Effects And Mechanism Of The Estrogen-Receptor-Antagonist-Like Effect In Triptolide

Objective: Triptolide is the strongest biological activity of substances which isolated out from Tripterygium, and it is currently also the main component of the quality control standards in Tripterygium preparations with biological activity of immunosuppression, anti-inflammatory, and antitumor. The antitumor effect of triptolide has the characteristics of high efficiency, broad spectrum, which especially has a good curative effect for gynecological tumors associated with estrogen. Therefore triptolide is considered to be promising antitumor drugs. Our topic is to investigate the effect of estrogen-receptor-antagonist-like effects in triptolide on the immature mice, and explore the related mechanism through estrogen receptor pathway, and at the same time verify its function and mechanism in vitro. Our study will provide the possible experimental support for possible mechanisms and the clinical application of gynecological tumors treated by triptolide.Method: Estrogen-receptor-antagonist-like effects of triptolide were studied in vitro and vivo, and then explore its mechanism of action. In vivo: By using the immature mice model we explore the effect of estrogen receptor antagonist in triptolide. 21-day-old Kunming healthy female immature mice were fed adaptively two days. Then they were randomly divided into ten groups: Control Group, ICI Group, TP50(50 μg/kg) Group, TP100(100 μg/kg) Group, TP200(200 μg/kg) Group, E2(17b-estradiol) Group、E2+ ICI Group, E2+TP50 Group(TE50),E2+TP100 Group(TE100), E2+TP200 Group(TE200). ICI Group injected intraperitoneally 500 μg/kg fulvestrant suspension. Triptolide each dose group respectively treated by triptolide. E2 Group were given with 100 μg/kg 17 b-estradiol by gavage. E2 + ICI Group were while giving fulvestrant and 17 b-estradiol, TE50, TE100 and TE200 Group were while giving triptolide and 17 b-estradiol, there are at least 0.5 h between two drugs were given. And Control Group treated with isovolumetric distilled water per day for 7 days. On 7 days, the rat blood samples were withdrawn by eyeball-plucking method and a centrifuge separates the serum from the blood. The activities of E2 of the blood samples were evaluated by ELISA Kit. All rats were then sacrificed and their uterus and vagina tissues were separated. The histology and morphology of their uterus and vagina was observed by HE and immunohistochemistry technique and Western blot were used to detect the distribution and expression of ERa and ERb in rat uterus and vagina. Meanwhile the gene expression of ERa and ERb were detected by real-time polymerase chain reaction. In vitro: Estrogen receptor-positive MCF-7 breast cancer cell viability was measured by MTT assay to discover the effect of triptolide. The logarithmic growth phase MCF-7 cell were digested by trypsin enzyme to single cell suspension and inoculated in 96 well plates. For 24 h to be adherent cells, each dose of triptolide group were treated with the corresponding concentration of triptolide and control group was treated with equal volume of DMEM high glucose medium. Each group have 3 holes. After culturing 48 h measured absorbance, compute the mean growth rate and screen the effective concentration range. Using luciferase assays to detect the proliferation of triptolide on HEK-293 cells stably transfected with ERa-ERE-luciferase reporter gene. The logarithmic growth phase cell were digested by trypsin enzyme to single cell suspension in DMEM without phenol red and inoculated in 96 well plates. For 24 h to be adherent cells, each dose of triptolide group were treated with the corresponding concentration of triptolide and control group was treated with equal volume of medium. Each group have 3 holes. After 24 h, transfer to a white microplate after lysis and measure the luciferase activity immediately. Detect the binding strength between TP and ERa/b in Moe docking molecular docking technology. According binding score, the more negative the representative of the stronger binding. ERs binding kits were used to verify the binding capacity between triptolide and estrogen receptor. Triptolide was gradient dilution and formulated corresponding solution in accordance with the instructions. Then added them into 384 blackboard, incubated 2h after mixing in the dark at room temperature. At last it was detected at a wavelength of 485/535 nm. The Western blot were used to detect the expression of ERa and ERb in MCF-7. Meanwhile the gene expression of ERa and ERb were detected by real-time polymerase chain reaction.Result: 1. The anti-estrogen-like effect of triptolide in immature mice: TP can reduce the uterus coefficient of immature mice, while can reduce the increased effect of E2 in uterine factor. TP can reduce the content of E2 in the serum of immature mouse, while can reduce the elevated role of E2. HE staining showed that TP100 and TP200 group significantly inhibit the growth of uterus and vagina in immature mouse, and compared with the E2 group, TE100 and TE200 group also has a prohibitive role in promoting effect of E2, the prohibitive effect is consistent with ICI. 2. The role of inhibition of growth on triptolide on breast cancer MCF-7 cell:MTT assay in vitro showed that triptolide can significantly inhibit the proliferation of MCF-7 cells in the range of 10~100 n M concentration, while inhibit the promotion of E2 on MCF-7 cell. The effect is consistent with ICI, which works best when concentration is 40 n M. The results prompted that TP had an anti-estrogenic effect. 3. The down-regulating of the expression of estrogen receptors by triptolide in uterus and vigina of immature mice: The results in vivo suggest that triptolide played its estrogen-receptor-antagonist-like effects by reducing the expression of estrogen receptor. Results in IHC and Western Blot showed, TP100 and TP200 group can significantly reduce the expression of ERa and ERb in the uterus and vagina, and ERb slightly weaker than ERa, the effect was consistent with ICI; while TE100 and TE200 group which can show a similar effect of E2 + ICI group significantly inhibited the expression of ERs, and thee express in uterus is stronger than in vagina. The result at the genetic level suggested that in the uterus and vagina of immature mice compared with Con group, the ERa and ERb expression of ICI group was significantly lower, and TP100 and TP200 group are also reduced expression. The expression of E2 group was obviously strengthened, and compared with E2 group, TE100 and TE200 ERa and ERb expression were significantly reduced. 4. The down-regulating of the expression of estrogen receptors by triptolide in vitro: TP and ERa and ERb has strongly binding capacity by molecular docking test results. In the luciferase assay, triptolide can significantly inhibit the expression of ERa in the range of 10~100 n M concentration, while inhibit the promotion of E2 on expression of ERa. The effect is consistent with ICI, which works best when 40 n M. The results showed that TP had an estrogen-receptor-antagonist-like effect. The binding experiments on TP and ERs binding kits showed that TP existed the competitive binding effect of ERa and ERb in the dose range, as the concentration increases, the combination is enhanced. 5. The inhibition of the expression of estrogen receptors by triptolide in MCF-7: Western Blot and Q-PCR results prompted TP can significantly inhibit the estrogen receptor in the level of protein and gene expression in MCF-7, and inhibited the promotion expression of the E2.Conclusion: The paper found that triptolide has anti-estrogenic effects in vivo and in vitro and verified the mechanism of the estrogen receptor pathway. first proposed triptolide estrogen receptor antagonist like effects. Then it found triptolide can competitively bind to estrogen receptors and inhibit its expression. This article was first proposed that triptolide had the same effect of estrogen receptor antagonist, meaned it had estrogen-receptor-antagonist-like effect. The development of the majority of gynecological tumors was closely linked with estrogen and its receptor. Therefore, further study triptolide on gynecological cancer mechanism of action, provide the basis for the treatment of gynecological cancer, for selective treatment of gynecological cancer will have important significance.