Mechanism Research Of Electroacupuncture Combined With Bushenhuoxue Decoction On Uterine Receptivity After Ovulation Induction In PCOS Rats

ObjectiveEndometrial receptivity is refers to the ability of endometrium receptivity to accept embryonic implantation. According to the difficult of the endometrial receptivity in PCOS patients after ovulation, progynova(estrogen) is frequently-used to increase the endometrial thickness, but the clinical effect is not satisfactorily. The curative effect of Chinese medicine can reduce the side effect of Western medicine.PCOS endometrial dysplasia and is accompanied by the abnormally expression of ER, PR, Integrinavb3 and HOXA10, on the basis of previous studies, this subject is focused on the endometrial receptivity after ovulation, to research the combination of acupuncture with medicine on endometrial receptivity of PCOS in different period after ovulation induction(after ovulation, periimplantation period, early pregnancy).For clinical prevention and treatment of polycystic ovary syndrome, increase pregnancy rate and decrease the rate of abortion has important theoretical significance and practical value.Methods450 clean level female rats of 21 days age(plan of 310,result of loss supplement of animals during experiment), at 24 days age, 40 subcutaneous injections of sesame oil as the normal control group(G), model rat subcutaneous injections of DHEA 6mg/100 g weight/day+0.2ml sesame oil, continuously inject 20 days. Made 270 rats die successful were randomly divided into model group(M), clomiphene group(CC), clomiphene + progynova group(CC+PVG), clomiphene + EA group(CC+A), clomiphene + kidney blood activating Decoction group(CC+M), Clomiphene + acupuncture medicine group(CC+M+A), 45 rats in each group. At 80 days age, except normal group, taking 5 rats in each group do vaginal smear observation of estrus, and measured serum to confirm molding success. Besides that, each intervention were 80 days of age, clomiphene gavage: 10mg/100 g intragastric administration, each rat(1ml/100g) by gavage; progynova 0.104mg/kg, each mouse(1ml/100g) by gavage; traditional Chinese medicine granule according to the proportion of 1:1, 12 g was dissolved in 30 ml of distilled water, each rat(1ml/100g) by gavage, are gavage for 5 days. Electroacupuncture: selection of Guan yuan, bilateral Sanyinjiao acupoint, uterus, then HANS-200 electro acupuncture, continuous wave, frequency of 2Hz, strength is 1m A, power 20 min, 1/d, 5 days of continuous treatment.10 rats in each group in 5 days of treatment do vaginal smear examination, found that keratinocyte prompt ovulation day after the rats were killed, serum, removal of bilateral ovarian uterus, anatomical observation and weighing. The left uterine fixed in paraformaldehyde, used for HE staining, the right uterus scraping endometrial-80 degrees Celsius to preserve the reserve do detection of protein and gene. Besides that, 30 experimental rats in each group at 6:00 pm and male rats according to the proportion of 2:1 cage, as early as 8:00 the next day vaginal smear to check, found that sperm records is the first day of pregnancy(D1), 10 rats each at D4, D5, D8 in pregnancy were harvested at 4:00 in the afternoon, bilateral uterine, record number and the number of implantation the blastocyst in pregnant rats. Under the light microscope observation of endometrial morphology and micro vascular density, scanning electron microscope observation, drink process. Expression of SP by immunohistochemistry, RT-PCR, Western-blotting detection of ER、 PR、 HOXA10 integrin 3 protein.Results1. Comparison of ovulation rate and pregnancy rate after ovulation.Normal group is normal ovulation. ovulate, Group CC+M+A implantation rate was 88%, close to the normal group 100%.2. Endometrial receptivity is influenced after ovulation.2.1 Morphological comparison of endometrium: CC+M+A group is not obvious different than normal group(P>0.05), it’s comparison between other groups on equivalent diameter, area and endometrial thickness of glands and lumens has statistical significance(P<0.05).2.2.RT-PCR result: comparison of ER, PR, HOXA10, Integrinavβ3m RNA in each group, normal group and CC+M+A group increased significantly(P<0.05), there is no obvious difference among CC+M, CC+A, CC+PVG groups(P>0.05). comparison of ER and Integrinavβ3m RNA, there is no obvious difference among CC+PVG, CC+M, CC+A, CC+M+A group(P>0.05);These four groups significantly increased than normal group and model group, CC group, have statistical significance.Comparison of PR and HOXA10 m RNA: CC+M+A increased significantly than CC+PVG group, model group, CC group, has statistical significance(P<0.05).2.3.Western-blotting result:comparison of ER、PR、HOXA10 in RT-PCR result are the same.Integrinavβ3 comparison, CC+M+A group increased significantly than CC+PVG, CC, model group, has statistical significance(P<0.05).2.4. Effect of electroacupuncture combined with Bushenhuoxue decoction on endometrial receptivity after ovulation: morphology of endometrium, CC+M+A group is no difference from normal group, means endometrial receptivity is good, via measuring protein and gene. Expression of ER、PR、HOXA10、Integrinavb3 in CC+M+A group is lower than normal group, but increased significantly than other groups.3. pregnant rats D4, D5 days(periimplantation uterus endometrial receptivity) comparison index3.1.The rat uterine pinopodes compared to the situation: there is no significant difference between the normal group and the CC+M+A group(P>0.05) of endometrial pinopodes fully developed high enough.3.2.Immunohistochemical comparison: expression of ER、PR、HOXA10、Integrinavb3 in normal group and CC+M+A group are all positive expression and there is no significant difference between the two groups.3.3.RT-PCR results: comparison of ER、PR、HOXA10、Integrinavb3 m RNA in each group,there was no significant difference between the normal group and the CC+M+A group(P>0.05). Comparison of ER, PR m RNA in each group: comparing with CC+A group, CC+M group increased significantly(P<0.05), significantly different from CC+M group, CC group, model group(P<0.01); comparison of HOXA10, Integrinavβ3 in each group: comparing with CC+A group, CC+M group, CC group increased obviously(P<0.05), compared with the CC+PVG group, model group increased significantly(P<0.01).3.4.Western-blotting results: comparison of ER、PR、HOXA10、Integrinavb3 protein in each group, there was no significant difference between the normal group and the CC+M+A group(P>0.05). Comparison of ER, PR protein in CC+A group: obviously increased(P<0.05), and CC+PVG,CC+PVG, CC, model group were significant differences(P<0.01); HOXA10、Integrinavb3 protein comparison: with CC+M, CC+A group significantly increased(P<0.05), there was significant with the increase of CC+PVG, CC, model group(P<0.01).3.5 Effect of electroacupuncture combined with Bushenhuoxue decoction on endometrial receptivity during peri-implantation in rats. The rat uterine pinopodes compared to the situation, there is no significant difference between the normal group and the CC+M+A group(P>0.05) of endometrial pinopodes fully developed high enough, means endometrial receptivity is good. The expression of ER、PR、HOXA10、Integrinavb3 in CC+M+A group and normal group is obviously high, and their expression is significantly higher than other groups via SP immunohistochemistry, RT-PCR 、 Western-blotting test.4 pregnant rats D8 days(early pregnancy) compared to endometrial receptivity index of uterus4.1.Immunohistochemical comparison: normal group and CC+M+A group, no significant difference between the two. ER, PR HOXA10、Integrinavb3 expression the CC+A group, CC+M group, CC+PVG group, the three groups had difference(P<0.05); there was statistical significance compared with CC group, the model group(P<0.01). 4.2.RT-PCR: comparison of ER、PR、HOXA10、Integrinavb3 m RNA in each group, the normal group compared with CC+M+A group, no obviously difference(P>0.05); normal group compared with the CC+PVG,CC,model group increased significantly(P<0.01), ERm RNA comparison, significantly increased than CC+A group(P<0.05). Comparison of Integrinavb3m RNA in each group: significantly increased than the CC+A group and the CC+M group(P<0.05), HOXA10 m RNA,PRm RNA Comparison: compared with CC+M group there is a significantly increasing(P< 0.05).4.3.Western-blotting results: comparison of ER、Integrinavb3、HOXA10 protein in each group, the CC+M+A group significantly increased than the normal group(P<0.05) There is a significantly difference compare with CC+M、CC+A、CC+PVG、CC、model groups(P<0.01). Each PR protein comparison: there is no obviously difference between CC+M+A group and normal group(P>0.05),but compared with CC+M group increased significantly(P<0.05), and CC+A, CC+PVG, CC, model group group were significant differences(P<0.01).4.4 Effect of electroacupuncture combined with Bushenhuoxue decoction on endometrial receptivity during early pregnancy in rats. SP Immunohistochemical comparison, compared with normal group, CC+M+A group has a higher expression, via RT-PCR、Western-blotting test, the expression of ER、PR、HOXA10、Integrinavb3 in CC+M+A group is obviously high, and significantly increased than other groups.ConclusionIn the each period, after ovulation, periimplantation period and early pregnancy, electro acupuncture combined with Bushen Huoxue decoction has effect on improving the uterine receptivity. The decoction with the function of nourishing kidney and promoting blood circulation, electro acupuncture improved endometrial receptivity than western medicine of progynova, not only promoted the endometrial receptivity, but also ovulation rate in PCOS rat at the same time, this is related to the mediation mechanism in PCOS rats, the pregnancy rate is associated with up regulation of the endometrium estrogen receptor, progesterone receptor, AV integrin beta 3, homeobox gene A10 and the level of m RNA to achieve.