| Endometriosis is a common and frequently-occurring gynecological disease which defined as endometriotic tissues growing outside the normal uterine cavity.Endometriosis often causes dysmenorrhea, dyspareunia and infertility. The incidence has showed rising trend in recent years. Endometriosis is not only wide range of lesions, morphological diversity, but also has malignant biological characteristics, such as transfer, infiltration and recurrence. Because the pathogenesis of endometriosis has not been fully clarified leading to poor targeted therapies, clinical treatment shows some features with high recurrent, serious side effects at present.This object focused on the study of the theoretical foundation about the treatment by Guiling xiaoyi Decoction. By established the in-vitro cultural method of purified stromal-cell, we observed the effect of drug serum of Guiling xiaoyi Decoction on morphology and MMP-9 secretion of stromal cells in vitro. We have already confirmed that Guiling xiaoyi decoction could inhibit IL-6 and IL-8 secretion of endometrial stromal cells in vitro from patient with endometriosis. In order to explore the role of IL-6, IL-8 in the process of endometriosis and the mechanism of Guiling xiaoyi Decoction on endometriosis, further studies were carried out whether there are correlation between IL-6, IL-8 and MMP-9 secretion of endometrial stromal cells, based on the preparatory work.1 The study of the theoretical basis of the treatment of Guiling xiaoyi decoction for dometriosisAccording to the cardinal symptoms of endometriosis, endometriosis belongs to those of algomenorrhea, abdominal mass and infertility in traditional Chinese medicine. This disease is due to malfunction of the dominating reproduction of the kidney, frequent sexual intercourse during menstruation and postpartum,which lead to the injury of Chong and Ren meridians and failure of the functions of storing and discharging of the uterus. The function of kidney which is not sufficient is the radical factor of occurrence of this disease. Blood stasis is an important factor for occurrence and development of the disease. So the treatment for endometriosis should be based on the concept of enhancing warming kidney and dispersing blood stasis.Guizhi fuling pill originates from Jinkui yaolue. Zhongjing Zhang, the author of the book,designed this prescription to mildly remove mass in the abdomen,disperse blood stasis so as to prevent abortion which was due to the abdominal mass from endangering the fetus.If the prescription was utilized mainly in order to remove blood stasis and disperse the mass in the abdomen,there may be some shortage of the strength of the prescription. So,first more drugs having the property of?enhancing blood circulation and resolving blood stasis should be added to the prescription.Second, the prescription should be modified according to syndrome differentiation. Therefore,Guiling xiaoyi Decoction was designed which combines the Guizhi fuling pill with Xianlingpi to strengthen warming kidney, and Xuejie to disperse blood stasis. That the vitro experimental studies on the treatment of endometriosis were carried out with Guiling xiaoyi Decoction provided a basis for clinical rational use.2 In-vitro culture method of stromal-cell established initiallyWe collected endometria from patients who suffered from endometriosis or uterine fibroids, and recieved operation in the first people's Hospital of Yangzhou and Chinese medicine hospital from October in 2008 to April in 2009. Stromal cells were isolated and subcultured from endometria. The cell proliferation was observed by microscope, and the purity coefficient of cells was characterized with immunohistochemistry method.The results showed that human endometrial stromal cells could be successfully isolated and cultured by combination of enzyme digestion, sieve separation, centrifugal separation and differential adhesion method, with 88.89% success rate. The cells could be subcultured for 4-5 generations, and survived for 4-5 weeks. Under our experiment condition, the endometrial stromal cells in vitro have good proliferative capacity and the stromal cells from both endometriosis and non-endometriosis can be successfully isolated and cultered (P>0.05). Furthermore, the derived time of endometrial stromal cells has an influence on the survival rate and proliferative capacity (P<0.05). Based on our experimental, an effective research platform can be provided for the study on endometriosis pathogenesy and pharmacodynamics.3 The effect of Guiling xiaoyi decocti on MMP-9 secretion of endometrial stromal cells and the correlation between IL-6, IL-8 and MMP-9 secretion of endometrial stromal cellsWe prepared drug serum of Guiling xiaoyi Decoction to interfere with the endometrial stromal cells in vitro extracted from the patients with endometriosis, and then observed the cell morphologic changes under microscope. In the meantime, we collected cell culture supernatant at 24h,48h,72h after treatment with drug serum, and tested the concentration of MMP-9 by the method of ELISA respectively.The medium supplemented with different concentrations of IL-6, IL-8 was prepared to culture the endometrial stromal cells extracted from the patients with endometriosis, as well as the cell morphologic changes were observed under microscope. Besides, we collected cell culture supernatant at 24h after treatment with drug serum, and detected the concentration of MMP-9 by the method of ELISA .Through detection, we confirmed that the serums of Guiling xiaoyi Decoction could inhibit MMP-9 secretion of endometrial stromal cells(P<0.01), and the effect of inhibition is more and more obvious with the concentration increasing and the time extending. Nevertheless, IL-6 could stimulate MMP-9 secretion(P<0.01), with the stimulation depending on the concentration of IL-6. The results of experiment indicated the correlation between IL-6 and and MMP-9 secretion of endometrial stromal cells. Guiling xiaoyi Decoction could not only inhibit directly but also inhibit indirectly MMP-9 secretion of endometrial stromal cells by inhibiting IL-6 secretion, controlling the occurrence and development of endometriosis. The results of the experiment showed that IL-8 didn't have significant effect on MMP-9 secretion of endometrial stromal cells(P>0.05). IL-8 doesn't produce high level of MMP-9 to promote the planting of ectopic endometrium.
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