| Kidney stores essence and controls the reproductivity. Liver stores blood and controls conveyance and dispersion. Basing on three aspects as followed: first, ‘liver and kidney homologous’. Kidney stores essence, liver stores blood. The generation of blood depends on the Qi transformation of kidney essence, and the essence also needs the nourishment of blood. Second, the relation of Kidney and Liver is mother and child. Illness of mother vescera can affect the child one and vice versa. In clinical practice, it is first to treat the gynecological diseases from the point of view of Kidney. That Kidney and liver are treated at the same time is also essential. Third, kidney and liver are closely related to the menstrual cycle. The storing and discharging of Chong and Conception Channels depends upon of kidney to make the menstrual cycle. The relationship between kidney governing storage and liver controlling conveyance, dispersion is both opposite and complementary. It is only when the function of liver controlling conveyance and dispersion is normal that the menstruation occurs regularly. Kidney and liver are closely associated with the menstrual irregularities. Menstruation disorders including Menstruation at irregular intervals, profuse menstruation, prolonged menstruation, flooding and spotting and even infertility resulting from the diseases mentioned above are all related to the delayed and defective repair of the shed endometrium. The clinical effect of treating menstrual disorder through Kidney and Liver is obvious.In 1984, Finn CA etc. established the first mouse menstrual model. The menstrual like changes in mouse uterus occurs by artificial stimulation to make the uterine decidualize, and then withdrawal of progesterone. This erected a good platform for the research of endometrium. We copied the mouse menstrual model by sequential treatment of hormone in pilot trial and observed the mouse endometrial histomorphology at 0h, 8h, 12 h, 16 h, 24 h, 32 h, 40 h, 48 h, 72 h after the progesterone withdrawal following. The histology of endometrium in mouse had demonstrated that the endometrium showed the similar characteristics as the human one in proliferation, secretory stage, breakdown and repair. 12-16 h after progesterone withdrawal, the endometrium began to breakdown. The uterine bleeding was most intense 24 h after progesterone withdrawal and the formation of new luminal epithelium can be observed. 72 h after progesterone withdrawal the uterus is completely reepithilialized, repaired and represent a normal, non-decidualized morphology. To further explore the effects on endometrial repair and mechanism by Bushen formula and Tiaogan formula. We selected 24 h, 32 h, 40 h, 48 h and 72 h as the observation point. On the basis of mouse menstruation model, we compared the effects of three formulae by Bushen formula, Tiaogan formula, Bushen Tiaogan formula(Bushen formula plusing Tiaogan formula) on the endometrial repair. Part Ⅰ Morphological effects of Bushen formula, Tiaogan formula onendometrial repair in mouse menstrual modelObjective: To compare morphological effects on the endometrial repair in mouse menstrual model by Bushen formula, Tiaogan formulaMethods:Two hundred female C57BL/6J mice of 7-8 week of age were randomly divided into the control group, Bushen formula group, Tiaogan formula group, Bushen Tiaogan formula group(Bushen formula plus Tiaogan formula) 50 in each group. Mouse menstrual model was copied: one week after ovariectomy, mice were ovariectomized. After 7 days, mice received s.c.(subcutaneously) injections of 100 ng estradiol in arachis oil on three consecutive days. On day 7, a progesterone releasing silastic tube was implanted s.c. into the back of mice followed by further applications of 5ng estradiol and 50 ng P. 5ng of estradiol in arachis oil was injected s.c. on the subsequent days. On the day of the final estrdiol injection, 15μl of arachis oil was injected into the lumen of the right uterine of each mouse to induce decidualization. The left horn remained untreated as a control. The time at which each mouse injected with sesame oil was recorded.And 49 h later the P implant was removed to initiate P withdrawal. The time of implant removal was regarded as the starting point(0h). Bushen formula group, Tiaogan formula group and Bushen Tiaogan group were treated with Bushen formula, Tiaogan formula, Bushen Tiaogan formula respectively on day 10. The control group was treated with distilled water. All treatment ended on day 14. All groups were intragastrically administered with 0.4ml liquor. Mice were killed at the time of 24 h,32h,40 h,48h,72 h.The uteri were harvested, photographed. Blood was taken from the mice at indicated points of time for estimation of serum estradiol concentration. Tissue sections were analyzed using a scoring system to analyze the morphological differences.Results: 1 Comparion ofserum estradiol concentrations of mice in every group at 24 h, 32 h, 40 h, 48 h, 72 h after progesterone withdrawal 1.1 Serum estradiol concentrations at different time point in the same groupSerum estradiol concentrations were increased(P<0.05) at 24 h, 32 h compared with that at 48 h, and was decreased(P<0.05) at 72 h in control group. In Bushen group, serum estradiol concentration was gradually increased(P<0.05), and were increased(P<0.05) at 48 h and 72 h than that at 24 h, and in Tiaogan group was increased(P<0.05) at 24 h than those at 32 h, 40 h. Serum estradiol concentration was the highest at 32 h. 1.2 Serum estradiol concentrations at the same time point in different groupsCompared with control group, serum estradiol concentrations were significantly decreased(P<0.05) in all experimental groups at 24 h and 40 h and were significantly decreased(P<0.05) in Bushen group and Tiaogan group and were significantly increased(P<0.05) in Bushen Tiaogan group at 32 h. Among every experimental group, serum estradiol concentrations were the lowest in Bushen group at 24 h, and were the highest in Bushen Tiaogan group at 32 h. 2 Comparison of morphological grading of endometrium in mouse enstrual model in every group at 24 h, 32 h, 40 h, 48 h, 72 h after progesterone withdrawal 2.1 Morphological gradings of endometrium at different time point in the same groupMorphological gradings of endometrium in all groups were the highest(P<0.05) at 48 h, 72 h. 2.2 Morphological gradings of endometrium at the same time point in different groupsMorphological grading of endometrium was significantly decreased(P<0.05) in Bushen Tiaogan group compared with that in Bushen group and was significantly increased(P<0.05) compared with that in Tiaogan group as the extension of time and had no significant difference between Tiaogan group and control group(P>0.05).Conclusions: The experimental results showed that the level of E2 had no significant effect on the endometrial repair. Bushen Tiaogan formula can accelerate the repair of endometrium, but the effect was inferior to Bushen formula, and there was no obvious effect on repair of endometrium in Tiaogan formula. Part Ⅱ Comparison of effects on expression of CXCL2, Bcl-2 protein inmouse menstrual model by Bushen formula, Tiaogan formulaObjective: To explore the effects of Bushen formula, Tiaogan formula, Bushen Tiaogan formula on the expression of CXCL2, Bcl-2 protein in mouse menstruation model. To compare the mechanisms of these three formulae in influencing the endometrium repair.Methods: Groups and materials are the same as those in part Ⅰ. Some uterine tissue was fixed in phosphate buffered formalin to prepare for the immunohistochemical examination. Other tissue was snap frozen in liquid nitrogen. Western blot was used to examine the protein expression of CXCL2 and Bcl-2. Immunohistochemical method was used to detect the expression of CXCL2 and Bcl-2 protein. 1Expression of CXCL2 protein in the endometrium of mice in every group at 24 h, 32 h, 40 h, 48 h, 72 h following progesterone withdrawl 1.1 Expression of CXCL2 protein in the endometrium of mice at different time point in the same groupExpression of CXCL2 protein was the higest at 32 h, 48 h in all groups. 1.2 Expression of CXCL2 protein in the endometrium of mice at the same time point in different groupsExpression of CXCL2 protein in all experimental groups was increased(P<0.05) compared with control group at all time points. Among every experimental group, expression of CXCL2 protein was significantly decreased(P<0.05) successively in Bushen group, Tiaogan group and Bushen Tiaogan group at 24 h, 48 h, 72 h and was the lowest at 32 h and was the highest at 40 h in Bushen Tiaogan group. 2 Expression of Bcl-2 protein in the endometrium of mice in every group at 24 h, 32 h, 40 h, 48 h, 72 h following progesterone withdrawl 2.1 Expression of Bcl-2 protein in the endometrium of mice at different time point in the same groupExpression of Bcl-2 protein in all groups was the highest at 48 h. 2.2 Expression of Bcl-2 protein in the endometrium of mice at the same time point in different groupsExpression of Bcl-2 protein was increased in all experimental groups when compared with control group at all time points. Among every experimental group, expression of Bcl-2 protein was the lowest in Bushen Tiaogan group at 24h(P<0.05) and was significantly decreased(P<0.05)successively in Tiaogan group, Bushen group, Bushen Tiaogan group at 32 h, and was the highest in Bushen Tiaogan group at 40h(P<0.05), and was significantly decreased(P<0.05) successively in Tiaogan group, Bushen group, Bushen Tiaogan group at 48 h, and was highest in Bushen group at 72h(P<0.05).Conclusions: The mechanism of affecting the repair of endometrium by Bushen formula and Bushen Tiaogan formula was similar. Both Bushen Results: formula and Bushen Tiaogan formula could accelerate the repair of endometrium by inceasing the expression of CXCL2 and Bcl-2 protein in mouse endometrium, and could affect the repair of endometrium from the point of view of inflammatory response factor. The effect of Bushen formula was superior to Bushen Tiaogan formula. Bushen formula not only can increase the expression of CXCL2 and Bcl-2 protein, but also maintain a longer period of time. Tiaogan formula could also increase the expression of CXCL-2 and Bcl-2 protein, but had no obvious effect on the repair of endometrium. Part Ⅲ Comparison of effects on expression of VEGF, Ang-2, VEGFR2protein in mouse menstrual model by Bushen formula, TiaoganformulaObjective: To explore the effects of Bushen formula, Tiaogan formula, Bushen Tiaogan formula on the expression of VEGF,Ang-2,VEGFR2 protein in mouse menstruation model. To compare the mechanisms of these three formulae in influencing the endometrium repair.Methods: Groups and materials are the same as those in part Ⅱ. Examined protein expression of VEGF,Ang-2 and VEGFR2 with Western blot and detect the expression of VEGF,Ang-2 and VEGFR2 protein with immunohistochemical method.Results: 1 Expression of VEGF protein in the endometrium of mice in every group at 24 h, 32 h, 40 h, 48 h, 72 h following progesterone withdrawl 1.1 Expression of VEGF protein in the endometrium of mice at different time point in the same groupExpression of VEGF protein was the highest at 48 h in all groups. 1.2 Expression of VEGF protein in the endometrium of mice at the same time point in different groupsExpression of VEGF protein was increased(P<0.05) in all experimental groups compared with control group at all time points. Among every experimental group, expression of VEGF protein was significantly decreased(P<0.05) successively in Tiaogan group, Bushen group, Bushen Tiaogan group at 24 h, and was lowest(P<0.05) in Tiaogan group at 32 h, and was significantly decreased(P < 0.05) successively in Bushen Tiaogan group, Tiaogan group, Bushen group at 40 h, and was the lowest in Bushen Tiaogan group at 48h(P<0.05), and was the highest in Bushen group at 72h(P<0.05). 2 Expression of Ang-2 protein in the endometrium of mice in every group at 24 h, 32 h, 40 h, 48 h, 72 h following progesterone withdrawl 2.1 Expression of VEGF protein in the endometrium of mice at different time point in the same groupExpression of Ang-2 protein was the highest at 48 h in all groups. 2.2 Expression of Ang-2 protein in the endometrium of mice at the same time point in different groupsExpression of Ang-2 protein was increased in all experimental groups compared with control group at every time point. Among every experimental group, expression of Ang-2 protein was the highest in Tiaogan group at 24 h, and was significantly decreased(P<0.05) successively in Bushen group, Bushen Tiaogan group and Tiaogan group at 32 h, and was higest in Bushen Tiaogan group at 40h(P<0.05), and was significantly decreased(P<0.05) successively in Bushen group, Tiaogan group, Bushen Tiaogan group at 48 h, 72 h. 3 Expression of VEGFR2 protein in the endometrium of mice in every group at 24 h, 32 h, 40 h, 48 h, 72 h following progesterone withdrawl 3.1 Expression of VEGFR2 protein in the endometrium of mice at different time point in the same groupExpression of VEGFR2 protein was the highest at 48 h in all groups. 3.2 Expression of VEGFR2 protein in the endometrium of mice at the same time point in different groupsExpression of VEGFR2 protein was increased in all experimental groups at every time point compared with control group. Among every experimental group, expression of VEGFR2 protein was highest at 24h(P<0.05), and was the lowest at 32 h in Tiaogan group(P<0.05). Expression of VEGFR2 protein was the lowest at 40 h and was the highest at 72 h in Bushen group(P<0.05), and was the lowest in Bushen Tiaogan group at 48h(P<0.05).Conclusions: Both Bushen formula and Bushen Tiaogan formula could accelerate the repair of endometrium by inceasing the expression of VEGF, Ang-2 and VEGFR2 protein in the endometrium. The mechanism may be the two formulae affected the expression of CXCL2,Bcl-2,Ang-2, VEGFR2 protein by increasing the expression of VEGF protein. The effect of Bushen formula was superior to Bushen Tiaogan formula. This may be associated with that Bushen formula could increase the expression of VEGF, Ang-2 and VEGFR2 protein and maintain a longer period of time. Tiaogan formula could also increase the expression of VEGF, Ang-2 and VEGFR2 protein, but had no obvious effect on the repair of endometrium. A further study is needed to explore the function mechanism. |
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