| BACKGROUND:Adenomyosis(AM)is a kind of estrogen endometrial glands and stroma invasion into the uterine muscle colonization and proliferation and the formation of dependent diseases.In recent years,its incidence is rising,has become a common gynecological diseases.The main clinical symptom of AM is the progressive dysmenorrhea and infertility.The incidence of dysmenorrhea was as high as 77.8%.Affect the patient’s life,work,etc.seriously.At present,the only curative treatment for AM is hysterectomy,but it is not easy to be accepted by patients,especially the young patients with fertility requirements.Western medicine has large side-effects,during medication pregnant is not allow,and after stopping drug Dysmenorrhea and other symptoms are easy to relapse.Therefore,it is urgent to find an effective therapy.The traditional Chinese medicine shows a unique advantage in such as relieving or eliminating the symptoms of dysmenorrhea,improving the patient’s quality of life and fertility,minoring side-effects,stabling long-term efficacy etc.Studies have shown that tumor suppressor gene(p53)and its mutants,epidermal growth factor(EGFR),pathway signal transducer and activator of transcription 3(STAT3)play an important role in the pathogenesis of AM.This study intends to observe the expression changes of related factors of the P53/EGFR/STAT3 signaling pathway in the lesions of nude mice treated with the Modified Shaoyaogancao Decoction by animal experiment.In order to clarify the mechanism of the Modified Shaoyaogancao Decoction in the treatment of AM.It is of great theoretical and practical significance for AM in guiding the clinical medication,enriching the theory of traditional Chinese medicine,researching and developing of new drugs.OBJECTIVE:1 To establish an ideal mouse model of human AM,in order to study the pathogenesis,pathological features and explore the effective treatment of AM.2 In this study,we aimed to observe the regulating effect of miR-27a on related factors of the P53/EGFR/STAT3 signaling pathway in the lesions of nude mice under the Modified Shaoyaogancao Decoction by animal experiment.In order to elucidate the mechanism of the Modified Shaoyaogancao Decoction in the treatment of AM.It can provide the scientific basis in deep understanding of the etiology and pathology of AM,guiding the clinical medication,researching and developing of new drugs.METHODS:1 Establish the human AM nude mouse model by intraperitoneal transplantation,and appraisal modeling results by detecting the vimentin and keratin with HE staining and immunohistochemistry.2 The method of establishing the nude mouse model of human AM was used to establish the sham—operation group as control.The nude mice were randomly divided into sham-operation group,blank model group,mifepristone group,high dose group of the Modified Shaoyaogancao Decoction,middle dose group of the Modified Shaoyaogancao Decoction and low dose group of the Modified Shaoyaogancao Decoction,drug intervention respectively,collect experimental samples until 3th week.3 Measure the lesion tissue’ s maximum diameter(L)and maximum transverse diameter(W)of nude mice in each group,calculate the lesion volume(V),V = L*W2/2,compare the difference of the volume in each group.4 Immunohistochemical method was used to detect the expression of p53 protein in the lesions of nude mice in each group.5 Western Blot was used to detect the expression of p53-273H,c-Src,LIF,EGFR,p-EGFR,STAT3,p-STAT3,COX-2 in the lesions of nude mice in each group.6 RT-qPCR was used to detect the expression of p53-273HmRNA,miR-27a,EGFR mRNA and LIFR mRNA in the lesions of nude mice in each group.RESULTS:1 The appearance,size,histology and the expression of vimentin and keratin in the nude mouse model of human AM established by intraperitoneal implantation are in line with the characteristics of AM,which can be considered as a successful nude mouse model of human AM.2 There was a significant difference between the groups in the volume of the intraperitoneal transplanted tissue in nude mice in each group(P<0.05).The volume of the intraperitoneal transplanted tissue in nude mice in the blank model group was larger than that in the other groups(P<0.05).There was no significant difference between the mifepristone group and all the Modified Shaoyaogancao Decoction groups(P>0.05).3 There was a significant difference between the groups in the expression of p53 protein of the intraperitoneal transplanted tissue in nude mice which were detected by immunohistochemical method(P<0.01).The expression of P53 protein of the transplanted tissue in nude mice in the sham-operation group was lower than that in other groups(P<0.01).The expression of P53 protein of the transplanted tissue in nude mice in the blank model group was higher than that in other groups(P<0.05).4 There was a significant difference between the groups in the expression of P53-273H,c-Src,LIF,EGFR,p-EGFR,STAT3,p-STAT3,COX-2 of the intraperitoneal transplanted tissue in nude mice which were detected by Western Blot(P<0.01).The expression of P53-273H protein in the blank model group was higher than that in the other groups(P<0.05).The expression of EGFR in the sham-operation group was lower than that in other groups(P<0.05).The expression of c-Src in the blank model group was higher than that in the other groups(P<0.05).The expression of P-STAT3 in the sham-operation group was lower than that in other groups(P<0.05).There was no significant difference between the mifepristone group and all the Modified Shaoyaogancao Decoction groups in the expression of P53-273H,c-Src,STAT3,P-STAT3 protein(P>0.05).5 There was a significant difference between the groups in the relative expression level of p53-273HmRNA,miR-27a,EGFR mRNA and LIFR mRNA of the intraperitoneal transplanted tissue in nude mice which were detected by RT-qPCR(P<0.05).The relative expression level of miR-27a mRNA in mifepristone group was the highest,and the difference was statistically significant(P<0.05).The expression of EGFR mRNA in the high dose group of the Modified Shaoyaogancao Decoction and the middle dose group of the Modified Shaoyaogancao Decoction was lower than that in the blank model group(P<0.05).The relative expression level of P53-273H mRNA in the low dose group of the Modified Shaoyaogancao Decoction was lower than that in mifepristone group(P<0.05).The relative expression level of P53-273H mRNA in the blank model group was higher than that in other groups(P<0.05).The expression of LIFR in the blank model group was higher than that in the other groups(P<0.05).6 Analysis of the mechanism of the regulation of P53/EGFR/STAT3 signaling pathway by miR-27a,P53-273H gene was moderate negatively correlated with miR-27a,conform to the reference document[172],mutant P53-273H is able to bind directly to the DNA region upstream of the transcription start site of miR-27a to inhibit the expression of miR-27a.MiR-27a was slightly negatively correlated with other genes,while P53-273H was slightly correlated with other genes.CONCLUSION:1 To establish the nude mouse model of human AM by intraperitoneal implantation,the modeling method has good stability,high repeatability,and it can also simulate the natural growth process of an AM lesions and the changes of the peritoneal microenvironment.It is a way to improve the success rate of AM modeling,and it also a new method accord with human body mechanism,it can provide an ideal animal model for exploring the pathophysiology and treatment of AM.2 The Modified Shaoyaogancao Decoction can effectively reduce the volume of the lesions in the intraperitoneal of the nude mouse model of human AM,reduce the expression of P53,EGFR,P-EGRG,STAT3,P-STAT3,COX-2 protein in the lesions.It can inhibit the proliferation of AM cells and reduce the angiogenesis in the lesions.Therefore,the application of the Modified Shaoyaogancao Decoction in the treatment of AM can limit or delay the development and invasion of AM.3 All the Modified Shaoyaogancao Decoction groups can up regulation of miR-27a,and inhibit all genes and proteins in P53/EGFR/STAT3 pathway.As a compound in the multi targets,the Modified Shaoyaogancao Decoction effects the expression of multiple genes and proteins by its complex mechanism.This study provides a theoretical basis and scientific basis for further research and development of the Modified Shaoyaogancao Decoction. |
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