in the periodontal treatment, also the effect of inflammatory medium. Scholars found that Radix scutellariae and Realgar could inhibit many kinds of bacteria and virus. Researchers also found that Chinese medicine could promote proliferation and attachment to root surface of cultured. Nowadays, the research on the local use of Chinese herbal medicine still remains in medical effects of single medicine, whereas complex prescription and its pharmaceutical mechanisms are still unknown.
Based on the above backgrounds, we investigated Erhuansan of a new Chinese medicine. This study used diagnosis based on overall analysis of patients' condition, mixed Radix scutellariae and Realgar in the reasonable proportion, then made ustained release preparation.We evaluated Erhuansan sustained-release preparation's to eliminate inflammation and promote period on talregeneration in animal and in human body, and observed the activity of inhibiting inflammation animal experiment and clinical application. This research provided experimental evidence in developing effective and practical Chinese herbal preparation on cervical erosion treatment. It will contribute to the step research on effect and pharmaceutical mechanisms of Chinese drugs Erhuansan to the cervical erosion.and provide the theory instruction for pharmaceutical application.
The paper is divided into three parts as follows.
Part one Research on the Animal Model with Colpitis and Cervicitis in Wistar rats
Objective To build the model with colpitis and cervicitis in Wistar rats.
Methods 15 normal Wistar rats and 54 animal models were studied. Preparing animal models as following, 20%Phenol mucilage are made of Phenol 4ml, Arabia balbta 4ml and distilled water 12ml. In the pathological model of cervical inflammation in rats set up by deep injection of phenol mucilage 1.5ml into every rat vagina by catheter for every two days, total five times. The change of the pudendum is observed for ten days. Then the model rats were killed, the weight of the rat’s body and inflammatory cells of its cervix were taken and quantified, and its index examined and calculated by pathology.
Results In model groups, the rats lost their appetite, reduced movement and the weight of them. At the same time, we also found that perineum tissue swelled and excreted pusappeared in the vagina orifices. Putrescence or desquamation appeared in the vagina and cervical mucous membrane. Under the vagina and cervical mucous membrane, inflammation cells soakage and acestom a hyperplasia were discovered.
Conclusion We could successfully establish the wistar rat model with colpitis and cervicitis by deep injection of 20% phenol mucilage 1.5ml into every rat vagina by catheter, five times. It was very convenient and done repeatedly.
Part two Study on the curative effect and mechnism of Chinese medicine Erhuansan on the animal model with cervicitis
Objective To research on effect and pharmaceutical mechanisms of Chinese drugs Erhuansan to animal model with cervicitis.
Methods After injecting phenol slurry for five times, we operated to the rats. Five normal Wistar rats and six animal model rats were observed. The rats of the positive and negative control groups were treated by Xiaomishuan and blank matrix suppository. The rats of ErhuansanⅠand ErhuansanⅡgroups were treated by 30 and 45 times of normal clinical human dose. The expression of PAF and PGE2 were remarkably decreased by radiommunoassay. The protein levels and mRNA of EGF and PDGF were determined by immunohistochemistry and the reverse transcriptional polymerase chain reaction (RT-PCR). The expressions of EGFR were assessed by Western blot method.
Results The Pathomorphology of positive control group and ErhuansanⅠ, ErhuansanⅡgroups were all changed better than that of the negative ones (the model group). The inflammation cells were reduced significantly in the former. The configuration of squamous cells was normal. It also showed that Erhuansan could improve the degree of Wistar rat model with colpitis and cervicitis. It could alleviate the signs and symptoms of them, such as swelling, discharge in the vagina orifices and putrescence, desquamation appeared in the vagina and cervix mucous membrane.
After treatment, the contents of PAF and PGE2 were (745.22±153.10, 624.55±70.21; 614.65±128.53, 594.52±143.46). The level decreased significantly in the Erhuansan treated group in comparison with the blank treated group(P<0.05).The content of them in ErhuansanⅠand ErhuansanⅡg roups were close to those of the normal control group.
The expressions of EGF and PDGF were significantly increased in ErhuansanⅠandⅡgroup compared to the normal ones(P<0.05). There were no differences between the negative control group and normal one. The transcription levels of the EGF mRNA in ErhuansanⅠandⅡgroup were significantly higher than that of normal groups(4.53±0.39vs 1.87±0.40; 4.88±0.28 vs 1.87±0.40, P<0.01). However, the expression levels of PDGF mRNA were higher in cervix tissue compared to control as assessed by RT-PCR(2.94±0.42vs 1.55±0.52; 3.51±0.29 vs 1.55±0.52, P<0.05). In negative control groups EGF and PDGF in cervix tissues were all no significantly than those in normal one(2.33±0.57, 1.43±0.45, P>0.05). The concentrations of EGFR in Erhuansan groups were significantly higher than those in normal one and negative one.
Conclusion Erhuansan can promote the healing of cervical erosion on rats. After treatment, the content of PAF and PGE2 were lower and the expressions of EGF, EGFR and PDGF in cervititis tissue were increased.The balance of inflammation medium was setted up.This was probably the mechanisms of Erhuansan on the animal model with cervicitis.
Part three Clinical Study of Erhuansan of chines medicine preparation in cervical erosion
Objective To study the curative effect of Erhuansan in cervical erosion .
Methods 60 women with cervical erosion were investigated. Every one was treated by Erhuansan for every two days, seven times for each period. On the basis of pathological changes, it was given two to four periods of treatment. During the treatment, the changes of cervix surface were watched by naked eye and colposcopy, and examined the content of EGF and PDGF in cervix secretion by enzyme linked immunosorbent assay.
Results The curative rates of mild, moderate and severe cervical erosion cases were 94.4%, 86.9%, 73.7% respectively. And the ones of three types were 96.1%, 90.4%, 53.8%. The total efficient rate was 85%. After treatment the content of EGF was increased (97.45±20.12)in cervix mucus. There was no change in serum.The same thing was to the content of PDGF. In cervix mucus, the content of EGF and PDGF in cervix tissues were all significantly higher than before(P<0.05).
Conclusion The efficient rates of Erhuansan on cervical erosion cases were high. This method was safe, easy, efficient, Patients were taken pleasure in it. It can promote the re-epithelium.
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