Effect Of Modified Shaoyao-Gancao Decoction On Adenomyosis By Regulating P53 Function And Improving Sensitivity To Ferroptosis

Objective1.To study the resistance of both eutopic and ectopic endometrium of AM to ferroptosis,eutopic and ectopic endometrium tissues of AM patients were collected.2.To observe the sensitizing effect of Modified Shaoyao-Gancao Decoction on ferroptosis,the human dirived AM nude mice model was established by the method of tumorbearing.3.To verify whether the sensitizing effect of Modified Shaoyao-Gancao Decoction on ferroptosis were based on P53/SLC7A11/GPX4 mechanism,primary endometrial stromal cells were extracted and cultured from ectopic endometrial tissues of AM patients.Methods1.Clinical researchPatients who underwent hysterectomy due to AM and SIL in the First and Third Affiliated Hospital of Guangzhou University of Chinese Medicine from August 2020 to August 2021 were selected as the case group and the control group respectively.Baseline data,endometrium and myometrium were collected from both groups.The mRNA and protein expressions of ferroptosis-related factors in endometrium and myometrium were determined by RT-qPCR and WB.IHC was used to observe the locations of ferroptosisrelated proteins in AM ectopic endometrium.The contents of glutathione(GSH),malondialdehyde(MDA)and Fe in endometrium and myometrium were detected by colorimetry.2.Nude mice tumor-bearing experimentForty-five female SPF BALB/C-nu mice were purchased,and 10 mice were randomly selected as the operation control group with myometrium tissues from SIL patients transplanted into their abdominal.The remaining 35 mice were transplanted with AM ectopic endometrium in order to construct the human derived AM nude mice model.At the end of the modeling,5 AM model mice were randomly selected for identification,and the remaining 30 AM nude mice were randomly divided into AM model group,Modified Shaoyao-Gancao Decoction group,Modified Shaoyao-Gancao Decoction+Fer-1 group,with 10 mice in each group.The operation control group and AM model group were given normal saline intragastric administration daily.The Modified Shaoyao-Gancao Decoction group was given Modified Shaoyao-Gancao Decoction intragastrically every day.Modified Shaoyao-Gancao Decoction+Fer-1 group was intraperitoneally injected with ferroptosis inhibitor Fer-1 every other day on the basis of the former group.After treatment,the general situation,changes of both body weight and tumor size of nude mice before and after intervention were recorded.RT-qPCR and WB were used to detect the mRNA and protein expressions of ferroptosisrelated factors in tumor.The contents of GSH,MDA and Fe in tumor were detected by colorimetric method.3.AM primary endometrial stromal cell experimentPrimary endometrial stromal cells were extracted and cultured from ectopic endometrum of AM patients,which were identified by cellular immunochemistry.The cells were divided into vehicle group,Modified Shaoyao-Gancao Decoction group,Modified Shaoyao-Gancao Decoction+ferroptosis inhibitor Fer-1 group and Modified ShaoyaoGancao Decoction Decoction+P53 inhibitor PFT-α group.The 24h migration rate,fluorescence value after loading DHE and relative expression levels of MutP53 and P53/SLC7A11/GPX4 proteins in each group were observed after corresponding treatments.In another experiment,WB method was conducted to determine the relative protein expression levels of(MutP53)P53/SLC7A11/GPX4 in primary cells after treated with Modified Shaoyao-Gancao Decoction 0h,6h,12h,18h and 24h respectively.The Modified Shaoyao-Gancao Decoction meaned serum of rat after taking Modified Shaoyao-Gancao Decoction in this part of the study.Results1.The resistance to ferroptosis in eutopic and ectopic endometrium of AM patients(1)The baselines of AM group(n=13)and SIL group(n=11)were basically the sameThere were no significant differences in age,BMI,pregnancy,childbirth and abortion times between 2 groups(P>0.05).(2)Abnormal expression of mRNA and proteins of ferroptosis signaling axis P53/SLC7A11/GPX4 and related factors in AM eutopic and ectopic endometriumCompared with SIL endometrium,the relative expressions of P53,FTL,TFRC and DMT1 mRNA and protein in AM eutopic endometrium were increased,and the relative expression of GPX4 mRNA in AM eutopic endometrium was increased,all with statistical significance(P<0.05).The same results were seen in AM ectopic endometrium when compared with SIL myometrium.(3)Protein localizations of ferroptosis signaling axis P53/SLC7A11/GPX4 and related factors in AM ectopic endometriumP53 and GPX4 were widely expressed in both epithelial and stromal cells of AM ectopic endometrium.FTL was selectively expressed in ectopic endometrial stromal cells,while ectopic endometrial epithelial cells did not express FTL.The expression of TFRC and DMT1 proteins were localized in the ectopic endometrial epithelial cells,and no obvious staining was observed in the stromal cells.(4)Overloaded iron,increased lipid peroxidation levels,and enhanced antioxidant capacity were seen both in eutopic and ectopic endometrium of AMCompared with SIL endometrium,the content of Fe,MDA and GSH in AM eutopic endometrium were increased with statistically significant differences(P<0.05).The same results were seen in AM ectopic endometrium when compared with SIL myometrium.2.Modified Shaoyao-Gancao Decoction shrank tumor in AM nude mice by inducing ferroptosis(1)Identification of AM nude mice modelAt the end of the 4th week of modeling,5 AM model mice were randomly selected for HE staining,and typical AM island ectopic lesions were seen in the tumor under microscope,indicating that the modeling was successful.(2)Ferroptosis was involved in the effect of Modified Shaoyao-Gancao Decoction on AM tumorCompared with AM model group,the size of the tumor in the Modified Shaoyao-Gancao Decoction group was reduced,and the difference was statistically significant(P<0.05).Modified Shaoyao-Gancao Decoction reduced the tumor volume of AM mice.Compared with the Modified Shaoyao-Gancao Decoction group,the tumor size in the Modified Shaoyao-Gancao Decoction group+Fer-1 group increased,and the difference was statistically significant(P<0.05),indicating that the effect of Modified Shaoyao-Gancao Decoction on reducing the tumor size in AM mice was related to ferroptosis(which could be rescued by Fer-1).(3)Modified Shaoyao-Gancao Decoction enhanced sensitivity to ferroptosis in tumors of AM mice by regulating P53/SLC7A11/GPX4 axis and related factorsCompared with operation control group,the relative expression level of P53 mRNA and protein decreased in AM model group,and the difference was statistically significant(P<0.01).The relative expression levels of SLC7A11,GPX4,FTL,TFRC and DMT1 mRNA and proteins increased,with statistical significance(P<0.01).These results of AM nude mice was consistent with the trend of clinical results of AM patients.Compared with AM model group,the relative expression of P53 mRNA in Modified Shaoyao-Gancao Decoction group increased,and the difference was statistically significant(P<0.01).The relative expression levels of SLC7A11,GPX4,FTL,TFRC and DMT1 mRNA and proteins decreased,and the difference was statistically significant(P<0.01).These results suggested that Modified Shaoyao-Gancao Decoction positively regulated P53 and negatively regulated SLC7A11,GPX4,FTL,TFRC and DMT1.Compared with Modified Shaoyao-Gancao Decoction group,the relative expression levels of GPX4 mRNA and protein increased,as well as SLC7A11,FTL,TFRC and DMT 1 proteins in the Modified Shaoyao-Gancao Decoction+Fer-1 group,while the relative expression level of P53 protein decreased,all with statistical significance(P<0.01).These results suggested that ferrroptosis inhibitor Fer-1 reversed the effect of Modified ShaoyaoGancao Decoction on P53/SLC7A11/GPX4 and related factors.(4)Modified Shaoyao-Gancao Decoction improved sensitivity to ferroptosis in AM tumor by increasing lipid peroxidation level and decreasing antioxidant capacityThe content of MDA in the tumor of Modified Shaoyao-Gancao Decoction group was the highest and significantly higher than other three groups(P<0.01).The content of GSH in the tumor of Modified Shaoyao-Gancao Decoction group was lower than AM model group(P<0.01).The content of Fe in the tumor of AM model group,Modified ShaoyaoGancao Decoction group and Modified Shaoyao-Gancao Decoction+Fer-1 group were significantly higher than operation control group(P<0.01).3.Modified Shaoyao-Gancao Decoction serum regulated the function of P53/SLC7A11/GPX4 signalling axis and enhanced the sensitivity to ferroptosis in primary endometrial stromal cells.(1)Identification of endometrial stromal cellsThe primary cells extracted in this study were high-purity endometrial stromal cells identified by immunocytochemistry.(2)Modified Shaoyao-Gancao Decoction serum reduced the 24h migration rate of primary endometrial stromal cells by regulating P53Compared with vehicle group,the 24h migration rate of Modified Shaoyao-Gancao Decoction group was decreased,and the difference was statistically significant(P<0.01).Compared with Modified Shaoyao-Gancao Decoction group,the 24h migration rate of Modified Shaoyao-Gancao Decoction+Fer-1 group and the Modified Shaoyao-Gancao Decoction+PFT-α(P53 inhibitor)group increased,and the differences were statistically significant(P<0.01).The Modified Shaoyao-Gancao Decoction reduced the 24h migration rate of cells,which is related to the mechanism of ferroptosis and the function of P53.(3)Modified Shaoyao-Gancao Decoction serum increased the level of oxidative stress in primary endometrial stromal cells by regulating P53Compared with vehicle group,the DHE fluorescence intensity of cells in Modified Shaoyao-Gancao Decoction group was increased,and the difference was statistically significant(P<0.01).Compared with Modified Shaoyao-Gancao Decoction group,DHE fluorescence intensity of cells in both Modified Shaoyao-Gancao Decoction+Fer-1 group and Modified Shaoyao-Gancao Decoction+PFT-α group were decreased,and the difference was statistically significant(P<0.01).Modified Shaoyao-Gancao Decoction increased the level of oxidative stress in cells,which is related to the mechanism of ferroptosis and the function of P53.(4)Modified Shaoyao-Gancao Decoction serum enhances sensitivity to ferroptosis in primary endometrial stromal cells based on P53/SLC7A11/GPX4 signal axis①Modified Shaoyao-Gancao Decoction serum enhanced sensitivity to ferroptosis in primary endometrial stromal cells by regulating P53/SLC7A11/GPX4 axisCompared with vehicle group,the relative expression levels of MutP53,SLC7A11 and GPX4 proteins in Modified Shaoyao-Gancao Decoction group were decreased,and the relative expression levels of P53 protein were increased,both with statistical significance(P<0.01).Compared with Modified Shaoyao-Gancao Decoction group,the relative expression levels of MutP53,SLC7A11 and GPX4 proteins increased while P53 protein decreased in Modified Shaoyao-Gancao Decoction+Fer-1 group,with statistical significance(P<0.05).Same change was seen in Modified Shaoyao-Gancao Decoction+PFT-α group(P<0.01).②The sensitization effect of Modified Shaoyao-Gancao Decoction serum on primary endometrial stromal cells to ferroptosis was related to the durationThe relative expression levels of MutP53 protein,SLC7A11 protein and GPX4 protein decreased while P53 protein increased with the prolongation of Modified Shaoyao-Gancao Decoction within 24h.Conclusion:1.The the function of P53 was insufficient in the eutopic and ectopic endometrial tissues of AM patients,and the inhibitory function of P53 on SLC7A11/GPX4 was lost.AM eutopic and ectopic endometrium were overloaded with iron and had elevated lipid peroxidation levels,but their ability to resist ferroptosis was enhanced.2.Modified Shaoyao-Gancao Decoction reduced the size of AM tumor in nude mice,and the therapeutic effect was related to the improvement of lipid peroxidation level,reduction of antioxidant capacity,reduction of iron storage capacity of AM cells,and enhancement of sensitivity to ferroptosis through regulation of P53/SLC7A11/GPX4 pathway and related factors.3.Modified Shaoyao-Gancao Decoction serum restored the functional status of P53,increased the level of oxidative stress in endometrial stomal cells,and improved the sensitivity of endometrial stromal cells to ferroptosis.Modified Shaoyao-Gancao Decoction serum induced ferroptosis in endometrial stromal cells via P53/SLC7A11/GPX4 signaling pathway.