| Objectives:Gastric administration of Tripterygium Glycoside (TG) Pill was given tothe rats to establish ovarian dysfunction model through which to observe theeffects TiaoJingZhuYun decoction on reproductive system of rat with ovariandysfunction and study futher the mechanism of this decoction in strengtheningovarian reserve, improving follicle qualilty and enhancing the endometriumreceptivity.Furth more, verified clinical curative effect on infertility caused byDecreasing Ovarian Reserve (DOR) and follicle development dysfunction canprovide a scientific evidence to some degree for the research in developingpatterned Chinese herbal products.Methods：The placebo group (P group) were perfused with physiological saline bydoes in10000mg/(kg·d), TG were administered to the rest for2consecutiveweeks to copy the model of low reproduction for follicle developmentdysfunction caused by DOR deemed as insufficiency of kidney. In the meantime, conduct viginal exfoliate cells test to observe the change in estrus so asto ensure whether the model was induced successfully. Next, the model-induced rats were randomized into model control group(M group), weartern medicine treated group (W group), Chinese herb treatedgroup (H group), combination group (C group). Then, P group and M groupwere perfused with physiological saline by does in10000mg/(kg·d). Whereas,conduct hormone replacement therapy (HRT) and ovarian stimulation in Wgroup. It comes specificly as below: implement HRT in1stestrus, perfuse ratswith Estradiol Valerate Tablete by does in0.12mg/(kg·d) for5sucessive daysbut add Progesterone Capsule by does in18mg/(kg·d) on4thday, thenadminister Clomifene Citrate Tablet to rats by does4.5mg/(kg·d) to conductovarian stimulation therapy for2consecutive days before the next proestrus.H group were ferfused with TiaoJingZhuYun complex priscription powder bydoes in5250mg/(kg·d) for10sucessive days. C group were treated withcombination of Chinese herb&Werstern drugs, and the does was the same asthe former2groups respectively.Adopting Enzyme Linked Immunosorbent Assay (ELISA) andChemiluminescence Immuno Assay (CLIA) after the therapy to detect thelevel of Follicle Stimulating Hormone(FSH)、 Luteinizing Hormone (LH)、Estradiol (E2)、 Progesterone (P) and Anti-Müellerian hormone (AMH)respectively. Taking advantage of Pathology to study the shape andPathological change of rats’ ovaries ang uteries, and make a statistics of thecount of follicles on each rank and corpus luteum. Observing the change onthe level of protein expressed of ovarian relevant cytokines by means ofWestern Blot. Watching the expression condition of ovarian cellspro-apoptotic gene Bax and the change on apoptosis rate throughImmunohistochemical Method and TdT-mediated Dutp Nick-End Labeling(TUNEL). Detecting the expression of endometrium Calcitonin inperi-implantation by Real-Time Quantitative Polymerase Chain Reaction(RT-PCR) to inspect the change of its receptivity comes to the final step. Results:1. Impact on the reproductive hormone in serum: FSH and LH of Mgroup were higher than that of P group; whereas, E2and AMH were lowercompared with P group, which maked a statistical significance (P<0.01). Thehormone level of W group and H group has recovered to the normalrespectively after the therapy, FSH and LH decended but E2and AMH rised incomparision with M group, which counted a lot in statistic, on the contrast, thedifference with P group didn’t make any sense.2. Influence on the organ index: There was statistical significancebetween the P group and M group refer to the ovary and womb index whichwere lower in M group than in P group. But index in W group and H grouphas got up, not making a statistical significance (P>0.05) when compared withP group after the therapy.In contrast to M group, it did not (P<0.05).3. Effect on the microstructure of ovary and womb: a scale of primitivefollicle can be spotted in HE section by light microscope in ovarian cortex of Pgroup. The size and shape of follicle on every stage which developed normallyvaried. Oocytes were both large and round that were in distinct outline, andcorpus luteum in large amount can be caught.Follicles on each stage whichhad a short diameter were barely spotted in M group. Layers of granulosa cellsin follicle had reduced in comparision with other groups. There were also alarge sun of atrefic follicles and corpus albicans and infrequent corpus luteum.The medella of ovary were intensive and its blood vessels were fewer. Firstand second rank follicles in W group and H group developed in the normalway and the condition oocytes were the same as P group but atrefic folliclescan be seen and abundance in the blood vessels of medulla.Endometrial epithelial cells of P group and H group arranged orderly,glands and matrix were abundant.Whereas, the cells of M group were indisorder with decreasing glands and sparse papilledema matrix as well as massive infiltrating ensinophilic granulocyte. But the situation in W groupwere opposite.4. Impact on the count of follicle and corpus luteum: The developing andmature follicles has reduced with infrequent corpus luteum and increasingatrefic follicles in M group which maked a great statistical significance(P<0.01) compared with P group. After therapy, the developing and maturefollicles has increased obviously and atrefic follicles has reduced, improving alot in comparision with M group (P<0.01) and making any sense opposed to Pgroup (P>0.05). What was emphasize was that the developing follicles in Wgroup were up apparent than that in P group and H group (P<0.01), but thedistinction of mature follicles and atrefic follicles as well as corpus luteum canbe neglected (P>0.05).5. Influence on cytokines in ovary: The level of quantitative protein beingencoded of cytokines such as Stem Cell Growth Factor (SCF)、HumanGrowth Differentiation Factor-9(GDF-9) and Bone Morphogenetic Protein-15(BMP-15) has clearly decended opposed to P group, making a statisticalsignificance (P<0.05). Even though the expressin of three cytokines in Wgroup and H group has been evidential up-regulated, the distinct with P groupmake any sense (P>0.05).6. Effect on the expression of ovarian cell pro-apoptotic gene Bax: Thepositive expression ratio of ovarian cell pro-apoptotic gene Bax in P groupwere manifest lower than the rest, making a statistical significance with Mgroup and W group (P<0.01) but a bit difference compared with H group(P<0.05). The positive expression rate of pro-apoptotic gene Bax in W groupand H group were much lower than that in M group after the therapy. Thoughthe divergence between M group has statistical significance (P<0.01), thedifference between W group and H group can be ignored (P>0.05). 7. Impact on the apoptosis rate of ovarian cell: The apoptosis rate ofovarian cell in P group was at the bottom among all the groups and thedivergence was much evidential compared to the other groups respectively(P<0.01). When the W group ang H group got treated even though theapoptosis rate has descended a lot in comparision with M group (P<0.01) anddifference made no sence between the two groups (P>0.05), there was stillstatistical significance (P<0.01) opposed to P group.8. Influence on the content of serum Progesterone in peri-implantation:There was no manifest differense among groups. Divergence between Pgroup、H group and W group can be neglected (P>0.05).9. Effected on the expression of endometrium CT mRNA inperi-implantation: The expression level of endometrium CT mRNA of Wgroup in peri-implantation was the lowest in comparision with P group、Cgroup and H group,making a great statistical significance (P<0.01). Specificly,the expression level of H group was a bit higer than that of P group and Cgroup, but the difference between the three groups maked no sense (P>0.05).Conclusions:Professor Shengyang Xu clings to the perspective that it is primary insufficiency of kidney Essence and Qi and partly hepatic stagnation whenmerged with luteal insufficiency based on syndrome differentiation thatcontributes to infertility as the result of DOR and follicle developmentdysfunction. So, he puts forwards the decoction of regulate menstruation forpregnancy promotion, a creative achievement from his perspective which isfocused on nourishing kidney to foster the development of follicle, smoothingliver to improve the function of corpus luteum, and enhancing theendometrium receptivity by strengthening the spleen as the therapy of this disease or supplementary for In Vitro Fertilization And Embryo Transfer(IVF-ET).This experimental research show that TiaoJingZhuYun decoction canregulate the disordered hormorne level, renovate model rats injured womb andovarian tissue as well as mitigate pathological change of microstructure bymeans of Hypothalamus-Pituitary-Ovary Axis. What’s more, it’s able to fosterthe development of follicle and corpus leteum, reduce the amount of atreficfollicle, accumulate the protein that relevant cytokines in ovarian encodes toregulate the function of model rats autocrine and paracrine as well asjustacrine, depress apoptosis of rats granulosa cells, slacken atrefic follicle toadjust it to cultivate in forward direction. Finally, TiaoJingZhuYun decoctioncan upgrade the expression of endometrium CT mRNA in peri-implantationfor enhancing the endometrium receptivity which can lay the foundation ofembryo implantation. |
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