Effect Of Diazepam On Rat Cervical Ripening During Late Pregnancy

ObjectiveAt the end of pregnancy, during the ripening process, the cervix changes its biochemical composition and structure to permit delivery. Recent research has proposed that these structural alterations may be due to changes in the water content of the extra cellular matrix ( ECM ) , alterations in the proteoglycan content, and/or changes in the activity of collagenases. MMPs are suspected to contributed to overall tissue remodeling in the uterus, the fetal membranes, and in the cervix during pregnancy. MMP - 8 and MMP - 9 concentrations have been shown to increase in the lower uterine segment during labor in parallel with changes in cervical dilatation. However, the exact MMPs and TIMPs that are present in the human cervix in pregnancy, and the mechanism by which their activity is controlled in cervical ripening are not fully understood.As we all know that diazepam is capable of inducing human cervical ripening. But the mechanisms how diazepam is involved in the ripening process and how it interacts with other mediators of cervical ripening remain unclear. Since the cervix undergoes a remodeling process before the onset of both term and pre-term partiturition that is characterized by decreased tensile strength and a change in texture, moreover, now the study of cervical ripening has been limited largely to animal models because of safety concerns that are related to tissue sampling during human pregnancy. So we measured the cervical tensile strength by cervical creep method and investigated the expression of MMP - 9/TIMP - 1 protein in the pregnancy rat cervix during normal pregnancy and in response to diazepam administration to determine whether MMP -9/TIMP - 1 expression is involved in the cervical ripening process.MethodsThe Wistar rats were obtained from the Second Affiliated Hospital Animal Lab of the Chinese Medical University. The day after mating was considered day of gestation in the presence of a vaginal plug. Animals that were used to study diazepam - induced cervical ripening were pretreated with different dose. Animals were killed on gestational days 13,16,20 and 21. The cervix was separated from the uterus;care was taken to exclude the more muscular protein above the mainly fibrous cervix. The tissue was rinsed in normal saline solution then used for measuring the cervical tensile strength or frozen in liquid nitrogen and stored at - &0X1 until it was used for protein isolation.Cervical tensile strength was determined by the cervical creep method. A 5- mm long cylindrical segment of cervix was suspended in a heated 37 T! water bath that contained Dulbeccoftiodified Eagle medium. The rat of cervical distension under a constant 50 - g load was determined with a linear variable differential transformer.Western blots cervical protein were obtained by homogenizing tissue and centrifuged. The supernatant was quantified by Brad - Fold method. Protein (lOjxg) were resolved on a 10% sodium dodecylsulfate - poly aery lamide gel and then transferred electrophoretically to nitrocellulose membranes. Immunob-lots were performed with rabbit anti - MMP9 polyclonal antibodies (Chemicon International Inc. , Temecula, CA, USA;cat. no. AB 19016)at a dilution of 1:1000,rabbit anti - TIMP - 1 polyclonal antibodies at a dilution of 1:1000( Chemicon International Inc. , Temecula, CA, USA;cat. no. AB 800) , rat anti- 3 - actin monoclonal antibodies at 1:2000 dilution ( Sigma Chemical Company. , St. Louis, MO). And horseradish peroxides - conjugated anti - rabbit and anti -rat secondary antibody(Sigma Chemical Company. , St Louis, Mo) at 1: 5000 dilution. The relative density of protein bands was determined with ECL Western Blotting Detection System ( Amersham, Japan) and LUMINESCENT IMAGE ANALYZER LAS - 1000PLUS (FUJIFILM. , JAPAN). Blots were then probed for f$ - actin and assayed to control for differences in loading and transferefficiency.ResultCervical Tensile StrengthCervical tensile strength decreased with decreased with advancing gestation. The decrease on day 20 was significant compared with day 13. Medium -dose and high - dose Diazepam applied to the cervix on day 20 of gestation decreased cervical tensile strength was significant compared with normal pregnancy. Diazepam was administered in mid - gestation, day 13, no significant difference in cervical tensile strength was detected.MMP - 9/TIMP-1 Protein ExpressionImmunoblots that were performed to detect MMP - 9 protein demonstrated 2 bands that represented the inactive proenzyme and the activated enzyme. Blots were also evaluated for (3 - actin to adjust for loading and transfer efficiency. The expression of MMP - 9/TIMP - 1 protein on day 20 was significantly greater than what was significantly greatly than what was detected on day 13. Total MMP - 9 protein was unchanged after treatment with diazepam. The active form of MMP - 9 was increased significantly after medium - dose and high - dose diazepam treatment.ConclusionMMP - 9/TIMP - 1 expression parallels changes in cervical tensile strength through pregnancy. After the administration of Medium - dose and high - dose diazepam can decrease cervical tensile and induce the activation of matrix metal-loproteinase - 9 but does not affect matrix metalloproteinase - 9 protein expression of rat cervix during late pregnancy. Medium - dose and high - dose diazepam can also increase TIMP - 1 protein expression of rat cervix during late pregnancy. Further studies evaluating the mechanisms involved in diazepam - induced cervical ripening are required.