The Influence Of Nicotine On Endometrial Stromal Cells Mechanical Characteristics And Mechanism Research

Endometriosis is a frequent disease witch affects about 10-15% women in reproductive age. The clinical manifestation included:dysmenorrhoea, pelvic mass and infertility. It has seriously influenced the quality of life and fertility function in women. Until now the effect of drug treatment to this disease is not good, the recurrence rate of conservative surgery is high. Recent research showed that the onset risk of Endometriosis can decreased 50% in the women who have a smoke. But the cause is still not clear. The research of it will make the pathological and physiological mechanism be understood further, and bring new ideas into prevention and therapy.To investigate the protective action of Nicotine on Endometriosis, in the first part of this article the uterus of mice in which RFP have been transferred were transplanted to the celiac peritoneum of castrated nude mice. Four groups were built:control group (C), estrogen group (E), estrogen plus Nicotine group (E+N) and estrogen plus progestogen group (E+P). Estradiol benzoate, Nicotine and hydroxyprogesterone caproate were added respectively, then the fluorescence intensity and dimension of celiac focus were investigated. At the same time, the levels of serum estradiol were investigated by RIA. Results:the total focus fluorescence intensity of E+N group was lower than E group, in which a obvious statistical significance was shown (P< 0.05). But the level of serum estradiol of E+N group was found not lower than E group. It was concluded that Nicotine can inhibit the increasing of Endometriosis focus in vivo, and it was not found that the level of serum estradiol of Nicotine group decreased in this model.To prove Nicotine carried out protective action on Endometriosis through classical estrogen receptor signaling pathway or not, in the second part of this article the primary endometrial stromal cells culture and treatment of Nicotine and estrogen were performed. Endometrial stromal cell growth and expression level of estrogen regulating genes CCND1, MYC, PGR,estrogen receptors ESR1, ESR2, GPER and aromatase were tested. Results:It was not found that Nicotine can inhibit the expression level of ERE report gene, cell growth, estrogen controlling gene,estrogen receptor and aromatase. So, Nicotine did not carry out protective action to endometriosis through inhibiting classical estrogen receptor signaling pathway.To prove Nicotine carried out protective action in Endometriosis by acting on the mechanical state of uterus endometrial stromal cells or not, in the third part of this article gel contraction experiment, cell adhesion experiment and living cell motility experiment were performed to investigate the effect of Nicotine and estrogen on the mechanical state of NESC and EuESC, EcESC. Results:It was found that the gel contraction ability of EuESC was higher than NESC, and cell motility ability of EuESC, EcESC were higher than NESC. Nicotine can inhibit the gel contraction which was promoted by estrogen but in EuESC the phenomenon was not observed. In the cell motility experiment the effect of Nicotine on the three kind of cells was not obvious. It was concluded that the effect of cell motility inhibition and cell adhesion promoting of Nicotine on NESC may inhibit the migration and invasion of ESC that influence the occurrence and development of Endometriosis.To investigate the mechanism of inhibition in ESC motility and adhesion after Nicotine treatment, in the fourth part of this article we observed the effect of Nicotine and estrogen on RhoA activity of NESC, EuESC, EcESC, and surface adhesion by pull-down and immunofluorescence method. Results:The RhoA activity level of EuESC was higher than NESC. Nicotine can inhibit the RhoA activity of EuESC, but it became not obvious after estrogen treatment. The levels of F-ACTIN and Vinculin in NESC, EuESC and EcESC increased gradually, and the effect of Nicotine administration on F-ACTIN was not obvious but can inhibit Vinculin whether estrogen was added or not. It was concluded that the cell motility of NESC, EuESC and EcESC increased gradually, Nicotine carried out protective action on Endometriosis by inhibiting RhoA activity through influencing cell motility and adhesion.In conclusion, Nicotine can influence the mechanical state by inhibiting RhoA activity. It is one of the causes in the protective action of Nicotine on Endometriosis that inhibition stromal cell motility and promoting cell adhesion. In this research, inhibition of nicotine on classical estrogen receptor signaling pathway was not found. The cell motility in NESC, EuESC and EcESC increased gradually showed the disorder of cell mechanical state in Endometriosis. So, ESC mechanical property is a new target for prohibition and treatment of endometriosis without disturbing the function of HPO axis in women.