Clinical Research And In Vitro On Treatment Of Endometriosis By Huazhuo Jiedu Huoxue

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 .