化浊解毒活血方治疗子宫内膜异位症的临床及体外研究
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摘要
目的:
⒈大约7%~10%的生育年龄妇女患有子宫内膜异位症,其中71%~87%患者合并有慢性盆腔疼痛,65.6%~77.3%患者合并有痛经。可见,慢性盆腔疼痛和痛经是子宫内膜异位症患者最常见、最痛苦的临床症状之一,严重影响了患者生活质量。然而对于疼痛,目前尚缺乏可靠的评价指标及有效的治疗手段。因此,本部分研究旨在探讨化浊解毒活血方对子宫内膜异位症伴疼痛患者的治疗作用及可能的作用机制。
(1)检测子宫内膜异位症伴疼痛患者月经期血浆孤啡肽(OFQ)及前列腺素(PGF2a、PGE2、6-keto-PGF1a、TXB2 )的含量变化,并探讨其临床意义,以及化浊解毒活血方对它们的影响。
(2)观察化浊解毒活血方对子宫内膜异位症伴疼痛患者的疼痛改善及不良反应、血液流变学、血清内分泌激素的影响,从而探讨化浊解毒活血方可能的作用机制。
⒉虽然我们大都知道,血管内皮生长因子(VEGF)在子宫内膜异位症发生、发展中起着极其重要的作用,但是,VEGF受体特别是VEGFR-1(Flt-1)并未引起足够重视,而可溶性受体sFlt-1与VEGF之间的关系及它们在子宫内膜异位症血管形成中的作用是非常值得研究的课题,目前尚未见报道。
关于子宫内膜异位症的治疗,至今也未找到一种好的治疗手段。现今常用的药物如口服避孕药、达那唑、促性腺激素释放激素激动剂(GnRH-a)等,通过降低体内雌激素状态而达到治疗目的,因此副作用较多且较严重。如服用达那唑导致的男性化,应用GnRH-a导致骨质疏松等围绝经期综合征表现等,这就预示着患者只能短期用药,而一旦停药,5年后50%患者复发。值得庆幸的是,中医药具有很多优势。著名中医药专家李佃贵教授根据多年经验积累,从“浊、毒、瘀”论治,采用化浊解毒活血方治疗子宫内膜异位症,取得了很好疗效。本部分研究旨在探讨子宫内膜异位症患者血清、腹腔液中游离VEGF、sFlt-1的水平和临床意义,以及化浊解毒活血方对它们的影响。
⒊为了从细胞水平进一步研究化浊解毒活血方的作用机制,建立体外细胞模型是非常必要的。并利用患者的药物血清进行干预,观察化浊解毒活血药物血清(简称HJH血清)对异位子宫内膜细胞生长、增殖、凋亡及分泌VEGF的影响。
方法:
第一部分:子宫内膜异位症无疼痛对照组25例,子宫内膜异位症轻度疼痛、中重度疼痛各31例。近3个月未曾用其它药物(如达那唑等)治疗。给予化浊解毒活血中药(藿香、佩兰叶、清夏、陈皮、茯苓、胆南星、白花蛇舌草、红景天、田基黄、丹参、元胡、五灵脂、当归)水煎400ml,分早、晚各服200ml,3个月1疗程。治疗前后观察下列指标:
1.采用放射免疫法(RIA)测定血浆OFQ及PGs(PGF2a、PGE2、6-keto-PGF1a、TXB2)含量。
2.分别在治疗1、2、3个月后,采用视觉模拟疼痛评分(VAS)对痛经及慢性盆腔疼做出评价,同时进行副作用观察。
3.分别在治疗前、治疗后3个月,测定血液流变状态及血清FSH、LH、PRL、P及E2水平。
第二部分:对28例子宫内膜异位症患者,采用酶联免疫吸附法(ELISA)检测血清、腹腔液中游离VEGF和sFlt-1的水平。设正常对照组10例。
第三部分:采用化浊解毒活血方,治疗子宫内膜异位症患者20例,应用ELISA法检测血清游离VEGF和sFlt-1的水平,并计算VEGF活性指数。
第四部分:体外研究
1.子宫内膜异位症患者1名,口服化浊解毒活血方水煎剂后,采血制备成药物血清(简称HJH血清);健康志愿者1名,采血制备成空白血清。
2.离体培养20例子宫内膜异位症患者的异位子宫内膜细胞。
3.对离体培养的子宫内膜异位症患者的异位子宫内膜细胞,采用HJH血清(10%HJH、20%HJH)进行干预,氮蓝四唑盐比色法(MTT)观察异位内膜细胞的生长曲线变化;流式细胞技术(FCM),观察HJH作用24h、48h后异位内膜细胞增殖和凋亡情况;ELISA法测定HJH作用48h后VEGF浓度。
结果:
第一部分:
⒈87例子宫内膜异位症患者中有85例完成了本项观察。
子宫内膜异位症疼痛患者经期血浆OFQ、PGF2a、PGE2、6-keto-PGF1a、TXB2含量均显著高于无疼痛患者(P<0.01),6-keto-PGF1a /TXB2比值显著低于无疼痛患者(P<0.01);子宫内膜异位症中重度疼痛组血浆OFQ、PGF2a、PGE2含量均明显高于轻度疼痛组(P<0.05)。子宫内膜异位症疼痛组经化浊解毒活血中药治疗后,OFQ、PGF2a、PGE2含量显著下降,6-keto-PGF1a水平显著升高(P<0.01)。
⒉62例子宫内膜异位症伴疼痛患者中有45人接受并完成本向观察。
治疗第1~2周有三分之一病人出现轻度腹泻,大便次数增多至每日2-3次,继续服药症状消失。未出现其它任何不良反应。复查肝、肾功能正常。化浊解毒活血中药治疗第1个月后,痛经及慢性盆腔疼痛即有明显改善,随着治疗时间的延长,疼痛评分逐渐下降,其中治疗后2个月的VAS评分明显低于治疗前(P<0.05)。治疗3个月后,痛经VAS评分由治疗前的(6.23±2.12)下降到(4.01±1.34),慢性盆腔疼痛由治疗前的(4.92±1.67)下降到(4.47±1.52)(P<0.01)。
⒊62例子宫内膜异位症伴疼痛患者中资料齐全者32例。
经化浊解毒活血治疗后发现,子宫内膜异位症伴疼痛患者血液粘滞度和红细胞压积均有明显降低,并有统计学意义(P<0.01)。
⒋62例子宫内膜异位症伴疼痛患者中资料齐全者30例。
经化浊解毒活血治疗后发现,子宫内膜异位症伴疼痛患者血清FSH、LH、PRL、E2水平有不同程度的下降(P<0.05),对T和P的影响无统计学意义。
第二部分:28例子宫内膜异位症患者腹腔液、血清中VEGF、sFlt-1及VEGF活性指数的表达情况。
1.子宫内膜异位症患者腹腔液VEGF、sFlt-1水平明显高于对照组﹙P<0.01)。Ⅲ~Ⅳ期VEGF水平高于Ⅰ~Ⅱ期﹙P<0.05)。对照组、Ⅰ~Ⅱ期和Ⅲ~Ⅳ期VEGF活性指数分别为0.310、0.276和0.273。
2.子宫内膜异位症增生期腹腔液VEGF及sFlt-1水平均明显高于分泌期﹙P<0.01),增生期VEGF活性指数为0.191,分泌期为1.185。
3.子宫内膜异位症患者血清VEGF、sFlt-1水平明显高于对照组﹙P<0.01),且Ⅲ~Ⅳ期VEGF、sFlt-1水平高于Ⅰ~Ⅱ期﹙P<0.01)。对照组、Ⅰ~Ⅱ期和Ⅲ~Ⅳ期VEGF活性指数分别为0.366、0.380和0.508。
4.子宫内膜异位症增生期血清VEGF及sFlt-1水平均明显高于分泌期﹙P<0.01),增生期VEGF活性指数为0.531,分泌期为0.368。
第三部分:化浊解毒活血中药治疗后,子宫内膜异位症患者血清VEGF的下降率为100%。血清VEGF含量由治疗前的(268.1±30.1)pg/ml,下降到治疗后(268.1±30.1)pg/ml,具有统计学意义(P<0.01)。血清sFlt-1含量治疗后为(616±41.1)pg / ml,虽然较治疗前(637±24.3)pg/ml略有下降,但无统计学意义(P>0.05)。VEGF活性指数治疗后为0.371,较治疗前(0.421)有所下降。
第四部分:体外研究部分。20例内异症患者异位内膜细胞经培养,11例成功,9例失败,成功率为55%。
1.HJH血清对体外培养的异位子宫内膜细胞的抑制作用明显高于空白血清对照组(P<0.05)。HJH血清作用后的细胞数,呈现完全的浓度和时间相关性,即随着HJH浓度增加和作用时间的延长,异位内膜细胞生长率呈下降趋势,MTT法显示细胞生长曲线出现下降改变。10%HJH血清和20%HJH血清之间,作用24h(第5日)和作用48h(第6日)之间比较,均有统计学意义(P<0.05)。
2.HJH血清作用24h后HJH组细胞增殖活性均高于空白对照组(P<0.05),但10%HJH与20%HJH两个浓度之间无差别。HJH血清作用48h后,HJH组增殖活性明显下降并低于对照组,且呈浓度依赖性,即20%HJH组增殖活性(0.735±0.059)低于10%HJH组(0.846±0.048),并有统计学意义(P<0.01)。
3.HJH血清作用48h后,流式细胞技术结果显示,10%HJH、20%HJH组的细胞凋亡指数分别为37.25±8.1和47.7±8.7,明显高于空白对照及相同浓度的空白血清对照,并且20%HJH组凋亡指数明显高于10%HJH组,差异有统计学意义(P<0.05)。
4.HJH药物血清作用48h后,ELISA法测定VEGF显示,10%HJH、20%HJH组的VEGF含量分别为(175.4±24.3)pg/ml和(135.4±26.1)pg/ml,与空白对照及空白血清对照比较,HJH血清组VEGF量明显降低,且VEGF在20%浓度时比10%浓度下降明显,并有统计学意义(P<0.05)。
结论:
⒈血浆OFQ和PGs水平与子宫内膜异位症患者疼痛的发生密切相关。血浆OFQ的测定简单方便,有望成为评价子宫内膜异位症疼痛程度及临床疗效的指标。
⒉化浊解毒活血方治疗子宫内膜异位症疼痛的作用机制:化浊解毒活血方可恢复患者正常气血运行,改善血液流变学,降低血液粘稠度,调整内分泌紊乱,降低血浆OFQ及PGF2a、PGE2水平,从而缓解疼痛,改善临床症状。
⒊本研究首次观察了VEGF及sFlt-1在子宫内膜异位症患者腹腔液、血清中的含量,并采用二者的比值计算了VEGF活性指数,从一全新角度探索研究了VEGF在子宫内膜异位症血管形成中的作用机制认为:
(1)sFlt-1可能存在中和、下调VEGF的作用。
(2)VEGF、sFlt-1在子宫内膜异位症腹腔液和血清中高表达,并与病情的进展程度成正相关。
(3)联合测定血清中VEGF、sFlt-1及VEGF活性指数,有助于子宫内膜异位症诊断、疗效判断及预后监测。
(4)VEGF及sFlt-1有可能成为阻断子宫内膜异位症血管形成的靶点。
⒋化浊解毒活血方治疗子宫内膜异位症的抗血管形成机制:
(1)化浊解毒活血方可降低子宫内膜异位症患者血清VEGF含量及VEGF活性指数。
(2)化浊解毒活血方可通过抑制VEGF的分泌,抑制异位子宫内膜细胞生长并诱导其凋亡,从而促使异位内膜萎缩,以实现对子宫内膜异位症的治疗作用。
Objective:
1. Endometriosis affects approximately 7%–10% of all women.71%–87% of patients with chronic pelvic pain(CPP),and 65.6%～77.3%of patients with dysmenorrhea. Chronic pelvic pain and dysmenorrhea are the hallmark symptoms experienced by affected women.Their survival quality is affected seriously.However,to pain,we have no found a better index to evaluate it, and effective method to treat.So,in this part of study,based on the treatment of endometriosis-associated pain with Huazhuo Jiedu Huoxue Recipe(HJH),to investigate the mechanism of pains in endometriosis.
(1) Detect the changes of plasma orphanin FQ and prostaglandins (PGF2a、PGE2、6-keto-PGF1a and TXB2 ) from patients of endometriosis with pain during menstruation,and the effect of Huazhuo Jiedu Huoxue Recipe on them. aim at understanding their clinical significance.
(2) Patient’s degree of pain and side effects will be observed.In addition, the blood hemorrheology and serum endocrine hormones are going to be monitored before and after the administration at the conclusion of the study.
2. Although we all know that vascular endothelial growth factor(VEGF) play a most important role in development of endometriosis, its receptor especially VEGFR-1(Flt-1) has not been payed enough attention. Further more, soluble form of VEGF receptor (sFlt-1) was seldom reported. Actually, to research about the relationship between VEGF and sFlt-1 from the viewpoint of angiogenesis is very valuable.
About the treatment of endometriosis,we also have had no nice method. Although there are several medical treatments available including oral contraceptives, danazol,and GnRH agonists,etc,these treatments attempt to create a hypoestrogenic state since estrogen is believed to be a contributing factor to endometriosis.Masculinizing side effects have essentially rendered Danazol a treatment of the past.Menopausal side effects,includ-ing osteoporosis,that are associated with GnRH agonists make them only short-term solutions for the treatment of endometriosis.The recurrence rate as long as 50% after stopping the treatment. Luckly, Traditional Chinese Medicine can bring us many advantages.The famous professor Diangui Li according to his experience based on“turbidity,toxin,stasis”.After have Huazhuo Jiedu Huoxue Recipe used on patients with endometriosis, nice clinical effects were found. In this part, levels of free VEGF and sFlt-1 in peritoneal fluid and serum will be measured,as well as the effect of Huazhuo Jiedu Huoxue Recipe on them.
3. To understand the mechanism of Huazhuo Jiedu Huoxue Recipe in treating endometriosis better, studying in vitro is very necessary.By serum pharmacology,to determine the effect of Huazhuo Jiedu Huoxue on the growth, proliferation, apoptosis,and VEGF secretion of cultured ectopic endometrial cells with endometriosis .
Methods:
Part one: 31 patients of endometriosis( EM) with moderate or severe pain ( severe group) , 31 patients with slight pain(slight group) and 25 patients without pain (control group) were studied. They were treated with Huazhuo Jiedu Huoxue Recipe (composed of Huoxiang, Peilan, Qingxia, Chenpi, Fuling, Baihuasheshecao, Hongjingtian, Tianjihuang, Danshen , Yuanhu, Wulingzhi and Danggui, etc).Decoction 400ml ,at 2 dose of 200ml,b.i.d. 3 months constituting a therapeutic course and these patients without taking western medicine,such as danazol,within and before 3 months.Before and after 3 months of the treatment, the clinical data would be obtained as follows: Radioimmunoassay(RIA) could be used to evaluate the concentrations of plasma orphanin FQ and prostaglandins(PGs),such as PGF2a、PGE2、6-keto-PGF1a and TXB2 during menstruation.Patient’s degree of pain would be quantified by visual analogue pain scale at baseline,months 1,2,and3,respectively.Also side effects were observed. In the meantime, the blood hemorrheology and serum endocrine hormones were monitored before and after the administration at the conclusion of the study.
Part 2: Levels of free VEGF and sFlt-1 were measured by enzyme-linked immunosorbent assay (ELISA) in peritoneal fluid and serum from 28 subjects with surgically confirmed endometriosis, and 10 controls with no clinical evidence of the disease and other diseases. Meanwhile, we calculated a VEGF activity index by means of the ratio VEGF/ sFlt-1.
Part 3: Levels of free VEGF and sFlt-1 were measured by enzyme-linked immunosorbent assay in serum from 20 cases of endometriosis after the administration with Huazhuo Jiedu Huoxue Recipe. A VEGF activity index also be calculated.
Part 4: To investigate the influence of HJH on the growth of ectopic endometrial cells with endometriosis in vitro, we observed the growth of cells by MTT method firstly,then, the apoptosis and proliferate was assessed by flowcytometry, and VEGF was quantified in the media by ELISA.Blank serum from a healthy volunteer was prepared. A patient with endometriosis was given Huazhuo Jiedu Huoxue(HJH)decoction for three days in order to prepare serum containing HJH in the early morning of the fourth day after preprandial administration of HJH. Ectopic tissue was obtained from 20 patients with endometriosis. The stromal and epithelial cells of ectopic were isolated and cultured in vitro. The cells were divided into five groups randomly and treated with different culture medium on the fourth day. Blank control group was in serum-free condition.Blank serum groups were given blank serum at the concentrations of 10% and 20% respectively and HJH-containing serum groups were given HJH-containing serum at the concentrations of 10% and 20% respectively. After being cultured for 24 hours and 48 hours, the apoptosis and proliferate was assessed by flowcytometry, and VEGF was quantified in the media by ELISA after 48 hours.
Results:
Part 1:
1. 85 of 87 cases finished the biochemical indices.The levels of OFQ、PGF2a、PGE2、6-keto-PGF1a and TXB2 in plasma during menstruation were significantly higher in patients of endometriosis with pain than that of no pain(P<0.01), while the ratio of 6-keto-PGF1a versus TXB2 was much lower in that of patients with pain(P<0.01);The plasma OFQ、PGF2a and PGE2 in severe pain group was significantly higher than the slight pain group (P<0.05). Plasma OFQ、PGF2a and PGE2 contents were significantly decreased , and plasma 6-keto-PGF1a level was markably increased after the treatment with Huazhuo Jiedu Huoxue Recipe (P<0.01) .
2. 45 of 62 cases with pain finished the observation. The pain with endometriosis was improved markably by Huazhuo Jiedu Huoxue. Effects were seen as early as 1 month after the treatment .The average pain score demonstrated a falling trends along with the time . There was a reduction in pelvic pain and dysmenorrhea when comparing the pain scores between the pre- and post-treatment. After 3 months of the treatment ,the dysmenorrhea score decreased from(6.23±2.12)to(4.47±1.52)and chronic pelvic pain(CPP)score decreased from(4.92±1.67)to(4.47±1.52),(P<0.01). About one third patients had mild diarrhea or defecation 2-3times a day within primary 1-2 weeks,but gradually disappeared.they had no other side-effects.Their functions of liver and kidney are all normal.
3. 32 of 62 cases with pain finished the test. Huazhuo Jiedu Huoxue Recipe was able to regulate blood viscosity and packed cell volume. There was a significant difference(P<0.01).
4. 30 of 62 cases with pain finished the test. Levels of follicule-stimulating hormone,luteotropic hormone, prolactin and estradiol were regulated after the treatment by Huazhuo Jiedu Huoxue(P<0.05), but the effects on progestin and testosterone were not found.
Part 2:
1. Free VEGF and sFlt-1 in peritoneal fluid from 28 cases of patients with endometriosis were observed.The results were as follows:(1) We found higher VEGF and sFlt-1 concentration in endometriotic lesions than controls﹙P< 0.01)and VEGF concentration in stagesⅢ~Ⅳwas higher than in stagesⅠ~Ⅱ﹙P<0.05). VEGF activity index in controls, stagesⅠ~Ⅱand stagesⅢ~Ⅳwith endometriosis was 0.310, 0.276 and 0.273, respectively. (2)VEGF and sFlt-1 concentration also were higher in proliferative phase than in secretory phase in endometriotic lesions(P<0.01),and VEGF activity index was 0.191 in proliferative phase and 1.185 in secretory phase with endometriosis.
2. Free VEGF and sFlt-1 in the serum from 28 cases of patients with endometriosis were observed.
(1) We found higher VEGF and sFlt-1 concentration in endometriotic lesions than controls﹙P<0.01). meanwhile,VEGF and sFlt-1 concentration in stagesⅢ~Ⅳwas higher than in stagesⅠ~Ⅱ﹙P<0.05). VEGF activity index in controls, stagesⅠ~Ⅱand stagesⅢ~Ⅳwith endometriosis was 0.366, 0.380 and 0508, respectively.
(2)VEGF and sFlt-1 concentration also were higher in proliferative phase than in secretory phase in serum from patients with endometriosis(P<0.01),and VEGF activity index was 0.531 in proliferative phase and 0.368 in secretory phase with endometriosis.
Part 3:
The concentration of free VEGF in serum from patients with endometriosis decreased completely after the treatment with Huazhuo Jiedu Huoxue Recipe. The concentration of free VEGF decreased from(268.1±30.1)pg /ml to(268.1±30.1)pg/ml with significant difference(P<0.01).Although sFlt-1 concentration also droped from (637±24.3)pg / ml to( 616±41.1)pg / ml,there was no significant difference(P>0.05).VEGF activity index was 0.421 pre-trentment and 0.371 post-treatment.
Part 4:
Ectopic endometrial cell with endometriosis was successful at a rate of 55% in vitro.
(1) The effect of HJH on growth curve of cultured cells was evaluated by MTT method.The cultured cells had a time-dependent and dose-dependent correlation .When the cells were treated by different dose of HJH(10%、20%) from the patient bearing endometriosis, the growth of cells was inhibited strongly. Also, the growth of cells showed a gradual decline after treating 24 hours and 48 hours by HJH serum with significant difference(P<0.05).
(2) The proliferate was assessed by flowcytometry,after being cultured for 24 hours and 48 hours in HJH combined with the serum at different concentration of HJH serum. The proliferate in HJH Surprisingly had a short-time increase at first 24 hours than controls( P<0.05),but there was no significant difference between 10% HJH and 20% HJH. Again 24 hours with the HJH serum, the proliferation of cells in 20%HJH (0.735±0.059)was significantly suppressed comparing with 10% HJH(0.846±0.048) and controls( P<0.01 ).
(3) The apoptosis was also assessed by flowcytometry,after being cultured for 48 hours in HJH combined with the serum at different concentration of HJH.The cells showed apoptosis was 47.7±8.7 in 20%HJH and 37.25±8.1 in 10%HJH. The cells apoptosis index in HJH was higher than in controls(P<0.05) and had a dose-dependent manner(P<0.05).
(4) After being cultured for 48 hours in HJH combined with the serum at different concentration of HJH serum. The level of VEGF was (175.4±24.3)pg/ml in 10%HJH and (135.4±26.1)pg/ml in 20%HJH. The level of VEGF in HJH was lower than in blank controls ( P < 0.05 ) and also in a dose-dependent manner(P<0.05).
Conclusions :
1.Pain was associated with plasma orphanin FQ and prostaglandins in patients of endometriosis with dysmenorrheal and chronic pelvic pain. Furthermore, the detection of plasma orphanin FQ may be a effective method to evaluate pains of endometriosis and clinical effects of treatment as it is very simple to perform.
2.The mechanism of Huazhuo Jiedu Huoxue releasing pains of patients with endometriosis may be as follows: Huazhuo Jiedu Huoxue exerts its effects on endometriosis with pains by clearing out the menstrual period blood stasis and edema in ectopic endometria, lowering biood viscosity, improving microcirculation and tissue oxygen supply, regulating endocrine disorder, also make plasma OFQ、PGF2a and PGE2 contents significantly decrease by promoting decomposition and metabolism of them.
3. It was the first time that free VEGF、sFlt-1 and VEGF activity index in peritoneal fluid and serum from patients with endometriosis were observed.From this new viewpoint,we think that roles of VEGF in endometriosis may be as follows:
(1) sFlt-1 may be has the role of down regulate VEGF .
(2) We found higher concentration of VEGF and sFlt-1 in peritoneal fluid and serum from patients with endometriosis.Forthermore,there was a positive correlation with clinical stages of endometriosis
(3) It Can be of benefit to diagnosis, evaluation of clinical effects and prognosis to associate with detecting VEGF、sFlt-1 and activity in serum.
(4) May be,VEGF and sFlt-1 will become the target of treatment from the viewpoint of angiogenesis in the future. 4.The mechanism of Huazhuo Jiedu Huoxue treating patients with endometriosis may be as follows:
(1) Huazhuo Jiedu Huoxue can down regulate VEGF and VEGF activity index in serum of patients with endometriosis.
(2) Huazhuo Jiedu Huoxue can adjust secretion of VEGF , inhibit the cell proliferation, induce apoptosis of ectopic endometrial cells,and finaly make atrophy of ectopic endometrium .
⒈大约7%~10%的生育年龄妇女患有子宫内膜异位症,其中71%~87%患者合并有慢性盆腔疼痛,65.6%~77.3%患者合并有痛经。可见,慢性盆腔疼痛和痛经是子宫内膜异位症患者最常见、最痛苦的临床症状之一,严重影响了患者生活质量。然而对于疼痛,目前尚缺乏可靠的评价指标及有效的治疗手段。因此,本部分研究旨在探讨化浊解毒活血方对子宫内膜异位症伴疼痛患者的治疗作用及可能的作用机制。
(1)检测子宫内膜异位症伴疼痛患者月经期血浆孤啡肽(OFQ)及前列腺素(PGF2a、PGE2、6-keto-PGF1a、TXB2 )的含量变化,并探讨其临床意义,以及化浊解毒活血方对它们的影响。
(2)观察化浊解毒活血方对子宫内膜异位症伴疼痛患者的疼痛改善及不良反应、血液流变学、血清内分泌激素的影响,从而探讨化浊解毒活血方可能的作用机制。
⒉虽然我们大都知道,血管内皮生长因子(VEGF)在子宫内膜异位症发生、发展中起着极其重要的作用,但是,VEGF受体特别是VEGFR-1(Flt-1)并未引起足够重视,而可溶性受体sFlt-1与VEGF之间的关系及它们在子宫内膜异位症血管形成中的作用是非常值得研究的课题,目前尚未见报道。
关于子宫内膜异位症的治疗,至今也未找到一种好的治疗手段。现今常用的药物如口服避孕药、达那唑、促性腺激素释放激素激动剂(GnRH-a)等,通过降低体内雌激素状态而达到治疗目的,因此副作用较多且较严重。如服用达那唑导致的男性化,应用GnRH-a导致骨质疏松等围绝经期综合征表现等,这就预示着患者只能短期用药,而一旦停药,5年后50%患者复发。值得庆幸的是,中医药具有很多优势。著名中医药专家李佃贵教授根据多年经验积累,从“浊、毒、瘀”论治,采用化浊解毒活血方治疗子宫内膜异位症,取得了很好疗效。本部分研究旨在探讨子宫内膜异位症患者血清、腹腔液中游离VEGF、sFlt-1的水平和临床意义,以及化浊解毒活血方对它们的影响。
⒊为了从细胞水平进一步研究化浊解毒活血方的作用机制,建立体外细胞模型是非常必要的。并利用患者的药物血清进行干预,观察化浊解毒活血药物血清(简称HJH血清)对异位子宫内膜细胞生长、增殖、凋亡及分泌VEGF的影响。
方法:
第一部分:子宫内膜异位症无疼痛对照组25例,子宫内膜异位症轻度疼痛、中重度疼痛各31例。近3个月未曾用其它药物(如达那唑等)治疗。给予化浊解毒活血中药(藿香、佩兰叶、清夏、陈皮、茯苓、胆南星、白花蛇舌草、红景天、田基黄、丹参、元胡、五灵脂、当归)水煎400ml,分早、晚各服200ml,3个月1疗程。治疗前后观察下列指标:
1.采用放射免疫法(RIA)测定血浆OFQ及PGs(PGF2a、PGE2、6-keto-PGF1a、TXB2)含量。
2.分别在治疗1、2、3个月后,采用视觉模拟疼痛评分(VAS)对痛经及慢性盆腔疼做出评价,同时进行副作用观察。
3.分别在治疗前、治疗后3个月,测定血液流变状态及血清FSH、LH、PRL、P及E2水平。
第二部分:对28例子宫内膜异位症患者,采用酶联免疫吸附法(ELISA)检测血清、腹腔液中游离VEGF和sFlt-1的水平。设正常对照组10例。
第三部分:采用化浊解毒活血方,治疗子宫内膜异位症患者20例,应用ELISA法检测血清游离VEGF和sFlt-1的水平,并计算VEGF活性指数。
第四部分:体外研究
1.子宫内膜异位症患者1名,口服化浊解毒活血方水煎剂后,采血制备成药物血清(简称HJH血清);健康志愿者1名,采血制备成空白血清。
2.离体培养20例子宫内膜异位症患者的异位子宫内膜细胞。
3.对离体培养的子宫内膜异位症患者的异位子宫内膜细胞,采用HJH血清(10%HJH、20%HJH)进行干预,氮蓝四唑盐比色法(MTT)观察异位内膜细胞的生长曲线变化;流式细胞技术(FCM),观察HJH作用24h、48h后异位内膜细胞增殖和凋亡情况;ELISA法测定HJH作用48h后VEGF浓度。
结果:
第一部分:
⒈87例子宫内膜异位症患者中有85例完成了本项观察。
子宫内膜异位症疼痛患者经期血浆OFQ、PGF2a、PGE2、6-keto-PGF1a、TXB2含量均显著高于无疼痛患者(P<0.01),6-keto-PGF1a /TXB2比值显著低于无疼痛患者(P<0.01);子宫内膜异位症中重度疼痛组血浆OFQ、PGF2a、PGE2含量均明显高于轻度疼痛组(P<0.05)。子宫内膜异位症疼痛组经化浊解毒活血中药治疗后,OFQ、PGF2a、PGE2含量显著下降,6-keto-PGF1a水平显著升高(P<0.01)。
⒉62例子宫内膜异位症伴疼痛患者中有45人接受并完成本向观察。
治疗第1~2周有三分之一病人出现轻度腹泻,大便次数增多至每日2-3次,继续服药症状消失。未出现其它任何不良反应。复查肝、肾功能正常。化浊解毒活血中药治疗第1个月后,痛经及慢性盆腔疼痛即有明显改善,随着治疗时间的延长,疼痛评分逐渐下降,其中治疗后2个月的VAS评分明显低于治疗前(P<0.05)。治疗3个月后,痛经VAS评分由治疗前的(6.23±2.12)下降到(4.01±1.34),慢性盆腔疼痛由治疗前的(4.92±1.67)下降到(4.47±1.52)(P<0.01)。
⒊62例子宫内膜异位症伴疼痛患者中资料齐全者32例。
经化浊解毒活血治疗后发现,子宫内膜异位症伴疼痛患者血液粘滞度和红细胞压积均有明显降低,并有统计学意义(P<0.01)。
⒋62例子宫内膜异位症伴疼痛患者中资料齐全者30例。
经化浊解毒活血治疗后发现,子宫内膜异位症伴疼痛患者血清FSH、LH、PRL、E2水平有不同程度的下降(P<0.05),对T和P的影响无统计学意义。
第二部分:28例子宫内膜异位症患者腹腔液、血清中VEGF、sFlt-1及VEGF活性指数的表达情况。
1.子宫内膜异位症患者腹腔液VEGF、sFlt-1水平明显高于对照组﹙P<0.01)。Ⅲ~Ⅳ期VEGF水平高于Ⅰ~Ⅱ期﹙P<0.05)。对照组、Ⅰ~Ⅱ期和Ⅲ~Ⅳ期VEGF活性指数分别为0.310、0.276和0.273。
2.子宫内膜异位症增生期腹腔液VEGF及sFlt-1水平均明显高于分泌期﹙P<0.01),增生期VEGF活性指数为0.191,分泌期为1.185。
3.子宫内膜异位症患者血清VEGF、sFlt-1水平明显高于对照组﹙P<0.01),且Ⅲ~Ⅳ期VEGF、sFlt-1水平高于Ⅰ~Ⅱ期﹙P<0.01)。对照组、Ⅰ~Ⅱ期和Ⅲ~Ⅳ期VEGF活性指数分别为0.366、0.380和0.508。
4.子宫内膜异位症增生期血清VEGF及sFlt-1水平均明显高于分泌期﹙P<0.01),增生期VEGF活性指数为0.531,分泌期为0.368。
第三部分:化浊解毒活血中药治疗后,子宫内膜异位症患者血清VEGF的下降率为100%。血清VEGF含量由治疗前的(268.1±30.1)pg/ml,下降到治疗后(268.1±30.1)pg/ml,具有统计学意义(P<0.01)。血清sFlt-1含量治疗后为(616±41.1)pg / ml,虽然较治疗前(637±24.3)pg/ml略有下降,但无统计学意义(P>0.05)。VEGF活性指数治疗后为0.371,较治疗前(0.421)有所下降。
第四部分:体外研究部分。20例内异症患者异位内膜细胞经培养,11例成功,9例失败,成功率为55%。
1.HJH血清对体外培养的异位子宫内膜细胞的抑制作用明显高于空白血清对照组(P<0.05)。HJH血清作用后的细胞数,呈现完全的浓度和时间相关性,即随着HJH浓度增加和作用时间的延长,异位内膜细胞生长率呈下降趋势,MTT法显示细胞生长曲线出现下降改变。10%HJH血清和20%HJH血清之间,作用24h(第5日)和作用48h(第6日)之间比较,均有统计学意义(P<0.05)。
2.HJH血清作用24h后HJH组细胞增殖活性均高于空白对照组(P<0.05),但10%HJH与20%HJH两个浓度之间无差别。HJH血清作用48h后,HJH组增殖活性明显下降并低于对照组,且呈浓度依赖性,即20%HJH组增殖活性(0.735±0.059)低于10%HJH组(0.846±0.048),并有统计学意义(P<0.01)。
3.HJH血清作用48h后,流式细胞技术结果显示,10%HJH、20%HJH组的细胞凋亡指数分别为37.25±8.1和47.7±8.7,明显高于空白对照及相同浓度的空白血清对照,并且20%HJH组凋亡指数明显高于10%HJH组,差异有统计学意义(P<0.05)。
4.HJH药物血清作用48h后,ELISA法测定VEGF显示,10%HJH、20%HJH组的VEGF含量分别为(175.4±24.3)pg/ml和(135.4±26.1)pg/ml,与空白对照及空白血清对照比较,HJH血清组VEGF量明显降低,且VEGF在20%浓度时比10%浓度下降明显,并有统计学意义(P<0.05)。
结论:
⒈血浆OFQ和PGs水平与子宫内膜异位症患者疼痛的发生密切相关。血浆OFQ的测定简单方便,有望成为评价子宫内膜异位症疼痛程度及临床疗效的指标。
⒉化浊解毒活血方治疗子宫内膜异位症疼痛的作用机制:化浊解毒活血方可恢复患者正常气血运行,改善血液流变学,降低血液粘稠度,调整内分泌紊乱,降低血浆OFQ及PGF2a、PGE2水平,从而缓解疼痛,改善临床症状。
⒊本研究首次观察了VEGF及sFlt-1在子宫内膜异位症患者腹腔液、血清中的含量,并采用二者的比值计算了VEGF活性指数,从一全新角度探索研究了VEGF在子宫内膜异位症血管形成中的作用机制认为:
(1)sFlt-1可能存在中和、下调VEGF的作用。
(2)VEGF、sFlt-1在子宫内膜异位症腹腔液和血清中高表达,并与病情的进展程度成正相关。
(3)联合测定血清中VEGF、sFlt-1及VEGF活性指数,有助于子宫内膜异位症诊断、疗效判断及预后监测。
(4)VEGF及sFlt-1有可能成为阻断子宫内膜异位症血管形成的靶点。
⒋化浊解毒活血方治疗子宫内膜异位症的抗血管形成机制:
(1)化浊解毒活血方可降低子宫内膜异位症患者血清VEGF含量及VEGF活性指数。
(2)化浊解毒活血方可通过抑制VEGF的分泌,抑制异位子宫内膜细胞生长并诱导其凋亡,从而促使异位内膜萎缩,以实现对子宫内膜异位症的治疗作用。
Objective:
1. Endometriosis affects approximately 7%–10% of all women.71%–87% of patients with chronic pelvic pain(CPP),and 65.6%～77.3%of patients with dysmenorrhea. Chronic pelvic pain and dysmenorrhea are the hallmark symptoms experienced by affected women.Their survival quality is affected seriously.However,to pain,we have no found a better index to evaluate it, and effective method to treat.So,in this part of study,based on the treatment of endometriosis-associated pain with Huazhuo Jiedu Huoxue Recipe(HJH),to investigate the mechanism of pains in endometriosis.
(1) Detect the changes of plasma orphanin FQ and prostaglandins (PGF2a、PGE2、6-keto-PGF1a and TXB2 ) from patients of endometriosis with pain during menstruation,and the effect of Huazhuo Jiedu Huoxue Recipe on them. aim at understanding their clinical significance.
(2) Patient’s degree of pain and side effects will be observed.In addition, the blood hemorrheology and serum endocrine hormones are going to be monitored before and after the administration at the conclusion of the study.
2. Although we all know that vascular endothelial growth factor(VEGF) play a most important role in development of endometriosis, its receptor especially VEGFR-1(Flt-1) has not been payed enough attention. Further more, soluble form of VEGF receptor (sFlt-1) was seldom reported. Actually, to research about the relationship between VEGF and sFlt-1 from the viewpoint of angiogenesis is very valuable.
About the treatment of endometriosis,we also have had no nice method. Although there are several medical treatments available including oral contraceptives, danazol,and GnRH agonists,etc,these treatments attempt to create a hypoestrogenic state since estrogen is believed to be a contributing factor to endometriosis.Masculinizing side effects have essentially rendered Danazol a treatment of the past.Menopausal side effects,includ-ing osteoporosis,that are associated with GnRH agonists make them only short-term solutions for the treatment of endometriosis.The recurrence rate as long as 50% after stopping the treatment. Luckly, Traditional Chinese Medicine can bring us many advantages.The famous professor Diangui Li according to his experience based on“turbidity,toxin,stasis”.After have Huazhuo Jiedu Huoxue Recipe used on patients with endometriosis, nice clinical effects were found. In this part, levels of free VEGF and sFlt-1 in peritoneal fluid and serum will be measured,as well as the effect of Huazhuo Jiedu Huoxue Recipe on them.
3. To understand the mechanism of Huazhuo Jiedu Huoxue Recipe in treating endometriosis better, studying in vitro is very necessary.By serum pharmacology,to determine the effect of Huazhuo Jiedu Huoxue on the growth, proliferation, apoptosis,and VEGF secretion of cultured ectopic endometrial cells with endometriosis .
Methods:
Part one: 31 patients of endometriosis( EM) with moderate or severe pain ( severe group) , 31 patients with slight pain(slight group) and 25 patients without pain (control group) were studied. They were treated with Huazhuo Jiedu Huoxue Recipe (composed of Huoxiang, Peilan, Qingxia, Chenpi, Fuling, Baihuasheshecao, Hongjingtian, Tianjihuang, Danshen , Yuanhu, Wulingzhi and Danggui, etc).Decoction 400ml ,at 2 dose of 200ml,b.i.d. 3 months constituting a therapeutic course and these patients without taking western medicine,such as danazol,within and before 3 months.Before and after 3 months of the treatment, the clinical data would be obtained as follows: Radioimmunoassay(RIA) could be used to evaluate the concentrations of plasma orphanin FQ and prostaglandins(PGs),such as PGF2a、PGE2、6-keto-PGF1a and TXB2 during menstruation.Patient’s degree of pain would be quantified by visual analogue pain scale at baseline,months 1,2,and3,respectively.Also side effects were observed. In the meantime, the blood hemorrheology and serum endocrine hormones were monitored before and after the administration at the conclusion of the study.
Part 2: Levels of free VEGF and sFlt-1 were measured by enzyme-linked immunosorbent assay (ELISA) in peritoneal fluid and serum from 28 subjects with surgically confirmed endometriosis, and 10 controls with no clinical evidence of the disease and other diseases. Meanwhile, we calculated a VEGF activity index by means of the ratio VEGF/ sFlt-1.
Part 3: Levels of free VEGF and sFlt-1 were measured by enzyme-linked immunosorbent assay in serum from 20 cases of endometriosis after the administration with Huazhuo Jiedu Huoxue Recipe. A VEGF activity index also be calculated.
Part 4: To investigate the influence of HJH on the growth of ectopic endometrial cells with endometriosis in vitro, we observed the growth of cells by MTT method firstly,then, the apoptosis and proliferate was assessed by flowcytometry, and VEGF was quantified in the media by ELISA.Blank serum from a healthy volunteer was prepared. A patient with endometriosis was given Huazhuo Jiedu Huoxue(HJH)decoction for three days in order to prepare serum containing HJH in the early morning of the fourth day after preprandial administration of HJH. Ectopic tissue was obtained from 20 patients with endometriosis. The stromal and epithelial cells of ectopic were isolated and cultured in vitro. The cells were divided into five groups randomly and treated with different culture medium on the fourth day. Blank control group was in serum-free condition.Blank serum groups were given blank serum at the concentrations of 10% and 20% respectively and HJH-containing serum groups were given HJH-containing serum at the concentrations of 10% and 20% respectively. After being cultured for 24 hours and 48 hours, the apoptosis and proliferate was assessed by flowcytometry, and VEGF was quantified in the media by ELISA after 48 hours.
Results:
Part 1:
1. 85 of 87 cases finished the biochemical indices.The levels of OFQ、PGF2a、PGE2、6-keto-PGF1a and TXB2 in plasma during menstruation were significantly higher in patients of endometriosis with pain than that of no pain(P<0.01), while the ratio of 6-keto-PGF1a versus TXB2 was much lower in that of patients with pain(P<0.01);The plasma OFQ、PGF2a and PGE2 in severe pain group was significantly higher than the slight pain group (P<0.05). Plasma OFQ、PGF2a and PGE2 contents were significantly decreased , and plasma 6-keto-PGF1a level was markably increased after the treatment with Huazhuo Jiedu Huoxue Recipe (P<0.01) .
2. 45 of 62 cases with pain finished the observation. The pain with endometriosis was improved markably by Huazhuo Jiedu Huoxue. Effects were seen as early as 1 month after the treatment .The average pain score demonstrated a falling trends along with the time . There was a reduction in pelvic pain and dysmenorrhea when comparing the pain scores between the pre- and post-treatment. After 3 months of the treatment ,the dysmenorrhea score decreased from(6.23±2.12)to(4.47±1.52)and chronic pelvic pain(CPP)score decreased from(4.92±1.67)to(4.47±1.52),(P<0.01). About one third patients had mild diarrhea or defecation 2-3times a day within primary 1-2 weeks,but gradually disappeared.they had no other side-effects.Their functions of liver and kidney are all normal.
3. 32 of 62 cases with pain finished the test. Huazhuo Jiedu Huoxue Recipe was able to regulate blood viscosity and packed cell volume. There was a significant difference(P<0.01).
4. 30 of 62 cases with pain finished the test. Levels of follicule-stimulating hormone,luteotropic hormone, prolactin and estradiol were regulated after the treatment by Huazhuo Jiedu Huoxue(P<0.05), but the effects on progestin and testosterone were not found.
Part 2:
1. Free VEGF and sFlt-1 in peritoneal fluid from 28 cases of patients with endometriosis were observed.The results were as follows:(1) We found higher VEGF and sFlt-1 concentration in endometriotic lesions than controls﹙P< 0.01)and VEGF concentration in stagesⅢ~Ⅳwas higher than in stagesⅠ~Ⅱ﹙P<0.05). VEGF activity index in controls, stagesⅠ~Ⅱand stagesⅢ~Ⅳwith endometriosis was 0.310, 0.276 and 0.273, respectively. (2)VEGF and sFlt-1 concentration also were higher in proliferative phase than in secretory phase in endometriotic lesions(P<0.01),and VEGF activity index was 0.191 in proliferative phase and 1.185 in secretory phase with endometriosis.
2. Free VEGF and sFlt-1 in the serum from 28 cases of patients with endometriosis were observed.
(1) We found higher VEGF and sFlt-1 concentration in endometriotic lesions than controls﹙P<0.01). meanwhile,VEGF and sFlt-1 concentration in stagesⅢ~Ⅳwas higher than in stagesⅠ~Ⅱ﹙P<0.05). VEGF activity index in controls, stagesⅠ~Ⅱand stagesⅢ~Ⅳwith endometriosis was 0.366, 0.380 and 0508, respectively.
(2)VEGF and sFlt-1 concentration also were higher in proliferative phase than in secretory phase in serum from patients with endometriosis(P<0.01),and VEGF activity index was 0.531 in proliferative phase and 0.368 in secretory phase with endometriosis.
Part 3:
The concentration of free VEGF in serum from patients with endometriosis decreased completely after the treatment with Huazhuo Jiedu Huoxue Recipe. The concentration of free VEGF decreased from(268.1±30.1)pg /ml to(268.1±30.1)pg/ml with significant difference(P<0.01).Although sFlt-1 concentration also droped from (637±24.3)pg / ml to( 616±41.1)pg / ml,there was no significant difference(P>0.05).VEGF activity index was 0.421 pre-trentment and 0.371 post-treatment.
Part 4:
Ectopic endometrial cell with endometriosis was successful at a rate of 55% in vitro.
(1) The effect of HJH on growth curve of cultured cells was evaluated by MTT method.The cultured cells had a time-dependent and dose-dependent correlation .When the cells were treated by different dose of HJH(10%、20%) from the patient bearing endometriosis, the growth of cells was inhibited strongly. Also, the growth of cells showed a gradual decline after treating 24 hours and 48 hours by HJH serum with significant difference(P<0.05).
(2) The proliferate was assessed by flowcytometry,after being cultured for 24 hours and 48 hours in HJH combined with the serum at different concentration of HJH serum. The proliferate in HJH Surprisingly had a short-time increase at first 24 hours than controls( P<0.05),but there was no significant difference between 10% HJH and 20% HJH. Again 24 hours with the HJH serum, the proliferation of cells in 20%HJH (0.735±0.059)was significantly suppressed comparing with 10% HJH(0.846±0.048) and controls( P<0.01 ).
(3) The apoptosis was also assessed by flowcytometry,after being cultured for 48 hours in HJH combined with the serum at different concentration of HJH.The cells showed apoptosis was 47.7±8.7 in 20%HJH and 37.25±8.1 in 10%HJH. The cells apoptosis index in HJH was higher than in controls(P<0.05) and had a dose-dependent manner(P<0.05).
(4) After being cultured for 48 hours in HJH combined with the serum at different concentration of HJH serum. The level of VEGF was (175.4±24.3)pg/ml in 10%HJH and (135.4±26.1)pg/ml in 20%HJH. The level of VEGF in HJH was lower than in blank controls ( P < 0.05 ) and also in a dose-dependent manner(P<0.05).
Conclusions :
1.Pain was associated with plasma orphanin FQ and prostaglandins in patients of endometriosis with dysmenorrheal and chronic pelvic pain. Furthermore, the detection of plasma orphanin FQ may be a effective method to evaluate pains of endometriosis and clinical effects of treatment as it is very simple to perform.
2.The mechanism of Huazhuo Jiedu Huoxue releasing pains of patients with endometriosis may be as follows: Huazhuo Jiedu Huoxue exerts its effects on endometriosis with pains by clearing out the menstrual period blood stasis and edema in ectopic endometria, lowering biood viscosity, improving microcirculation and tissue oxygen supply, regulating endocrine disorder, also make plasma OFQ、PGF2a and PGE2 contents significantly decrease by promoting decomposition and metabolism of them.
3. It was the first time that free VEGF、sFlt-1 and VEGF activity index in peritoneal fluid and serum from patients with endometriosis were observed.From this new viewpoint,we think that roles of VEGF in endometriosis may be as follows:
(1) sFlt-1 may be has the role of down regulate VEGF .
(2) We found higher concentration of VEGF and sFlt-1 in peritoneal fluid and serum from patients with endometriosis.Forthermore,there was a positive correlation with clinical stages of endometriosis
(3) It Can be of benefit to diagnosis, evaluation of clinical effects and prognosis to associate with detecting VEGF、sFlt-1 and activity in serum.
(4) May be,VEGF and sFlt-1 will become the target of treatment from the viewpoint of angiogenesis in the future. 4.The mechanism of Huazhuo Jiedu Huoxue treating patients with endometriosis may be as follows:
(1) Huazhuo Jiedu Huoxue can down regulate VEGF and VEGF activity index in serum of patients with endometriosis.
(2) Huazhuo Jiedu Huoxue can adjust secretion of VEGF , inhibit the cell proliferation, induce apoptosis of ectopic endometrial cells,and finaly make atrophy of ectopic endometrium .
引文
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18 Sharon E, Maynard, Jiang Y M, Jime M, et al. Excess placental soluble fms-like tyrosine kinase 1(sFlt 1) may contribute to endothelial dysfunction, and proteinuria in preeclampsia.J. Clin.Invest,2003,111:649-558
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