【作者】 凌娜；

【导师】 洪家铁；

【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】 1研究目的慢性盆腔炎是妇科临床的常见病，多发病，据调查：在我国育龄女性中，约41%女性曾经患有妇科炎性疾病，已婚女性更是高达70%。其主要症状表现为腹痛、腰骶痛、白带量多，可导致不孕症，异位妊娠等并发症，严重影响妇女身心健康。西医多采用抗生素结合物理治疗等方法，效果不佳；中医采用中药口服、灌肠、外敷、针灸等综合疗法，取得了一定的效果，但临床辨证治疗主要以清热解毒及活血化瘀通络为主。我们在长期的中医治疗临床实践中，结合病人的临床症状体征，对慢性盆腔炎的病机提出了自己的见解，认为慢性盆腔炎中多数人是由于肾阳不足、脾气虚弱及瘀血邪毒滞留于冲任胞宫所致，是本虚标实之证。本研究基于以上理论，本着扶正祛邪的原则，在《金匮要略》及《傅青主女科》经方的基础之上，采用温补脾肾、活血化瘀，祛湿解毒的方法，自拟妇炎宁汤来治疗慢性盆腔炎。在前期动物试验已经证实了妇炎宁汤能够降低模型大鼠血清中IL-2、6水平，从而提高慢性盆腔炎大鼠的免疫功能。本项研究是通过对慢性盆腔炎及阳虚型慢性盆腔炎大鼠模型的其他细胞因子如TNF-α、IFN-γ、IL-10、NK细胞的变化，在免疫学的层面科学论证妇炎宁汤对慢性盆腔炎的免疫调节作用，以期为探讨慢性盆腔炎的用药治疗规律提供新的治疗思路和理论依据，同时也进一步证明了由于慢性盆腔炎病程久，久病多虚，易导致阳气损伤，因此治疗上采用温补脾肾之阳，可以振奋阳气，补益虚损，鼓邪外出，是有效的治疗方法。2材料与方法2.1实验材料2.1.1实验动物SPF级Wistar雌性大鼠120只（辽宁中医药大学实验动物中心提供）。2.1.2实验药品及试剂白介素-10(IL-10)、IFN-γ：ELISA检测试剂盒；抗大鼠肿瘤坏死因子-α(TNF-α）抗体、抗大鼠IFN-γ抗体试剂盒；肿瘤坏死因子-α(TNF-α）及IFN-γ引物；RT-PCR试剂盒购自TAKARA公司提供；抗CD16-FITC及抗CD56-PE抗体；菌种：大肠杆菌、金黄色葡萄球菌、乙型溶血性链球菌。2.2造模2.2.1慢性盆腔炎模型大肠杆菌、金黄色葡萄球菌、乙型溶血性链球菌按2：1：1比例，配制成浓度为30亿个/ml的混合细菌溶液，用眼科手术器械分别在子宫角近输卵管处进针，向输卵管－卵巢方向缓慢注射，每侧注射混合菌液0.05ml后关腹。然后将子宫回纳入腹腔，1号丝线，连续缝合腹壁和皮肤。空白对照组，按上法打开皮肤，不做任何处理，缝皮。2.1.2阳虚慢性盆腔炎动物模型将慢性盆腔炎造膜后的大鼠适应性喂养3天后，随机选取慢性盆腔炎模型大鼠36只，用地塞米松注射液2mg，每日一次肌注。连续7天，中间休息1天，共肌注16次。模型动物出现行动迟缓，神志萎靡，蜷缩，拱背，下肢水肿，畏寒，体毛稀疏尤以尾部突出证明其阳虚模型造模成功。2.3分组及给药方法2.3.1分组随机分成空白对照组8只，其余112只分别进行慢性盆腔炎造模。然后随机选出慢性盆腔炎造模大鼠36只造成阳虚型慢性盆腔炎的模型。慢性盆腔炎大鼠共分为模型组、中成药康妇炎胶囊组、西药左氧氟沙星组、妇炎宁低剂量组、妇炎宁中剂量组和妇炎宁高剂量组。而阳虚慢性盆腔炎大鼠模型分成3组，分别是阳虚型模型组，简称阳虚组；阳虚型妇炎宁汤治疗中剂量组，简称阳虚中剂量组和阳虚型妇炎宁汤治疗高剂量组，简称阳虚高剂量组。加上空白对照组，共10个实验组。2.3.2给药方法(1)正常对照组：8只，予以蒸馏水灌胃。(2)模型组：10只，予以蒸馏水灌胃。(3)中成药康妇炎胶囊组：9只，0.04g/只溶于2ml蒸馏水中灌胃。(4)西药左氧氟沙星组：10只，0.006g/只溶于2ml蒸馏水中灌胃。(5)妇炎宁汤低剂量组：11只，1.62g生药浓煎成2ml/只/天进行灌胃。(6)妇炎宁汤中剂量组：12只，3.24g/天，生药浓煎成2ml/只/天进行灌胃。(7)妇炎宁汤高剂量组：12只，6.48g/天，生药浓煎成2ml/只/天进行灌胃。(8)阳虚模型组：10只，予以蒸馏水2ml/只灌胃。(9)阳虚中剂量组：10只，3.24g/天，生药浓煎成2ml/只/天进行灌胃。(10)阳虚高剂量组：10只，6.48g/天，生药浓煎成2ml/只/天进行灌胃。2.4实验方法（1）HE染色来观察各组大鼠子宫的组织形态学的变化。（2）酶联吸附法（ELISA法）检测各组大鼠血清中IL-10、IFN-γ的表达。（3）免疫组化法测量各组大鼠子宫组织中TNF-α的表达情况。（4）RT-PCR法测量各组大鼠子宫组织中TNF-α、IFN-γ的表达情况。（5）称大鼠的脾重/体重。（6）流式细胞仪检测大鼠脾内NK细胞的比例。2.5实验结果2.5.1病理大体病理及光镜下均显示慢性盆腔炎及阳虚型慢性盆腔炎经妇炎宁汤中、高剂量组灌胃治疗后炎症明显减轻。大体病理显示妇炎宁中、高剂量组、阳虚型中、高剂量组可见子宫无明显充血，大小、形态、质地基本正常，盆腔无明显充血迹象，与周围组织无明显粘连，与模型组、阳虚模型组、康妇炎胶囊组、左氧氟沙星组相比有显著差异。光镜下病理显示妇炎宁中、高剂量组、阳虚型中、高剂量组子宫结构基本正常，仅见少量炎细胞浸润，无明显腺体扩张及纤维细胞增生，与模型组、阳虚模型组、康妇炎胶囊组、左氧氟沙星组相比有显著差异。2.5.2ELISA法血清IL-10测定：妇炎宁高剂量组、阳虚高剂量组分别与模型组相比均有显著性差异，且与左氧氟沙星组有明显差异；在本实验的观察中我们可以发现，妇炎宁汤可以降低慢性盆腔炎模型和阳虚型慢性盆腔炎模型大鼠血清IL-10的表达，这与许多专家报道的有所不同。如果从炎症的角度考虑，IL-10主要由Th2细胞分泌，在炎症的初期主要是由Th1细胞占优势，而到了慢性阶段，为了防止过度免疫造成的细胞损伤，Th1/Th2出现了漂移， Th2细胞应该逐渐增强，因此IL-10应该呈现上升趋势，但是如果从病理纤维化的角度考虑，IL-10是主要的致纤维化因子，IL-10的降低有助于减轻过度纤维化，从而减轻慢性炎症的病理变化。血清中IFN-γ在各组之间的差异不明显；IFN-γ/IL-10比值的变化，妇炎宁及阳虚高剂量组与左氧氟沙星组相比有明显差异。因为IFN-γ在各组之间的差异不明显，考虑IFN-γ/IL-10的变化主要是由于IL-10的变化造成。血清测定IFN-γ组间没有差异，而在PCR法中出现IFN-γ明显降低，因为基因表达往往在蛋白表达之前，提示本次试验造模时间有可能较短，另外治疗时间也相对较短，因此血清没有出现明显变化，提示我们在今后的实验中要延长相应的时间安排，那么在细胞因子的表达上会有更明显的变化。2.5.3免疫组化法测量子宫组织中TNF-α模型组大鼠和正常组大鼠、妇炎宁高剂量组和阳虚高剂量组的TNF-α表达有明显差异，说明妇炎宁汤可以降低TNF-α在细胞内的表达，在阳虚模型中尤为明显。2.5.4PCR法测量子宫组织中TNF-α、IFN-γ在降低TNF-α表达方面，康妇炎胶囊、阳虚中、高剂量有明显差异较左氧氟沙星组有明显差异；TNF-α是重要的促炎因子，而且还是重要的致纤维化的细胞因子。妇炎宁汤可以明显降低TNF-α的表达，无论是在抗炎与促炎的平衡当中还是降低纤维化的角度，对于慢性盆腔炎的模型的治疗都是十分有益的。在降低IFN-γ表达方面，妇炎宁汤中、高剂量与模型组相比有显著性差异，说明妇炎宁汤在对影响IFN-γ表达方面有明显作用，正常组与西药左氧氟沙星组有明显差异（P＜0.01），提示抗生素对于慢性炎症模型中降低IFN-γ表达的效果不理想；正常组与康妇炎胶囊、妇炎宁低剂量组有明显差异（P＜0.05），说明单纯清热利湿，活血化瘀的中成药对于慢性炎症模型中降低IFN-γ表达的效果也不好，甚至妇炎宁低剂量组虽然也含有补益脾肾阳气之品，但由于含量低，而且活血化瘀和清利湿热的药物浓度也降低，因此疗效欠佳。而妇炎宁汤中高剂量组、阳虚中高剂量组均可以明显降低IFN-γ的表达，但这种差异但在并没有在阳虚中高剂量组和妇炎宁中高剂量组之间产生，说明所有慢性盆腔炎由于病情迁延均会造成脾肾阳气不足，因此运用补益脾肾阳气兼活血化瘀的妇炎宁汤均可以取得很好的疗效。2.5.5流式细胞仪检测脾内NK细胞的比例妇炎宁中高剂量和阳虚型中高剂量与模型组和阳虚模型组之间均没有明显差别（p＞0.05）。考虑是否与造模时间和试验用药时间比较短有关。3实验结论（1）混合菌液法及地塞米松注射法成功建立了慢性盆腔炎及阳虚型慢性盆腔炎大鼠模型。（2）通过观察妇炎宁汤对慢性盆腔炎及阳虚型慢性盆腔炎大鼠模型细胞因子IL-10、IFN-γ、TNF-α表达的影响，证实了以温补脾肾，利水活血为主组成的中药复方妇炎宁汤，具有较好的疗效。（3）本实验进一步证实了温补脾肾法是治疗慢性盆腔炎的有效法则，而妇炎宁汤是治疗慢性盆腔炎的有效方剂，在目前市场上几乎所有治疗相关疾病的中成药均为以清热利湿和活血化瘀为主要治则的局面下，本方在慢性盆腔炎的治疗领域具有广阔的应用前景。更多还原

【Abstract】 1ObjectiveChronic pelvic inflammation is a common and frequently-occurring disease ofgynecology in clinic. According to the investigation，there are about41%females at thereproductive ages in China have been suffering from gynecological inflammatory diseases.The incidence of married women even reaches up to70%. The main manifestations includeabdominal pain，pain in lumbosacral region，increased leucorrhea and complications such asinfertility or ectopic pregnancy will be developed. All these seriously influence the physicaland metal health of women. In Western medicine，anti-inflammation and physical therapy arecommonly used but the effect is not satisfactory. TCM therapy，including oral administration，enema，external application of Chinese drugs and acupuncture and moxibustion，has obtainedexact effect. However，the syndrome differentiation in clinic is mainly clearing heat andeliminating toxins as well as activating blood circulation to resolve blood stasis and dredgingcollaterals. Through clinical practice for a long time and combined with the symptoms andsigns of patients，our department has proposed the unique view of pathogenesis of chronicpelvic inflammation and believes that most patients are caused by kidney-Yang deficiency，spleen-Qi deficiency and blood stasis as well as evils in Chong，Ren meridians and uterus，which belongs to syndrome of root deficiency and branch excess. Therefore，based on thetheory mentioned above，according to the principle of supporting vital-Qi and expelling evils，and combined with the classical prescriptions in Jinkui Yaolue and Fuqingzhu Nuke，self-made Fuyanning Decoction which can warmly supplement spleen and kidney，activateblood circulation to resolve blood stasis and dispel damp and eliminate toxins is used to treatchronic pelvic inflammation. During the experimental researches in early time，it has beenproved that Fuyanning Decoction can reduce the levels of IL-2and IL-6in serum of rats withchronic pelvic inflammation so as to improve the immune function. This research is to discussthe immune regulating function by observing the levels changes of TNF-α，IFN-γ，IL-10and NK in chronic pelvic inflammation rats with or without Yang deficiency，expecting to providenew ideas and theory basis for medication laws，Chinese drugs mechanism，developing new，convenient and practical drugs with high effect and low toxicity. Meanwhile，it also can provethat due to long term of chronic pelvic inflammation，Yang-Qi can be easily damaged andthus warmly supplementing spleen and kidney can invigorate Yang-Qi to dispel evils，whichis an effective method.2. Materials and Methods2.1Materials2.1.1AnimalsA total of120SPF level Wistar female rats were used（supplied by Experimental AnimalCenter of Liaoning University of Traditional Chinese Medicine）.2.1.2Drugs and ReagentsIL-10，IFN-γ：ELISA test kit；anti-body of TNF-α，anti-body kit of IFN-γ；primer ofTNF-α and IFN-γ；RT-PCR test kit from TAKARA company；anti-bodies of CD16-FITC andCD56-PE；bacteria strain：bacillus coli，Staphylococcus aureus，Beta-hemolytic streptococcus.2.2Modeling2.2.1Chronic Pelvic Inflammatory Disease ModelEscherichia coli, Staphylococcus aureus, andβ-hemolytic streptococcus，at a ratio of2:1:1, formulated the mixed bacterial solution at a concentration of3billion/mL. The solutionwas injected through the uterine horn near the fallopian tubes with ophthalmic instruments tothe direction of fallopian tubes to ovary. After0.05mL solution injected to both sides，theabdomen should be sutured. The uterus should be back in the abdominal activity. Theabdominal wall and skin were sutured with No.1silk thread. The bland control group openedthe abdominal skin without any interventions and then sutured.2.1.2Chronic Pelvic Inflammatory Disease of Yang Deficiency ModelThe above model rats were adaptive fed for3days and36of them were randomlyselected.2mg of Dexamethasone injection was given by an intramuscular way once a day，lasting for7days. There was one-day rest. All together there were16times of injection.Tardy，sluggish，curled，back arch，lower limb swelling，fear of cold as well as sparse bodyhair specially tail hair，all these indicated the successful modeling of chronic pelvic inflammation diseases of Yang deficiency model.2.3Groups and Administration2.3.1Groups8rats were put into bland control group randomly. The other112rats were for thechronic pelvic inflammation disease model. And then36rats of these models were selectedout to establish the model with Yang deficiency.The chronic pelvic inflammation disease model rats were divided into model group，Kangfuyan Capsule group，Levofloxacin group，low，middle and high doses of Fuyanninggroups. The models of Yang deficiency were divided into Yang deficiency model group(Yangdeficiency group)，middle dose of Fuyanning for Yang deficiency model group（Yangdeficiency with middle dose group）and high dose of Fuyanning for Yang deficiency modelgroup（Yang deficiency with high dose group）. There were all together10groups，adding theblank control group.2.3.2Administration(1)normal control group：8rats were given distilled water by gavage.(2)model group：10rats were given distilled water by gavage.(3) Kangfuyan Capsule group：9rats were given Kangfuyan Capsule，0.04g in2mLdistilled water for each by gavage.(4) Levofloxacin group：10rats were given Levofloxacin，0.006g in2mL distilled waterfor each by gavage.(5) low dose of Fuyanning group：11rats were given Fuyanning with1.62g crude drugsdecocted2mL for each by gavage every day.(6) middle dose of Fuyanning group：12rats were given Fuyanning with3.24g crudedrugs decocted2mL for each by gavage every day.(7) high dose of Fuyanning group：12rats were given Fuyanning with6.48g crude drugsdecocted2mL for each by gavage every day.(8) Yang deficiency group：10rats were given distilled water by gavage.(9) Yang deficiency with middle dose group：10rats were given Fuyanning with3.24gcrude drugs decocted2mL for each by gavage every day.(10) Yang deficiency with high dose group：10rats were given Fuyanning with6.48g crude drugs decocted2mL for each by gavage every day.2.4Methods(1)The tissue morphological changes of uterus in rats were observed by HE staining.(2)The levels of IL-10and IFN-γwere tested by ELISA method.(3)The TNF-α expression in uterus tissues of rats were determined byImmunohistochemistry method.(4)The expressions of TNF-α and IFN-γ in uterus tissues of rats were measured byRT-PCR method.(5)The ratio of spleen weight/body weight was recorded.(6)The proportion of NK in spleen of rats was tested by flow cytometry.2.5Results2.5.1Pathological ResultsEther general pathological observation or light microscope view， it showed theinflammation was obviously relieved both in chronic pelvic inflammation rats with or withoutYang deficiency in middle and high dose of Fuyanning Decoction groups. The generalpathological observation showed：the uterus had no obvious congestion，and the size，shapeas well as texture of uterus in middle and high dose of Fuyanning groups and middle and highdose groups with Yang deficiency. There was no congestion in pelvic cavity and there was onobvious adhesion with peripheral tissues. There was obvious significant difference whencompared with model，Yang deficiency model，Kangfuyan Capsule and Levofloxacin groups.Under the light microscopic view，the uterus tissue was basically normal in middle and highdose of Fuyanning Decoction group and middle and high dose groups with Yang deficiency.There was only a few of infiltration of inflammatory cells. There was no obvious enlargementof glands or fibrous cell proliferation. There was significant difference when compared withmodel，Yang deficiency model，Kangfuyan Capsule and Levofloxacin groups.2.5.2ELISA MethodFor testing IL-10level in serum：compared with model group respectively，FuyanningDecoction high dose group or Yang deficiency high dose group had significant difference，and there was obvious difference compared with Levofloxacin group. We have found thatFuyanning Decoction can reduce the IL-10expression in serum of chronic pelvicinflammation rats and those model rats with Yang deficiency，which was different from some reports of experts. Considering from the inflammation，IL-10is mainly secreted by Th2cell.At the early stage of inflammation，Th1cell was in the dominate. However，at the chronicstage，Th1/Th2happened to drift in order to avoid cell damage due to excessive immune. Th2becomes strong gradually. Therefore，IL-10should has an increasing trend. But if consideringfrom the pathological fibrosis，IL-10is main fibrosis factor and can help to reduce theexcessive fibrosis so as to reduce the pathological changes of chronic inflammation.IFN-γ levels in serum in groups had no difference. For the ratio change of IFN-γ/IL-10，compared with Levofloxacin group，Fuyanning Decoction high dose group and Yangdeficiency high dose group had obvious difference. Since there was no obvious difference ofIFN-γ among groups，it was considered that IFN-γ/IL-10change was mainly caused by IL-10change. IFN-γ among groups had no difference tested by blood serum test. However，whentested by PCR method，there was obvious decrease of IFN-γ. Gene expression is mainlybefore protein expression，indicating that this model time may be short，furthermore，thetreatment time was also short，and thus there was no obvious change in serum. It is indicatedthat we should prolong the corresponding time in the future experiment so that cell factorexpression will be changed more obviously.2.5.3TNF-α in Uterus TissuesModel group and normal rats had obvious difference on TNF-α expression. FuyanningDecoction high dose group and Yang deficiency high dose group had obvious difference onTNF-α expression，indicating that Fuyanning Decoction can reduce the intra-celluarexpression of TNF-α，especially in the Yang deficiency model group.2.5.4TNF-α and IFN-γ in Uterus TissuesFor reducing TNF-α expression，Kangfuyan Capsules，Yang deficiency middle and highdose groups had obvious difference and compared with Levofloxacin group，there wasobvious difference. TNF-α is one of the important proinflammatory factors as well as theimportant fibrosis-inducing cell factor. Fuyanning Decoction can obviously reduce the TNF-αexpression. Either for the balance of anti-inflammation and pro-inflammation or for thefibrosis reducing， Fuyanning Decoction has significance of treating chronic pelvicinflammation.For reducing IFN-γ expression，Fuyanning Decoction middle and high dose groups compared with model group，there was obvious significance，indicating Fuyanning Decoctionhad obvious effect on IFN-γ expression. There was obvious difference between normal groupand Levofloxacin group（P＜0.01），indicating that the effect of antibiotics on chronicinflammations was not satisfactory. There was obvious difference between normal group andKangfuyan group as well as between normal group and Fuyanning Decoction low dose group（P＜0.05），indicating that Chinese patent only for clearing heat and damp and activatingblood circulation to resolve stasis can not obtain satisfactory effect either. Fuyanning highdose group and middle and high dose groups with Yang deficiency all can obviously reduceIFN-γ expression. But there was no difference，indicating that prolonged chronic pelvicinflammation may lead to spleen and kidney Yang deficiency. Therefore， FuyanningDecoction which can supplement spleen and kidney Yang combined with activate bloodcirculation to resolve stasis can obtain better effect.2.5.5Ratio of NK in SpleenThere was no obvious difference when Fuyanning Decoction middle and high dosegroups compared with model and Yang deficiency model groups（p＞0.05）. Yang deficiencymiddle and high dose groups had no obvious difference compared with model and Yangdeficiency model group（sp＞0.05）. This may be related to the short duration of modeling andmedication.3Conclusion(1)The chronic pelvic inflammation model and the chronic pelvic inflammation modelwith Yang deficiency were successfully established by injecting mixed bacteria anddexamethasone.(2)Through observing the influence of Fuyanning Decoction on expressions of IL-10，IFN-γand TNF-α in rats with chronic pelvic inflammation and those with Yang deficiency，it has beenproved that Fuyanning Decoction，which consists of drugs for warmly supplementing spleen andkidney as well as promoting urination and activating blood circulation，has better effect.(3)This research has further proved that the method of warmly supplementing spleen andkidney is the effective treatment principle and Fuyanning Decoction is the effectiveprescription. At present，almost all the Chinese patent drugs for this disease are guided by theprinciple of clearing heat and damp and activating blood circulation to resolve stasis， indicating this prescription has broad application prospect in treating chronic pelvicinflammation.更多还原