Inhibition Of Neiyixiao On Xenograft Endometriosis Based On Wnt Signaling Pathway

BackgroundEndometriosis is a disease caused by the active endometrial cells growing outside the endometrium.Endometriosis is considered as blood stasis syndrome with treatment of activating blood and dissolving stasis in traditional Chinese medicine.Neiyixiao,a new anti-endometriosis Chinese compound,is composed of ligustrazine,ferulic acid and tetrahydropalmatine from famous gynecological prescription“Foshousan”.In previous studies,Neiyixiao inhibited the autograft endometriosis,but with unclear effect on xenograft endometriosis.Network pharmacology is benefit to analyze the mechanism of Neiyixiao on endometriosis systematically.Invasion and metastasis are considered as one of the important steps in the development of endometriosis.Epithelial mesenchymal transformation is closely related to invasion and metastasis,which can be promoted by Wnt signaling pathway.In this paper,the therapeutic effect of Neiyixiao on xenograft endometriosis was observed.The regulations of Neiyixiao were investigated in Wnt singaling pathway,epithelial mesenchymal transformation,invasion and metastasis.Then network pharmacology was ulteriorly used to explore the potential mechanism of Neiyixiao on endometriosis.It will provide the theoretical basis of Neiyixiao on endometriosis.ObjectiveThe fluorescent xenograft endometriosis model was used to verify whether the mechanism of Neiyixiao on endometriosis is related to regulation of Wnt signaling pathway to effect epithelial mesenchymal transformation,and further inhibition of invasion and metastasis in endometrium.Then,the network pharmacology was used to further explore the potential targets of Neiyixiao inhibiting endometriosis.Methods1.Improvement of the fluorescent xenograft endometriosis modelBilateral uteri were cut into 4 mm²-sized tissue blocks from C3H ^tdTomato female mice at the age of 8?10 weeks.Then the tissues were transplanted into the subcutaneous abdomen of nude mice,with injection of 2 mg·kg^-1 estradiol every 5days.After 28 days of transplantation,the fluorescence intensity of ectopic lesions were observed by the in vivo imaging system.Volume of ectopic lesions were measured by a vernier caliper,and calculated according to the formula:volume=0.52×length×width×height.The relationship was constructed between fluorescence intensity and volume in ectopic lesions.The structures of ectopic lesions were observed by HE staining,and the expression of ENDO-I protein was detected by immunohistochemistry.2.Efficacy index evaluationAfter 28 days of transplantation,animals with successful transplantation were randomly divided into 4 groups according to the fluorescence intensity,including endometriosis model group,the low,medium,high Neiyixiao groups,and gestrinone group.Normal female C3H ^tdTomato mice were treated as control group.The control group and endometriosis model group were treated with 0.5%CMC-Na.Neiyixiao groups were administrated with 90,180 and 360 mg·kg^-1 Neiyixiao respectively.Then2 mg·kg^-1 gestrinone were given in the gestrinone group.After oral administration for28 days,the fluorescence intensity and volume of ectopic lesions were detected.The morphology of ectopic endometrium was observed by HE staining.E2 and PROG levels in the serum were measured by ELISA.3.Detection of invasion and metastasis and epithelial mesenchymal transformationRNA and protein were extracted from normal endometrium in control group,ectopic lesions in model and Neiyixiao groups.The genes expression of invasion and metastasis and epithelial mesenchymal transformation were detected by RT-qPCR respectively,such as MMP-2,MMP-9,TIMP-1,and E-cadherin,N-cadherin,Vimentin,Twist,Snail,Slug,ZEB1.The MMP-2,MMP-9,TIMP-1 proteins expression of invasion and metastasis were inspected by Western blot.E-cadherin,N-cadherin,Vimentin,Snail proteins expression of epithelial mesenchymal transformation were also inspected by Western blot respectively.4.Detection of Wnt signaling pathwayRNA and protein were extracted from normal endometrium in control group,ectopic lesions in model and Neiyixiao groups.The levels of APC,GSK3?,?-catenin,c-Myc and CyclinD1 mRNA of Wnt signaling pathway were examined by RT-qPCR respectively.The expression of Wnt3a,GSK3?,p-GSK3?,?-catenin,p-?-catenin and c-Myc proteins in Wnt signaling pathway were detected by Western blot respectively.5.Network pharmacology study of inhibition of Neiyixiao on endometriosisFirstly,TCMID,TCMSP and SEA databases were used to collect the targets of Neiyixiao,while OMIM,DisGeNET and GEO databases were used to collect the targets of endometriosis.Then the“Neiyixiao ingredients-targets”and“Neiyixiao targets-endometriosis targets”networks were built by Cytoscape 3.5.0 software respectively.The Network Analyzer plugin was used to analyze the topological parameter of the network.The key targets were screened with the value of degree and betweenness more than the average in the network.Then,key targets were inputted in DAVID and PANTHER databases for GO and pathway enrichment analysis.Finally,the molecular docking between Neiyixiao and key targets were validated by Sybyl-X2.1.1 software.Results1.Fluorescent xenograft endometriosis model improvementAfter 28 days of transplantation,strong fluorescence with intensity?9.58E+6were observed from the ectopic lesions in the subcutaneous abdomen of nude mice.Ectopic lesions presented transparent vesicles,with abundant blood vessels on the surface and effusions inside visibly.The height of effusions inside were?1 mm,and the volume of ectopic lesions were?4 mm³.Furthermore,the linear relationship between fluorescence intensity and volume of ectopic lesions was y=862154x+6.13E+6,R²=0.9005.In HE staining,there were endometrial epithelial cells,glands and mesenchymal cells in ectopic tissues.ENDO-I protein expressed in the cytoplasm of ectopic endometrium interstitial cells according to the results of immunohistochemical.In a word,fluorescent xenograft model of endometriosis was successfully improved.2.Inhibition of Neiyixiao on endometriosisAfter 28 days of oral administration,the fluorescence intensity and volume of ectopic lesions did not change significantly in endometriosis model group,compared with pre-administration?P>0.05?.Neiyixiao significantly diminished fluorescence intensity and volume of ectopic lesions?P<0.05?.Meanwhile,gestrinone significantly decreased fluorescence intensity and volume of ectopic lesions?P<0.01?.In HE staining,ectopic endometrium in model group had a structure similar to the normal one.The regular monolayer epithelial cells were columnar.Moreover,thicker endometrium walls were observed,with more glands,rich blood vessels and inflammatory cells in ectopic endometrium.Using 360 mg·kg^-11 Neiyixiao,ectopic endometrium walls were becoming thinner.Numbers of glands and blood vessels were significantly reduced,and inflammatory cells were also decreased.Serum levels of E2 and PROG in the model group were significantly increased compared with the control group?P<0.05?,while Neiyixiao significantly diminished the serum levels of E2 and PROG vs model group?P<0.05?.3.Neiyixiao inhibited invasion and metastasis and epithelial mesenchymal transformation?1?The mRNA levels of MMP-2 and MMP-9 in model group were significantly increased?P<0.01?,while the mRNA level of TIMP-1 was significantly decreased compared with control group?P<0.05?.Compared with model group,Neiyixiao remarkably down-regulated the expression of MMP-2 and MMP-9 genes?P<0.05?,and remarkably up-regulated TIMP-1 gene expression?P<0.05?.The MMP-2 and MMP-9 proteins were significantly aggrandized in model group?P<0.05?,while TIMP-1 protein was significantly reduced vs control group?P<0.05?.Neiyixiao remarkably inhibited MMP-2 and MMP-9 proteins?P<0.05?,and promoted TIMP-1protein remarkably compared with model group?P<0.05?.?2?The mRNA level of E-cadherin in model group was significantly down-regulated?P<0.01?,while the mRNA levels of N-cadherin,Vimentin,Twist,Slug,Snail and ZEB1 were significantly up-regulated compared with control group?P<0.05?.Neiyixiao obviously augmented the expression of E-cadherin gene vs model group?P<0.05?,while lessened the expression of N-cadherin,Vimentin,Twist,Slug,Snail and ZEB1 genes obviously?P<0.05?.Compared with control group,the level of E-cadherin protein was significantly lower?P<0.05?,while the levels of N-cadherin,Vimentin,Snail proteins were significantly higher in model group?P<0.05?.The expression of E-cadherin protein was remarkably increased by Neiyixiao?P<0.05?,while the expression of N-cadherin,Vimentin,Snail proteins were remarkably reduced compared with the model group?P<0.05?.4.Neiyixiao regulated Wnt signaling pathwayRemarkably lower mRNA levels of APC and GSK3?were found in model group?P<0.05?,with higher mRNA levels of?-catenin,c-Myc and CyclinD1 than those in control group?P<0.05?.On the contrary,Neiyixiao significantly promoted the expression of APC and GSK3?genes?P<0.05?,while prevented the expression of?-catenin,c-Myc and CyclinD1 genes significantly vs model group?P<0.05?.Compared with control group,the protein levels of GSK3?and p-?-catenin were significantly down-regulated?P<0.05?,while the protein levels of?-catenin,p-GSK3?,c-Myc and Wnt3a were significantly up-regulated?P<0.05?.The expression of GSK3?and p-?-catenin proteins were obviously put on?P<0.05?,while the expression of Wnt3a,p-GSK3?,?-catenin,and c-Myc proteins were cut down obviously by Neiyixiao vs model group?P<0.05?.5.Network pharmacology study of Neiyixiao inhibition on endometriosisA total of 275 Neiyixiao targets and 401 endometriosis targets were collected.There were 22 common targets between them.The“Neiyixiao ingredients-targets”and“Neiyixiao targets-endometriosis targets”network were constructed respectively.Then22 common targets and their neighbors were selected for a core protein-protein interaction?CPPI?network.66 key targets were obtained.GO enrichment analysis showed that 66 key targets were mainly involved in several biological processes,including the negative regulation of apoptosis,DNA-templated,and metabolic process,etc.They mainly existed in the nucleus,cytoplasm and cytomembrane.Molecular functions embodied protein binding,enzyme binding and signal transducer activity,etc.Pathway enrichment analysis manifested that 66 key targets were mainly participated in PI3K-Akt signaling pathway,Estrogen signaling pathway,TNF signaling pathway,Apoptosis signaling pathway,Wnt signaling pathway and VEGF signaling pathway,etc.In molecular docking,ferulic acid had a good binding activity with IL6,MMP-2,and a certain binding activity with TP53,MMP-9,VEGFA.Tetrahydropalmatine owned good binding activity with MMP-2,IL8,and binding activity with VEGFA,TIMP-1.None of the 13 targets had binding activity with ligustrazine.ConclusionIn summary,In vivo experiment,the relationship between fluorescence intensity and volume of ectopic lesions was constructed in fluorescent xenograft endometriosis model,R²=0.9005.Neiyixiao significantly reduced the fluorescence intensity and volume of ectopic lesions,improved the structure of ectopic endometrium tissues,and reduced E2 and PROG levels in serum.The mechanism of Neiyixiao on endometriosis may be related to regulation of Wnt signaling pathway to effect epithelial mesenchymal transformation,and further inhibition of invasion and metastasis in endometrium.Through network pharmacology research,66 key targets of Neiyixiao on endometriosis were obtained,including MMP-2,MMP-9 and TIMP-1.They involved in multiple signaling pathways including Wnt signaling pathway.Furthermore,ferulic acid and tetrahydropalmatine had good binding activities with several key targets including invasion and metastasis factors.