| Research Objective:Endometriosis （EMS） is regarded as one of the most complicated diseases which endanger women’s health even nowadays in the 21st century. Roughly about 10% of all women at childbearing age have suffered from this disease. The mechanism of its development is not clear, and there is no guaranteed cure from the Western medicine. It’s now already a hot topic on how to diagnose and treat endometriosis whose rates of both occurrence and recurrence are all very high. A great deal of efforts has been made in recent years to develop effective therapies from all possible angles. The potential application of Chinese traditional medicine （TCM） on this disease has obviously become the focal point in public eye. One of the methods from TCM’s point of view is to improve blood circulation in order to remove blood stasis. However, the development of EMS is hormone-dependent, and its pathological process is complicated and unclear. During our long term clinical practice, we have achieved ideal results by using the method of kidney-invigorating plus period-regulation （proposed by Prof. Guicheng Xia） to treat patients with endocrine disorders. We therefore propose to apply the method of sequentially nourishing kidney plus softening hard masses and blood stasis for the purpose of correcting the endocrine disorder in EMS.Our research begins from the analysis of the EMS development, focusing on the important points along the "3A" process of the disease, as well as the factors involved in its aggression process. In animal model study, we have checked the effect of the two therapeutic treatments, dissipating blood stasis and masses （including using soups of nourishing both yin and yang） in addition to sequentially reinforcing kidney （such as using method for swelling dispersing and analgesia）, on the expression of MMP-2 and TIMP-2 in the ectopic endometrial tissues of rats suffered from EMS. By comparing their expression variation in different groups of animals, treated vs untreated, ectopic endometrial tissues vs healthy tissues, we are trying to demonstrate the role of MMP-2 and TIMP-2 in the process of endometriosis, to uncover the mechanism at the molecular and cellular level of the effect of the kidney-sequential-reinforcing plus hardness-softening and blood-stasis-resolving method in EMS treatment. In addition, we have also compared the pathomorphological changes of EMS rats （experimental groups vs control group） under the electron microscope in order to confirm the therapeutic effects of the kidney-sequential-reinforcing plus hardness-softening and blood-stasis-resolving methods in EMS treatment. These studies have offered experimental evidence in supporting the combination approaches to treat EMS.Methods:The EMS model system in rat was established by using self-endometrium implantation approach. Based on this model system, its, the EMS rat was fed, according to its oestrus cycle, with medicines for kidney-sequential-reinforcing plus hardness-softening and blood-stasis-resolving （mixed group）, and the control groups were rats with the following different treatments: kidney-sequential-reinforcing plus hardness-softening only, hardness-softening and blood-stasis-resolving only, no treatment （normal rat）, and no treatment （EMS rat）. 4 weeks post treatment, the animals were sacrificed at their oestrus to collect tissues from ectopic and eutopic endometrium for future analysis. We then carried out the following experiments on these tissues: 1. Observing morphological changes of their pathological tissues; 2. Using immunoblotting to examine the protein levels of MMP-2 and TIMP-2 in ectopic and eutopic endometrium of the EMS rat; 3. Checking their transcript abundance of MMP-2 and TIMP-2 gene by using quantitative real-time PCR.SPSS11. 5 （Statistical Package for the Social Science. 11.5） was used for data statistic analysis.Results:1. Results of pathological studyHisotopathological study showed that the gland epithelial cells in the endometrium of control rats were arranged in columns, and the intimal glands were abundant. In comparison, although the gland epithelial cells in the ectopic endometrium of the experimental rats were arranged in columns, their intimal glands were scarce, their glandular lumens were big with the secretion of epithelia and the angiectasis in the surrounding regions. In addition, we also observed the following: stale hemorrhage on the side lines of the ectopic endometrium, fibrous tissue hyperplasia, and the increased amount of inflammatory cells in the mesenchymes. As to the groups with variety of treatments, the interior membranes became thinner, the epithelial cells of endometrium appeared cubic shape, dwarf cubic shape, or flat monolayer, the gland number of endometrium was decreased, glandular lumens were shrunk; angiectasis was not observed, nor was the hemosiderin deposition. Among them, the interior membranes of the ectopic endometrium were the thinnest in the group of rats treated with medicines for both kidney-sequential-reinforcing plus hardness-softening and blood-stasis-resolving （mixed group）, most of their epithelial cells of endometrium appeared dwarf cubic shape or flat monolayer, in comparison with other groups.The electronic microscopy showed that the epithelial cells in the endometrium of the rats from the control group （normal rats with no treatment） were arranged in tight columns, and the tight connection of the cells and dense microvilli could be observed in the surface area close to the lumens; the border lines of the glandular epithelia cells, with the normal size, were visibly clear; their nucleus appeared normal in size with fine border lines of the nuclear membrane; the amount of the rough ER and lysosomes was much less; the nucleus were located at the bottom of the cytoplasms, the nuclear membranes appeared smooth and the heterochromatins were evenly distributed. As to the model group （EMS rats with no treatment）, the epithelial cells in the endometrium were arranged in tight columns, and the tight connection of the cells and dense microvilli could also be observed in the surface area close to the lumens; however, their glandular lumens were much narrower, and microvilli were partly detached; the border lines of the glandular epithelia cells were basically clear; the number of mitochondria in the cytoplasm were abundant; their nuclear membranes had fine border lines, with several deep sunken notches on some of them, and some heterochromatins were aggregated close to the nuclear membranes. For the experimental groups （EMS rats with different treatment）, the epithelial cells in the endometrium appeared cubic shape and tightly arranged, and the linking complexes between cells were poorly developed; the microvilli were very few inside the glandular lumens; the number of mitochondria in the cytoplasm were abundant, most of which appeared expanding round shaped without majority of their ridges, bright and empty-looking; the amount of the rough ER and lysosomes was very few; nucleus were located at the bottom of the cytoplasms, with several deep sunken notches on their membranes, and heterochromatins were evenly distributed. In particular, the intercellular side lines from the group with mix treatment were not clear, and their nuclei were significantly shrunk.2. Results of immunohistochemistryThe MMP-2 is usually expressed in the epithelial cells of endometrium, however, its expression was hardly detectable in the normal group of rats （control group）. The expression of MMP-2 in the ectopic endometrial cells of EMS rats （model group） was significantly higher than in the endometrial cells of normal rats （P<0.01）. In addition, the expression of MMP-2 could also be detected from granular and stromal cells of EMS rats （model group）. The staining area for MMP-2 in model group was significantly bigger than in the control group, and the staining was much darker, indicating higher protein expression level. As for the experimental groups, the staining areas for MMP-2 in the ectopic endometrial cells were shrunk, the staining was not even, and the color was faint, in comparison with the model group. Among them, the difference was much more significant in the groups with mixed treatment and treated with hardness-softening and blood-stasis-resolving method only （P<0.05）. In particular, the expression of MMP-2 in the group with mix treatment was the lowest.Similarly, TIMP-2 is expressed mostly in the glandular epithelial cells and stromal cells of endometrium, and its expression in these cells is usually high. TIMP-2 expression in the glandular epithelial cells and stromal cells of ectopic endometrium was much lower, but detectable. In comparison with normal control group, the staining areas for TIMP-2 in the ectopic endometrial cells in the model group were significantly shrunk, and the color was faint, indicating lower expression of TIMP-2. The staining areas for TIMP-2 in the experimental groups became bigger than in the model group. The staining color became darker, indicating an increased level of expression in the experimental groups. Among them, the difference was much more significant in the groups with mixed treatment and treated with hardness-softening and blood-stasis-resolving method only （P<0.05）, especially for the mixed treatment group.Based on the results above, we conclude: either of the two therapeutic methods in treating EMS, kidney-sequential-reinforcing and hardness-softening plus blood-stasis-resolving, is able to decrease the expression of MMP-2 and increase the expression of TIMP-2 in the ectopic endometrium of EMS rats, resulting in the correction of the imbalance of MMP-2/TIPM-2 ratio. The combination of these two methods, kidney-sequential-reinforcing and hardness-softening plus blood-stasis-resolving, has achieved the best result.3. Results of quantitative RT-PCRThere are basal level of MMP-2 mRNA and TIMP-2 mRNA in the eutopic endometrium of normal rats （control group）, and their amounts in ectopic endometrium of EMS rats （model group） are much higher. Although the increase of MMP-2 mRNA leads to the increase of TIMP-2 mRNA level, the magnitude of their increases is not the same. The level of TIMP-2 mRNA is higher than MMP-2 in normal group of rats, while the TIMP-2 mRNA is lower than MMP-2 in ectopic endometrium of EMS rats （model group）. Similarly, the ration of MMP-2 mRNA/TIMP-2 mRNA was further decreased with different magnitude in different experimental groups. The change of transcript level directly leads to the protein production. It’s therefore possible to correct the imbalance of MMP-2/TIMP-2 from their transcript level.Summary of results: the MMP-2 mRNA level was significantly decreased in the two experimental groups, EMS rats treated with kidney-sequential-reinforcing method and EMS rats treated with hardness-softening plus blood-stasis-resolving method, compared with model group. This observation is consistent with immunohistochemistry result, suggesting that the medical treatments from both methods have affected the MMP-1 and TIMP-2 production through their transcript level. However, the MMP-2 mRNA in rats treated with the combination of two methods （mixed group） was abnormally higher than in the model group, failing to show the expected therapeutic result. Since the data are way out of normal range, they are not reliable to be counted.Conclusion:1. We have successfully made a copy of animal models to study endometriosis, and observed significant pathological changes in the ectopic endometrium of EMS rats under the light as well as electron microscopes. The combination of kidney-sequential-reinforcing and hardness-softening plus blood-stasis-resolving methods was able to improve the histological and ultra-structural morphology of ectopic endometrium, and their immune pathological effects, resulting in effective therapy of this disease.2. The inference from our results is that MMP-2 mRNA/TIMP-2mRNA imbalance is the most important factor for endometriosis. The imbalance of MMP-2 / TIMP-2 caused by the increase of MMP-2 mRNA/TIMP-2 mRNA ratio might be the direct cause of endometriosis;3. Therapeutic methods of kidney-sequential-reinforcing and hardness-softening plus blood-stasis-resolving are able to lower the protein levels of MMP-2 and TIMP-2, correct MMP-2/TIMP-2 imbalance, to reduce the ectopic endometrial aggression and interfere with their cultivation, so as to treat this disease. It remains to be answered whether the effect of above treatments works through the transcription level of MMP-2 and TIMP-2.4. Based on animal model study, we are able to dissect the mechanism of the effect of therapeutic method （kidney-sequential-reinforcing and hardness-softening plus blood-stasis-resolving） on the "aggression" process of ectopic endometrium of EMS rats, and provide experimental evidence to establish more effective therapeutic methods to treat EMS. Therefore, our research has great potential for future clinical application. |
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