Based On GPER Signaling Pathway, The Estrogen-like Effect Of Siwutang And Its Molecular Mechanism Were Discussed

ObjectiveIn Taiping Huimin and prescription Bureau,Siwu soup was considered to be effective in treating many different kinds of diseases of women and became the basic prescription of various gynecologic diseases caused by Chong and Ren deficiency in later ages.As a classic prescription of traditional Chinese medicine,experimental studies in recent years have shown that the Siwu Decoction and its diffrent derived formular have certain estrogenic effects.At present,the research on its estrogenic effects focused mainly on the classical nuclear estrogen receptor subtype:ER alpha and ER beta.The molecular mechanism of the estrogenic effects via the membrane receptor pathway is not clear.As a new type of membrane estrogen receptor,G protein coupled estrogen receptor(GPER)attracts much attention recently.Different from the classical nuclear estrogen receptor,it can mediate the rapid non genomic effect of estrogen or indirectly participate in the regulation of gene transcription to affect the downstream signal molecule expression and play its regulatory function..The purpose of this experiment is to explore the estrogenic effects and their possible molecular mechanism of Siwu Decoction mediated by GPER.Methods1.40 immature SD rats were randomly divided into 4 groups according to the principle of random distribution law:normal control group,estrogen group,high dose and low dose Siwu groups.After 4 days of gavage with normal saline,estrogen and Siwu Decoction,the blood was taken from the abdominal aorta and the pharmacological serum was prepared.The primary osteoblasts were extracted from new born rats(rats osteoblasts,ROBs)within 72h by aseptic operation and enzyme digestion method.The morphological characteristics of ROBs were observed under microscope.ALP staining and alizarin red staining were used for identification of ROBs.The effect of serum containing Siwu Decoction on the proliferation of ROBs cells was detected by MTT assay.G1 and G15 were used as tools to detect the effect of Siwu Decoction via GPER signaling pathway.Also with G1 and G15 as tools,the expression of PI3K,AKT,p-Akt,EGFR,ERK and p-ERK in ROBs were detected by the cell immunofluorescence assay.2.40 immature SD rats were randomly divided into 4 groups according to the principle of random distribution law:normal control group,estrogen group,high dose and low dose Siwu groups.After gavage administration for 4 days,the rats were sacrificed.The uterus were weighed and uterus coefficient were calculated.The blood were collected for the preparation of serum pharmacological research.The proliferation rate of MCF7 cells influenced by Siwu Decoction was determined by MTT assay with the application of ERa and ER? antagonists MPP and PHTPP as tools.The protein expression of ERa and ER? and the ratio of ERa and ER? in MCF7 cells affected by Siwu Decoction were detected by western blotting.3.40 immature SD rats were randomly divided into 4 groups according to the principle of random distribution law:normal control group,estrogen group,high doseand low doseSiwu groups.After 4 days of gavage with normal saline,estrogen and Siwu Decoction,the blood was taken from the abdominal aorta and the pharmacological serum was prepared.The protein expression of PI3K and p-Akt in MCF7 cells affected by Siwu Decoction were detected by western blotting.With G15 as a tool,the RT-PCR experiment were performed to detect the changes of GPER,PI3K and p-Akt mRNA expressions in MCF7 cells affected by Siwu Decoction.Results1.Study on the estrogenic effects and its molecular mechanism of Siwu Decoction onosteoblast via GPER signaling pathwayAfter ALP staining,the positive reaction in ROBs showed as gray black particles or lump and strip precipitation.After alizarin red staining,the positive cells of calcium deposition in ROBs showed as orange red particles.Siwu Decoction could promote proliferation rate of ROBs(P<0.05 or P<0.01).Such effect could be inhibited significantly by G15(P<0.01)and enhanced significantly by G1(P<0.05 or P<0.01).The pharmacological serum of Siwu Decoction could improve the average fluorescence intensity of PI3K and p-Akt in ROBs(P<0.05 or P<0.01).Such effect could also be decreased significantly by G15(P<0.05 or P<0.01)and enhanced significantly by G1(P<0.05).The average fluorescence intensity of total AKT was similar in the five groups with no statistical significance(P>0.05).The average fluorescence intensity of EGFR and p-ERK in ROBs treated with Siwu Decoction increased significantly(P<0.01)(P<0.01).The expression intensity of EGFR was almost equal to that of the estrogen group after adding G1 and the expression intensity of p-ERK decreased significantly(P<0.05)after the addition of G15.The mean fluorescence intensity of total ERK in the five groups showed no statistical significance(P>0.05).2.Study on the estrogenic effects and its molecular mechanism of Siwu Decoction on ER positive cells via estrogen receptor subtypes Siwu Decoction could increase the uterus coefficient of rats(P<0.05 or 0.01).Siwu Decoction could also promote MCF7 cell proliferation rate(P<0.05 or 0.01)and it could be inhibited by MPP or PHTPP,especially by PHTPP(P<0.01).The pharmacological serum of Siwu Decoction could improve protein expression level of ERa and ER? in MCF7 cells and reduce the ratio of ERa and ER?(P<0.01).3.Study on the estrogenic effects and its molecular mechanism of Siwu Decoction on ER positive cells via GPER signaling pathway.Western blotting detection found that compared with the normal control group,the expression level of PI3K and p-Akt in MCF7 cells treated by Siwu decreased significantly(P<0.01).And the results also showed that the expression level of PI3K and p-Akt in MCF7 cells treated by Siwu Decoction were less than that of the estrogen group(P<0.01).RT-PCR detection showed that compared with the normal control group,the expression of GPER,PI3K and AKT mRNA in MCF7 cells reduced both in the high and low dose Siwu groups(P<0.01).After adding G15,PI3K and AKT mRNA expression increased significantly(P<0.05 or 0.01).Conclusions1.Siwu Decoction could play estrogenic effects through GPER pathway.It could improve ROBs proliferation and promote the expression level of GPER mediated downstream signal molecules including PI3K,p-Akt,EGFR.and p-ERK..That is to say,Siwu Decoction could play an estrogenic effect through the interaction between GPER/PI3K/AKT and GPER/EGFR/MAPK signaling pathway.2.Siwu Decoction showed estrogenic effects in animal experiment.It could affect the proliferation of ER positive cells through ER,mainly ER? pathway and change the ratio of ERa and ER?,to a certain extent,which inhibits the growth of estrogen dependent cells.3.Siwu Decoction could affect the proliferation activity of ER positive cells through GPER/PI3K/AKT pathway.That is to say,to a certain extent,the growth of estrogen dependent cells is inhibited by down regulation of the expression of PI3K and p-Akt.