【作者】 张军；

【导师】 姚昶；

【作者基本信息】 南京中医药大学 ， 中医外科学（专业学位）， 2020， 博士

【摘要】 背景:非哺乳期乳腺炎(non-puerperal mastitis,NPM)是目前发病率逐年上升的乳腺炎性疾病,临床以反复发作、迁延难愈为特点,现代医学尚无特别有效的治疗手段。对广大患者来说,该病病程长、难以治愈、且极易反复发作及多个窦道形成,严重影响了患者的生活质量,并增加了其家庭的经济负担;而手术又多会导致乳房严重变形、甚至乳房全切的地步,严重影响了美观,给患者造成了极大的心理影响,故非哺乳期乳腺炎成为目前急需研究、解决的疾病,而中医药治疗该病临床报道较多,具有自己的独特优势,但是尚缺乏在作用机制方面的探索。在临床实践中,我院长期运用清热化痰散结方治疗本病,具有很好的临床疗效,但缺乏具体的数据支持,故我们在分析总结非哺乳期乳腺炎临床危险因素的基础上,对清热化痰散结方改善临床疗效进一步验证,并探索清热化痰散结方的可能作用机制。本研究拟进一步对不同时期非哺乳乳腺炎腺体组织CD4+、CD8+、CD4+/CD8+及IgG、IgM的状态进行评估,并对治疗后腺体组织免疫指标、NF-kBp65表达强度及VEGF含量的影响进行观察,以此评价清热化痰散结方调控非哺乳期乳腺炎免疫反应进而控制病变进展、促进炎症消退的临床效果,并探索其可能的作用机制。目的:探索清热化痰散结方抑制非哺乳期乳腺炎局部组织免疫反应、促进炎症消退的临床效果及其探讨可能的作用机制。方法:依据非哺乳期乳腺炎发病危险因素:体重指数(BMI)、乳头溢液、乳头凹陷、流产次数、泌乳素水平,对照同时期住院的非哺乳期乳腺炎及健康女性体检者各60例,观察两组的差异。采取数字化非中心随机对照的原则,将60例非哺乳期乳腺炎患者分为治疗组和对照组,每组各30例,两组患者均经病理诊断为非哺乳期乳腺炎,该研究均经伦理委员会讨论通过并取得患者知情同意。治疗组年龄33.26±8.55岁,病程34.56±31.67天;对照组年龄34.63±7.44岁,病程33.23±31.02天;肿块直径:<2cm者1例,(?)2cm而<4cm者7例,(?)4cm而<5cm者6例,(?)5cm者16例;乳房局部皮肤不红者3例,皮肤鲜红者11例,皮肤稍红者8例,而皮肤紫红或暗红者8例;对照组肿块直径大小:<2cm者1例,(?)2cm而<4cm者5例,(?)4cm而<5cm者5例,(?)5cm者19例;乳房局部皮肤不红者4例,皮肤鲜红者13例,皮肤稍红者6例,皮肤紫红或暗红者7例。对照组予抗生素、外科换药(1次/日)、微波治疗(2次/日)、外敷青宝散(2次/日)等综合治疗;治疗组在一般综合治疗的基础上予口服清热化痰散结方(每付中药方煎成200ml汤剂,2次/日,100ml/次)。疗程为一个月,观察时点:治疗第1天及第30天分别填写临床症状、体征观察评分表,并分别对肿块患者行穿刺活检取炎性腺体组织、对脓肿患者切开引流时或窦道患者换药时剪取部分腺体组织,以酶联免疫吸附法(ELISA)检测IgG、IgM、VEGF的浓度,免疫组化法检测CD4+、CD8+及NF-kBp65表达的强度。结果:1危险因素:非哺乳期乳腺炎患者BMI值为17.33～35.29,平均为24.8±4.05。非哺乳期乳腺炎组中54.1%的患者体重过重或偏胖,与同期健康体检者比较,有统计学差异(p<0.05);非哺乳期乳腺炎组中38.33%的患者有乳头凹陷,较健康体检者乳头凹陷的比例高,但无统计学意义,P>0.05;非哺乳期乳腺炎组中25%的患者泌乳素水平偏高,而健康体检者中只有10%的患者泌乳素水平偏高,但两组相比较,无统计学意义(p>0.05)。2临床症状、体征评分及疗效:经1个月治疗后,治疗组疼痛评分及红肿评分(1.53±1.45、1.60± 1.52)较对照组(2.60±1.83、2.66±1.91)明显降低(P<0.05),具有统计学意义;治疗后治疗组肿块直径评分(4.00±2.16)较对照组(2.40± 1.92)明显降低,具有显著统计学意义(P<0.01);治疗组治疗后窦道评分(1.20±1.15)与对照组(1.23±1.16)相比不具有统计学意义(P>0.05)。总体疗效:治疗组有5例痊愈,显效8例,有效13例,无效4例,总疗效率为86.67%,对照组有2例痊愈,4例显效,10例有效,14例无效,总疗效率为53.33%,治疗组临床疗效评分(3.73±1.55)与对照组(2.20±1.60)相比具有统计学意义(P<0.05)。3炎症组织IgM、IgG、VEGF:治疗前非哺乳期乳腺炎单纯肿块者组织IgM、IgG含量最高,伴窦道者IgM、IgG的含量最低;而伴脓肿(溃破)者组织VEGF的含量最高,单纯肿块者组织VEGF的含量最低。治疗后治疗组组织中IgM、IgG的含量(51.45±25.78ug/g、996.23±331.17 ug/g)明显低于对照组(69.68±25.25 ug/g、1404.45+464.77 ug/g)(P<0.05),而 VEGF 的含量(263.41±215.99 ug/g)显著低于对照组(491.14±323.65 ug/g)(P<0.01),均具有统计学意义。4炎症组织CD4+T、CD8+T:治疗组治疗后炎症组织CD4+T免疫组化评分(2.70±2.31)较对照组(5.70±3.34)显著下降,CD8+T免疫组化评分(9.80±4.47)较对照组(5.40±2.90)则显著升高,均具有统计学意义(P<0.05)。高倍镜下病理切片表现:治疗前治疗组非哺乳期乳腺炎腺体间质内CD4+T及CD8+T 阳性细胞染色均较深,CD4+T阳性细胞比例高,CD8+阳性细胞占比较低,CD4/CD8比值高,治疗后CD4+T阳性细胞染色变浅,CD4+T阳性细胞占比降低,CD8+T 阳性细胞染色深,CD8+T阳性细胞占比升高,CD4/CD8比值降低。5炎症组织NF-KBp65:治疗组治疗后炎性腺体组织NF-KBp65免疫组化评分(2.07±0.97)较对照组(4.26±0.65)明显下降,具有统计学意义(P<0.05)。高倍镜下病理切片表现:治疗组治疗前非哺乳期乳腺炎乳腺导管内皮细胞的胞质或胞核内呈散在或聚集的棕黄色颗粒,染色深,NF-KBp65表达强;而治疗后胞膜或胞质内有少许棕黄色颗粒沉积,染色浅,NF-KBp65表达较弱。结论:1非哺乳期乳腺炎一般危险因素的观察结果表明体型过重或肥胖、高泌乳素血症、流产在一定程度上与非哺乳期乳腺炎密切相关,均值得生活及临床上重视,并采取生活护理、饮食指导、改变不良生活习惯、运动减肥、合适的哺乳时间、避免流产等措施,早干预、早预防,对减少非哺乳期乳腺炎的发生有一定的意义。2清热化痰散结方明显地减轻患者乳房疼痛症状,促进乳房皮肤红肿消退,缩小肿块,使窦道变浅,减轻了患者的痛苦,并提高了临床疗效,值得临床推广及进一步研究。3清热化痰散结方能显著降低非哺乳期乳腺炎组织中IgM及IgG的含量,可有效地降低炎症组织中的初次体液免疫应答及再次免疫应答,进而减轻了免疫应答对腺体组织的损伤;清热化痰散结方能减弱CD4+T的表达及增强CD8+T的表达,进而抑制非哺乳期乳腺炎组织中T淋巴细胞介导的免疫应答,并在一定程度上提升机体的免疫力,从而减轻临床症状、体征及促进恢复。4清热化痰散结方能减弱非哺乳期乳腺炎炎症组织中NF-kBp65的表达,其改善组织炎症及免疫可能是通过调控NF-kBp65通路活化介导实现的。更多还原

【Abstract】 Background:Non-lactating mastitis is a mastitis disease whose incidence is increasing year by year.Clinically,it is characterized by repeated attacks and difficult to cure.Modern medicine has no particularly effective treatment.For the majority of patients,the characteristics of the disease are long,difficult to cure,and easy to recur and multiple sinus formation severely affect the patients’ quality of life and increase the financial burden on their families.Surgery may lead to severe breast deformation or even a full breast cut,which seriously affects the aesthetics and has a great psychological impact on the patients.Therefore,non-lactating mastitis has become a disease that urgently needs to be studied and resolved.There are many clinical reports on the treatment of this disease by Chinese medicine,and each has its own unique advantages,but there is still no report on the exploration of mechanism of action.In clinical practice,our hospital has used Qingre Huatan Sanjie Recipe for the treatment of this disease for a long time.It has good clinical efficacy,but it lacks specific data to be supported.Therefore,based on the analysis and summary of the clinical risk factors of non-lactating mastitis,we further verified the clinical efficacy of Qingre Huatan Sanjie Recipe and explored the possible mechanism of Qingre Huatan Sanjie Recipe.This study also observes the clinical effects of glandular tissue immune indicators,NF-kBp65 expression intensity and VEGF content after treatment,in order to evaluate the effect of Qingre Huatan Sanjie Recipe on regulating the immune response of non-lactating mastitis to control the progression of disease and promote inflammation.Objective:To explore the clinical effect of Qingre Huatan Sanjie Recipe on inhibiting the local tissue immune response and promoting inflammation subsidence in non-lactating mastitis,and explore its possible mechanism.Methods:Based on the risk factors for non-lactating mastitis:BMI,nipple discharge,nipple depression,number of abortions,and prolactin levels,this study compared 60 cases of non-lactating mastitis with 60 healthy women.Based on the principle of randomized control,60 non-lactating mastitis patients were divided into a treatment group and a control group,with 30 cases in each group.Both groups of patients were diagnosed as non-lactating mastitis by pathology.The study was approved by the ethics committee and the patients’ informed consent was obtained.The age of the treatment group was 33.26±8.55 years,the course of disease was 34.56±31.67days;the age of the control group was 34.63±7.44 years,the course of disease was 3 3.23±31.02days;The diameter of the mass of the treatment group:1 case was less than 2cm,7 cases were 2cm or more and less than 4cm,6 cases were 4cm or more and less than 5cm,16 cases were 6cm or more;3 cases of localized breast with red skin,11 cases with bright red skin,and slightly 8 cases were red,and 8 cases were purple or dark red.The diameter of the mass of the control group:1 case was less than 2cm,5 cases were 2cm or more and less than 4cm,5 cases were 4cm or more and less than 5cm,19 cases were 6cm or more;4 cases of localized breast with red skin,13 cases with bright red skin,and slightly 6 cases were red,and 7 cases were purple or dark red.The control group was given comprehensive treatments such as antibiotics,surgical dressing changes(1 time/day),microwave treatment frequency(2 times/day),and topical application of Qingbao San(2 times/day).The treatment group was given Qingre Huatan Sanjie Recipe on the basis of general comprehensive treatment,each Chinese medicine prescription was fried into 200ml decoction,100ml orally twice a day.The course of treatment is one month,and the time of observation:fill in the clinical symptoms and signs observation scores on the 1st and 30th days of treatment,perform puncture biopsy on inflammatory glandular tissues in patients with masses,incision and drainage of abscesses and sinus replacement.Partial glandular tissues were cut from the patients during the drug change,and the concentrations of IgG,IgM,and VEGF were measured by enzyme-linked immunosorbent assay.The intensity of CD4+,CD8+,and NF-kBp65 expression was detected by immunohistochemistry.Results:1.Risk factors:The BMI value of patients with non-lactating mastitis is 17.33～35.29,with an average of 24.8±4.046.54.1%of patients in the non-lactating mastitis group were overweight or underweight.Compared with the health checkers group during the same period,there was a statistical difference(p<0.05).In the non-lactating mastitis group,38.33%of patients had nipple depression,which was higher than that of health checkers,but there was no statistical significance(P>0.05).In non-lactating mastitis group,25%of patients had high levels of prolactin in the peripheral blood,while only 10%of patients in the health checkers had high levels of prolactin.However,there was no statistical significance in the two groups(P>0.05)2.Clinical symptoms,physical scores and efficacy:After 1 month of treatment,the pain score and redness score(1.53±1.45,1.60±1.52)in the treatment group were significantly lower than those in the control group(2.60±1.83,2.66±1.91)(P<0.05),which was statistically significant.After treatment,the mass diameter score of the treatment group(4.00±2.16)was significantly lower than that of the control group(2.40± 1.92),which had significant statistical significance(P<0.01).The sinus score(1.20±1.15)in the treatment group was not statistically significant compared with the control group(1.23±1.16)(P>0.05).Overall effect:In the treatment group,5 cases were cured,8 cases were markedly effective,13 cases were effective,and 4 cases were ineffective.The total efficacy rate was 86.67%.In the control group,2 cases were cured,4 cases were markedly effective,10 cases were effective,and 14 cases were ineffective.The total response rate was 53.33%.The clinical efficacy score of the treatment group(3.73±1.55)was statistically significant compared with the control group(2.20±1.60)(P<0.05).3.Before treatment,non-lactating mastitis patients with simple masses had the highest IgM and IgG content,and those with sinus tract had the lowest IgM and IgG content;while those with abscess(ulcer)had the highest VEGF content and those with simple mass had the lowest VEGF.After treatment,the contents of IgM(51.45±25.78 ug/g)and IgG(996.23±331.17 ug/g)in the tissues of the treatment group were significantly lower than those in the control group(69.68±25.25 ug/g,1404.45±464.77 ug/g)(P<0.05),and the content of VEGF(263.41±215.99 ug/g)was significant lower than that of the control group(491.14±323.65 ug/g)(P<0.01),all were statistically significant.4.CD4+T,CD8+T in inflammatory tissues:In the treatment group,the CD4+T immunohistochemical score(2.70±2.31)of the inflammatory tissue was significantly lower than that of the control group(5.70±3.34),and the CD8+T immunohistochemical score(9.80±4.47)was significantly higher than that of the control group(5.40±2.90).All of them have statistical significance(P<0.05).High power microscope pathological section:Before treatment,non-lactating mastitis gland interstitial cells showed deeper CD4+T and CD8+T positive staining,a higher proportion of CD4+T positive cells,a lower proportion of CD8+positive cells and the ratio of CD4/CD8 is high.After treatment,the staining of CD4+T-positive cells becomes lighter,the proportion of CD4+T-positive cells decreases,the staining of CD8+T-positive cells is deep,the proportion of CD8+T-positive cells increases and the ratio of CD4/CD8 decreases.5.NF-kBp65 in inflammatory tissues:After treatment,the NF-kBp65 immunohistochemical score of inflammatory glandular tissues in the treatment group(2.07±0.97)was significantly lower than that in the control group(4.26±0.65),which was statistically significant(P<0.05)).Pathological sections under high magnification:Before treatment,the cytoplasm or nucleus of non-lactating mastitis mammary duct endothelial cells in the treatment group showed scattered or aggregated brown-yellow particles with deep staining and strong NF-kBp65 expression.After treatment,only a few brown-yellow particles were deposited in the membrane or cytoplasm,and the staining was light,and the expression of NF-kBp65 was weak.Conclusion:1.Observation of general risk factors for non-lactating mastitis shows that overweight or obesity,hyperprolactinemia,and abortion are closely related to non-lactating mastitis to a certain extent,all of which are worth living and clinical attention and need to take Nursing,diet guidance,changing bad lifestyle habits,exercise to lose weight,proper breastfeeding time,avoid abortion and other measures.Early intervention and early prevention have certain significance for reducing the incidence of non-lactating mastitis.2.Qingre Huatan Sanjie Recipe can obviously alleviate the symptoms of breast pain in patients,promote the redness and swelling of breast skin,reduce the mass and lighten the sinus tract,reduce the patient’s pain and improve the clinical efficacy.The prescription is worthy of clinical promotion and further research.3.Qingre Huatan Sanjie Recipe can significantly reduce the content of IgM and IgG in non-lactating mastitis tissues,and effectively reduce the primary humoral immune response and re-immune response in inflammatory tissues,thereby reducing the damage of immune response to glandular tissues.Qingre Huatan Sanjie Recipe can attenuate the expression of CD4+T and enhance the expression of CD8+T,and then suppress the T lymphocyte-mediated immune response in non-lactating mastitis tissues,and enhance the body’s immunity to a certain extent.Finally,it can reduce the clinical symptoms and signs of non-lactating mastitis and promote recovery.4.Qingre Huatan Sanjie Recipe can attenuate the expression of NF-kBp65 in glandular tissues of non-lactating mastitis,and its inhibition of tissue inflammation and immune regulation may be achieved by regulating the activation of NF-kBp65 pathway.更多还原