The Mechanism Of Huoxue Xiaoyi Granule In The Treatment Of Endometriosis Based On The Changes Of MicroRNA Expression Profiles

Bankground:HuoXueXiaoYi granule with the effect of promoting blood circulation to eliminate the stasis is prescribed for the patients with endometriosis of qi stagnation and blood stasis.The scientific development of Chinese traditional medicine embodies the inheriting and innovation.In this study,we try to find out the mechanism of TCM formula with activating blood circulation to dissipate blood stasis,deepening the understanding of mechanism of Huoxue xiaoyi granule treatment of endometriosis based on these researches.We hope more patients with endometriosis benefit from good prevention and treatment measures,though with the existing western medicine and surgical treatment now.Objectives:1.Endometriosis belongs to the category of ancient disease“Zhengjia",this paper combines the ancient literature analysis and data mining technology to analysis the characters of ancient gynecological literature in the treatment of ZhengJia,to sum up the theoretical basis of TCM treatment of EMs and the laws of TCM prscriptions,to have the systematic knowledge of TCM theory.2.To observe the clinical efficacy of Huoxue Xiaoyi granule in treatment of patients with endometriosis,as well as the changes of serum microRNA gene expression profile,before and after TCM treatment,to explore the possible biological mechanism on the genetic level.3.To observe the changes of ectopic lesion volume,histological changes,serum and peritoneal fluid-related inflammatory factors and related protein and gene expression in rats with endometriosis,by establishing experimental rats model of endometriosis.To investigate the possible molecular mechanism of Huoxue Xiaoyi granule treatment of endometriosis.Methods:1.Retrieval the ancient gynecological recipe information in treatment of ZhengJia from "Chinese Medical Code",entering the traditional Chinese medicine transmission support system.Analysis the TCM formula information with application of data mining methods,including improve mutual information,complex system entropy clustering,unsupervised entropy hierarchy clustering.2.In line with the inclusion criteria,do not meet the exclusion criteria of endometriosis patients was recruited,with Huoxue Xiaoyi granule treatment for 3～6 months,before and after treatment,the clinical symptoms and signs will be evaluated.After the treatment,5 patients with good efficacy were randomly selected;venous blood was collected before and after treatment.Mononuclear lymphocytes were isolated and total RNA was extracted.Real-timeq PCR array microarray was used to perform miRNA detection,and the differentially expressed genes were analyzed by bioinformatics.3.60-day-mature SD female rats were randomly divided into seven groups:Chinese medicine high,middle and low dose group,Diphereline group,model group,Sham operation group,blank control group,each group of 8 rats.Administration of treatment for 3 weeks,to observe the changes in the relevant indicators between the groups.Result:Literature research:1.In this study,a total of 108 recipes were collected from the treatment of gynecological gynecological prescriptions,involving 188 herbs.According the frequency of herbs in more than 8 times,the top 15 are Angelica,cassia twig,radix paeoniae rubra,radix rehmanniae,rhubarb,rhizome,Rhizoma Chuanxiong,Rhizoma sparganii,peach kernel,licorice,ginger,Rhizoma Corydalis,radix,Radix Aucklandiae,cortex moutan.The frequency of thoese herbs is much higher,mainly with the function of nourishing blood,invigorate the circulation of blood,warm yang,regulate the flow of vital energy and remove obstruction.2.The frequency of the top 10 common combination of herbs are "Angelica,Guixin" "red peony,angelica" "Chuanxiong,angelica" "Corydalis,Angelica""Triangular,Curcuma" Curcuma,Angelica" "Rhizoma Chuanxiong,Guixin","Rhizoma Curcuma,Qingpi".Angelica as the core is the most common used:combined with Guixin with the function of warm yang,combined with red peony,Radix Rehmanniate with the method of nourishing blood,combined with triangular,Curcuma with the law embodies the stasis-breaking,and combination of Qingpi,Corydalis,Chuanxiong with the law of regulating vital energy qi,and peach kernel,Achyranthes bidentata reflects the method of removing stasis.3.Network display map found that the commonly used core herbs to treat Zhengjia,mainly with fuction of activating blood circulation,combined with enriching blood,nourishing qi,warming the channels,regulating vital energy qi,clearing stasis.Clinical observation:1.102 cases of patients successfully completing the 3～6 month course of treatment,its clinical total effective rate were 85.30%.2.After 3～6 month course of treatment,the ovarian cysts were smaller than those before treatment,but the first 3-month course of treatment,Cysts tend to reduce faster.(p<0.05).3.With the prolonging of treatment time,the degree of dysmenorrhea was relieved,the time of dysmenorrhea was shorter,the difference was statistically significant(p<0.05);during 3～6 months of treatment,patients with TCM symptoms were alleviated,and compared with untreated,the difference was statistically significant(p<0.05).4.31 patients with menorrhagia in the recruited 102 patients,there were significant differences at different time points to measure menstruation(F=19.648,p<0.01);after treatment with 6 menstrual cycles,more menstrual flow was significantly reduced;menstrual volume decreased to normal level after 4 menstrual cycles.11 patients with a small amount of menstrual flow in the recruited 102 patients,menstrual condition improved after treatment with TCM,but there was no significant difference(F=1.084,p=0.382).5.After 6 months of treatment,CA125 was lower than before(F=7.101,p=0.024).The overall trend of CA199 was lower(F=10.008,p=0.008)than before,the difference was statistically significant(p<0.05).6.The serum TNF-α abnormalities in 2 cases(2.5%),after 6 months treatment,the TNF-a was abnormal in 1 cases,the treatment difference was not statistically significant(p>0.05).The patients in the study,after the treatment of 3～6 menstrual cycle,the indicators of kidney function were normal,there was no statistically significant difference(p>0.05),electrocardiogram,blood and urine routine showed no obvious abnormalities.7.A total of 5 differentially expressed qRT-PCR were identified by microRNA.The up-regulated gene expression was miR-202-3p,miR-9-5p,miR-155-5p,and the down regulated gene expression was miR-216a-5p,miR-518a-5p.To focus on prolactin signaling pathway,Ras signaling pathway,Rap1 signaling pathway,Hippo signaling pathway,estrogen signaling pathway,neurotrophic factor signaling pathway,ErbB signaling pathway,thyroid hormone signaling,phosphatidylinositol signaling,MAPK signaling pathway,TGF-beta signaling pathway,FoxO signaling pathway,cancer and other signals pathway.The enrichment pathway was mainly related to cell adhesion,migration and apoptosis partly.Experimental Study:1.The cyst of endometriosis model rats in Chinese medicine high dose and middle dose group and Diphereline group volume significantly reduced or disappeared,compared with before administration,the difference was statistically significant(p<0.05);After the HE staining,tissue classification method by semi quantitativeto determine the severity of endometriosis:endometriosis score of Diphereline group and high dose of Chinese medicine group was significantly lower than the model group,the difference was statistically significant(p<0.05).The low dose group and middle dose group had no obvious advantage in reducing the score of endometriosis(p>0.05).2.The Real-time PCR on gene detection:the normal group compared with model group,NFKB,PIK3R3,RAS gene expression of model groupwas significantly increased,there Were significant differences(2-ΔΔCt>1);after treatVent,high dose of CM group compared with the model group,the gene expression of NFKB PIK3R3,RASwas significantly down,there was a significant difference(2-ΔΔCt>1);mid-dose of CM groupcompared with model group,NFKB,PIK3R3 gene expression was significantly down,there was a significant difference(2-ΔΔCt>1);RAS gene expression falling,but no significant differences(2-ΔΔCt<1);after treatment,low-dose of CM group compared with the model group,NFKB,PIK3R3,RAS gene expression was significantly down,but no significant differences(2-ΔΔCt<1).3.By using ELISA double antibody sandwich detection technology,related cytokines concentration were detected:the concentration of IL-2,IL-6,IL-1B in peritoneal fluid and serum of model group is significantly higher than that in normal group,and the sham operation group and treatment group(p<0.05).In Huoxuexiaoyi high dose group and Diphereline group,the concentration of IL-2,IL-6,IL-1β decreased significantly(p<0.05)Conclusion:Huoxue xiaoyi formula is the effective prescription in the treatment of endometriosis,which based on the understanding of the ancient literature,following the law of prescription,with inheritance and development.The clinical observation shows that the treatment of endometriosis with the method of promoting blood circulation and removing blood stasis has good efficacy,good safety and no obvious adverse reaction.The experimental study shows that huoxue xiaoyi granule inhibited the growth of endometriosis lesions.The possible molecular mechanism is up-regulated expression of miR-9-5p,lowered its target genes NFKB,PIK3R3,through the "Ras-PIK3R3/Akt" signaling pathways inhibits ectopic cell growth,proliferation and metastasis.There also exists dose dependent effect.The efficacy of CM high dose group was significantly better than the low dose group.The herbs for promoting blood circulation also play an important role by reducing the levels of cytokines IL-2,IL-6,IL-1β,improving the immune environment and regulating the inflammatory response.There is actually a multi-target therapeutic role of Huoxue xiaoyi formula.