Effects Of Lipopolysaccharide And Relaxin On Uterine Involution And PI3K/Akt Signal Pathway In Postpartum Mice

The physiological process of the female animal’s uterus returning to pre-pregnancy state within a certain period of time after delivery is called uterine involution,including a series of changes such as the reduction of uterine volume and weight,uterine muscle contraction,endometrial hyperplasia and vascular repair Uterine subinvolution will prolong the calving interval and reduce production efficiency.There are many factors which can affect the function of the uterus after parturition in dairy cows.Endometritis and poor uterine contraction are main factors of poor uterus recovery.At present,estrogen,prostaglandin,trace element selenium/chromium and traditional Chinese herbs are mainly used for treatment at home and aboard.However,the mechanism of uterine involution is not clear,and the effects of these medicines are not obvious or have huge individual differences.Lipopolysaccharide(LPS)is the main toxic component of gram-negative bacteria,which can induce inflammation of endometrial cells in vitro.Relaxin(RLX)can inhibit uterine contraction in pregnancy and promote collagen degradation in the reproductive system,finally lead to subinvolution of uterus.In this experiment,we established two kinds of uterine subinvolution models by uterus perfusion in postnatal mice By comparing the changes of several kinds of steroid hormones and inflammatory cytokines in mice with normal uterine involution,comparing the effects of LPS and RLX on apoptosis and the expression of apoptosis related factors mRNA and protein in PI3K/Akt signal pathway,we can explore the role of these hormones and factors in the process of uterine involution,and provide the basic theory for uterine subinvolution.Through the study of related factors and proteins changes in the uterine tissue of postpartum mice,the mechanism of uterine involution was found out,which is of great significance for the prevention and treatment of uterine subinvolution in dairy cows.The main research contents and results are as follows1.The effects of LPS and RLX on uterine of postpartum mice105 postpartum mice were divided into three groups:PBS、LPS(20 μL,2.5 mg/mL)and RLX(2 mL,1.25 μg/mL),35 mice in each group.The postpartum day was 0th day These mice uterus were perfused in every other day including 0th day、2nd day、4th day、6th day and 8th day after birth.Each group in different days had 7 mice.Blood was collected in next day morning,and mice were killed after that.These collected mice uterus were weighed and measured.At the same time,HE staining was performed to observe the changes of uterus.The results showed that the uterus weight of RLX treated mice was significantly higher than that of the control group in the middle uterine involution period(P<0.05).Length and width of uterus were significantly higher in later period of uterine involution than that of the control group in the same period(P<0.01);the uterus body width of LPS treated mice was only significantly higher in later period of involution than that of the control group(P<0.01),Whereas it had no significant effect on length and weight.HE staining microscopy showed that LPS and RLX could both lead to poor uterine involution,in which LPS mainly led to endometrial epithelial renewal delay and affected arterial blood flow,etc.Whereas in RLX group,the involution defects are mainly concentrated in the aspects of incomplete contraction of the uterine body and the timely discharge of lochia2.LPS and RLX treat the regulatory effect secretion of steroid hormones and inflammatory factors in postpartum miceThe secretion of estradiol(E2)and progesterone(P4)in serum and tumor necrosis factor-α(TNF-α)and interleukin-1 β(IL-1 β)in uterine tissue were measured by ELISA The results showed that,compared with the control group,estrogen in LPS group and RLX group were both significantly higher than that in the control group within 8 days after delivery,and the difference between RLX group and PBS group was extremely significant(P<0.01);the secretion of progesterone was not significantly affected by LPS group,while the concentration of progesterone in RLX group increased significantly within 0-4 days.In addition,TNF-α concentration in LPS group was significantly higher than that in control group(P<0.05),while IL-1 β concentration in RLX group was significantly lower than that in control group(P<0.05)3.Effects of LPS and RLX on apoptosis and expression of apoptosis related genes in PI3K/Akt signaling pathwayTUNEL method was used to detect the apoptosis of endometrial cells.The results showed that there were more apoptosis cells in the endometrium and myometrium of LPS group and the endometrium of RLX group.The expressions of apoptosis related genes in PI3K/Akt signaling pathway were detected by real time PCR,and the expressions of apoptosis related protein were detected by immunohistochemistry and Western blot.The results showed that the mRNA and protein expressions of Bax and FoxOlwere significantly increased in LPS group(P<0.01);the mRNA expressions of Akt,Bim and Bcl-2 were significantly increased in the end of involution(P<0.05);the protein expression of Bim was significantly increased(P<0.05).In RLX group,mRNA expression of Akt was significantly increased(P<0.01);mRNA expression of Bax was significantly decreased(P<0.01)in the early stage of involution,and significantly increased in the middle and late stage(P<0.01);mRNA expression of FoxO1 was significantly increased(P<0.05)and protein expression was significantly decreased(P<0.01);The m RNA expression of Bcl-2 decreased significantly(P<0.01),and increased significantly in the middle and late stage(P<0.05);the protein expression of Bim increased significantly in the early stage of involution(P<0.01)In conclusion,LPS can induce inflammatory reaction and even lead to endometritis By down regulating the expression of Akt in PI3K/Akt signal pathway,it can relieve the inhibition of FoxO1,regulating the apoptotic signal.And up regulate the expression of Bax,promoting the apoptosis of endometrial cells,especially endometrial epithelial cells,so that they cannot complete the renewal and repair in time,which will lead to poor uterine involution.RLX can promote the apoptosis of uterine cells by up regulating the expression of Bax in the middle and late postpartum period.At the same time,it can affect the contractile ability of the uterine muscles,so that the contents such as lochia cannot be discharged in time,and RLX can inhibit the secretion of IL-1 β,which may be related to some anti-inflammatory mechanisms.