Effects Of ROS Inhibitor NAC On NF-κB-involved Endometrial Tissue Breakdown Under The Support Of Progesterone In Mice

The uterus, as a unique reproductive organ, is guided by the steroidhormones progesterone(P4) and estrogen(E2) which act primarily through andsubsequently regulate expression of their specific(progesterone receptor(PR)and estrogen receptor(ER) respectively). During women’s reproductive phasethe endometrium undergoes repetitive cycles of proliferation, differentiation,breakdown and repair preparing the uterus for implantation and growth ofembryo. And the endometrial tissue is the only periodic trauma, quicklyrepaired and without scar by itself. Progesterone withdrawal is well acceptedas a trigger for the abortion the initiation of menstruation, and plays a vitalrole in rodent delivery. Before delivery in rodents, the decrease of P4 inmaternal serum causes uterine contractions and successful delivery. But inhuman and guinea pig, the concentrations of P4 are high throughout pregnancy.Observers noted that the expression of PR-A rapidly increased, and P4 wasantagonised at the receptor level. That is to say there is functionalprogesterone withdrawal(FPW).P4 is responsible for maintaining decidualization. However, our previousstudy observed that the decidual endometrium disintegrated and OS-NF-κBsignal activated when P4 maintaining in mouse model and cell model. Wehypothesized that FPW occurs in P4 maintaining and the activation ofOS-NF-κB signal leads to endometrial tissue breakdown and apoptosis.Objectives: The aim of the present study is to evaluate the molecularmechanisms of endometrial tissue breakdown by treatment with ROS inhibitorN-Acetyl-L-cysteine(NAC) under the support of P4.Methods: Fourty virgin female C57BL/6 mice, 8-10 weeks old, wereovariectomized under diethyl ether-induced anesthesia and allowed to recoverfor one week. Mice were then sequentially given steroid hormones whereinthe first of three days, all mice were subcutaneously(s.c.) injected with 100 ngof 17 β-estradiol(E2) in arachis oil at AM 9:30. After the mice were rested for3 days, progesterone implants were inserted into the back of the mice and 5 ngE2 and 50 ng of P4 in arachis oil were also instantaneously injected s.c. On day8 and 9, 5 ng of E2 was also injected s.c. at AM 9:30, while on day 9, fifteenmicroliters of arachis oil was injected into the uterus to induce decidualization.After two days(i.e. on day 11), the injected horn had undergone extensivedecidualization(regarded as 0 h). NAC was given to twenty mice throughintraperitoneal injection at 48 h, 60 h, 72 h. Mice were sacrificed at 0, 24, 48,60, 72 and 96 h and uterine horns were harvested for further analysis. Afterbeing weighed, tissue was frozen immediately in liquid nitrogen, before beingstored at-80℃. The expression of NF-κB was evaluated by western blotting inthe nucleus and cytoplasm. The interaction of NF-κB and PR was tested byCo-immunoprecipitation(Co-IP). The activity of superoxide dismutase(SOD)and the level of O2·- in uterus tissues were estimated by spectrophotometerusing commercially available kits. Uterus tissues were fixed with 4% bufferedparaformaldehyde, tissue sections were prepared for hematoxylin and eosin(H&E), to perform histological studies. And other sections prepared forimmunohistochemical staining to observe the position of NF-κB and PR.Results:1 Determination of P4 in serumThe results determined that the levels of P4(about 40 ng/ml) did not showsignificant difference, at the different time points.2 Morphological and histological changes in uterusMorphologically, the uterine horns were pink in color and occuredhyperaemia and edema over time at 0-48 h. At 60-72 h the uteri were crimsonand the edema was exacerbated. Meanwhile, the bleeding spots and crimsoncontents were observed. At the point of 96 h, the uteri color were dark red;after traversed, the dark red contents flowed quickly. These were similar to themorphologic changes of uterus at 16 h after progesterone withdrawal. At 96 hafter NAC treatment, the uteri color was shallow and the degrees ofhyperaemia and edema were relief.Histologically, the degree of endometrial decidualization was relativelysufficient and the structure of endometrium was intact at 0-48 h. But there is afew karyopyknosis in the center of decidual zone over time. At 60-72 h, thenumber of karyopyknosis of stromal cells was increased in the central region.And the endometrial breakdown was observed in some part area. At the pointof 96 h, the number of karyopyknosis of stromal cells was significantlyincreased in the central region, and in peripheral area we observed the obviousphenomenon of endometrial breakdown, similar to 16 h after progesteronewithdrawal. At 96 h after NAC treatment, the number of karyopyknosis ofstromal cells was significantly decreased and the endometrial breakdown wasnot occured.3 Changes of O2·- and SOD in uterus homogenateThe results showed that the level of O2·- and the activity of SOD almostdid not change in P4 maintained within 48 h, but at the point of 72 h, the levelof O2·- and the activity of SOD were significantly increased. As the markers ofoxidative stress, it is shown that the oxidative stress reaches a high level in P4 maintained 72 h. At 96 hours after NAC treatment, the level of O2·- wasdecreased, but the activity of SOD was increased. These result showed thatNAC inhibits the oxidative stress in the process of tissue breakdown.4 Activation of NF-κBImmunohistochemical analysis was performed to verify the NF-κBtranslocation. At 0 h, NF-κB p65 was mostly located in the cytoplasm and waslow in abundance. But p65 was distinctly translocated from the cytoplasm tonucleus with time. In order to further confirm the activation of NF-κB, thelevel of NF-κB p65 was analyzed to assess its translocation from cytoplasm tonucleus. Western blotting analysis showed that p65 in nucleoprotein graduallyincreased and peaked at 72 h. In a parallel comparison, the cytoplasmic levelsof p65 decreased within 72 h.5 Interaction between PR and NF-κBImmunohistochemical analysis was also perforemed to locate the PRprotein. The results showed that PR was expressed in nucleus. To furtherprove the role of the PR-p65 interaction in the process of endometrial tissuebreakdown, Co-IP analysis was performed using tissue extracts. The lysateswere subjected to immunoprecipitation with anti-p65 antibody, followed bywestern blotting analysis with either anti-PR antibody. The combination ofboth increased gradually with time and reached a peak at 72 h. The dataindicated that the physical interaction between PR and p65 exists in vivo.Conclusions: Our results suggested that the decidual endometriumbreakdown was associated with the activation of OS-NF-κB signal pathway inprogesterone maintaining. Under the support of progesterone, oxidative stresswas activatied and then oxidative stress lead to the expression of NF-κB p65 increased. P65 combined P4 can inhibit the combination of P4 and PR,resulting in functional progesterone withdrawal and thus participate in theprocess of endometrial breakdown.