A Preliminary Study Of Conditioning Liver And Kidney With Periodic Treatment On Menstrual-like Mouse Model

Endometrium undergoes cyclical changes of proliferation, differentiation and disintegration, repair by the impact of hormones, which as a target tissue of ovarian steroid hormones control. Barriers to any part of the menstrual cycle will cause the entire pathological change, so menstrual-linked disorders affect physical and mental health of women. Endometrial cyclical periodic changes and follicular maturation has important significance on reproductive. Factors of endometrium were the key failure in IVF-ET.The study of mechanism of menstrual still has many problems in suspension and unsolved, as the characteristic change of endometrial reflects the running mechanism of menstruation, the lack of menstruation model is the main bottleneck of in-depth study on it. Therefore, our study group introduced the menstrual-like mice model to study the impact of conditioning liver and kidney with periodic treatment(CLKPT) on it, and to establish a theoretical basis for clinical treatment of menstrual disorders.In menstruation, many basic research of menstrual-like model including uterine condition of decidual, hormone changes in serum, the expression of inflammatory cytokines, and the change of the blood vessels, the expression of disintegration related regulatory factors of MMPs, and NF-κB pathway is involved in a variety of endometrial tissue extracellular matrix degradation as well as. Our study is on the basis of successful replication mouse model of menstrual-like and with traditional Chinese medicine(TCM) impact on it for the first time. In our study, to observe the morphological changes of the uterus, uterus index, vaginal smear test, E2 and P levels in serum. And ER-α, PR, VEGF/VEGFR2 in proliferation and secretion phases were detected by Immunohistochemical method, Western blot and Real-time PCR. In the phase of shedding, the expression of MMP3, MMP9, P50 and P65 were observed by Immunohistochemical staining, the expression of MMP3 and MMP9 were detected by Real-time PCR. The expression levels of MMP3, MMP9, nucleoprotein of P50 and P65 were determined by Western blot method. In different phases in the lining of the uterus was bounded, to study the mechanism of CLKPT on endometrial from the organization, molecular, genetic level and provide the experimental basis. Part One The theoretical discussion of conditioning liver and kidney with periodic treatmentObjective: to study the theoretical basis, phasing principle, the principle of CLKPT, and the based source of prescription and application in this study were discussed.Methods: There are corresponding theories of “Kidney Qi-Tian Gui-Chong Ren-Uterus” in TCM menstruation mechanism and H-P-O axis adjusts the uterus cyclical changes in the endometrium of modern medicine neuroendocrine system. The significance for the treatment of menstrual disorders by cyclical changes in the characteristics of the menstrual period could conform to the proliferation, differentiation, and periodic changes of disintegration repair in endometrium. This is also the embody principles of “Suit measures to differing conditions in terms time” and the combination of diseases and syndromes in TCM. The theory of “Kidney is the foundation of the innate, and of reproduction”, “Liver is the innateness in the women’s physiology” and “Woman with blood foundation” plays key roles in female physiology and reproductive. The CLKPT is with “period” for the principle, and to reflect the clinical significance of “Regulate the menstrual function is good for pregnancy and diseases treatment” by combination of disease, symptom and time.Conclusion: The conditioning liver and kidney periodic treatment is based on the characteristics of menstruation periodic changes, and it provide important clinical and research significance for menstrual disorders treatment.Part Two Effect of conditioning liver and iddney with perodic treament on uterine morphology in menstruall-like modelObjective: To discuss CLKPT on the endometrial proliferation, secretion,disintegrating of cyclical changes by observing effect of CLKPT on uterine horns, uterus index and endometrial morphology in menstrual-like model.Methods:1 Animal grouping and model replication: 200 SPF nonpregnant female C57BL / 6J mice, 8 to 10 weeks old, weighing18~20g, were randomly divided into Sham group(n=40), Control group(n=80), TCM group(n=80). Except the mouse of Sham group, the others were established model of menstrual-like. Animals were ovariectomized under anaesthesia and then sequentially treated with steroid hormones, on Days 1, 2 and 3 of the study all mice were subcutaneously(S.C.) injected daily with 100 ng of 17β-estradiol(E2) in arachis oil at 09:30h. They were on Days 4, 5 and 6 without any treated. On Day 7, 50 ng of progesterone(P) and 5ng of E2 in arachis oil were injected S.C.at 09:30h and at the same time P implants were inserted into the back of each mouse. On Days 8 and 9, 5ng of E2 in arachis oil were injected S.C. at 09:30h,while on Day 9 at 11:30h, 20μL of arachis oil was injected into the lumen of the left uterine horn of each mouse to induce decidualization through a dorsal incision, however, as a negative control the right horn was not treated. The P implants were removed 49 h later and as the 0h. The Sham group without ovariectomized and only laparotomy, closed abdomen was done, and the relative test for model successing was detected.2 Method of administration: Respectively, during the days of 1 to 4, the mice were oral administered with Bushen Recipe, days of 5 to 9, Tiaogan Recipe oral administered, and during the days of 9 to 12, the mice were oral administered with Huoxue Recipe in TCM model. The dosage was0.2ml/10 g.3 Design of sampling: The mice were sacrificed at day 4th for T1 point;the day 9th for T2 point; and at 0h, 8h, 12 h, 16 h, 24 h and 32 h after P withdrawal the uterine horns were harvested at each time interval.4 Index of detection: The general conditions in mice, the morphology of uterine horn, vaginal cytology smear, the uterus index were detected, and cross Part Two Effect of conditioning liver and kidney with periodic treatment on uterine morphology in menstrual-like model section of uterine were prepared respectively for H&E staining.Results:(1) The general condition in mice: the surgical wound recovered well, mental state was well, and response to external stimuli was sensitive, fur was shiny, daily diet and water were stable, the body weight was stable before and after steroid hormones treated. After laparotomy, the uterine horn ligation no significant inflammatory response, uterine horn was in good condition.(2) The morphology of uterine horn: with the time changing over, the uterine horn showed no significantly change in Sham group. The uterine horn enlargement and with liquid filling, peripheral vascular intensive was clearly visible at T1 point, which effect by E2 only in Control group. The entire uterus was shown red and no significant enlargement at T2 point, which effect by combined E2 and P. The experimental side uterine horn showed pink with luminal thickening and swelling at 0h point. Compared with 0h, the horn showed no significantly changes, the experimental side color deepened and showed dark red, swollen significantly enlarged of lumen at 16 h. From 24 h to 32 h time points, the experimental side of uterine horns showed dark purple color, the degree of swelling reduced. Compared with the same time of Control group, the experimental side of the uterine horn presented a similar trend; however, there were significant differences in different time points, the changes at 0h time point was similar with 0h of Control group, the degree of swelling showed obvious at 12 h, and the whole bleeding process was shortened, the experimental side showed clearly repair appearance at 32 h point.(3) The situation of vaginal smears: with the time going on, there were no red blood cells in Sham group. The red blood cells were not detected during 0h and 8h in Control group. The percentage of positive mice increased from 12 h, and the percentage was 87.5% at 24 h, 88.9% at 32 h, the most number of red blood cells was at 24 h time point. There were red blood cells detected at 0h in TCM group, the percentage was 87.5% at 16 h, 77.8% at 32 h, and decreased at 32 h, the highest point was at 16 h. Compared with the Control group, the rate of endometrial disintegration increased significantly, the duration of breakdown shortened significantly in TCM group.(4) The changes of uterus index: Compared with the sham group at the same time point, the both sides of uterus index were significantly increased(P<0.05) at T1 and T2 points, the left side of uterus index were significantly increased(P<0.05) from 0h to 32 h with decidualization, the right showed no significant difference(P>0.05); compared with the right side, the left uterus index were significantly increased(P<0.05) in both model and TCM group from 0h to 32 h points. The highest value was at 16 h in model and at 12 in TCM group; compared with the Control group, the left uterus index were significantly increased from T1 to 12h(P<0.05), significantly reduced at 32h(P<0.05).(5) The changes of uterine morphological: with the time changing, the tissue epithelial was integrity, functional layer cells tightly packed, and stromal cells showed no significant changes in Sham group, indicating decidualization not happened in the horns. The endometrial showed monolayer epithelial structure, with high quality nuclear nucleus of oval shape, and the matrix cell were loosed at T1 point. The endometrium was false stratified change, and secretory cells were observers at T2 point; compared with the Control group, the endometrial changes were more obviously in TCM group. After P withdrawal, the uterine cavity changed narrow due to interstitial decidua cells thickening the tissue, and cell hypertrophy with cytoplasm stained red at 0h. The interstitial decidua cells shrinkage, cytoplasmic and chromatin were dense, eosinophilic cell staining enhanced and karyopycnosis at 8h; from 12 h to 16 h, cell interstitial were infiltration by leukocytes, decidualized stromal cells necrosis and bleeding; from 24 h to 32 h, the endometrial shedding and bleeding with leukocyte infiltration and emergence of a large number of necrotic tissue. The experimental side of uterine tissue trended similar in TCM and Control groups, however, there were significant differences changes at each time points, with similar changes at 0h in TCM and 8h in control, and cytoplasmic staining red; the most bleeding at 12 h, the boundaries between disintegration and basal layer showed more clear; the luminal epithelial cell showed papillary hyperplasia, endometrial had the significantly repaired change at 32 h.(6) The change of duration of endometrium breakdown: Compared with the Control group, the parameters of the uterus showed similar trends with Control group in TCM group, nevertheless, the peak of each parameter in TCM group ahead 4 hours than the control, the endometrial repair features observed significantly at 32 h time point, indicating Huoxue Recipe can significantly shorten the duration of endometrium breakdown.Conclusion: Bushen Recipe could significantly promote the proliferation changes of endometrium in the proliferative phase; Tiaogan Recipe could significantly contribute to the cellular differentiation of endometrium in secretory phase; Huoxue Recipe could significantly accelerate the disintegration of the endometrium, and significantly shorten the breakdown duration. Part Three Effect of conditioning liver and kidney with periodic treatment on E2, P, VEGF and its receptors in endometrial proliferation and secretion phaseObjective: To investigate the effects of Bushen and Tiaogan Recipe on E2/ER-α, P/PR and VEGF/VEGFR2 of endometrial and to explore the correlation of Bushen and Tiaogan treatment on the proliferation and secretion phase.Methods: The methods of animal grouping, model establishment and administration were the same to Part two. The E2 and P in serum were detected by radioimmunoassay, and the protein and m RNA expression of ER-α, PR and VEGF/VEGFR2 were detected by immunohistochemistry, Western blot and Real-time PCR respectively.Results:(1) Comparison of serum E2 and P: compared with the Sham group, serum content of E2 and P were increased significantly(P<0.05). Compared to the Control group, serum content of E2 and P were increased, but there was no significant difference(P>0.05).(2) Result changes of Immunohistochemistry: The positive signal of ER-α mainly located in the cytoplasm of interstitial cells, and the expression became strengthen in epithelial and interstitial cells at T2 than T1. The positive signal of PR was weaker in the cytoplasm and nucleus at T1, and expressed in epithelial cells at T2.The positive signal of VEGF and VEGFR2 were strong in both epithelial and interstitial cells at T1 and T2 point.Compared with the mice in the Control group,the expressions were increased in TCM group.(3)The protein expression of ER-α,PR,VEGF/VEGFR2 detected by Western blot:Compared with the T1 time point,the protein expression of ER-αwas decreased;the PR and VEGF were enhanced obviously(P<0.05);and the VEGFR2 was decreased significantly(P<0.05)at T2 points in Control group.Compared of the mice in Control group at the same time point,the protein expression of ER-α,PR and VEGF/VEGFR2 were increased significantly(P<0.05).(4)The m RNA expression of ER-α,PR and VEGF/VEGFR2 detected by Real-time PCR:Comparison with the sham group,the m RNA expression of ER-α,PR and VEGF/VEGFR2 were enhanced significantly(P<0.05).Compared with the T1 time point,the m RNA expression of ER-αwas decreased;the PR and VEGF m RNA were enhanced obviously(P<0.05);and the VEGFR2 m RNA was decreased significantly(P<0.05)at T2 points in Control group.Compared of the mice in Control group at the same time point,the m RNA expression of ER-α,PR and VEGF/VEGFR2 were enhanced obviously(P<0.05)Conclusion: The status and function of endometrial in proliferative and secretory phase were adjusted by Bushen Recipe and Tiaogan Recipe. The mechanism was not the serum hormones increased simply, might be the expression of ER-α, PR and VEGF/VEGFR2 were enhanced by TCM treatment. Part Four Effect of conditioning liver and kidney with periodic treatment on the expression of MMP3 and MMP9 in endometrial menstrual phaseObjective: To observe the phase changes in expression of MMP3 and MMP9 in menstrual, and to investigate the mechanism of Huoxue Recipe effect on endometrial disintegration.Methods: The methods of animal grouping, model establishment and administration were the same to Part two. The protein and m RNA expression of MMP3 and MMP9 were detected by immunohistochemistry, Western blot and Real-time PCR method respectively.Result:(1) Result changes of Immunohistochemistry: with the time going on after progesterone withdrawal, the expressions of MMP3 and MMP9 were were detected in the right side of Control and TCM group. The positive signal of MMP3 mainly located in the stromal cells of decidualization extracellular matrix and basal layer, with the extension of the stromal cells in the decidua over time, the positive signals became strengthen and the peak expression at 16h; from 24 h to 32 h, the positive signal expressed in a large area of the extracellular matrix shedding of endometrial stromal cells. The positive signal of MMP9 mainly located in the interstitial cells, the expression was weak at 0h after progesterone withdrawal, and intensity of expression crescendo with time. A large of area of endometrial disintegration happened at 16 h, the positive signal became stronger in decidua region. Compared with the same time point in the Control group, the expression of the scope and intensity was significantly enhanced at 0h to 16 h, and the strongest point at 12 h, and the expression of the strength diminuendo after 16 h, at 32 h significantly decreased.(2) The protein expression of MMP3 and MMP9 detected by Western blot: Compared with 0h, the protein expression of MMP3 significantly increased at 8h-16h(P<0.05) in control group, significantly decreased at 32h(P<0.05), the peak was at 16 h. Compared with 0h, the protein expression was significantly increased at 8h-16h(P<0.05) in TCM group, while significantly decreased at 32h(P<0.05), the peak was at 12 h. Compared with 0h, the protein expression of MMP9 was significantly upregulated at 8h-32h(P<0.01) in control group, the peak was at 16 h. Compared with 0h, the protein expression was significantly increased at 8h-16h(P<0.01) in TCM group, while significantly decreased at 32h(P<0.01), the peak was at 12 h. It indicates that MMP participate disintegration of endometrial shedding and bleeding. The expression was ahead of 4h in TCM, and the Huoxue Recip accelerated the disintegration of endometrium.(3) The m RNA expression of MMP3 and MMP9 detected by Real-time PCR: Compared with 0h, the m RNA expression of MMP3 and MMP9 were significantly increased at 8h to 24 h in Control group, the peak was at 24 h. Compared with the same time point in Control group, the expression was significantly increased(P<0.05) in TCM at 0h to 16 h, and the peak was at 12 h, the expression was significantly down-regulated at 32h(P<0.05).Conclusion: Huoxue Recipe could regulate the protein and m RNA expression of MMP3 and MMP9 at 12 h in the early of endometrial disintegration, and the expression peak was advanced, but down-regulated at in the late of shedding and early of repair phase. Moreover, Huoxue Recipe shortened the duration of endometrium breakdown. Part Five Effect of conditioning liver and kidney with periodic treatment on the expression of NF-κB signaling pathway in endometrial menstrual phaseObjective: To observe the expression of nucleoprotein of P50 and P65 in menstrual, and investigate the regulation mechanism of activating and closure of NF-κB signaling pathway.Methods: The methods of animal grouping, model establishment and administration were the same to Part two. The expression by immunohistochemistry, and the nucleoprotein of P50 and P65 were detected by Western blot.Results:(1) Result changes of Immunohistochemistry: the expression of MMP3 and MMP9 were observed in the right side of Control and TCM group. The positive signal of P50 and P65 mainly located in cytoplasm, and transferred to nucleus at 16 h, indicated that NF-κB signaling pathway was in inactive state at 0h, and activated at 16 h. Compared with the Control group, the positive signal was expressed in nucleus of decidua interstitial cell at 0h; the expression of peak was at 12 h, but positive signal located in cytoplasm at 8h and 24 h, indicating that NF-κB signaling pathway was activated at beginning, followed by a buffer phase, and then in a strong activation state, finally closed timely.(2) The protein expression of P50 and P65 detected by Western blot: After progesterone withdrawal, Compared with 0h, the nucleoprotein expression of P50 was significantly upregulated at 8h-32 h in control group(P<0.01). There was a highly expressed for P50 nucleoprotein at 0h in TCM; Compared with 0h, the expression was significantly increased at 12h(P<0.05), while was significantly decreased at 24 h and 32h(P<0.01). Compared with 0h, the nuclear protein expression of P65 significantly increased at 12 h, 16 h in control group(P<0.05), little change in the remaining points; There was a highly expressed for P50 nucleoprotein at 0h in TCM; compared with 0h, the nuclear protein expression significantly decreased at 8h and 24h(P<0.05), and 32h(P<0.01).Conclusion: Huoxue Recipe could regulate the P50 and P65 into and out of the nucleus rapidly, and the NF-κB signaling pathway activated quickly and closed timely by TCM, and thus made the endometrium has a quickly and completely breakdown in favor of endometrial repair.