| Purpose: 1.Through literature search,to sort out the ancient and modern doctors and medical research on endometriosis,to explore the principle and law of moxibustion in the treatment of endometriosis.2.Induce the establishment of an endometriosis mouse model through an improved autotransplantation method.On the basis of verifying that the drug-separated cake moxibustion has a good analgesic effect on endometriosis dysmenorrhea and inhibits the growth of ectopic endometrium,explore the drug-separated cake The effect of moxibustion on the expression of miRNA in endometriotic mice was verified by PCR to detect its effect on the expression of PI3K/Akt signaling pathway.3.Comprehensive literature and experimental research,through the study of the relationship between meridians,acupoints,fascia,miRNA,and signal pathways,explore the meridian mechanism of meridian cake moxibustion against cell adhesion,cell growth and proliferation,and anti-angiogenesis.The specific mechanism of drug-separated moxibustion at Guanyuan acupoint in the treatment of endometriosis.Methods: 44 SCID mice,five in a cage,cut one uterus into fragments of 5 × 5mm,fixed the inner side of the uterus on the abdominal wall of the other side,fasted for one day after operation and continuous intramuscular injection of penicillin for three days to prevent infection.Seven days later,two mice were randomly selected.Laparotomy showed that the ectopic endometrium in the lesion was enlarged,a translucent vesicle with a diameter of 5～8mm,with fluid accumulation and neovascularization.It shows that the modeling is successful.The successful mice were randomly divided into model group(group A),western medicine group(group B)and herbal cake-separated moxibustion group(group C),with 12 mice in each group and 4 mice in each cage.Each group was given medicine once a day for two weeks from the 14 th day after the end of the model.The model group was perfused with the same volume of normal saline with No.8 gastric perfusion needle.In the herbal cake-separated moxibustion group,Chinese herbs such as aconite,safflower and angelica were powdered in proportion and mixed with yellow rice wine.The medicinal cake with the specification of 10 mm × 10 mm × 4.5mm was made by self-made mold,and the moxa made by the mold was used to moxibustion at Guanyuan point,each mouse moxibustion was 4 strong.The western medicine group was perfused with Danazol solution according to the body mass of 36mg/ kg.The analgesic effect of medicine-separated cake moxibustion was recorded during the treatment.after the treatment,the size of ectopic endometrial tissue in each group was measured,and the ectopic endometrial tissue in each group was extracted and preserved and detected.High-throughput sequencing method was used to screen the ectopic endometrium differentially expressed miRNAs,in each group of EMs experiment to predict the downstream target genes of miRNA.The target genes were matched with miRNAs by MAGIA2 software and Pearson correlation coefficient,and the key miRNAs and target genes identified above were verified by qRT-PCR method.The differential expression of genes in the regulatory network was analyzed,and the function of the selected miRNA was analyzed by DAVID software,and the significantly enriched GO entries and KEGG path map were obtained by hypergeometric test.Westernblotting technology was used to detect the expression of PI3K-Akt-mTOR signal pathway and related factors.Results: 1.The total number of writhing reactions in the drug-separated cake moxibustion group showed a downward trend.The number of mice with writhing reactions,the number of writhing and the incidence of writhing were all decreasing,and the data were statistically significant,indicating that the drug-separated Cake moxibustion has obvious analgesic effects on dysmenorrhea in mice.2.The average growth inhibition rate of the drug-separated cake moxibustion group was 86.28%,which was better than the 78.65% of the western medicine group.The drug-separated cake moxibustion had a more obvious effect on the growth inhibition of the ectopic endometrium.3.Differentially expressed miRNA Compared with the model group,the medicine-separated cake moxibustion group screened out 4 down-regulated miRNAs: miR-6538,miR-3470 a,miR-2137,miR-5126.Compared with the western medicine group,the medicine cake moxibustion group screened out 9 down-regulated miRNAs: miR-499-5p,miR-133b-3p,miR-496a-3p,miR-206-3p,miR-21a-5p,miR-3473 b,miR-493-5p,miR-133a-3p,miR-487b-3p.Four up-regulated miRNAs were screened: miR-183-5p,miR-205-5p,miR-181a-2-3p,miR-200c-3p.Compared with the model group,the western medicine group screened out 10down-regulated miRNAs: miR-21a-5p,miR-3473 b,miR-483-3p,miR-6240,miR-3963,miR-3970,miR-15a-5p,let-7i-5p,miR-2137,miR-5126.Seven up-regulated miRNAs were screened: miR-195a-3p,miR-615-3p,miR-708-3p,miR-296-3p,miR-671-3p,miR-298-5p,miR-181a-2-3p.4.PCR verification of miRNA Through miRNAs jointly regulated by each group,miR-2137,miR-5126,miR-3963,miR-6240,miR-1981-5p,miR-708-3p,miR-3473 b,miR-206-3p,miR-The ten miRNAs 21a-5p and miR-181a-2-3p,after real-time fluorescence quantitative detection,screened out 4 miR-708-3P,miR-5126-3P,miR-1981-5P,miR-206-3p miRNA,it was finally determined that the two miRNAs,miR-708-3P and miR-5126-3P,met the conditions.5.PCR to verify mRNA The mRNAs regulated by miR-708-3P and miR-5126-3P selected by PCR are Igf1 r,Mapk1,VEGF,Ppp2r5 c,Magi2,Wnt7 a,Nprl3,Rps6ka2,Igf1,and Sos1 for real-time fluorescence quantitative detection,combining miRNA and mRNA The negative regulatory relationship of Vegf,Ppp2r5 c,Wnt7a,Rps6ka2 these four target genes were screened out.6.Western Blot detects PI3K-Akt-mTOR protein WB detected the expression levels of the corresponding PI3 K,Akt,and mTOR proteins in each group.Compared with the model group,the expression levels of the three proteins in the medicated cake moxibustion group all decreased and were statistically significant,while the western medicine group had three protein expression levels.The expression level also decreased,but the decrease of PI3 K protein did not reach statistical significance,and the decrease of Akt and mTOR was statistically significant.Conclusion: 1.The medicine-separated cake moxibustion has obvious analgesic effect on endometriosis mice and the inhibitory effect of ectopic endometrial tissue.2.Medicinal cake moxibustion can regulate the miRNA and corresponding target genes of endometriosis mice,and then regulate the PI3K/Akt/mTOR signaling pathway,which has a therapeutic effect on ectopic endometrial tissue.3.Medicinal cake moxibustion at Guanyuan point can regulate miRNA,target genes and PI3K/Akt/mTOR signaling pathways on the fascia,and play the role of anti-cell adhesion,anti-cell growth and proliferation,and anti-angiogenesis,and then adjust Chong and Ren two meridians,cure endometriosis. |
PDF Full Text Request