Study On Effects And Mechanism Of Neiyixiao On Ectopic Endometrium Proliferation Through Notch Pathway

BackgroundEndometriosis（EMS）is a benign gynecological disease in which endometrioid tissue adheres to and grows outside of the uterine.The incidence among women of childbearing age is about 6-10%.AlthoughEMS pathogenesis has not been clarified,the reflux theory of menstruation is most known theory proposed by Sampson in 1920.According to this theory,endometrial tissue is transported from fallopian tube to outside areas of uterine,such as pelvic cavity,ovary,and peritoneum.Adherence and blood supply are build up,with proliferating endometrial cells.Thus endometriosis lesions are established.In this theory,ectopic proliferation is the important step in EMS.Activated Notch pathway promotes proliferation,otherwise,inactivated Notch pathway inhibits proliferation.It is suggested that Notch pathway may play a key role in EMS.As the famous gynecological prescription on EMS,Foshou san has the effects of invigorating blood and fusing stasis.Ferulic acid and ligustrazine are the active ingredients of Foshou san.Both of them join with tetrahydropalmatine to form Neiyixiao,a new Chinese medicine monomer compound created by our team.In previous studies,Neiyixiao effectively inhibitted EMS occurrence.However,it is unknown whether Neiyixiao has an inhibitory effect on ectopic proliferation,and whether it is related to Notch pathway.The purpose of this study is to explore the mechanism of Neiyixiao on proliferation through Notch pathway in EMS.It will provide a basis for EMS treatment.PurposeTo explore whether its mechanism depends on regulating Notch pathway,then suppressed Ki67 and PCNA.This study provides a theoretical basis for EMS treatment.To explore whether regulation of Notch pathway on proliferation is based on Ki67,and PCNA.It need to be verified the Neiyixiao effects on EMS proliferation.Methods1.Autologous EMS model establishment and group administration in SD ratsEMS model was established by autologous transplantation in estrus SD rats.After 28days of transplantation,the growth and volume of the heterotopic intima were observed.The height of effusion was greater than or equal to 2 mm.The graft volume was greater than or equal to 8 mm³.The successfully established rats were randomly divided into four groups,EMS,45,90,and 180 mg·Kg^-1 Neiyixiao groups.Another 6 female SD rats were treated as control without transplantation.After 28 days of continuous administration,the ectopic endometrium was observed by laparotomy,and measured with volume size.2.Xenograft EMS model establishment and group administration in nude miceBilateral uteri of estrus C3H mice were cut into 4 mm² tissue,and transplanted subcutaneously into nude mice abdomen.After 28 days,the ectopic growth was observed.There were nodular protrusions in the abdomen of the nude mice.Effusion height was greater than or equal to 1 mm.The ectopic volume was calculated to be greater than or equal to 4 mm³.The successfully established mice were randomly divided into EMS,90,180,and 360 mg·Kg^-1 Neiyixiao groups.Another 6 C3H female mice were treated as control without transplantation.After 28 days of continuous administration,the appearance of the ectopic was observed and measured with volume size.3.Effect of Neiyixiao on ectopic proliferation in vivoTotal RNA and protein were extracted from normal endometrial tissue of control group,or from ectopic tissues in EMS and Neiyixiao groups in autologous and xenograft EMS models,respectively.The effect of Neiyixiao on proliferation factors,Ki67 and PCNA,were detected by RT-qPCR and Western blot.4.Neiyixiao regulation on cell proliferation in vitroHuman endometriosis-derived eutopic endometrium stromal cells hEM15A and endometrial adenocarcinoma cells HEC1-B were selected.First,the effect of Neiyixiao on proliferation was detected by plate cloning and MTT assay.Then,after treated with Neiyixiao,Ki67 and PCNA,were investigated by RT-qPCR and Western blot.5.The role of Notch pathway in proliferation inhibition by NeiyixiaoIn autograft,xenograft EMS models,and hEM15A,HEC1-B cells,Notch1,NICD1,Notch2,NICD2,Hes1 and Hey1 in Notch pathway were observed with RT-qPCR and Western blot using Neiyixiao.Jagged1 or DAPT was used as Notch pathway agonist or inhibitor,seperately combined with Neiyixiao.The changes of Notch pathway and proliferation factors were detected by RT-qPCR and Western blot.Proliferation ability was analyzed by plate cloning and MTT assay in hEM15A and HEC1-B cells.Results1.Neiyixiao inhibited ectopic volume in vivo（1）In autologous EMS model,there was no significant difference in ectopic volume among all groups before administration（P>0.05）.After continuous gavage of Neiyixiao for 28 days,ectopic volume were significantly reduced,with less blood vessels on the surface and adhesion area by laparotomy.Compared with pretreatment,ectopic volume in 45 mg·Kg^-1 Neiyixiao group was significantly decreased to 83.40±6.99mm³（P<0.05）.Ectopic volumes in 90 and 180 mg·Kg^-1 Neiyixiao groups were reduced to 41.10±20.99 and 16.33±10.40 mm³（P<0.01）.（2）In xenograft EMS model,there was no obvious difference in ectopic volume in all groups before treatment（P>0.05）.After 28 days of continuous administration,the nodular protrusion in abdomen was remarkablly reduced in Neiyixiao groups.Adhesion area between ectopic and surrounding tissue was refined,Blood vessels on the surface became less.Compared with pretreatment,ectopic volume was significantly decreased to 10.01±5.97 and 12.56±7.65 mm³ in 90 and 360 mg·Kg^-1 Neiyixiao groups（P<0.05）,respectively.Ectopic volume was significantly reduced to 12.56±7.65 mm³ in 180mg·Kg^-1 Neiyixiao group（P<0.01）.2.Inhibitory effect of Neiyixiao on proliferation in ectopic endometriumBoth in autologous and xenograft EMS models,the mRNA levels of Ki67 and PCNA were significantly up-regulated in EMS group than those in control group（P<0.01）.Neiyixiao obviously down-regulated Ki67 and PCNA gene by RT-qPCR（P<0.01）.In autologous EMS model,protein levels of Ki67 and PCNA were significantly increased in EMS group than those in control group（P<0.05）.The protein levels of Ki67 and PCNA were significantly decreased in 45,90 mg·Kg^-1 Neiyixiao groups than those in EMS group（P<0.05）.In xenograft EMS model,Ki67 and PCNA protein were promoted in EMS group than those in control group（P<0.01）.Neiyixiao diminished Ki67 and PCNA protein（P<0.05）.3.Neiyixiao inhibited cell proliferation in vitroIn hEM15A and HEC1-B cells,the clones became smaller and fewer among Neiyixiao groups than those control group in plate cloning test（P<0.05）.Using Neiyixiao for 6 days,hEM15A cell number was significantly decreased in 960μg·m L^-1Neiyixiao group（P<0.05）.Meanwhile,625,1250μg·m L^-1 Neiyixiao groups had significantly inhibitory effect on HEC1-B cells in MTT assay（P<0.05）.In hEM15A and HEC1-B cells,the mRNA levels of Ki67 and PCNA were significantly down-regulated in Neiyixiao groups than those in control group by RT-qPCR（P<0.05）.The Ki67 and PCNA protein were obviously reduced in Neiyixiao groups than those in control group by Western blot（P<0.05）.4.Neiyixiao down-regulated Notch pathway（1）Neiyixiao inhibited Notch pathway in vivoBoth in autologous and xenograft EMS models,the mRNA levels of Notch1,Notch2,Hes1 and Hey1 were significantly up-regulated in EMS group than those in control group（P<0.05）.The mRNA levels of Notch1,Notch2,Hes1 and Hey1 were obviously down-regulated in Neiyixiao groups than those in EMS group by RT-qPCR（P<0.05）.Notch1,NICD1,Notch2,NICD2,Hes1 and Hey1 protein were significantly increased in EMS group than those in control group（P<0.05）.Neiyixiao obviously reduced Notch1,NICD1,Notch2,NICD2,Hes1 and Hey1 protein by Western blot（P<0.05）.（2）Neiyixiao arrested Notch pathway in vitroIn hEM15A and HEC1-B cells,the mRNA levels of Notch1,Notch2,Hes1 and Hey1 were significantly down-regulated in Neiyixiao groups than those in control group by RT-qPCR（P<0.05）.However,low concentration of Neiyixiao induced Notch2 and Hey1 gene expression in hEM15A cells（P<0.05）and Notch1 gene expression in HEC1-B cells（P<0.05）.The protein levels of Notch1,NICD1,Notch2,NICD2,Hes1and Hey1 were significantly reduced in Neiyixiao groups than those in control group by Western blot（P<0.05）.（3）Neiyixiao inhibited proliferation through Notch pathwayAfter treatment with the Notch pathway agonist Jagged1,the protein levels of Notch1,NICD1,Notch2,NICD2,Hes1 and Hey1 were up-regulated.Notch pathway was activated.Then Ki67 and PCNA increased,thus cell proliferation was promoted.When treated with Neiyixiao and Jagged1,Notch pathway activation was restricted,accompanied with low expression of Notch1,NICD1,Notch2,NICD2,Hes1,and Hey1in hEM15A and HEC1-B cells.Moreover,the downstream proteins,Ki67 and PCNA decreased compared with the Jagged1 group.It indicated that Neiyixiao antagonized Notch pathway activation,and weakened proliferation promotion by Jagged1.After using Notch pathway inhibitor DAPT,the protein levels of Notch1,NICD1,Notch2,NICD2,Hes1 and Hey1 were down-regulated.Notch pathway was inhibited.Furthermore,Ki67 and PCNA were reduced to inhibit cell proliferation.Combined DAPT with Neiyixiao,Notch1,NICD1,Notch2,NICD2,Hes1,and Hey1 decreased in hEM15A and HEC1-B cells.Then,Ki67 and PCNA were much more down-regulated with proliferation suppression compared with the DAPT group.It indicated that Neiyixiao showed synergy influence with DAPT,promoting Notch pathway inactivation,and inhibiting proliferation.ConclusionIn conclusion,Neiyixiao inhibits the proliferation of ectopic endometrium in vivo and in vitro.Its mechanism may be related to down-regulating Notch pathway.