| Objective1. To revealed the mechanism of MAPK/ERK signaling pathways in adenomyosis, we conducted a clinical research to investigate the differences of the expression level of MAPK signaling pathway factor ERK1 mRNA, ERK2 mRNA and MEK-2, ERK1/2, and inner nuclear transcription factor (NF-kB, c-Fos, c-Jun) in the endometrium between the adenomyosis women and the healthy people.2.The cell experiment part was conducted to observe the impact of Modified Shaoyaogancao decoction containing serum, Modified Shaoyaogancao decoction water extract (high and low concentration), the monomer paeoniflorin or glycyrrhizin (high and low concentration) and peony glycyrrhizin (high and low concentration) applied to in vitro human adenomyosis cells MAPK/ERK signaling pathway factor ERK1 mRNA, ERK2 mRNA and MEK-2, ERK1/2, the nucleus transcription factor (NF-kB, c-Fos, c-Jun). It clarified the intervention mechanism of Modified Shaoyaogancao decoction compounds and monomer on adenomyosis, and lay the foundation for the clinical syndrome differentiation.Methods1. Fluorescence quantitative PCR-probe method and the Western blotting was adopted to detect the differences of the expressional level of MAPK/ERK signaling pathway factor ERK1 mRNA, ERK2 mRNA and MEK-2, ERK1/2, and inner nuclear transcription factor (NF-kB, c-Fos, c-Jun) in the endometrium between the adenomyosis group and the control group.2. Fluorescence quantitative PCR-probe and Western blot was adopted to detect the impact of the monomer paeoniflorin, glycyrrhizin (high and low concentration), peony glycyrrhizin(high and low concentration) and serum containing(high and low concentrations) against the human tumor cells of adenomyosis MAPK/ERK signaling pathway factor ERK1 mRNA, ERK2 mRNA and MEK-2, ERK1/2, the nucleus transcription factor (NF-kB, c-Fos, c-Jun). The blank control group, the inhibitor group and positive drug control group were set.Results1. The clinical trial part:The expression level of Adenomyosis group endometriosis ERK2 mRNA was higher than the control group, the difference was statistically significant CP<0.05). As per the Adenomyosis group, the expression level of Endometriosis MEK-2, ERK1/2, c-Fos was higher than the control group, and the difference was statically significant (P<0.05). The difference of the index between Adenomyosis group and the control group was not statically significant.2. The cell experiment part:(1) The effect brought by Peony and licorice flavored soup and the whole side of human serum containing to adenomyosis foci of cell proliferation:Compared to the blank control group, the 10% serum containing group and the 20% serum containing group were able to Inhibit tumor cell proliferation adenomyosis, and the difference was statistically significant (P<0.05). The difference between the 10% serum containing group and the 20% serum containing group was not statically significant (P>0.05). Screened 10% serum containing serum concentration of the drug.(2) The effect brought by serum-containing group to Adenomyosis lesion cell ERK1 mRNA, ERK2 mRNA:Compared to the blank control group, the expression level of ERKl mRNA in the U0126 group (inhibitor group) was significantly lower, the difference was statically significant (P<0.05). The difference between other groups were not statically significant (P>0.05). Compared to the blank control group, the expression level of ERK1 mRNA in the 10% serum containing group, mifepristone group and the U0126 group were lower, and the difference was statically significant (P<0.05).Compared to the 10% serum containing group, the expression level of ERK2 mRNA in the U0126 group was significantly lower, and the difference was statically significant (P<0.05)(3) The effect brought by Chinese medicine monomer and the expression level of the whole side water extract group to the Adenomyosis lesion cell ERK1 mRNA, ERK2 mRNA:Compared to the blank control group, the expression level of ERK1 mRNA in the Liquiritin high concentration group, peony liquiritin high concentration group and the U0126 group were lower, and the difference was statically significant (P<0.05), while the difference between the three was not statically significant. Comparing the Paeoniflorin high concentration group to the Peony liquiritin low concentration group and the whole side of high concentration group, the expression level of ERKl mRNA in the Paeoniflorin high concentration group was lower, the difference was statically significant (P<0.05) Compared to the blank control group, the expression level of ERK2 mRNA in the Liquiritin high concentration group, peony liquiritin high concentration group, U0126 group were lower, the difference was statically significant (P<0.05), while the difference between the three was not statically significant. Compared to Paeoniflorin low concentration group, glycyrrhizin low concentration group, the whole side of high concentration group and the whole side of low concentration group, the expression level of ERK2 mRNA in the Mifepristone group was lower, and the difference was statically significant (P<0.05) Compared to the All parties high concentration group, the expression level of the ERK2 mRNA in the Paeoniflorin high concentrations, low concentrations of paeoniflorin, glycyrrhizin low concentration group were lower, and the difference was statically significant (P<0.05)(4) The effect brought by the Containing serum group to the expression level of Adenomyosis lesion cells MEK-2, ERK1/2, NF-k B, c-Jun, C-Fos:Compared to the 10% serum containing group and the blank control group, the expression level of ERK1/2 in the U0126 group was significantly lower, and the difference was statically significant (P< 0.05), while the difference between other group were not statically significant. Compared to the blank control group, the expression level of NF-k B in the U0125 group and the 10% serum containing group was significantly lower, and the difference was statically significant (P<0.05), while the difference between the two groups were not statically significant. Compared to the 10% of normal rat serum and the mifepristone group, the expression level of NF-k B in the 10% serum containing group was significantly lower, and the difference was statically significant (P<0.05). Comparing the blank control group to the expression level in C-Jun group, the difference was not statically significant.P>0.05). Compared to the 10% serum containing group, the expression level of c-Fos was significantly lower, and the difference was statically significant (P<0.05), while compared to the blank control group, the difference was not statically significant (P>0.05)(5) The effect brought by Chinese medicine monomer and the whole side water extract group to the expression of Adenomyosis lesion cells MEK-2, ERK1/2, NF-k B, c-Jun, C-Fos:Compared to the blank control group, the expression level of MEK-2 and NF-k B in the Liquiritin high concentration group, peony liquiritin high concentration group, U0126 group were significantly lower, and the difference was statically significant (P< 0.05), while the difference between the three was not statically significant. Compared to the blank control group, the expression level of ERKl/2 in the Peony liquiritin high concentration group, U0126 group (inhibitor group) were significantly lower, and the difference was statically significant (P<0.05). Compared to All parties high concentration group, the expression level of ERKl/2 in the Liquiritin high concentration group was significantly lower, and the difference was statically significant (P<0.05). Comparing the Paeoniflorin high concentration group, Paeoniflorin low concentration group, peony liquiritin low concentration group, the whole side of high concentration group and high concentration liquiritin group, the expression level of NF-k B in the high concentration liquiritin group was lower, and the difference was statically significant (P<0.05) Comparing the Paeoniflorin high concentrations, low concentrations of paeoniflorin, glycyrrhizin low concentration group, peony liquiritin low concentration group, the whole side of high concentration group, the whole side of low concentration group, mifepristone group and peony liquiritin high concentration group, the expression level of NF-k B in the Peony liquiritin high concentration group was significantly lower, and the difference was statically significant (P<0.05). Comparing the c-Jun, c-Fos group to the blank control group, the difference was not statically significant(P>0.05).Conclusions1. The expression level of MAPK signaling pathways related factors in the endometriosis of the Adenomyosis women like Gene ERK2, protein MEK-2, ERK1/2, c-Fos were higher than in that in endometrium of healthy women. This indicates the activation of the Factors in MAPK signaling pathways cause abnormal cell proliferation and apoptosis, which is related to the occurrence and development of adenomyosis.2. Modified Shaoyaogancao decoction can inhibit cell proliferation while interventing In vitro tumor cells adenomyosis, which in turn inhibited the progress of Adenomyosis. This is due to the chemical composition of the traditional Chinese medicine and the multi-target effect. Impacts of Modified Shaoyaogancao decoction containing serum, monomer and water extracts varied as below:①Modified Shaoyaogancao decoction containing serum:10% serum containing gene expression by reducing ERK2, protein NF-k B, ERK is the last step of activation of phosphorylation of MAPK/ERK signaling pathway. Activated ERK can enter karyon from cytoplasm and further activate expression of transcription factors. serum with 10% Modified Shaoyaogancao decoction can inhibit expression of ERK2 and its transfer into karyon, therefore decreased expression of NF-k B inside nucleus, which would lower abnormal activation of relative factors of MAPK/ERK signaling pathway, inhibit excessive proliferation of focal cells of adenomyosis.② Monomer and water extracts of Modified Shaoyaogancao decoction:High concentrated paeoniflorin with liquiritin can inhibit ERK1, ERK2, MEK-2, ERK1/2 and NF-k B. High concentrated liquiritin can inhibit ERK1, ERK2, MEK-2 and NF-k B. Both high concentrated paeoniflorin with liquiritin or only liquiritin can regulate abnormal signal of factors in MAPK/ERK signaling pathway successively, so as to inhibit over-activation of pathway which would lead to abnormal excessive proliferation. The regulation of water extracts of Modified Shaoyaogancao decoction on MAPK signaling pathway was subtle. Effect of paeoniflorin with liquiritin was better than only liquiritin, which was consistent to the theory of Chinese medicine in drug compatibility.③ Inhibitor U0126 and mifepristone the positive control:U0126 is the inhibitor of conduction of kinase cascades in MAPK signaling pathway. It decreased expression of NF-kB through decreasion of MEK-2 and ERK1/2. Mifepristone used widely in treating adenomypsis. As the positive control, it can lower expression of ERK2, but no others.④c-Fos, c-Jun were not sensitive to all forms of Modified Shaoyaogancao decoction. c-Fos、c-Jun may be regulated by many signaling pathways. Modified Shaoyaogancao decoction may increased c-Fos、c-Jun by other pathways, which may counteract the inhibition by MAPK signaling pathway.3. The role of MAPK signaling pathway in the pathogenesis of adenomyosis is being increasingly recognized, along with the gradual awareness depth, it will bring a brighter future to the adenomyosis treatment. |
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