Effect Of Jiawei Shaoyao Gancao Decoction On Adenomyosis Via ER And MiR-21 Mediated Ras/Raf Pathway

Objective1. Through the collection of Uterine tissue specimens of patients with dysmenorrhea uterine adenomyosis, observe expression of miR-21, aromatase (P450arom), estradiol(E2), estrogen receptor(ER) and Ras/Raf/P-C-Raf signal pathway of protein in uterine adenomyosis eutopic endometrium and ectopic lesion tissue, to explore the correlation between the mechanism of uterine adenomyosis dysmenorrheal and miR-21, P450arom, ER and Ras/Raf cell signaling pathway factor; Through cultivation of adenomyosis patients with dysmenorrheal lesion cells by in vitro, To detect inhibition rate and miR-21 expression levels of estrogen activation and estrogen deprivation state.2. Through cultivation of adenomyosis patients with dysmenorrheal lesion cells by in vitro, to study the effect of Jiawei Shaoyao Gancao Decoction pharmacological serum, or the monomer on cell proliferation and apoptosis and the expression of miR-21,aromatase(P450arom), estrogen receptor(ER)、E2the Ras/Raf/P-C-Raf signaling pathway related factor in uterine Adenomyosis foci cells,Expect to elucidate the treatment of adenomyosis dysmenorrheal deep mechanisms from molecular gene level;determined by pharmacodynamic screening of the effective part, to provide the basis and experimental foundation of Optimization of prescription and Chinese medicine decoction on adenomyosis dysmenorrheal.Methods:1. Select 60 cases wax block from patients with adenomyosis dysmenorrhea who hospitalized in the First Affiliated Hospital of Guangzhou University of Chinese Medicine for hysterectomy uterine from 2006 January-2014 June, with 59 cases wax block from Patients with cervical lesions(CIN)for hysterectomy as control group in the same period. Observe expression of miR-21, aromatase (P450arom), estrogen receptor (ER) and Ras/Raf/P-C-Raf signal pathway of protein in uterine adenomyosis eutopic endometrium、ectopic lesion tissue and normal endometrium and myometrium by immunohistochemical method;Select uterine tissue specimens patients with adenomyosis dysmenorrhea who hospitalized in the First Affiliated Hospital of Guangzhou University of Chinese Medicine for hysterectomy uterine from 2013 January-2014 June, with Patients with cervical lesions(CIN) for hysterectomy as control group in the same period, detect expression of E2、P-c-Raf、miR-21 in uterine adenomyosis eutopic endometrium、ectopic lesion tissue by ELISA, Western blot, real time fluorescent quantitative(probe),using patients with cervical lesions(CIN) for hysterectomy as control group in the same period. Through cultivation of adenomyosis patients with dysmenorrheal lesion cells by in vitro, to detect cell inhibition rate and miR-21 expression levels for Estrogen activation and deprivation, blocking estrogen receptors, inhibit the expression of Ras by using CCK-8 method and real-time fluorescence quantitative probe method.2. Using mmunocytochemistry, enzyme linked immunosorbent assay, real time fluorescent quantitative(probe), flow cytometry, CCK-8 method assay Shaoyao Gancao decotion Shaoyao Gancao decotion pharmacological serum or the monomer for people of adenomyosis lesion cell miR-21、estrogen receptor(ER)、 aromatase(P450arom)、E2、Ras/Raf/P-C-Raf signaling pathway related factor proliferation and apoptosis. Using mifepristone、blank medium and blank serum as control group.Results:1. Expression of E2, ER, P450arom, Ras, Raf in adenomyosis and normal uterine tissue:the expression levels of E2、ER、P450arom in adenomyosis ectopic lesions and eutopic endometrium are significantly higher than what in normal uterine tissue and there was no statistical significance of E2 between adenomyosis ctopic lesion tissue and noral endometrium; P450arom mainly expressed in the cytoplasm, the expression of it between adenomyosis eutopic and ectopic endometrium was no statistical significance (P> 0.05); The expression levelof ER which mainly expressed in the nucleus in adenomyosis ectopic lesions significantly higher than its level in eutopic endometrium; Ras, Raf expressed mainly in the cytoplasm and cell membrane, the expression of it in adenomyosis ectopic lesions and eutopic endometrium are significantly higher than what in normal Normal myometrium and endometrium,and the overall level in the adenomyosis and normal uterine are the same.2. Expression of PC-Raf in adenomyosis and normal uterine tissue: the expression level of PC-Raf in adenomyosis eutopic endometrium is significantly higher than what in normal uterine endometrium (P<0.05);The expresstion levels of P-C-Raf in Adenomyosis ectopic lesions, normal myometrium and endometrium were not significantly different (P> 0.05), and also there was no significant difference between Adenomyosis eutopic myometrium and normal myometrium (P>0.05)3. The cell inhibitor rate of FTS+E2 (RAS inhibitor+estradiol) group was higher than FTS (RAS inhibitor) group (P< 0.05);Both of ICI182780 (ER) inhibitor group and ICI182780+E2 group can inhibit uterine adenomyosis cells proliferation, the difference between them was No statistical significance (P>0.05).The cell inhibitor rate of estrogen deprivation+ICI182780 group was higher than Estrogen deprivation group,, the difference was statistical significance (P<0.05);Estrogen activated state can promote uterine adenomyosis cells proliferation; E2 (estradiol activates) group could improve the expression levels of mi.R-21 of the adenomyosis cells, Compared with ICI182780+E2 group and the contrast group, there was statistical significance (P< 0.05); Compared to ICI182780+E2 group and the contrast group, there was no statistically significant difference (P>0.05).The expression level of mi.R-21 was the lowest, compared to the estrogen deprivation group and control group there was statistically significant (P< 0.05)4. The influence of Jiawei Shaoyao Gancao Decoction pharmacological serum, or the monomer on human adenomyosis lesion cell proliferation inhibition and apoptosis:Jiawei Shaoyao Gancao Decoction high and low concentration group of 24h and 48h, Jiawei Shaoyao Gancao Decoction pharmacological scrum, mifepri stone group 24h and 48h, Paeoniflorin high and low concentration group of 24h, Paeoniflorin liquiritin high and low concentration group of 24h can enhance the apoptosis rate of cells in adenomyosis lesions (P>0.05); In addition to liquiritin high concentration group of 24h group, the blank serum group 24h and 48h,the other groups were significantly inhibited cell proliferation, and Jiawei Shaoyao Gancao Decoction with high concentrations of 48h which was no statistical significance when Compared with Gancao Decoction with high concentrations of 24h has the highest cell proliferation inhibition rate and apoptosis rate than other groups; Compared with jiawei Shaoyao Gancao Decoction pharmacological serum of 24h, jiawei Shaoyao Gancao Decoction pharmacological serum of 48h and mifepristone group of 24h and 48h were worse on the apoptosis rate, and there was no statistical significance in Jiawei Shaoyao Gancao Decoction high and low concentration group of 24h, Jiawei Shaoyao Gancao Decoction low concentration of 48h, paeoniflorin high concentration of 24h, paeoniflorin liquiritin high concentration of 24h;Compared with mifepristone group of 24h, paeoniflorin high and low concentration of 48h、 liquiritin high and low concentration of 48h, paeoniflorin liquiritin high and low concentration of 48h were worse on the apoptosis rate, and there was no statistieal significance in Jiawei Shaoyao Gancao Decoction Pharmacological serum、mifepristone group、paeoniflorin high and low concentration group of 48h、paeoniflorin high concentration of 24h、liquiritin low concentration of 48h、paeoniflorin liquiritin high concentration of 48h on the cell proliferation inhibition rate. Among the groups of drug intervention time in 24h, both Jiawei Shaoyao Gancao Decoction high concentration group and Jiawei Shaoyao Gancao Decoction pharmacological serum group were higher than other groups on cell apoptosis rate;the inhibition efficiency of Jiawei Shaoyao GancaoDecoction high concentration group was highest than other groups; Between each monomer high and low concentration group, there were statistical difference on cell proliferation inhibition rate. Among the groups of drug intervention time in 48h, the cell inhibition and apoptosis rate of Jiawei Shaoyao Gancao Decoction high concentration group was highest than other groups;Compared with drug intervention time of 48h, Jiawei Shaoyao Gancao Decoction Pharmacological serum of 24h、Paeoniflorin high and low concentration group of 24h, paeoniflorin liquiritin high and low concentration group of 24h were better on cell apoptosis rate;There was no statistical significance between Paeoniflorin low concentration group of 24h and 48h on cell inhibition rate.5. Jiawei Shaoyao Gancao Decoction pharmacological serum can significantly reduce E2, Ras, Raf expression level of uterine adenomyosis lesions cells, when 24h group of it can reduce ER expression level, which compared with mifepristone group the difference was statistically significant (P< 0.05). Both of Jiawei Shaoyao Gancao Decoction pharmacological serum and mifepristone group can significantly reduce expression levels of P450arom in human uterine adenomyosis lesions cells (P<0.05), and Jiawei Shaoyao Gancao Decoction pharmacological serum was better than the other gourps(P<0.05);Groups of Chinese medicine monomer can significantly reduce levels of ER, P450arom, Ras of adenomyosis lesions cell, the effect of Paeoniflorin high concentration Paeoniflorin liquiritin high concentration group was best; Paeoniflorin high concentration group of 24h、Paeoniflorin liquiritin high concentration group of 24h、Paeoniflorin liquiritin low concentration group of 48h were better than mifepristone group on reducing the level of Ras (P<0.05). liquiritin high and low concentration group、 Paeoniflorin liquiritin high and low concentration group were better than other groups on reducing the level of Raf. Jiawei Shaoyao Gancao Decoction high and low concentration group can suppress the level of ER、P450arom、 Ras、Raf of human uterine adenomyosis lesions cells, which was better than mifepristone group; Neith Jiawei Shaoyao Gancao Decoction high and low concentration group nor mifepristone group can recude the level of E2 of adenomyosis lesions cells.6. The influence of Jiawei Shaoyao Gancao Decoction、Jiawei Shaoyao Gancao Decoction pharmacological serum on the expression of P-C-Raf of human adenomyosis lesions cells:Among the groups of Jiawei Shaoyao Gancao Decoction high concentration group、Jiawei Shaoyao Gancao Decoction pharmacological serum goup、mifepristone group、Blank control group, Jiawei Shaoyao Gancao Decoction high concentration group of 48h can reduce significantly the expression level of P-C-Raf of human adenomyosis lesions cells when other groups can not (P>0.05)7. The influence of Jiawei Shaoyao Gancao Decoction、Jiawei Shaoyao Gancao Decoction pharmacological serum on the expression of miR-21 of human adenomyosis lesions cells:Among the groups of jiawei Shaoyao Gancao Decoction high and low concentration group、Jiawei Shaoyao Gancao Decoction pharmacological serum goup of 48h、mifepristone group、Blank control group, There were no statistical Significance in jiawei Shaoyao Gancao Decoction high concentration group、mifepristone group of 24h and Jiawei Shaoyao Gancao Decoction low concentration group of 48h, which can reduce significantly the expression level of miR-21 of human adenomyosis lesions cells (p<0.05). Conclusions:1. Increased Expression of E2, ER,P450arom, Ras/Raf/P-C-Raf was in the adenomyosis;miR-21 was expressed in adenomyosis normal uterus tissue, and there there was no significant difference between them. Except combine with ER to exert biological effects, E2 may also through the RAS/Raf signaling pathway mediated by promote uterine adenomyosis cells proliferation. E2 and ER upregulate the expression levels of miR-21 of uterine adenomyosis cells, and ER was the main influencing factor which can also combine with E2 to work.2. ER and the mir-21-mediated Ras/Raf/P-C-Raf signaling pathway by involved in the occurrence and development of uterine adenomyosis in the following aspects:① The was a positive feedback between P450arom and E2. E2 combine ER to participate in the development of adenomyosis by inducing alteration of the target gene and promoting cell proliferation. ②E2 tcaused abnormal cell proliferation and induced disease binded membrane receptor by activating Ras/Raf/P-C-Raf signaling pathway to induce phosphorylation of nuclear transcription factor FOS, Jun and NF kappa β.3. Jiawei Shaoyao Gancao Decoction maybe Through inhibiting the proliferation and inducing apoptosis to promote uterine adenomyosis lesions decreased or even regression, playing a therapeutic effect on uterine adenomyosis dysmenorrheal, in which the Jiawei Shaoyao Gancao Decoction high concentration group play the biggest role, the effect was dependent on concentration;Followed by Jiawei Shaoyao Gancao Decoction low concentration and its drug serum group, while each monomer in the bad situation.We conclude that Jiawei Shaoyao Gancao Decoction can treat AD effectively though the comprehensive effect of multiple targets and multiple pathways of the Chinese herbal monomer in the compatibility of the constituents. The next step of experiment can be further to screen concentration of Chinese herbal monomer intervention of adenomyosis foci cells.4. Jiawei Shaoyao Gancao Decoction pharmacological serum、the monomer and Jiawei Shaoyao Gancao Decoction can significantly reduce expression levels of Er, P450arom, RAS, RAF of people adenomyosis cells, the effect was better than that of mifepristone. Jiawei Shaoyao Gancao Decoction pharmacological serum can obviously reduce the E2 level of adenomyosis lesions, which can inhibite the positive feedback loop between E2 and P450arom to block the oclassical pathway and non classical pathway of E2, inhibiting angiogenesis to treat adenomyosis and dysmenorrheal..Jiawei Shaoyao Gancao Decoction and its monomer had no obvious inhibition to the level of E2 suggest that they possible reduce level of ER and block binding of E2 and ER, while suppressing the RAS/Raf signaling pathway, and play a role in deterring the neovascularization. The effect of Jiawei Shaoyao Gancao Decoction high concentration group was better than other groups among the jiawei Shyaoyao Gancao Decoction groups; The effect of paeoniflorin liquiritin high concentration group was the best among the monomer groups.5. Jiawei Shaoyao Gancao Decoction high concentration of 48h can blocking the Ras/Raf signaling pathway to inhibit the occurrence and development of uterine adenomyosis by Reducing the expression of P-C-Raf in adenomyosis.6. Jiawei Shaoyao Gancao Decoction high concentration group, mifepristone group of 24h, Jiawei Shaoyao Gancao Decoction low concentration of 48h could both block the positive feedback between miR-21 and Ras to inhibit Ras/Raf signaling pathway to promote cell apoptosis of adenomyosis ectopic lesion and lessen lesions so that inhibit the development of adenomyosis.overall, Jiawei Shaoyao Gancao Decoction can treat adenomyosis dysmenorrheal patients from the multi way, multi link, multi targets by intervene in signal transportation network formed by ER and miR-21 Mediated Ras/Raf Signal pathway.