The Study Of Jiawei Shaoyao Gancao Decoction On Adenomyosis Via P53/EGFR/STST3 Pathway

ObjectiveTo explore the mechanism of Jiawei Shaoyao Gancao Decoction on adenomyosis via miR-27a mediated P53/EGFR/STST3 pathway towards a further understanding of how Jiawei Shaoyao Gancao Decoction works on adenomyosis from the molecular gene level.And in this way,we may screen the effective fraction to provide the experimental basis for optimizing the prescription.Methods:1.Select 10 cases from patients with adenomyosis who hospitalized in the First Affiliated Hospital of Guangzhou University of Chinese Medicinef for hysterectomy uterine from 2015 September to 2016 May and culture the cell in-vitro.2.Adenomyosis cells were identified by immunohistochemical staining.After 24h and 48h,cells proliferation was detected by MTT assay,and migration was investigated by wound-healing test.Western blot assay was used to determine the expression levels of p53-273H,LIF,EGFR,p-EGFR,c-Src,STAT3,p-STAT3 and COX-2.And the levels of p53-273HHmRNA,LIFR mRNA,miR-27a and EGFR mRNA were tested by RT-qPCR.Result:1.In this study,10 cases of adenomyosis were collected.After primary cell culturing for 24h,the interstitial cellswere outspread,straight andfusiform.The glandular epithelial cells were swirling,and smooth muscle cells,which were fusiform or oval,grew radially and parallelly.2.All cases of primary cultured cells were adenomyosis cells which were identified by immunocytochemical method.3.After 24h and 48h culturing,the situation of proliferation inhibition rate(IR%)was:high,medium and low concentration of Jiawei Shaoyao Gancao decoction groupsshowed a higher proliferationinhibitionratecompared with the positive group and the control group with significant difference(P<0.01).4.The high,medium and low concentration of Jiawei Shaoyao Gancao decoction groups’ cell apoptosis rate werehigher than the control groupand the rate of high concentration of Shaoyao Gancao decoction group were significantly differentin related to the positive group(P<0.05)and the control group(P<0.01).At the same time,the cells in the G1/S phase of the high,medium and low concentration ofjiawei Shaoyao Gancao decoction groups were significantly higher than those in the control group(P<0.01).5.By culturing 24h and 48h,Thecell migration were inhibited in the group of high,medium and low concentration of Jiawei Shaoyao Gancao decoction.Using Image J software to measure the distance of cellsmigration,the wound-healing rate showed:The high,medium and low concentration of Jiawei Shaoyao Gancao decoction groups can effectively inhibit adenomyosis cells from migrating.Andthere was a statistical difference(P<0.05)compared to the control group and positive group.But there was no statistical difference between high,and medium concentration of Jiawei Shaoyao Gancao decoction(P>0.05).6.The high,medium and low concentration of Jiawei Shaoyao Gancao decoction groups had a higherexpression ofmiR-27a gene,while a lower expression ofp53-273HmRNA,LIFR mRNA and EGFR mRNA,with respect to the control group(P<0.05,the difference was statistically significant).The correlation within miR-27a,p53-273H mRNA,LIFR mRNA and EGFR mRNA was examined by Spearman method.Then it showed that miR-27a was a significant negative correlation with p53-273H mRNA,LIFR mRNA and EGFR mRNA,and there was a positive correlation within p53-273H mRNA,LIFR mRNA and EGFR mRNA.7.The expression of p53-273H,LIF,c-Src,EGFR,p-EGFR,STAT3,p-STAT3 and COX-2 in the high,medium and low concentration of Jiawei Shaoyao Gancao decoction groups were significantly decreased compared with control group.Further more,the high concentration of Jiawei Shaoyao Gancao decoction group was a statistical different with the control group(P<0.05)after 24h and 48h,while the difference was statistically significant with the positive group after 48h(P<0.05).Conclusion:By upregulatingmiR-27a,Jiawei Shaoyao Ganchao Decoction inhibited the expressions of EGFR mRNA and p53-273H mRNA so that it decreased the expressions of p53-273H,LIF,c-Src,EGFR,p-EGFR,STAT3,p-STAT3 and COX-2 protein in P53/EGFR/STAT3 signal pathway.It may be the the mechanism of how Jiawei Shaoyao Gancao Decoction inhibiteadenomyosis cell proliferation and promote apoptosis and reduce cell migration rate.