| Objective: To explore the mechanism underlying the effects of Juantong Decoction(a traditional Chinese medicine)on the occurrence and development of endometriosis through intervening the PI3K/Akt/mTOR signaling pathway by comparing the intervening effects of Juantong Decoction and LY294002,the PI3 K pathway inhibitor,on model rats with endometriosis so as to provide new ideas regarding prevention and treatment of endometriosis with traditional Chinese medicine(TCM),and provide a point of penetration in respect of elucidation of TCM mechanism using model scientific theory.Methods: A rat model of endometriosis was established using SD rats and autotransplantation of endometrial tissue,and the effects of different doses of Juantong Decoction on the rat model was observed.1 Rat model establishmentFemale rats with a oestrous cycle were chosen and randomly divided into 6 groups by weight with 8 ones in each group,including the normal sham-operation group,the model control group,the high-,middle-and low-dose Juantong Decoction groups,and the western medicine(LY294002)group.Rats in all groups were subject to establishment of the endometriosis model except those in the normal group who underwent abdominal incision,the sham operation.Abdominal incision and observation was done 4 weeks after the modeling and the ectopic tissue was taken for pathological examination to verify the success of modeling.2 Administration by groupsThe normal group: 1ml/100 g normal saline was given to rats via intragastric administration 4 weeks after operation,once per day for 4 weeks;The model group: Normal saline 1ml/100 g was given to rats via intragastric administration 4 weeks after operation,once per day for 4 weeks;Juantong Decoction 42.9g/kg,14.3g/kg and 4.8g/kg as per 4 times,2 times and 1 time the equivalent of the clinical adult dosage were given to the high-,middle-and low-dose Juantong Decoction groups respectively 4 weeks after operation for 4 weeks;The western medicine: Intraperitoneal injection of LY294002 dissolved in DMSO(0.04g/kg)was done 5 weeks after modeling,once per week for 4 weeks.3 Indexes measuredRat fasted for 12 hours 4 weeks after intragastric administration by groups,and were sacrificed after etherization and blood collection from abdominal aorta.The morphology and size of rat ectopic foci were observed and measured,and in-situ and ectopic endometria were dissected and placed into liquid nitrogen and 4% paraformaldehyde and electron microscopy fixative,respectively.Modern molecular biology technologies such as transmission electron microscope,ELISA,immunohistochemistry and real-time PCR were used to measure the levels of IL-1?,TNF-?,VEGF and MMP-2 in the rat serum as well as the expression of proteins PI3 K,Akt and mTOR and the relative amount of p70s6 KmRNA in in-situ and ectopic endometria.Results:1 General conditions of rats in all groupsIntramuscular injection of penicillin was administered to rats in the first three days after modeling to prevent infection;rats had fewer activities,most with messy and lusterless hair;all had moist wounds which were treated by topical iodophor sterilization.Progynova water solution was administered to rats via intragastric administration 4-9 days after modeling,and some rats showed abdominal distension,decreased food intake and watery excrement(in most cases).Rats were fed normally 10 days to 4 weeks after modeling;they had significant weight gain,a few had abdominal wound suture dehiscence,and sterilizaiton or re-suturing was done as necessary.Rats in the normal sham-operation group had bright,clean and tidy hair,and sparkling eyes;they showed no obvious adverse reactions during feeding.Rats in other model groups dispalyed dirty and disorder hair,some with slower action,glassy eyes and intermittent decrease in food intake and looking tired.Among them,one rat in the high-dose Juantong Decoction died because the trachea was choked with the decoction during intragastric administration.2 Observation of ectopic endometria of all groups under light microscope and transmission electron microscopeAppendix 2.5 shows the pathological images of ectopic endometria under light microscope 4 weeks after administration for all groups.For the normal group,the endometrial glandular cells were arranged in good order,glands and mesenchymal cells grew well;for the model group and low-dose Juantong Decoction group,there were clearly more glands,most epithelia had vacuoles under the nucleus,mesenchymal cells were dense,and obvious neovascularization existed;for the high-dose Juantong Decoction group and the western medicine group,glands and mesenchymal cells in the ectopic endometrium decreased and fibrillation was observed.Appendix 2.1 shows the transmission electron microscope images of ectopic endometria for rats in all groups.The normal group had normal organelles;for the model group,glandular epithelial cells were arranged in good order,with microvilli,normal cell plasma morphology;for the high-dose Juantong Decoction group and the western medicine group,glandular epithelial cells were atrophied or not in good order,microvilli were deceased or absent,cell nuclei were pyknotic,mitochondria were decreased,and some mesenchymal cells were apoptotic;for the low-dose Juantong Decoction group,glandular epithelial cells were in irregular arrangement,microvilli were reduced,and swelling mitochondria were seen.3 Comparison of TNF-a,MMP-2,VEGF,IL-1? levles in rat serum between all groupsResults show that serous NF-a,MMP-2,VEGF and IL-1? levels rose significantly in the model group and the low-dose Juantong Decoction group as compared to the normal group(P < 0.05),whereas there were no significant differences in those levels between the normal group and the middle-and high-dose Juantong Decoction and the western medicine groups(P>0.05);the post-intervention serous TNF-a,MMP-2,VEGF and IL-1? levels were significantly different between the model group and the middleand high-dose and the western medicine groups(P<0.05)but not notably different between the model group and the low-dose group(P > 0.05);effects in the middle-and high-dose and the western medicine groups were better than in the low-dose group(P<0.05),but showed no significant differences between the middle-and high-dose and the western medicine groups(P>0.05).4 Expression of proteins PI3 K,Akt and mTOR in rat endometrial tissue for all groupsThe content of proteins PI3 K,Akt and mTORlevels increased significantly in the model group and the low-dose group as compared to the normal group(P<0.05),whereas there were no significant differences between the normal group and the middle-and high-dose and the western medicine groups(P>0.05);the post-intervention expression levels were significantly different between the model group and the middle-and high-dose and the western medicine groups(P<0.05)but not notably different between the model group and the low-dose group(P>0.05);effects in the middle-and high-dose and the western medicine groups were better than in the low-dose group(P<0.05),but showed no significant differences between the middle-and high-dose and the western medicine groups(P>0.05).5 Result analysis of p70S6 k mRNA in in rat endometrial tissue for all groupsThe real-Time PC technique was used to measure the expression level of p70S6 k mRNA in the endometrial tissue of the normal and the model groups.The expression level of p70S6 k mRNA was measured by comparing the p70S6 k mRNA and the ?-Actin mRNA in the specimens.Results show that,compared to rats in the normal group,the relative expression amount of p70S6 k mRNA rose significantly in all model rats,especially in the model and the low-dose groups(P<0.05),whereas there were no significant differences between the normal group and the middle-and high-dose and the western medicine groups(P>0.05);the post-intervention expression levels were significantly different between the model group and the middle-and high-dose and the western medicine groups(P < 0.05)but not notably different between the model group and the low-dose group(P > 0.05);effects in the middle-and high-dose and the western medicine groups were better than in the low-dose group(P<0.05),but showed no significant differences between the middle-and high-dose and the western medicine groups(P>0.05).Conclusion:1 Juantong Decoction can promote circulation and remove stasis to alleviate pain,warm kidney and invigorate spleen to remove dampness,and it has definite curative effects for endometriosis;2 Juantong Decoction can reduce the content of serous TNF-a,MMP-2,VEGF and IL-1? in model rats;3 Juantong Decoction can reduce the expression level of proteins PI3 K,AKT and mTOR and the p70s6K-mRNA in the ectopic endometrial tissue of model rats;4 Juantong Decoction can produce therapeutical effects on model rats with endometriosis through inhibiting the activation of the PI3K/AKT/mTOR signaling pathway. |
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