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本试验以交感肾上腺系统为切入点,探究制动应激影响胚胎着床的作用机制。选用8周龄雌性CD1小鼠,妊娠E1时开始应激,并连续制动应激直至E7(4h/d),取妊娠母小鼠血浆、子宫组织等进行检查。结果发现,1E1母小鼠制动应激之后,血浆皮质酮和去甲肾上腺素含量、血糖水平提高,并诱导孕小鼠胚胎着床位点降低,以及性器官指数降低。2制动应激致E3-E7孕小鼠子宫内膜面积、子宫腺面积比例降低,诱导作为调节子宫内膜重建及血管发育的MMP-9蛋白以及VEGF和CD34阳性细胞降低,利用PCNA和TUNEL方法分别检测子宫内膜细胞的增殖与凋亡状态,发现制动应激诱导E3-E7孕小鼠子宫组织的MODPCNA/MODTUNEL比值下降,Caspase-3蛋白增加。3孕小鼠子宫内膜u NK细胞密度下降,肌层肥大细胞增加;T淋巴细胞的增殖活性及IL-2/IL-4比值降低。4孕小鼠子宫组织中GSH-PX、T-SOD和T-AOC含量降低,而MDA含量增加,且外源性H2O2作用于体外培养的孕小鼠子宫内膜基质细胞,细胞增殖活性降低并呈剂量依赖性。5孕小鼠子宫组织中p ERK1/2和p NF-κBp65蛋白表达增加,并激活了β2-AR mRNA的表达,且β2-AR阳性细胞分布于子宫肌层、腺上皮细胞、腔上皮细胞和蜕膜细胞中,以及体外分离的子宫内膜基质细胞中。6体外分离E5孕小鼠子宫内膜基质细胞,添加外源性β-AR激动剂异丙肾上腺素(ISO)使细胞增殖活性降低,同时Caspase-3的含量增加;添加腺苷环化酶激动剂FSK对ISO的刺激产生协同作用;而添加外源性β2-AR阻断剂(Butoxamine)和PKA抑制剂H-89,能显著逆转ISO对细胞的抑增殖与促凋亡作用;添加外源性MEK抑制剂PD98059和NF-κB抑制剂PDTC使细胞增殖活性增加,而Caspase-3的含量显著降低;同时在不同药物添加组中p ERK1/2表达的变化趋势与Caspase-3表达趋势一致。以上结果表明,制动应激激活孕小鼠交感肾上腺髓质系统,刺激皮质酮和去甲肾上腺素分泌,经β2-AR介导激活胞内c AMP以及下游PKA信号,直接激活或者间接通过诱导孕小鼠子宫局部的氧化应激产生ROS激活ERK1/2磷酸化,一方面直接激活NF-κB进入细胞核与靶基因结合,产生炎性细胞因子IL-2,使孕小鼠子宫局部Th1/Th2平衡偏向于Th1炎性状态,诱导着床期孕小鼠子宫的局部免疫平衡紊乱;另一方面增加孕小鼠子宫组织Caspase-3的蛋白表达,诱导子宫内膜细胞凋亡,阻滞子宫内膜的妊娠适应性重建,导致子宫内膜容受性的降低,从而不利于孕小鼠胚胎的着床和发育。
In this study, the sympathetic adrenal system as the starting point to explore the mechanism of brake stress affect embryo implantation. Eight-week-old female CD1 mice were used. Stress was induced during pregnancy E1, and continuous stress was induced until E7 (4h / d). The plasma and uterus tissues of pregnant mice were also examined. The results showed that the plasma corticosterone and norepinephrine levels, blood glucose levels were increased after 1E1 female mice were braked, and the implantation site of pregnant mice was decreased and the sexual organ index was decreased. 2 Braking stress reduced the area of endometrium and the ratio of uterine gland area in E3-E7 pregnant mice, and decreased the expression of MMP-9 protein and VEGF and CD34-positive cells as endometrial remodeling and angiogenesis. PCNA and TUNEL Methods The proliferative and apoptotic status of endometrial cells were detected respectively. It was found that the ratio of MODPCNA / MODTUNEL in the uterine tissue of E3-E7 pregnant mice induced by braking stress decreased and the protein of Caspase-3 increased. The density of NK cells in the endometrium of 3 pregnant mice decreased and the number of mast cells increased. The proliferation activity of T lymphocytes and the ratio of IL-2 / IL-4 decreased. The content of GSH-PX, T-SOD and T-AOC in the uterus of 4 pregnant mice decreased and the content of MDA increased, and the exogenous H2O2 acted on the endometrial stromal cells of pregnant mice in vitro and the cell proliferation activity decreased In a dose-dependent manner. The expression of p ERK1 / 2 and p NF-κBp65 in the uterus of 5 pregnant mice increased and activated the expression of β2-AR mRNA. The β2-AR positive cells distributed in the myometrium, glandular epithelial cells and luminal epithelial cells Decidual cells, and endometrial stromal cells isolated in vitro. 6 Isolation of endometrial stromal cells from E5-pregnant mice in vitro and the addition of exogenous β-AR agonist isoproterenol (ISO) reduced the cell proliferation activity and increased the content of Caspase-3. Addition of adenylyl cyclase FSK had a synergistic effect on the stimulation of ISO. However, the addition of exogenous β2-AR blocker and PKA inhibitor H-89 significantly reversed the inhibitory effect of ISO on the proliferation and apoptosis of cells. PD98059, a MEK inhibitor, and PDTC, an inhibitor of NF-κB, increased the cell proliferation and decreased the expression of Caspase-3. Meanwhile, the changes of p ERK1 / 2 expression in different drug-added groups were consistent with the tendency of Caspase-3 expression. The above results indicate that the braking stress activates the sympathetic adrenal system of pregnant mice to stimulate the secretion of corticosterone and norepinephrine, activates intracellular cAMP and downstream PKA signals via β2-AR, and directly activates or indirectly induces Oxidative stress in the uterus of pregnant mice produces ROS-activated ERK1 / 2 phosphorylation, which on the one hand directly activates NF-κB into the nucleus and binds to the target gene to produce inflammatory cytokine IL-2, which leads to local Th1 / Th2 The balance is biased toward the Th1 inflammatory state, which induces the local immune balance disorder in the uterus of pregnant mice at the implantation phase; on the other hand, it increases the protein expression of Caspase-3 in the uterus of pregnant mice, induces the apoptosis of endometrial cells, Pregnancy adaptive reconstruction of the membrane, leading to reduced endometrial receptivity, which is not conducive to implantation and development of embryonic mouse.