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医药导报, 2017, 36(12): 1350-1354
doi: 10.3870/j.issn.1004-0781.2017.12.004
少腹逐瘀汤对原发性痛经大鼠作用*
Effect of Shaofu Zhuyu Decoction on Primary Dysmenorrhea in Rats
袁立明1,, 周纤纤2, 杨梦洁1, 欧阳婉欣1, 贾诗杰1, 刘向光1, 毛豪初1, 陶晓军1,, 杨华中1

摘要:

目的 探讨少腹逐瘀汤对原发性痛经大鼠扭体行为、炎症反应及环氧化酶2(COX-2)表达的影响。方法 将50只SD雌性大鼠随机分为空白对照组,模型对照组,少腹逐瘀汤大、中、小剂量组,每组10只。采用苯甲酸雌二醇和缩宫素联合制备痛经大鼠模型,观察少腹逐瘀汤各剂量组对痛经大鼠扭体行为、子宫内膜炎症细胞含量变化、子宫平滑肌COX-2表达的影响。结果 模型对照组大鼠扭体潜伏期缩短,总分较高;子宫内膜及平滑肌可见大量以中性粒细胞为主的炎症细胞浸润;免疫组化子宫平滑肌细胞胞浆内可见棕黄色颗粒,COX-2表达呈阳性。与模型对照组比较,少腹逐瘀汤大、中剂量组SD大鼠扭体潜伏期增长,总分降低;子宫内膜及平滑肌可见极少量炎症细胞浸润,较模型对照组减少(P<0.01);COX-2表达减弱(P<0.01),其中以大剂量组最为显著。与模型对照组比较,小剂量组扭体潜伏期较长,总分较低(P<0.01),子宫内膜及平滑肌有部分炎症细胞浸润,差异无统计学意义(P>0.05);COX-2表达差异无统计学意义(P>0.05)。结论 少腹逐瘀汤治疗原发性痛经可能潜在机制为缓解疼痛、抑制炎症反应、下调COX-2的表达。

关键词: 少腹逐瘀汤 ; 痛经 ; 原发性 ; 环氧化酶2 ; 免疫组化

Abstract:

Objective To investigate the effect of the Shaofu zhuyu decoction on the changes of the body writhing behavior,inflammatory reaction and COX-2 expression of the rats with the primary dysmenorrhea. Methods Fifty SD female rats were randomly divided into blank control group,model control group,high,middle and low dose of Shaofu zhuyu decoction groups,10 in each group.Dysmenorrhea rat model was established by treating with estradiol benzoate and oxytocin.Effect of Shaofu zhuyu decoction of different doses on writhing behavior,changes of endometritis cells and COX-2 expression in uterine smooth muscle of dysmenorrhea rats were observed. Results In the model control group,latency of the body writhing behavior was shortened and the total score was high,many inflammatory cells (especially for neutrophils) infiltrated in endometrium and uterine smooth muscle,immunohistochemistry showed that brown granules were found in the cytoplasm of smooth muscle cells of uterus,and COX-2 expression was positive in uterine smooth muscle cells.As compared with the model control group,writhing latency increased,total score decreased in both the high and middle dose of Shaofu zhuyu decoction groups,and infiltration of a small number of inflammatory cells was seen in the endometrium and smooth muscle (P<0.01);COX-2 expression was decreased (P<0.01) especially for the high dose of Shaofu zhuyu decoction group.As compared with the model control group,the latency of the low dose of Shaofu zhuyu decoction group was significantly increased,and the total score was decreased (P<0.01),but there was no significant difference in inflammatory cell infiltration and COX-2 expression (P>0.05). Conclusion A potential mechanism by which Shaofu zhuyu decotion treats primary dysmenorrhea may be related with alleviating pain,inhibiting inflammatory responses,and down-regulating expression of COX-2.

Key words: decoction ; Dysmenorrhea ; primary ; Cyclooxygenase-2 ; Immunohistochemistry

原发性痛经(primary dysmenorrhea,PD)是指女性在月经前后及行经期间出现下腹疼痛、坠胀,而生殖器官无器质性病变的一种现象[1]。PD一般均认为应归咎于以下几种原因:内膜管型脱落(膜性痛经)、子宫发育不全、子宫屈曲、颈管狭窄等。西医多采用非甾体抗炎药或选择性的环氧化酶2(cyclooxygenase-2,COX-2)抑制药来缓解PD所引起临床症状,而中医在治疗该疾病具有疗效确切、不良反应少及方式方法多样等优点。依据辩证分型论治的观点,PD主要分为气滞血瘀、寒凝血淤、气血虚弱、肾虚血淤、气虚血淤五型,不同的中医证型具有不同的治疗方案[2]。少腹逐瘀汤作为一种传统的中药制剂,已广泛应用于寒凝血淤型PD的临床治疗当中,并且疗效得到同行的认可[3]。然而,关于少腹逐瘀汤治疗PD的作用机制及实验室相关证据相对较少。已有相关文献报道,PD可能与疼痛、炎症反应、COX-2的表达相关,这为本实验的开展提供一定的理论基础[4]。根据目前常用的PD模型建立方法,决定采用腹腔注射缩宫素与苯甲酸雌二醇联合来模拟痛经模型。笔者在本实验中通过观察少腹逐瘀汤对痛经大鼠扭体行为、子宫内膜炎症细胞含量变化和子宫平滑肌COX-2的表达的影响,进而研究少腹逐瘀汤镇痛效应及机制。

1 材料与方法
1.1 动物

雌性健康斯泼累格·多雷(SD)大鼠50只,SPF级,体质量(220±20) g,购自湖南斯莱克景达实验动物有限公司,实验动物合格证号: SYXK(湘)2014-0006。

1.2 药品

少腹逐瘀汤组成:当归9 g,赤芍6 g,川芎6 g,延胡索3 g,蒲黄9 g,干姜3 g,肉桂3 g,小茴香1.5 g,没药3 g,五灵脂6 g,购自湖南中医药大学第一附属医院药剂科,上述10味药,加10倍量水浸泡0.5 h,加热回流煎煮2次,第1次1.5 h,第2次1.0 h,合并煎煮液,浓缩备用。

1.3 主要试剂与仪器

抗体COX-2(D5H5)XPR Rabbit mAb,上海优宁维生物科技有限公司;规格:40 μL;批号:12282p。苯甲酸雌二醇,杭州动物药品场生产,规格:2 mL:4 mg,批号:160305。缩宫素,杭州动物药品场生产,规格:2 mL:10 U,批号:160222。

1.4 实验动物分组及模型制备

50只大鼠按体质量随机分为5组,实验组根据临床研究中使用PD的剂量,同时根据其他中药制剂的实验动物质量进行调整,以对实验组大鼠使用少腹逐瘀汤合适剂量,即少腹逐瘀汤大剂量组(4.8 g·kg-1·d-1)、少腹逐瘀汤中剂量组(2.4 g·kg-1·d-1)、少腹逐瘀汤小剂量组(1.2 g·kg-1·d-1)、 模型对照组(0.9%氯化钠溶液)和空白对照组(0.9%氯化钠溶液)。连续10 d对模型对照组和实验组皮下注射苯甲酸雌二醇,同步大鼠动情周期,同时提高子宫对缩宫素的敏感性(第1,10天皮下给药剂量为每只0.5 mg,其他时间均为每只0.2 mg);空白对照组每日予皮下注射等量0.9%氯化钠溶液。建模第10天,模型对照组和实验组皮下注射苯甲酸雌二醇1 h后,腹腔注射缩宫素(每只2 U);空白对照组腹腔注射缩宫素(每只2 U)。

1.5 大鼠扭体行为的观察

观察40 min内每只大鼠的扭体行为,应用SCHMAUSS等[5]行为学评分标准,将扭体行为分为4个等级:0级为正常姿势(平放于盒底或正常探查行为);1级为身体向左或右偏斜;2级为后肢伸展,后爪背屈,躯体伸展伴频繁的盆骨侧向旋转;3级为腹部肌肉收缩,伴躯体伸展和后肢伸展。行为学总分计算方法,0级:0分,1级:1分,2级:2分,3级:3分,将各级得分相加,即得到总分。记录扭体的潜伏期、次数,计算扭体总分。并且通过两组扭体行为的统计学分析来观察动物模型的建立成功与否。

1.6 大鼠子宫内膜炎症细胞含量测定

采用苏木精-伊红(HE)染色法检测大鼠子宫炎症细胞的含量。染色成功后将所有切片置于在同一条件下进行光学显微镜观察,每张切片随机取5个高倍视野,计算每平方毫米(mm2)内的中性粒细胞、嗜酸性粒细胞、单核巨噬细胞和淋巴细胞等炎症细胞数目。炎性细胞浸润子宫内膜层,组织取材时尽量靠近内膜层取材,同时不可避免的会夹带些子宫基层组织,计数时可以通过平滑肌细胞的形态来区分内膜层和肌层。

1.7 大鼠子宫平滑肌COX-2的含量测定

取出4%甲醛溶液固定24 h的子宫标本进行石蜡包埋,然后置于切片机上连续4 μm切片并贴片。放入60 ℃恒温箱烤片1 h后取出,采用免疫组织化学技术检测大鼠子宫平滑肌COX-2的表达情况。用磷酸盐缓冲溶液(PBS)代替一抗作为空白对照,染色成功后所有切片均在同一条件下进行光学显微镜观察。结果以胞质中有棕黄色或褐色颗粒状物的细胞为阳性细胞,每张切片随机选取高倍视野5个(×400),计算每平方毫米(mm2)内的平均阳性细胞数和阳性率。

1.8 统计学方法

采用SPSS20.0版统计软件进行统计学处理,所有数据以均数±标准差( x ¯ ±s)表示,各组间均数采用单因素方差分析(one-way ANOVA),两组间均数以最小显著性差异法(least-significant difference,LSD)检验,以P<0.05为差异有统计学意义。

2 结果
2.1 扭体行为的比较结果

表1。

表1 5组大鼠扭体行为的比较
Tab.1 Comparison of writhing times among five groups of rats x¯±s,n=10
组别 剂量/
(g·kg-1·d-1)
潜伏期/
min
40 min内扭体
数/次
总分/
空白对照组 8.90 ±1.85 8.80±1.93 17.30±2.54
模型对照组 6.00±1.05*1 18.30±2.21*1 37.40±7.12*1
少腹逐瘀汤
大剂量组 4.8 12.40±1.90*2 6.90±1.73*2 11.90±3.07*2
中剂量组 2.4 10.10±1.20*2 9.30±2.31*2 18.50±6.33*2
小剂量组 1.2 8.60±1.27*2 10.80±2.53*2 24.60±6.40*2
F 24.31 41.57 32.20

Compared with blank control group,*1P<0.01; compared with model control group,*2P<0.01

与空白对照组比较,*1P<0.01;与模型对照组比较,*2P<0.01

表1 5组大鼠扭体行为的比较

Tab.1 Comparison of writhing times among five groups of rats x¯±s,n=10

2.2 子宫炎症细胞含量比较

空白对照组未见明显炎症细胞;模型对照组可见大量炎症细胞浸润;少腹逐瘀汤大剂量组可见极少量炎症细胞;少腹逐瘀汤中剂量组可见较少量炎症细胞;少腹逐瘀汤小剂量组可见部分炎症细胞。见表2,图1。

表2 5组大鼠子宫内膜炎症细胞数的比较
Tab.2 Comparison of inflammatory cell numbers in uterus endometrium among five groups of rats 个,\(\overline{x}\)±s,n=10
组别 剂量/
(g·kg-1·d-1)
炎症细胞总数 中性粒细胞 嗜酸性粒细胞 单核巨噬细胞 淋巴细胞
空白对照组 15.00±18.64 9.60±11.65 0.20±1.08 0.80±1.29 4.00±4.97
模型对照组 93.00±7.63*1 60.90±4.79*1 1.50±0.42*1 6.10±0.42*1 24.70±2.11*1
少腹逐瘀汤
大剂量组 4.8 18.60±14.74*2 12.20±9.67*2 0.20±0.42*2 0.90±0.99*2 5.10±3.87*2
中剂量组 2.4 66.20±22.32*2 43.50±14.47*2 1.00±0.82*2 4.30±1.77*2 17.60±6.04*2
小剂量组 1.2 81.10±17.93*2 53.20±12.92*2 1.30±0.82*2 5.50±1.65*2 21.50±22.94*2
F 37.50 38.57 6.36 31.50 36.73

Compared with blank control group,*1P<0.01; compared with model control group,*2P<0.01

与空白对照组比较,*1P<0.01;与模型对照组比较,*2P<0.01

表2 5组大鼠子宫内膜炎症细胞数的比较

Tab.2 Comparison of inflammatory cell numbers in uterus endometrium among five groups of rats 个,\(\overline{x}\)±s,n=10

图1 5组大鼠子宫内膜炎症细胞的含量(HE染色,×400)
A.空白对照组;B.模型对照组;C.少腹逐瘀汤大剂量组;D.少腹逐瘀汤中剂量组;E.少腹逐瘀汤小剂量组

Fig.1 Inflammatory cells contents in uterus endometrium of five groups of rats (HE staining,×400)
A.blank control group;B.model control group;C.high-dose Shaofu zhuyu decoction group;D.medium-dose Shaofu zhuyu decoction group;E.low-dose Shaofu zhuyu decoction group

2.3 大鼠子宫平滑肌COX-2的表达情况比较

图2,表3。

图2 5组大鼠子宫平滑肌上COX-2表达(免疫组织化学法,×400)
A.空白对照组;B.模型对照组;C.少腹逐瘀汤大剂量组;D.少腹逐瘀汤中剂量组;E.少腹逐瘀汤小剂量组

Fig.2 COX-2 expression in uterine smooth muscle of five groups of rats (immunohistochemical staining,×400)
A.blank control group;B.model control group;C.high-dose Shaofu zhuyu decoction group;D.medium-dose Shaofu zhuyu decoction group;E.low-dose Shaofu zhuyu decoction group

表3 5组大鼠子宫平滑肌COX-2的表达比较
Tab.3 Comparison of COX-2 expression in uterine smooth muscle among five groups of rats x¯±s,n=10
组别 剂量/
(g·kg-1·d-1)
阳性细胞数/
阳性率/
%
空白对照组 13.40±13.46 6.00±6.29
模型对照组 77.60±19.27*1 35.00±8.49*1
少腹逐瘀汤
大剂量组 4.8 41.20±5.00*2 18.40±2.41*2
中剂量组 2.4 37.80±27.82*2 17.20±12.52*2
小剂量组 1.2 73.00±12.50*2 32.80±5.54*2
F 11.81 11.85

Compared with blank control group,*1P<0.01; compared with model control group,*2P<0.01

与空白对照组比较,*1P<0.01;与模型对照组比较,*2P<0.01

表3 5组大鼠子宫平滑肌COX-2的表达比较

Tab.3 Comparison of COX-2 expression in uterine smooth muscle among five groups of rats x¯±s,n=10

3 讨论

近期有相关学者发现,关于PD的病理生理学机制可能与下列相关因素有关:机械因素、内分泌因素及其他相关性因素[6]。针对其发病机制,目前相关学者在临床上开展了一系列关于PD的实验探讨,其中中药制剂少腹逐瘀汤得到大部分中医学者的认可,并已经广泛应用于临床实践当中。尽管如此,少腹逐瘀汤治疗PD的实验室数据仍略显不足[7-8]。针对这个现象,本研究从疼痛反应、炎症反应以及COX-2的表达着手进行实验,通过腹腔注射缩宫素引起大鼠子宫平滑肌剧烈收缩模拟痛经并观察其扭体行为,分别采用HE染色和免疫组织化学方法来显示炎症细胞在大鼠子宫内膜和COX-2在大鼠子宫平滑肌的分布情况。研究发现实验组大鼠扭体潜伏期延长、扭体次数、扭体总分与模型对照组比较均减少,表明少腹逐瘀汤对大鼠痛经能起到一定的镇痛效应。研究表明,痛经作为一种炎症反应过程,其发病机制可能与相关炎性因子有关[9]。本实验也显示少腹逐瘀汤剂量组大鼠子宫内膜及平滑肌内炎症细胞数目有不同程度的减少,这表明少腹逐瘀汤在一定程度上能够抑制炎症因子的增长,从而达到镇痛效应。

近年来,国内外学者对PD病理生理学方面的研究较多,目前主要认为PD与前列腺素以及与合成前列腺素相关的酶有关[10-12]。环氧化酶是在炎症反应中具有重要作用的酶[13]。COX-2属于环氧化酶家族中的诱导型酶,也是PGE2等炎症性PGs增加的主要原因。生理状态下,COX-2在大部分组织细胞中没有表达,但在机体出现组织损伤、炎症、肿瘤等情况下显著增强。本研究通过免疫组织化学方法探讨COX-2在大鼠子宫平滑肌内的表达,发现少腹逐瘀汤各剂量组对COX-2在大鼠子宫平滑肌中的表达有不同程度的抑制,并以高、中剂量组为佳,证明了少腹逐瘀汤对PD起到镇痛效应的另一种可能机制为抑制COX-2在大鼠子宫平滑肌内的表达。西医中选择性COX-2抑制药虽能够特异性抑制COX-2表达,但是可发生心血管不良事件,少腹逐瘀汤作为一种传统的中药制剂已经在临床实践中体现其优良的实用价值,然而其机制上的理论研究尚不充分,故本研究的开展为我国中药制剂的推广能够起到一定的启示作用。笔者在本研究中从剂量反应上揭示了少腹逐瘀汤对PD子宫平滑肌COX-2表达的情况,为中医药缓解PD的疼痛机制进行了进一步的阐述。

虽然本研究在少腹逐瘀汤对PD的镇痛效应方面上提供了一定的实验证据,但是对与PD密切相关的前列腺素PGE2和PGF分泌的影响不明确,尚需进一步研究。

The authors have declared that no competing interests exist.

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[5] SCHMAUSS C,YAKSH T L.In vivo studies on spinal opiate receptor systems mediating antinociception.II.Pharmacological profiles suggesting a differential association of mu,delta and kappa receptors with visceral chemical and cutaneous thermal stimuli in the rat.[J].J Pharm Exp Ther,1984,228(1):1-12.
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[6] 谢丹,居明乔,曹鹏,.炎症因子和原发性痛经相关性的研究进展[J].中国临床药理学与治疗学,2014,19(3):346-350.
原发性痛经是最常见的妇科疾病之一,因其严重影响工作和生活而备受关注。近年来国内外对其研究不断深入,发现炎性因子在原发性痛经的发病过程中发挥了重要作用。本文就炎性因子对原发性痛经的影响及原发性痛经的治疗新进展进行综述。
Magsci    
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[7] 孙桂芳,王佩娟.少腹逐瘀汤治疗原发性痛经的研究进展[J].现代中西医结合杂志,2012,21(11):1251-1252.
正原发性痛经指经期及其前后下 腹部痉挛性疼痛,而生殖器官并没有器质性病变。西医多用非甾体类抗炎药等治疗本病,但诸多不良反应限制了其应用。中医治疗本病经验丰富,形成了许多有效的 方剂,少腹逐瘀汤就是其中一首经典方。该方由清代名医王清任首创,见载于《医林改错》。方中小茴香、肉桂、干姜温经通络,通调气血,当归、川芎、赤芍、没
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[8] SU S,DUAN J,CUI W,et al.Network-based biomarkers for cold coagulation blood stasis syndrome and the therapeutic effects of Shaofu zhuyu decoction in rats[J].Evid Complem Altern Med : eCAM,2013,2013(2/4):901-943.
Abstract In this study, the reverse docking methodology was applied to predict the action targets and pathways of Shaofu Zhuyu decoction (SFZYD) bioactive ingredients. Furthermore, Traditional Chinese Medicine (TCM) cold coagulation blood stasis (CCBS) syndrome was induced in female Sprague-Dawley rats with an ice-water bath and epinephrine, and SFZYD was used to treat CCBS syndrome. A metabolomic approach was used to evaluate changes in the metabolic profiles and to analyze the pharmacological mechanism of SFZYD actions. Twenty-three potential protein targets and 15 pathways were discovered, respectively; among these, pathways are associated with inflammation and immunological stress, hormone metabolism, coagulation function, and glycometabolism. There were also changes in the levels of endogenous metabolites of LysoPCs and glucuronides. Twenty endogenous metabolites were identified. Furthermore, the relative quantities of 6 endogenous metabolites in the plasma and 5 in the urine were significantly affected by SFZYD (P < 0.05). The pharmacological mechanism of SFZYD was partially associated with glycerophospholipid metabolism and pentose and glucuronate interconversions. In conclusion, our findings demonstrated that TCM CCBS pattern induced by ice water and epinephrine was complex and related to multiple metabolic pathways. SFZYD did regulate the TCM CCBS by multitargets, and biomarkers and SFZYD should be used for the clinical treatment of CCBS syndrome.
DOI:10.1155/2013/901943      PMID:24288569      URL    
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[9] DAWOOD M Y.Primary dysmenorrhea: advances in path-ogenesis and management[J].Obstet Gyn,2006,108(2):428-441. [本文引用:1]
[10] HARELZ Z.Dysmenorrhea in adolescents and young adults: from pathophysiology to pharmacological treatments and management strategies[J].Exp Opin Pharmacoth,2008,9(15):2661-2672.
Abstract BACKGROUND: Dysmenorrhea is the most common gynecologic complaint among adolescent and young adult females. Dysmenorrhea is usually primary and is associated with normal ovulatory cycles and with no pelvic pathology. In approximately 10% of females with severe dysmenorrhea symptoms, pelvic abnormalities such as endometriosis or uterine anomalies may be found. OBJECTIVE: To review the current knowledge regarding the pathophysiology of dysmenorrhea, as well as review pharmacological treatments and strategies for management of dysmenorrhea in adolescent and young adult females. METHODS: Review of original articles on dysmenorrhea that have been published in the medical literature. RESULTS/CONCLUSIONS: Potent prostaglandins and potent leukotrienes play an important role in generating primary dysmenorrhea symptoms. Non-steroidal anti-inflammatory drugs (NSAIDs) are the most common pharmacologic treatment for dysmenorrhea. A loading dose of NSAIDs (typically twice the regular dose) should be used as initial treatment for dysmenorrhea, followed by a regular dose until symptoms abate. Adolescents and young adults with symptoms that do not respond to treatment with NSAIDs for three menstrual periods should be offered hormonal treatment such as combined estrogen/progestin oral contraceptive pills (OCPs) for three menstrual cycles. If dysmenorrhea does not improve within 6 months of treatment with NSAID and OCPs, a laparoscopy is indicated to look for endometriosis. The goal of pharmacological treatment for endometriosis is to block its abnormal positive feedback loop. The abnormal loop consists of high local levels of estrogen in the lesions, which induce transcription of COX-2 and synthesis of prostaglandin E(2.) This results in further expression and activity of aromatase and a further increase in estrogen.
DOI:10.1517/14656566.9.15.2661      PMID:18803452      URL    
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[11] CHAN W Y,DAWOOD M Y,FUCHS F.Prostaglandins in primary dysmenorrhea: comparison of prophylactic and nonprophylactic treatment with ibuprofen and use of oral contraceptives[J].Am J Med,1981,70(3):535-541.
DOI:10.1016/0002-9343(81)90576-3      URL    
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[12] DMITROVIC R,KUNSELMAN A R,LEGRO R S.Sildenafil-citrate in the treatment of pain in primary dysmenorrhea: a randomized controlled trial[J].Hum Reprod,2013,28(11):2958-2965.
Abstract STUDY QUESTION: Is a vaginal preparation of sildenafil citrate capable of alleviating acute menstrual pain in patients with primary dysmenorrhea (PD)? SUMMARY ANSWER: A vaginal preparation of sildenafil citrate is capable of alleviating acute menstrual pain in patients with PD with no observed adverse effects. WHAT IS KNOWN ALREADY: Oral preparations of nitric oxide (NO) donor drugs augment relaxant effects of NO on myometrial cells, reverse the vasoconstriction caused by prostaglandins and successfully alleviate pain, but the incidence of side effects is too high for routine clinical use. Sildenafil citrate inhibits type 5-specific phosphodiesterase (PDE5), thus preventing the degradation of cyclic guanosine monophosphate (cGMP) in the muscle and augmenting the vasodilatory effects of NO. Therefore, by inhibiting PDE5, the tissue remains relaxed and more blood can circulate through. It has been used previously in a vaginal form with no observed side effects, and it enhances endometrial blood flow. STUDY DESIGN, SIZE, DURATION: A double-blind, randomized, controlled trial comparing vaginal preparation of sildenafil citrate (100 mg single dose) to a placebo in 62 PD patients at the time of painful menstruation was conducted. The primary outcome was total pain relief over 4 consecutive hours (TOPAR4) comparing sildenafil citrate to placebo, where higher TOPAR4 scores represent better pain relief. Secondary outcomes were pain relief as measured by the visual analog scale (VAS) and uterine artery pulsatility index (PI). Subjects were recruited from December 2007 to January 2011. The trial was stopped due to closeout of the funding for the study. PARTICIPANTS, SETTINGS, METHODS: Participants were women in good health, were aged 18-35 years and suffered from moderate to severe PD. They were randomized to either vaginal placebo or 100 mg vaginal sildenafil citrate in a 1:1 ratio using random permuted blocks having a block size of 4. At baseline and 1, 2, 3, and 4 h post-treatment, patients were asked to provide assessment of their degree of pain using two scales: (i) pain on the 5-level ordinal scale used for TOPAR4 calculation and (ii) pain level on the VAS. The study ended 4 h after treatment initiation. MAIN RESULTS AND THE ROLE OF CHANCE: Twenty-five subjects completed the study. Using the TOPAR4 score, the sildenafil citrate group had significantly better pain relief compared with the placebo group [mean (SD): 11.9 (3.2) versus 6.4 (2.1), respectively; difference in means = 5.3; 95% CI: (2.9,7.6); P < 0.001)]. On the VAS, sildenafil citrate provided better pain relief than placebo at each time point. At the 2-h time point, the PI was significantly lower in the sildenafil citrate group compared with the placebo group [mean (SD): 1.6 (0.6) versus 2.3 (0.5), respectively; difference in means = -0.7; 95% CI: (-1.2, -0.1); P = 0.01)]. LIMITATIONS, REASONS FOR CAUTION: Since we did not meet our sample size due to the loss of funding and could not confirm our primary hypothesis, larger studies of longer duration, likely multi-center, are needed to confirm the findings from this study. WIDER IMPLICATIONS OF THE FINDINGS: A number of medications have been investigated to improve the treatment options for PD, but most have proven unsuccessful or to have an unfavorable risk/benefit ratio. Since PD is a condition that most women suffer from and seek treatment for at some point in their lives, our study offers hope that vaginal sildenafil citrate is a safe and effective option for patients who do not desire or are unresponsive to treatments now available on the market. STUDY FUNDING/COMPETING INTERESTS: Funding for this study was provided by National Institutes of Health (NIH) grants RO3 TW007438 and K24 HD01476. The authors report no relevant conflicts of interest. TRIAL REGISTRATION NUMBER: NCT00123162 (Clinical trials.gov).
DOI:10.1093/humrep/det324      URL    
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[13] COON D,GULATI A,COWAN C,et al.The role of cyclo-oxygenase-2 (COX-2) in inflammatory bone resorption[J].J Endod,2007,33(4):432-436.
Prostaglandin E2 (PGE(2)) is an important inflammatory mediator that plays an essential role in the development and progression of periradicular diseases. Cyclooxygenase-2 (COX-2) is the inducible enzyme responsible for increased PGE(2) levels during inflammation and other pathologic processes. The purpose of this study was to determine the role of COX-2-mediated PGE(2) synthesis in osteoclast formation in response to endodontic pathogens and materials. Primary osteoblast cultures and osteoclast cultures were prepared from COX-2 knockout (K/O) and wild-type (WT) littermates. These cultured cells were exposed to lipopolysaccharide (LPS) or root canal obturation materials including gutta-percha (GP), Resilon (RS), mineral trioxide aggregates (MTAs), and AH Plus (AH+). Osteoclast formation was evaluated using tartrate-resistant acid phosphatase (TRAP) staining. The expression of receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG) was determined by real-time polymerase chain reaction (PCR) analysis. It was found that in both WT and K/O cultures, treatment with LPS led to a marked increase in osteoclast formation. The number of osteoclasts formed was significantly lower in K/O cultures compared to WT cultures. Exposure to endodontic materials did not lead to any significant osteoclast formation. LPS and endodontic materials caused a decrease in both RANKL and OPG expression in WT cells. In K/O cells, the baseline levels of RANKL and OPG expression were dramatically decreased compared to the WT cells. In conclusion, COX-2-mediated PGE(2) expression is required for LPS-induced inflammatory bone resorption and maintaining the baseline level of RANKL and OPG expression. LPS-induced osteoclast formation may be independent of the RANKL pathway.
DOI:10.1016/j.joen.2006.12.001      PMID:17368333      URL    
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关键词(key words)
少腹逐瘀汤
痛经
原发性
环氧化酶2
免疫组化

decoction
Dysmenorrhea
primary
Cyclooxygenase-2
Immunohistochemistry

作者
袁立明
周纤纤
杨梦洁
欧阳婉欣
贾诗杰
刘向光
毛豪初
陶晓军
杨华中

YUAN Liming
ZHOU Xianxian
YANG Mengjie
OUYANG Wanxin
JIA Shijie
LIU Xiangguan
MAO Haochu
TAO Xiaojun
YANG Huazhong