Study On The Effect Of Leonurine On The Uterine Subinvolution Of Postpartum Mice Through PI3K/Akt Singnaling Pathway And Its Mechanism

In order to support successful pregnancy,the uterus of female livestock will undergo a series of physiological changes.After pregnancy,the uterus will return to its pre-pregnancy state and recover its function gradually.The uterine involution process involves anatomical,histological,and cytological changes that are key to the next pregnancy.Endometritis in cows can delay involution of the uterus,resulting in reduced reproductive performance.The clinical use of antibiotics and other treatments is not ideal for endometritis induced uterine involution.Motherwort is widely used in the treatment of gynecology and obstetrics diseases,and leonurine is the main active ingredient of motherwort,studies have found that leonurine has anti-apoptosis effect,and cell apoptosis is closely related to endometrial repair,one of the key links of uterine involution.PI3K/Akt signaling pathway regulates cell apoptosis through various reactions,which is closely related to cell apoptosis.Therefore,this study studied the mechanism of uterine subinvolution by infusing postpartum mice with leonurine.In this study,the time of establishing the model of uterine subinvolution in mice was firstly selected by injecting LPS into the uterus of postpartum mice.The experiment found that the model of uterine subinvolution in groups 1 and 3 days after LPS infusion was successfully established,and different concentrations of leonurine（0.5 mg/kg,1.5 mg/kg,4.5 mg/kg）were given to the groups for 1 or 3 days.Compared with the normal involution and subinvolution,the changes of steroid hormone in mice were observed,and the effects of leonurine on apoptosis and expression of apoptosis-related factors in PI3K/Akt,to explore the effect of leonurine on uterine subinvolution and the expression of apoptosis-related genes and proteins,so as to provide new ideas and theoretical support for the treatment of uterine subinvolution.The main findings are as follows:1.The time to establish the model of uterine subinvolution in mice36 pregnant female mice were randomly divided into two groups:model group（LPS group）and control group（normal saline）.18 mice in each group was infused with20μL LPS（5 mg/mL）or saline,and The day of production was recorded as day 0,and perfusion was performed on day 1,and the time of sampling was divided into 12 h（1 d）,3 d and 5 d after LPS/normal saline perfused.Six mice were killed in each group.Histopathological observation showed,in comparison with the control groups of 1 d and 3 d,endometrial continuity was interrupted,accompanied by hyperemia,bleeding and extensive inflammatory cell infiltration in the LPS groups.The endometrium of LPS group was intact in 5 d group,the situation of involution of uterus was comparable to the control group.By real-time PCR,we determined the level of Bax was remarkablely improved with the treatment of LPS in 1 d group（P<0.01）,and the level was also remarkablely improved with the treatment of LPS in 3 d group（P<0.05）,and after the treatment with LPS,it has no significant change with control group in 5 d group.According to histopathological observation and expression analysis of apoptosis-related gene Bax,the model of uterine subinvolution was established by LPS for 1 d and 3 d,and subsequent experiments were conducted.2.Effect of leonurine on uterine tissue of postpartum mice60 postpartum mice were randomly divided into two groups:treated with leonurine for 1 day and for 3 days.Each group had 30 mice,each group had five treatments,6mice in each treatment:（1）blank control group:saline 20μL,（2）LPS group:LPS（5mg/mL）20μL,（3）leonurine low dose group:LPS（5 mg/mL）20μL+leonurine（0.5mg/kg）was administered intragastrically.（4）leonurine middle dose Group:LPS（5mg/mL）20μL+leonurine（1.5 mg/kg）was administered intragastrically,（5）leonurine high dose group:LPS（5 mg/mL）20μL+leonurine（4.5 mg/kg）was administered intragastrically.The day of production was recorded as day 0,the uterus was perfused with LPS on day 1,12 h later,leonurine was given intragastric administration;and leonurine 1 d group:LPS was injected into uterus on day 1,and leonurine was intragastric12 h later.In the leonurine 3d group,the mice were intragastrically injected with LPS on day 1 and intragastrically injected with leonurine 12 h later.The mice were intragastrically intragastrically injected with leonurine 2 d and 3 d respectively.On the second day after the last intragastric treatment,the mice were anesthetized and sacrificed for blood and uterine tissue collection.The mass and other basic data of uterus were measured,and the uterus tissues were observed by HE staining.The results showed that the uterus wet weight index was significantly decreased compared with LPS group after Leo（1.5 mg/kg）and Leo（4.5 mg/kg）treatment（P<0.01 or P<0.05）,and the uterine body width of unilateral uterus was significantly decreased compared with LPS group after Leo（0.5 mg/kg,1.5 mg/kg,4.5mg/kg）（P<0.01 or P<0.05）in the groups of 1 d with leonurine.HE staining was used to score the uterine involution in each group according to the uterine involution standard scoring scale.It was found that LPS-infused uterine tissue showed delayed endometrial renewal,hyperemia and bleeding,which resulted in partial subinvolution of uterus.Leonurine treatment could promote endometrial renewal,tissue hyperemia and bleeding reduced,inflammatory cell infiltration decreased,and a dose-dependent.3.Effect of leonurine on estrogen and progesterone concentrations in postpartum miceBlood samples were collected from the mice,supernatants were separated and detected by Elisa.The results showed that leonurine had no significant effect on the concentration of estradiol（E₂）,and in leonurine treatment groups for 3 days,compared with control group,progesterone concentration in LPS group and Leo（0.5 mg/kg）group was significantly increased（P<0.05）,and compared with Leo（0.5 mg/kg）group,progesterone concentration in Leo（4.5 mg/kg）group was significantly decreased（P<0.05）.4.The effect of leonurine on the expression of genes related to PI3K/Akt pathwayThe expression of genes was detected by real-time PCR.The results showed that the expression of PI3K gene after LPS treatment was significantly increased compared with the control group（P<0.05）,and the expression of PI3K gene after Leo（4.5 mg/kg）treatment was significantly decreased compared with LPS group（P<0.01）and significantly decreased compared with Leo（0.5 mg/kg）group（P<0.05）in leonurine treatment groups for 1 day.In leonurine treatment groups for 1 and 3 days,the expression of Akt gene was significantly decreased in Leo（4.5 mg/kg）group compared with LPS group（P<0.001）,the expression of Akt gene in Leo（4.5 mg/kg）group was extremely significant or significantly decreased compared with Leo（0.5 mg/kg）group（P<0.01 or P<0.05）;Compared with the control group,Bax expression after LPS treatment was extremely significant or significantly increased（P<0.001 or P<0.05）,compared with LPS group,Bax expression after Leo（1.5 mg/kg）treatment was extremely significant or significantly decreased（P<0.001 or P<0.05）and Bax expression was significantly decreased after Leo（4.5 mg/kg）treatment（P<0.001 or P<0.01）;Compared with LPS group,Bcl-2 was significantly increased after Leo（4.5 mg/kg）treatment（P<0.05）.Immunohistochemical staining and western blot were used to detect the expression of apoptosis-related proteins.The results showed that apoptosis-related protein Bax was strongly expressed in the mucosal layer and serous layer of uterus in LPS group,but only in the mucosal layer after leonurine treatment,and the expression was weakened.In leonurine treatment groups for 1 and 3 days,the expression of Bax protein was significantly increased after LPS treatment compared with the control group（P<0.01）,compared with LPS group,Bax expression was significantly decreased in Leo（1.5mg/kg）and Leo（4.5 mg/kg）groups（P<0.001 or P<0.01 or P<0.05）.In conclusion,Leonurine can improve lipopolysaccharide-induced uterine subinvolution,inhibit the apoptosis of uterine cells,and promote the repair of endometrium,reduce congestion,bleeding,reduce inflammatory cell infiltration,which is related to the regulation of leonurine on PI3K/Akt signaling pathway.The results of this study provide a new idea and theoretical basis for leonurine in the treatment of post-natal uterine subinvolution.