Study On The Mechanism Of 1ysyl Oxidase Family In Endometrial Fibrosis After Injury

Background and aimIntrauterine adhesions(intrauterine adhesion IUA)were also called Asherm an syndrome,was first reported by Fritsch in 1894,In 1948,Asherman reported in detail for the first time is defined as: The uterine cavity due to partial or total occlusion of endometrial lesion,is often accompanied by abnormal menstruation,infertility,recurrent abortion and periodic abdominal pain and other clinical symptoms.In recent years,with the increase of intrauterine surgical operations and improvement of diagnosis methods of IUA,the incidence of IUA is increasing.The mechanical damage of endometrial base layer is the main cause of intrauterine adhesions,especially curettage surgery is considered to be the main cause during the pregnancy.The second is caused by endometrial infection.Although there are many treatments of IUA at present,the recurrence rate after treatment is high,especially severe intrauterine adhesions which often led to secondary infertility and recurrent abortion.Therefore,it is necessary to study the more effective treatment of intrauterine adhesions on the basis of the exact pathogenesis of intrauterine adhesions.The essence of intrauterine adhesions is endometrium fibrosis caused by the lesion of the basal layer of the endometrium.Studies found that there are a large number of extracellular matrix deposition in the IUA endometrium.The endometrium is gradually replaced by fibrous connective tissue,eventually forming intrauterine adhesion.Lysyl oxidase belong to copper-dependent amine oxidases,which can act on lysine residues of collagen and elastin resulting in molecular crosslinking,.There are a total of 5 members in the family of lysyl oxidase(LOX),lysyl oxidase like-1(LOXL1),lysyl oxidase like-2(LOXL2),lysyl oxidase like-3(LOXL3),lysyl oxidase like-4(LOXL4).Family members are involved in the formation of covalently crosslinking of collagen fibers and elastic fibers.The formation of covalent crosslinking is crucial to the stability of collagen fibers,which plays an important role in the fibrosis of many organs,such as the liver and the lungs.We speculate that the 1ysyl oxidase family is likely to play an important role in endometrial fibrosis.However,whether lysine oxidase family members are expressed in human and rat endometrium,and whether these enzymes are related to the fibrosis of endometrium have not yet been reported.TGF-beta 1 is considered to be a promoter of fibrosis.With its specific TGF-beta 1 receptor activation of TGF-beta /Smad2 pathway,TGF-beta 1 can induce the synthesis and secretion of collagen I and III,and can reduce the protease synthesis and secretion,promote protease inhibitor synthesis,promote extracellular matrix synthesis and inhibit its degradation,lead to tissue extracellular matrix accumulation and fibrosis in the end.Some studies have shown that TGF-?1 is mainly expressed in the epithelial cells of the endometrium.With the expression of TGF-?1 increasing in the endometrium of patients,endometrial fibrosis increased.After the use of specific blockers for Smad3,the expression of TGF-?1,Smad3 decreased,endometrial fibrosis decreased,TGF-?1/Smad signaling pathway plays an important role in endometrial fibrosis process.In this study we first detected the expression of lysyl oxidase family in human,rat normal,fibrosis endometrium and the fibrosis endometrium of model rats and explore the correlation between the expression of lysyl oxidase family and endometrial fibrosis by the use of lysyl oxidase family specific inhibitors ?-Aminoproprionitrile for intraperitoneal injection in the rat endometrial fibrosis model to inhibit lysyl oxidase family activity.Its activity was detected in the normal endometrium and fibrosis endometrium of rats after inhibition of ?-Aminoproprionitrile;By detecting the expression changes of TGF-beta 1/Smad signaling pathway key proteins were detected.Study is carried out on the relationship between lysyl oxidase family and TGF-beta 1/Smad signaling pathway in the process in endometrial fibrosis.We elaborate the molecular mechanism of the 1ysyl oxidase family members on the fibrosis of the endometrium,which provides effective targets for the clinical treatment of intrauterine adhesions.Methods1.Detecting the expression of lyamyl oxidase family in human normal endometrium and human endometrium1.1 Immunohistochemistry was used to detect the expression of lysyl oxidase family in human normal endometrium and fibrosis endometrium.1.2 The expression differences of lysyl oxidase family was detected in the normal endometrium and fibrosis endometrium of intrauterine adhesions patients by Western blot.2.Detecting the expression of lyamyl oxidase family in the normal rat endometrium and mechanical curettage endometrium at different time points.2.1 Wistar rats were randomly divided into 3 groups,the normal group(6 rats),the control group(24 rats)which were treated by mechanical curettage and the experimental group including 24 rats which were treated with 200mg/kg/d beta-aminoproprionitrile by intraperitoneal injection.2.2 The fibrotic endometrial rat models were established by mechanical curettage in the control group,then 6 rats were killed at 24 hours,48 hours,72 hours,7 days time points after mechanical curettage operation respectively to removed the bilateral uterine specimens.2.3 Immunohistochemical method was used to detect the expression of lysine oxidase family in the normal rats endometrium and mechanical curettage rat endometrium.2.4 Using mason(masson staining)to assess changes of the morphological and the amount of collagen changes after injury in each rat group respectively.2.5 Real-time quantitative PCR and Western blotting were used to detect the expression differences of lysine oxidase family mRNA and proteins in the normal endometrial and mechanical injury endometrium of Wistar rats at 24 hours,48 h hours,72 hours and 7 days time points.3.The effect on Wistar rat endometrial fibrosis and TGF-?1/Smad signaling pathway after lysyl oxidase activity inhibited by?-aminopropionitrile.3.1 The fibrotic endometrial rat models were established by mechanical curettage,then all these rats were treated with 200mg/kg/d ?-aminopropionitrile by intraperitoneal injection,6 rats were killed at 24 hours,48 hours,72 hours,7 days time points after mechanical curettage operation respectively to removed the bilateral uterine specimen s after anesthesia.3.2 Use lysyl oxidase activity assay kit to detect the lysyl oxidase activity in the normal rat endometrium,the rat endometrium after mechanical curettage(the control group)and the rat endometrium treated with 200mg/kg/d ?-aminopropionitrile(the experimental group).3.3 The changes of Histomorphology and collagen content of endometrium were evaluated by hematoxylin eosin and Masson staining.3.4 The content of hydroxyproline from insoluble collagen in the endometrium of each group was detected by hydroxyproline assay kit.The content of collagen can be reflected by the content of hydroxyproline.3.5 The expression of TGF-?1,Smad2,Smad3,p-Smad2 and p-Smad3 in the endometrium of each group was detected by immunohistochemistry.3.6 Western blot was used to detect the expression of Collagen I,TGF-?1,Smad2,Smad2,Smad3,p-Smad2 and p-Smad3 in endometrium of rats at different time points.3.7 The expression differences of TGF-?1,Smad2 and Smad3 at mRNA levels at different time points were detected by Quantitative Real-time PCR4.Statistical analysisThe data were represented by mean ± SD.Statistical analysis was carried out with the Statistical Program for Social Sciences software16.0 version.P<0.05 was considered statistically significant,and P < 0.01 was considered very significant.Results1.Lysine oxidase family are expressed in the normal and fibrosis human and also expressed in the normal rat endometrium and rat endometrium after mechanical curettage at different time points,and the content of mRNA and proteins of LOX and LOXL1 have a significant rise.There is difference between normal Wistar rat endometrium and rat fibrotic endometrium.1.1 Immunohistochemical results showed that LOX,LOXL1,LOXL2,LOXL3 and LOXL4 were expressed in normal endometrium,fibrotic endometrium,normal Wistar rat endometrium and rat fibrotic endometrium.1.2 Real-time quantitative PCR results showed that compared with normal endometrium,amount of LOX and LOXL1 mRNA in fibrotic endometrium increased significantly at 48 hours,72 hours,7 days time points(P<0.01).Amount of LOXL2 mRNA is increased at 72 hours and 7 day time points(P<0.05).1.3 Western blotting results showed that compared with normal endometrium,the amount of LOX and LOXL1 protein in fibrotic endometrium increased significantly(P<0.01),and the content of LOXL2,LOXL3 and LOXL4 showed no difference(P>0.05).Compared with the normal rat endometrium,the amount of LOX and LOXL1 in fibrotic rat endometrium increased significantly(P<0.01).2.The degree of endometrial fibrosis is reduced after lysine oxidase activity is inhibited2.1 Masson staining results showed:Compared with normal group rats after 72 hours and 7 days,the degree of Aniline blue in the mechanical injury endometrium increased significantly(P<0.01).The extent of Aniline blue in the experimental group rat endometrial which was treated with beta-aminopropionitrile decreased significantly at 72 hours and 7 days time points compared with the rats of control group.It is indicated that mechanical curettage can lead to endometrial fibrosis.The amount of collagen protein and degree of fibrosis in rat fibrotic endometrium decreased after treatment with beta-aminopropionitrile.2.2.Hydroxyproline test results showed that: the amount of hydroxyproline of insoluable collagen increased significantly in the uterus of control group at 72 hours and 7 days time points after curettage,(P<0.01),At 72 hours and 7 days time points after treatment with beta aminopropionitrile,the amount of hydroxyproline of insoluable collagen decreased significantly in rat uterus of experimental group(P<0.01).2.3.Western blotting results showed that the expression of Collagen I protein in uterus of control group increased significantly at 48 hours,72 hours and 7 days after curettage(P<0.01).The expression of Collagen I protein in rat uterus of experimental group decreased significantly at 48 hours,72 hours and 7 days post-treatment with betaaminopropionitrile.(P<0.01).3.The role of lysine oxidase in endometrial fibrosis is related to TGF-?1/Smad signaling pathway.3.1 Lysine oxidase activity test showed that the activity of lysine oxidase increased significantly at 72 hours and 7 days after mechanical curettage in rat uterus compared with the normal group rats(P<0.01).Compared with the same time point of control group rats,lysyl oxidase activity decreased significantly at 48 hours,72 hours and 7 days post-treatment with beta-aminopropionitrile(P<0.01).3.2 The results of HE staining showed that after the endometrial mechanical injury,the number of endometrial glands decreased,the glands were dilatate,endometrial tissue morphology is poor compare with the normal group rats.The morphology of endometrial tissue,gland,uterus cavity of the experimental group rats recovered better than that of the control group after treated with beta-aminopropionitrile at the same time point.3.3 Immunohistochemical results showed that TGF-?1,Smad2,Smad3,p-Smad2 and p-Smad3 were all expressed in normal rat endometrial tissue and fibrotic rat endometrium.3.4 Real-time fluorescence quantitative PCR test showed that the mRNA expression of TGF-?1 increased significantly at each experimental time point compared with the normal group rats(P<0.01).The mRNA expression of Smad2 and Smad3 increased significantly at 48 hours,72 hours,and 7 days after curettage in rats Compared with the same time point of control group rats(P<0.01).The mRNA expression of TGF-?1 decreased significantly at each experimental time point post-treatment with beta-aminopropionitrile compared with the control group rats(P<0.01).The mRNA expression of Smad2 and Smad3 decreased significantly at 48 hours,72 hours and 7 days post-treatment with beta-aminopropionitrile compared with the control group rats(P<0.01).3.5 Western blotting experiment showed that the expression of TGF-?1,Smad2,Smad3,p-Smad2 and p-Smad3 in the rat endometrium of the experimental group was lower significantly than those of the control group rats at 72 hours and 7 days after curettage(P<0.01).The expression levels of TGF-?1,Smad2,Smad3,p-Smad2 and p-Smad3 in the endometrium of the control group were higher significantly than those in the normal rat endometriumat at 72 hours and 7 days(P<0.01).Conclusion1.The lysine oxidase family is expressed in the normal human endometrium,the human intrauterine adhesion endometrium,the normal rat endometrium and the fibrotic rat endometrium.2.The expression of LOX and LOXL1 increasing significantly in human and rat fibrotic endometrium indicated that LOX and LOXL1 are closely related to the formation of endometrial fibrosis.3.LOX and LOXL1 are closely related to TGF-?1/Smad signaling pathway during the formation of endometrial fibrosis.