| Objectives: 1.To explore the relationship between moxibustion with medicine cake and endometriosis and miRNA through literature research on the name,etiology,pathogenesis and treatment of endometriosis.2.The differential expression of microRNA in mouse endometrial tissue was studied to find the relationship between differential expression genes and endometriosis.From the perspective of molecular biology,target genes that may be involved in regulation were searched to preliminarily understand the specific miRNA regulation mechanism involved in the regulation of endotopia.3.To study the biomolecular mechanism of differential expression of microRNA in isolated medicine cake moxibustion,observe the changes in the expression level of microRNA in endometriosis before and after treatment,discuss the relationship between the therapeutic mechanism of isolated medicine cake moxibustion and microRNA,and discuss the therapeutic target of isolated medicine cake moxibustion in EMs treatment from the level of microRNA.This paper discusses the mechanism of treating EMs with moxibustion with medicine cake,and provides objective experimental basis for treating EMs with moxibustion with medicine cake.Methods: the methods and principles of treatment of endometriosis were determined by literature discussion and database retrieval.Forty-twoSCID mice aged 6-8 weeks and weighing 15-20 g were selected and divided into five groups: blank group,model group,moxibustion group,western medicine group and moxibustion plus western medicine group.In addition to the blank group of 8 mice,the other 34 mice were prepared with EMs mouse model by autologous transplantation,and two EMs mice were randomly sacrificed seven days after modeling to see whether the modeling was successful.Building 14 days: every medicine cake moxibustion groups of mice after fixation in close yuan of acupuncture point for every cake moxibustion medicine,every hole moxibustion four strong,lasts about 10 minutes,1 time/day,for 14 consecutive days.Mice in the Danazol group were given Danazol suspension by gavage(36 mg /kg body mass daily)once a day for 14 consecutive days.Mice in the Danazol group were given Danazol suspension by gastric gavage first,and then moxibustion with Danazol suspension at guan yuan point,once a day,for 14 consecutive days.Both the normal group and the model group were given normal saline by intragastric administration of the same volume,once per day,for 14 consecutive days.Starting from the eighth day of treatment,each group was injected with oxytocin injection(36 mg /kg)one hour after treatment each day,and the number of writhing reactions of the mice was observed within 30 min for seven consecutive days.In the last 24 h after treatment,the mice were anesthetized with 10% chloral hydrate solution,endometrial tissue open find group(blank),ectopic endometrium tissue of growth after the rest of the four groups(building),with an eye under the camber of endometrial tissue,clean and filter paper in PBS buffer blot,quickly will be organized intoRNA preservation solution,in the refrigerator,inspection.Methods: 1.High-throughput sequencing method was used to analyze the differentially expressed microRNAs in each group,and a variety of bioinformatics software was used to predict the downstream target genes of differentially expressed miRNAs,and then GO,KEGG andother software were used to enrich and screen out the potential downstream regulatory pathways of differentially expressed microRNAs.2.Fluorescence quantitative PCR verification: based on miRNA sequencing prediction and screening results,real-time quantitative rt-pcr was used to detect the protein expression of vascular endothelial growth factor(VEGF)based on gene database.Western Blot: protein expressions of ERK1/2in five groups of endometrial tissues were detected.3.The differences and significance of miRNA expression were analyzed to search for miRNA that might be involved in the regulation of endometriosis by moxibustion with medicine cake.Results: 1.Endometriosis is a gynecological disease mainly characterized by "blood stasis".It is recommended to activate blood circulation to remove blood stasis,warm and clear the skin,and to treat endometriosis with moxibustion with medicine cake.2.Comparison of general conditions of mice: during the experiment,one mouse died in the model group,while the other group did not.The results of writhing body reaction showed that the moxibustion with medicine cake had analgesic effect on EMs mice.2.High throughput sequencing differential microRNA(1)compared with the model group,66 differentially expressed microRNA were screened(P<0.05),among which 31 were up-regulated and 35 were down-regulated,among which mir-21a-5p was the main down-regulated gene.Mir-223-3p,mir-142a-3p,mir-132-3p,mir-146b-5p;The upregulated genes mainly included mir-200b-5p,mir-200a-5p,mir-200b-3p,mir-429-3p,and mir-676-5p.(2)compared with the model group,4 differentially expressed microRNA were screened,with 0 upregulated and 4 down-regulated(P <0.05)as follows: mir-6538,mir-3470 a,mir-2137 and mir-5126,respectively.(3)thirteen differentially expressed micro rnas were screened from the western medicine group and the moxibustion group,of which 4 wereup-regulated and 9 were down-regulated(P <0.05)to down-regulate mmu-mir-499-5p,mir-133b-3p,mir-496a-3p,mir-206-3p,mir-21a-5p,mir-3473 b,mir-493-5p,mir-493-5p,mir-493-5p,mir-487b-3p.(4)compared with the model group,25 differentially expressed microRNA were screened,of which 7 were up-regulated and 18 were down-regulated(P <0.05).The up-regulated genes mainly included: mir-195a-3p,mir-615-3p,mir-708-3p,mir-296-3p,mir-671-3p,mir-298-5p,mir-181a-2-3p.Down-regulated genes mainly included: mir-21a-5p,mir-3473 b,mir-483-3p,mir-6240,and mir-3963.(5)one differentially expressed microRNA was screened between the western medicine plus cake moxibustion group and the cake moxibustion group,of which 0 were up-regulated and 1 was down-regulated.3.Fluorescence quantitative PCR technology and Western blot verification were used,which were consistent with miRNA high-throughput sequencing results.Down-regulated genes: miR-677-5p,miR-6240,miR-132-3p,miR-21a-5p,miR-21a-6p,miR-212-3p,miR-34a-5p,miR-486a-3p There are two genes up-regulated: miR-195a-3p and miR-298-5p,which are negatively correlated with the down-regulation results of VEGF PCR.Quantitative quantitative PCR confirmed that the protein expression of VEGF was consistent with the sequencing results.Western blot results showed that the protein expression level of ERK1/2 in normal endometrial tissue was higher than that in ectopic endometrial tissue(P<0.01),and the protein content of ERK1/2 was significantly lower than that in the control group(P<0.01)after the intervention of ERK1/2 in the cake moxibustion and Western medicine group(P<0.01),and the protein content of ERK1/2 in the Western medicine group was lower than that in the control group(P<0.05).conclusion:1.Meal-separated cake moxibustion has the effect of alleviating painsymptoms of EMs,and the analgesic effect of danazol is better.2.Drug-blocked moxibustion intervention may be involved in regulating differentially expressed microRNA.3.PCR verification is consistent with gene sequencing results,so it is inferred that these up-regulated and down-regulated genes may be involved in vascular endothelial factors regulating the growth and apoptosis of endometrial cells,and the expression of differential miRNAs may be an important mechanism for the occurrence of EMs. |
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