The Effect Of Guiling Xiaoyi Drug Serum On Apoptosis And The Activity Of NF-κB Of Eutopic Stromal Cell Of Endometriosis

Endometriosis is a common benign gynecological disease characterized with endometrial glandular epithelial cells and stromal cells present in those parts outside the uterine cavity. Those common clinical parts contain ovaries, fallopian tubes, bladder, myometrium and so on. Dysmenorrhea, chronic pelvic pain and infertility which caused by endometriosis affect the health and quality of life of the young and middle-aged women seriously, so this disease receives increasing emphasis from people. Experts and scholars of obstetrics and gynaecology are plagued by the diagnosis and treatment of the disease because of poor targeted therapies leaded by its unknown pathogenesis.By setting up the in-vitro cultural method of purified stromal cells, this object discussed the microscopic mechanism of Guiling xiaoyi Decoction. We observed the morphology, early apoptosis and the activity of NF-κB of stromal cells cultured in vitro under the influence of drug serum. There was evidence that Guiling xiaoyi decoction could inhibit the expression of IL-8of endometrial stromal cells in vitro drawn from patients with endometriosis. Based on this, our subjects studied whether there was correlation between the endometrial stromal cells’ IL-8expression and NF-κB activity in order to provide some theoretical support for Guiling xiaoyi Decoction in clinical treatment for endometriosis.1The study on the basic theory of traditional Chinese medicine of Guiling xiaoyi decoction for curing endometriosisThere was no record of the name of endometriosis in traditional Chinese medicine science. It belongs to those of algomenorrhea, infertility, abdominal mass and emmeniopathy in traditional Chinese medicine. At present, most scholars believe that enhancing warming kidney and dispersing blood stasis are two main methods of treating endometriosis. The deficiency of the kidney is the main factor of occurrence of this disease. Blood stasis is also an important pathogenic factor and pathological manifestation of the disease.Guiling xiaoyi Decoction was combined with the Guizhi fuling pill recorded in Jinkui yaolue by Zhongjing Zhang, Xianlingpi and Xuejie. Guizhi fuling pill was first used to remove mass in the abdomen mildly. My teacher added two herbs to enhance the strength of warming and blood circulation. The effect of clinical treatment of this decoction for endometriosis is significant. Through this experiment we did some studies on eutopic endometrial cells by the intervention of Guiling xiaoyi drug serum and provided a theoretical basis for clinical application.2Isolation and purification of human endometrial stromal cells cultured in vitroPatients with endometriosis who received operations from September in2011to May in2012in the first people’s Hospital of Yangzhou are our objects and we gathered normal endometrium as comparison. We isolated and cultured stromal cells by observing the growth under microscope and identified cell purity with immunohistochemistry method. According to our experience, we selected people whose endometrial thickness were at least5mm with preoperative B-ultrasonic as collected objects.The results of cell culture displayed that human endometrial stromal cells could be isolated and cultured successfully by using the main methods of enzyme digestion and sieve separation according to the existing experimental conditions and reference literature. These cells could survival for4-5generations by handing from generation to generation. Cell activity and the growth were not affected significantly by passage. By statistical analysis after our experiment we also found that both endometriosis and non-endometriosis could be isolated and cultured successfully under our experimental condition(P>0.05). Besides, the derived time of endometrial stromal cells had no markedly influence on the proliferative capacity and survival rate (P>0.05). Isolated and cultured endometrial stromal cells successfully provided a good platform for the next experiment.3The effect of Guiling xiaoyi drug serum on morphology and early apoptosis of eutopic stromal cells of endometriosisWe prepared the drug serum of Guiling xiaoyi Decoction,set the control groups, used the serum in a different drug concentration to affect the eutopic endometrial stromal cells and observed the morphological changes of cells under the influence of drugs; collected the cell culture supernatants after24,48,72hours and leaved it to the next step for detection; used the flow cytometry to measure the early apoptosis of eutopic endometrial stromal cells after the affection of24,48,72hours with different drug concentrations.4The correlation between the activity of NF-κB and IL-8expression and the effect of Guiling xiaoyi decoction on the activity of NF-kB of endometrial stromal cells from patients with endometriosisWe used TNF-a and TNF-a+PDTC with different concentrations to intervene human endometriosis eutopic endometrial stromal cells, observed the changes of cell morphology under microscope and collected the cell culture supernatants after24hours. Later, we used the measured values of NF-κB and IL-8by using the method of ELISA to analyze the relationship between them.By the method of ELISA we tested the activity of NF-κB respectively in cell supernatants after the action of drugs collected in the previous experiment.The results showed that there was correlation between the activity of NF-κB and IL-8expression in endometrial stromal cell supernatants affected by TNF-a and TNF-a+PDTC. This suggested the expression of IL-8in endometriosis may be associated with NF-κB. We also found that Guling xiaoyi Decoction could influence the activity of NF-κB in endometrial stromal cell supernatants. Because the previous studies have shown that Guiling xiaoyi Decoction could inhibit the expression of IL-8in endometrial stromal cells, we could say that the decoction may inhibit IL-8expression of endometrial stromal cells indirectly by NF-κB pathway to hinder the occurrence and development of endometriosis.